issn 2348-7410 prajnan o sadhona a science...
TRANSCRIPT
DEPARTMENT OF CHEMISTRY
FAKIR CHAND COLLEGE
PRAJNAN O SADHONA – A SCIENCE ANNUAL
VOL 2, 2015
ISSN 2348-7410
The Lycurgus Cup
Nanotube-Ice Cluster
Multi-walled Nanotubes
Quantum Dots
ISSN 2348-7410
Prajnan O Sadhona – A Science Annual
Volume 2, 2015
DEPARTMENT OF CHEMISTRY
FAKIR CHAND COLLEGE
DIAMOND HARBOUR
2
Prajnan O Sadhona – A Science Annual
Volume 2, 2015
Published by:
The Principal
Fakir Chand College
Diamond Harbour,
South 24 Parganas, West Bengal
January, 2015
Cover Design:
Dr. Prajnamoy Pal, Dr. Rana Karmakar
Dr. Moumita Sen Sarma, Dr. Tapas Kumar Mandal
Conceptualized from
Shashkov, E. V.; Everts, M.; Galanzha, E. I.; Zharov, V. P. Nano Letters 2008, 8, 3953-3958
http://worldofnanoscience.weebly.com/nanotube--carbon-fiber-overview.html
http://www.chem.uiowa.edu/people/tori-z-forbes
http://www.britishmuseum.org/explore/highlights/highlight_objects/pe_mla/t/the_lycurgus_cup.aspx
ISSN 2348–7410
Copyright © 2015. This is an open access journal. All open access journals are distributed under the terms of the
Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any
medium, provided that the original work is properly cited.
This journal is available online at website: http://www.fakirchandcollege.org
Prajnan O Sadhona ……., Vol. 2, 2015
3
EDITORIAL ADVISORY COMMITTEE
Editors: Dr. Prajnamoy Pal and Dr. Rana Karmakar
Associate Editors: Dr. Moumita Sen Sarma and Dr. Tapas Kumar Mandal
Editorial Board Members:
1. Dr. Subires Bhattacharyya Principal
Fakir Chand College, Diamond Harbour - 743331,
South 24 Parganas, West Bengal
2. Dr. Kalyan K. Mukherjee Professor in Chemistry
Jadavpur University, Jadavpur, Kolkata-700032
3. Dr. Debasish Bhattacharyya Chief Scientist
CSIR-Indian Institute of Chemical Biology
4, Raja S. C. Mullick Road
Jadavpur, Kolkata 700032
4. Dr. Debprasad Chattopadhyay
Deputy Director, ICMR Virus Unit
ID & BG Hospital, Beliaghata, Kolkata-700010
5. Dr. Debashis Bandyopadhyay Principal Scientist
Programme Management Division (CSIR)
Central Glass & Ceramic Research Institute
Jadavpur, Kolkata-700032
6. Dr. Pralay Das Assistant Professor & Scientist
CSIR-Institute of Himalayan Bioresource Technology
Palampur, Himachal Pradesh
4
Preface
Knowledge is defined as the familiarity, awareness or understanding of facts,
information’s, or skills acquired through experience or education by perceiving, discovering
or learning through theoretical or practical understanding of a subject. It can be implicit or
explicit understanding and formal or systematic. In philosophy, the study of knowledge is
called epistemology defined as "justified true belief" by Plato. Acquisition of knowledge
involves complex cognitive processes: perception, communication and reasoning. The
development of scientific method is usually based on the observable and measurable evidence
on the principles of reasoning and experimentation with collection of data, experimentation,
formulation and testing of hypotheses. Sir Francis Bacon described it as "knowledge is power",
while in modern society ‘repeated information is knowledge’ and repeated knowledge gives us
confidence; while repeated confidence leads to the success. In biological domains knowledge
must be usefully available to the system, though consciousness. Thus, in the age of information
technology, particularly the internet era, we still need printed information’s for the
dissemination of scientific knowledge through publication of Journals. As scientific knowledge
never claim to certainty, means that a scientist will never be absolutely certain about their
correctness until it is validated through everyday experience. It is thus an irony of scientific
method that one must doubt even when correct, in the hopes that this practice will lead to greater
convergence on the truth in general. Here comes the need of publications of scientific data for
its validation in the ‘human laboratory’ or nature.
Prajnan O Sadhona - A Science Annual is such a platform through which anybody
and everybody can express and nurture their ‘idea’ and ‘thoughts’ for self-realisation and
ultimate befit of the society. This Scientific Journal is metamorphosed from Spandan, the brain
child of the Department of Chemistry, Fakir Chand College, Diamond Harbour, West Bengal
since January 2013. In two years journey the infant is now ready to enter in the kindergarten
level and thus we appeal to the teaching and scientific community to extend their hands of co-
operation to nurture this child to its adulthood through love and affection to serve the humanity
at large.
Sd/-
Editorial Advisory Committee
Prajnan O Sadhona ……., Vol. 2, 2015
5
Contents
Research Article Pages
1. Optical control of excited state dynamics
Arnab Halder
2. Catalytic oxidation of alkanes and alcohols in presence of H2O2
using a polymer-anchored iron(III)-ferrocene complex
Kazi Tuhina
3. Terahertz time domain spectroscopy studies in liquids and solutions
Partha Dutta
4. Discussion on reciprocal graph from graph theoretical point of view
Piyali Ghosh
5. Studies on the anti-inflammatory effect of leishmanial lipid in vitro
and in vivo
Prajnamoy Pal, Subhadip Das, Nabanita Chatterjee,
Dipayan Bose and Krishna Das Saha
6. Studies on the effect of biofertilizer comprising of the plant growth
promoting rhizobacteria Bacillus thuringiensis on different types
of plants
Sandip Bandopadhyay and Swati Roy Gangopadhyay
Review Article
1. Synthesis and binding pattern of Calix[4]Pyrrole compounds
Debabrata Pal
2. Cytotoxic activity of organotin(IV) complexes - a short review
Moumita Sen Sarma
3. Quantum Dots and it method of preparations - revisited
Rana Karmakar
4. p300/CBP proteins: A HAT Trick to determine cell fate
Sanchari Kar
5. Vaccination and autism: a new perspective
Sanjukta Chaudhuri
6. Mechanism of action of antimicrobial peptides and proteins
Suranjana Chattopadhyay
7. Some synthetic and structural aspects of chromium nitrosyl
complexes
Swapna Ghosh
8. Biological importance of 4h-1-benzopyran and derivatives
Tapas Kumar Mandal
7 - 20
21 - 39
40 - 46
47 - 52
53 - 71
72 - 85
86 - 98
99 - 115
116 - 142
143 - 150
151 - 155
156 - 166
167 - 178
179 - 189
6
Halder, A.: Optical control of excited state dynamics
7
Research Article
Optical control of excited state dynamics
Arnab Halder
Department of Chemistry, Presidency University, 86/1 College Street, Kolkata – 700073, West
Bengal, India
Correspondence should be addressed to Arnab Halder; [email protected]
Abstract
In this article the author has reported on the field and one experimental result of optical
control of ultrafast processes with the help of programmable pulse shaping technique by use of
spatial light modulators. By modulating one or more laser pulse parameters, the shaped pulse
is generated. Multiparameter control shaped pulse is iteratively obtained through the feedback
mechanism and an automated optimization algorithm. I have presented an experimental result
of obtaining a shaped pulse that decreases the absolute magnitude of transmittance change of
a cyanine dye by controlling the decay pathway from S1 state to an energy state below S1 state.
Key Words: Optical control, Pulse shaping, Excited state dynamics
Introduction
The careful observation is the first step to understand nature. Advancement of ultrafast
laser technology enables us to observe with high spatial and temporal resolution. The next
should be the control of atoms and molecules involved in chemical reactions. The last two
decades saw remarkable progress in the development of experimental methods for the optical
control of chemical pathways and today, there is tremendous interest in the coherent control
process among researchers.1-7 Several experimental techniques have been developed for
various optical control concepts applicable to different quantum systems.1-3 Tsubouchi et al
examined a technique of complex shaping of mid-infrared femtosecond laser pulses towards
accurate and precise control of rovibrational wave packets of molecules in the ground
electronic state by a Germanium acousto-optics modulator.4 F van Hulst and coworkers
developed a universal method that allows full femtosecond pulse control in subdiffraction-
limited areas by exploiting the intrinsic coherence of the second harmonic emission from a
single nonlinear nanoparticle of deep subwavelength dimensions.5 Among the various
methods, femtosecond pulse shaping, one of the most powerful tools for the quantum control
Prajnan O Sadhona ……., Vol. 2, 2015
8
of photochemical reaction pathways, is now the most widely accepted tool by researchers. The
recent growth of coherent control studies is based on pulse shaping technology.2 By
phase/amplitude modulation, one may select the possible result of photoinduced physical or
chemical processes as optical phase properties influence the excited state. The femtosecond
pulse shaping is based on the use of spatial light modulator which is nothing but a spatially
patterned amplitude and phase masks modulates phase and amplitude of ultra short pulse.2 A
phase mask selectively delays colors and an amplitude mask shapes the spectrum. There are
three types of spatial light modulators: Liquid crystal arrays, Acousto-optic modulators,
Deformable mirrors. Among them, liquid crystal SLM is widely used due to its efficiency in
both phase and amplitude modulation.
Generally, an optimization algorithm is used to form a shaped laser pulse obtained by
automated search, via iteration loops, and then the phase-modulated laser pulse is utilized to
achieve the desired outcome.2-3 By manipulating coherent light-matter interaction with the
shaped pulse obtained by an appropriate program used with a feedback loop that optimizes the
pulse, various chemical dynamics may be controlled to produce many interesting results.3
Genetic algorithm (GA) is suitable for this purpose.9-11
Many groups are already succeeded to control different physical and chemical
processes. Using GA, Assion et al. selectively controlled the photodissociation reactions of
metal carbonyl complex in the gas phase.9 Taking practical importance into consideration, one
may be interested in relevant work in the liquid phase.10 Numerous experiments and
applications on a variety of condensed phase quantum systems have been realized using these
Figure 1: Schematic diagram of pulse shaping technique using SLM
Halder, A.: Optical control of excited state dynamics
9
control methods. The control of two-photon excitation efficiency,12-14 photoisomerization
(primary step of vision),14-16 bond activation,17 femtosecond photoassociation of thermally hot
atoms in the gas phase,18 molecular switching processes,19 and different photoinduced
processes9-10 has been demonstrated by many workers. Adaptive laser pulse shaping has
enabled to control the photophysical processes in complex molecules. Kuroda et al. were able
to enhance the emission quantum yield of a donor-acceptor macromolecule (a phenylene
ethynylene dendrimer tethered to perylene) by 15% through iterative phase modulation of the
excitation pulse and isolated the dominant elements underlying the control mechanism.20
Scientists have also started to control biological system. Prokhorenko et al. succeeded to
control the isomerisation of 13-cis retinal isomer of bacteriorhodopsin.21 Mayumi et al.
succeeded to control of ultrafast cis-trans photoisomerization of retinal in rhodopsin via a
conical intersection by optimally designed pulses, consist of shaping subpulses that prepare a
wave packet, which is localized along a reaction coordinate and has little energy in the coupling
mode, through multiple electronic transitions.22 Very recently, orbital and spin dynamics in a
solid-state defect has been optically controlled by using picosecond resonant pulses of light.23
Not only in ground state, but also, people have examined a technique of complex shaping of
mid-infrared femtosecond laser pulses towards accurate and precise control of wave packets of
molecules in the ground electronic state.24 However, much more information of the energy
levels and the excited state properties of the system is required to gain a thorough understanding
of the detailed mechanism.
Carbocyanine dyes are important for their use as saturable absorbers in mode-locked
laser systems.25 Because they have strong absorption bands ascribed to the S0 → S1 transition
in the visible and IR regions. Recently, Misawa and Kobayashi studied the dependence of wave
packet dynamics of a cyanine dye on the chirp rate of the excitation pulse because transient
processes depend on the temporal form of the optical pulse,26 and found a dramatic difference
in the temporal behaviour of the transmittance with negatively and positively chirped excitation
pulse. Using chirped laser pulse, they investigated the mechanism for the control of wave
packet dynamics. Very recently, Horikoshi et al. observed wave-packet dynamics, dependent
on the pulse chirp by developing a sensitive wave packet spectrometer.27 As many excited state
processes are closely related to vibrational wave packet dynamics, it may be used as a reference
for coherent control.28 Pulse shaping techniques are more efficient for the control scheme than
single-parameter control, such as the use of chirped excitation pulse. In this work, instead of
chirped pulse, we studied the effect of phase-controlled shaped pulse on the transient
Prajnan O Sadhona ……., Vol. 2, 2015
10
absorption of the cyanine dye, 3,3’-diethylthiatricarbocyanine iodide (DTTCI), in methanol
solution as the femtosecond shaped pulse is more selective than linear chirping. The basic aim
of this work was to observe the effect of shaped pulse used as the excitation pulse for S0 → S1
transition of the DTTCI molecule on the transmittance change of an ordinary probe pulse. Our
study may provide a crude idea of the control of the decay pathway from the S1 state of DTTCI
by adaptive pulse shaping. As already discussed above, spectral phase modulated laser pulse is
capable of controlling the desired target, and the main goal of this study was to observe the
effect of pulse shaping on the excited state dynamics of the DTTCI molecule. In this work, we
were able to control the said decay pathway using the pulse shaping technique. The most
important finding is that the absolute value of transmittance change is drastically decreased by
the shaped pulse.
Experimental
The femtosecond laser system consisted of a mode-locked Ti:sapphire laser (Mira Seed,
Coherent) pumped by an Nd:YVO4 laser (Verdi-6, Coherent). Center wavelength of the
exciting laser beam was 795 nm, pulse width was approximately 50 fs, and repetition rate was
76 MHz (Fig. 2). A 4f-configuration zero-dispersion compressor setup (1200 grooves/mm
grating) was used for spatial dispersion of the frequency spectrum and recollimation of the
laser pulse. The spatially dispersed laser pulse was focused on a liquid-crystal display (LCD)
in a spatial light modulator (SLM-256-NIR, CRI). Pulse shape control was achieved by
applying a specific voltage to each pixel in the LCD and modulating the phase spectra of the
input pulse in the frequency domain. Transmitted pulses were then reassembled to shaped pulse
with a cylindrical output lens and a grating. The zero-order reflection beam from the input
grating of the pulse shaper was used as the probe beam of the ordinary pump-probe experiment.
The beam of the shaped pulse was modulated at 672 Hz by a mechanical chopper, and scattered
light from the chopper was monitored by a photodiode and used to measure laser power. The
shaped pulse and the probe pulse were focused on the same position of a sample cell after
passing through optical delay lines. The intensity of the transmitted probe pulse was monitored
by a photomultiplier after passing through a filter and a monochromator, and the modulated
component corresponding to the transient absorption was subjected to phase-sensitive detection
using a lock-in amplifier. The emission intensity from the sample was also monitored by
another photomultiplier after passing through another monochromator and filter
simultaneously.
Halder, A.: Optical control of excited state dynamics
11
Figure 2: Experimental Set up for pulse shaping
During optimization, the blended crossover method and the minimal generation gap
method were adopted as the evolution operators in GA. Pulse shape was modified to optimize
the fitness, the ratio of laser power (ILP) to the absolute value of transmittance change (ITr), i.e.,
ILP / ITr, or the ratio of emission intensity at 850 nm (IEm) to the absolute value of transmittance
change (ITr), i.e., IEm / ITr.
DTTCI (Exciton) and methanol (Wako) were used as received. Methanol solution of 1
mM DTTCI was circulated in a 0.2 mm thick quartz flow cell.
Results and discussion
The temporal behaviour of the transmittance of the probe pulse was measured as a
function of delay time between pump pulse and probe pulse. The transmittance change at the
negative delay time corresponding to approximately 13.7 ns delay from the previous pump
pulse shows a negative value (Fig. 3a) that indicates the increase of absorbance by the pump
pulse irradiation.
Prajnan O Sadhona ……., Vol. 2, 2015
12
Figure 3: Transmittance change in the presence of DTTCI/methanol solution as function of
delay time with (a) single pulse and (b) shaped pulse
In the case of the pump-probe experiment that is related to only two level systems, S0
and S1, the transmittance change should be positive and converge to zero. In our experiment,
however, the transmittance change has a negative value and its magnitude at the positive delay
time is comparatively smaller than that at the negative delay time, -10 ps (Fig. 3a). This would
be ascribed to the presence of pump-induced absorption from an energy state that lies just
below the S1 state of the DTTCI molecule (Fig. 4) and has a lifetime much longer than 14 ns
(time interval between the two successive pump pulses). Hereafter, the long-lifetime energy
state is abbreviated as transit state.
Figure 4: Proposed scheme for excitation and decay pathway from S1 and transient absorption
of DTTCI molecule
From the temporal profile of the transmittance change, it is clear that after excitation
to the S1 state, transmittance is relatively increased as a result of ground state bleaching or
Halder, A.: Optical control of excited state dynamics
13
stimulated emission, and then the molecules start to decay to the transit state. However, the
magnitude of the transmittance change is small up to 10 ps (Fig. 2). This would be attributed
to the fact that the time constant for the decay pathway from the S1 state to the transit state is
much larger than 10 ps. The large negative value of the transmittance change at negative delay
time is due to the absorption of long-lifetime DTTCI molecules in the transit state and the
absolute value of the transmittance change is found to be directly proportional to the laser
power. In this context, it should be mentioned that the lifetime of DTTCI molecules in the S1
state is about 1.3 ns.29
In our optimization experiment, we succeeded in decreasing (approximately 0.64 times
in comparison with an ordinary single pulse) the absolute value of the transmittance change by
using the optimized shaped pulse (Table 1). We used the evaluation function to optimize the
excitation pulse shape is ILP / ITr at two different delay times (-10 ps and 1 ps) as our aim is to
change the contribution of long-lifetime components in the transit state. One significant
finding of our optimization experiment is that the maximum value of the evaluation function
is obtained within the range of 1.3-1.4.
Table 1: Magnitude of transmittance change, laser power, and emission intensity with single
pulse and shaped pulse optimized using the evolution function of ILP / ITr
Nature of Pulse
Laser
Power
(arb. unit)
(ILP)a
Emission
Intensity at
850 nm
(arb. unit)
(IEm) a
Absolute
Value of
Transmittance
Change
(arb. unit)(ITr) a
Em
Tr
I
I Em
LP
I
I
Tr
LP
I
I
Single Pulse 1 1 1 1 1 1
Shaped Pulse
(optimized at -10 ps) 0.85 0.84 0.64 1.32 0.99 0.75
Shaped Pulse
(optimized at 1 ps) 0.85 0.83 0.63 1.35 0.98 0.74
a Normalized to data corresponding to the single pulse
From Table 1, it is clear that the excitation pulse energy (laser power) is decreased to
approximately 0.85 times (compared to the single pulse) with the optimized pulse. This
decrease in laser power results in 0.84 times decrease in the emission intensity at 850 nm with
the shaped pulse (Table 1). In this context, it is noted that the value of IEm/ILP is 0.99, which
Prajnan O Sadhona ……., Vol. 2, 2015
14
means that the excitation efficiency of the optimized shaped pulse is the same as that of the
unshaped single pulse.
From the optimization experiment with the evaluation function IEm/ITr at the negative
delay time (-10 ps), we observed that the emission intensity at 850 nm is decreased to
approximately 0.9 times with the optimized excitation pulse as a result of the small decrease in
laser power, as we have already mentioned that the excitation efficiency remains unchanged
with the shaped pulse. However, the magnitude of change in the transmittance intensity is
decreased to approximately 0.65 times of that obtained by the single pulse and the maximum
value of the evaluation function is approximately 1.4. The change of IEm/ITr (also ILP/ ITr) is not
due to the intensity decrease of the pump pulse because the magnitude of transient absorption
is proportional to the pump intensity, as described above. This indicates that the genetically
evolved optimized laser pulse is capable of controlling the magnitude of transmittance change
at negative delay time. The decrease in the magnitude of transmittance change obtained by the
shaped pulse can be attributed to the presence of a relatively small number of DTTCI molecules
in the transit state. This may be due to the fact that the decay rate from the S1 state to the transit
state is decreased by the shaped pulse as the absorption of DTTCI molecules lying in the transit
state is responsible for the transmittance change at negative delay time.
On the assignment of the transit state, DTTCI molecule is known to undergo the trans-
cis photoisomerization following the S1←S0 transition.29 Namely, the DTTCI molecules
excited to the S1 state by the optimized laser pulse would have two different fates. One is the
return to the S0 state by radiative or non-radiative pathway, and another is the non-radiative
decay from S1 to the ground state of photoisomer. In a previous work, Fouassier et al. observed
the photoisomerization process in DTTCI molecules and reported the absorption maximum at
800 nm.30 Sahyun and Serpone reported the photoisomerization involving some radiationless
deactivation of photoexcited cyanine dyes.31 However, according to Kovalenko et al,
photoisomerization is usually observed when the molecules are excited at the blue end of the
absorption band with very high pulse energy.32 We excited DTTCI molecules at the red end
(795 nm) and the pulse energy of our setup is much lower (approximately 1 nJ) than the
required amount. It should be noted that Lee et al. did not observe the photoisomerization
process even with the pulse energy of approximately 40 nJ.33 In order to assign the nature of
the transit state, therefore, the further investigation using time-resolved spectroscopy is needed
and, we are unable to assign the nature of the transit state in this study.
If the decay rate from S1 to the transit state is decreased without affecting the
radiative/non-radiative pathway to the ground state, the population in the transit state will be
Halder, A.: Optical control of excited state dynamics
15
decreased. This leads to the decrease in the absolute value of the transmittance change at
negative delay time. In this context, one may think about the increase in emission intensity as
the rate of non-radiative decay pathway to the transit state is decreased with the shaped pulse.
However, if the shaped pulse opens some other relaxation pathway that competes with the non-
radiative decay pathway to the transit state, the population in that unknown state will be
decreased without hampering the radiative pathway, i.e., emission from S1. Thus, it may be
concluded that the shaped pulse obtained by GA and the feedback mechanism is capable of
decreasing the rate of decay from S1 to the transit state. However, we were unable to accelerate
this decay process by using ITr/ILP as the evaluation function.
The shape of the optimized pulse was derived by cross correlation with the ordinary
probe pulse (single pulse) using a second harmonic BBO crystal. Figure 5 shows the cross
correlation trace of the shaped pulse.
Figure 5: (a) Cross correlation trace of shaped pulse optimized at –10 ps delay with ordinary
probe pulse (single pulse) (b) Cross correlation trace of shaped pulse optimized
at 1 ps delay with ordinary probe pulse (single pulse)
Cross correlation traces of the shaped pulses optimized at – 10 ps and 1 ps delay times
are almost the same in shape (Fig. 5a and 5b). Clearly, the optimized pulse is very complex in
shape. Actually, the pulse envelope consists of four different groups of pulses, each of which
consists of 2-4 pulses. The interval between the small pulses is approximately 0.22 ps, whereas
that between two groups of pulses is approximately 0.67 ps.
Prajnan O Sadhona ……., Vol. 2, 2015
16
Fourier transform of the temporal profile of the cross correlation trace obtained by the
shaped pulse that controls the decay pathway from the S1 state, gave two intense peaks along
with few other weak peaks in the power spectrum in the frequency domain (Fig. 6).
Figure 6: (a) Fourier transform of temporal profile of the cross correlation trace obtained by
the shaped pulse optimized at -10 ps delay (b) Fourier transform of temporal
profile of the cross correlation trace obtained by the shaped pulse optimized at 1
ps delay
The peak frequency around 150 cm-1 is similar to the wave-packet oscillation of the
DTTCI molecule. Misawa and Kobayashi reported this type of vibration around 160 cm-1.26
According to them, this vibration is due to torsional motion of the bond connecting the two
rings and this type of vibrational motion may be coupled strongly with the electronic transition
of DTTCI molecules. In a very recent work, Misawa and co-workers reported a vibrational
wave packet having 4.5 THz frequency, i.e., 150 cm-1, in the excited state of the DTTCI
molecule.27
Another peak appearing around 50 cm-1 may be obtained by the Fourier transform of
some other oscillatory motions. We have already mentioned that the time interval between the
two groups of pulses is approximately 600-800 fs and this periodical motion can be attributed
to the second peak (at approximately 50 cm-1) in the frequency domain. There are two
candidates for the origin of the low frequency peak: one is the intermolecular librational motion
Halder, A.: Optical control of excited state dynamics
17
between solute and solvent molecules,34-36 and the other is low frequency intramolecular
vibrational motion, such as large amplitude motion.37-38 Regarding librational motion, optical
heterodyne detected Raman-induced Kerr effect spectroscopy (OHD-RIKES),34 dielectric
relaxation study,35 and terahertz time domain spectroscopy36 have demonstrated that the
spectrum in the frequency region lower than 100 cm-1 is the characteristic region of librational
motion as a result of solute-solvent interaction. Regarding the large amplitude intramolecular
vibrational motion, the phenyl groups at both ends of the stilbene molecule show vibrational
motion at frequencies lower than 100 cm-1.38 It is, therefore, natural to consider that the
vibrational motion involving benzothiazole groups at both ends of the DTTCI molecule has
frequencies lower than 100 cm-1. Thus, the peak at approximately 50 cm-1 can be interpreted
as a special kind of intermolecular vibration or a large amplitude intramolecular vibration that
decreases the rate of the decay pathway to the transit state. Since the Fourier spectrum of the
cross correlation trace of the complex shaped pulse gives a qualitative indication of different
types of vibrational dynamics of DTTCI molecules, we conclude that the different types of
oscillations may have some linkage to the decay pathway from S1 to the transit state.
Conclusions
Control of the molecules as well as of the chemical processes in nature may be the next
possible scientific goal and using programmable ultrafast Pulse Shaping technique many
physical as well as chemical processes have already been controlled.
We have obtained completely different transient absorption of DTTCI molecules by
using an optimized shaped pulse. The excited state dynamics of DTTCI is dependent on the
nature of the excitation pulse. The phase modulated pulse obtained from the optimization
experiment is capable of controlling the decay pathway from the S1 state to the transit state of
DTTCI, thereby decreasing the population in that energy state below the S1 state. In
consequence, the absolute value of the transmittance change is decreased by the GA optimized
pulse. The temporal behaviour of the optimized pulse is very complex in nature and different
periodical motions of that shaped pulse may be coupled with some intra and intermolecular
vibrational modes of DTTCI molecules and theses vibrational modes control the decay
pathway from S1 state.
References
1. Nurenberger, P.; Vogt, G.; Brixner, T.; Gerber, G. Femtosecond quantum control of
molecular dynamics in the condensed phase. Phys. Chem. Chem. Phys. 2007, 9, 2470.
Prajnan O Sadhona ……., Vol. 2, 2015
18
2. Winer, A. M. Femtosecond pulse shaping using spatial light modulators. Rev. Sci.
Instrum. 2000, 71, 1929.
3. Goswami, D. Optical pulse shaping approaches to coherent control. Phys. Rep. 2003,
374, 385.
4. Tsubouchi, M.; Momose, T. Pulse shaping and its characterization of mid-infrared
femtosecond pulses: Toward coherent control of molecules in the ground electronic
states. Opt. Comm. 2009, 282, 3757.
5. Accanto, N.; B Nieder, J.; Piatkowski, L.; Castro-Lopez, M.; Pastorelli, F.; Brinks, D.;
F van Hulst, N. Phase control of femtosecond pulses on the nanoscale using second
harmonic nanoparticles. Light: Science & Applications 2014, 3, e143.
6. Rice, S. A.; Zhao, M. Optical Control of Molecular Dynamics. Wiley, New York, 2000.
7. Rice, S. A.; Shah, S. P. Active control of product selection in a chemical reaction: a
view of the current scene. Phys. Chem. Chem. Phys. 2002, 4, 1683.
8. Brixner, T.; Damrauer, N. H.; Niklaus, P.; Gerber, G. Photoselective adaptive
femtosecond quantum control in the liquid phase. Nature 2001, 414, 57.
9. Assion, A.; Baumert, T.; Bergt, M.; Brixner, T.; Kiefer, B.; Seyfried, V.; Strehle, M.;
Gerber, G. Control of Chemical Reactions by Feedback-Optimized Phase-Shaped
Femtosecond Laser Pulses. Science 1998, 282, 919.
10. Levis, R. J.; Menkir, G. M.; Rabitz, H. Selective Bond Dissociation and Rearrangement
with Optimally Tailored, Strong-Field Laser Pulses. Science 2001, 292, 709.
11. Mizoguchi, R.; Onda, K.; Kano, S. S.; Wada, A. Optimization of resonant two-photon
absorption with adaptive quantum control. Rev. Sci. Instrum. 2003, 74, 2670.
12. Okada, T.; Otake, I.; Mizoguchi, R.; Onda, K.; Kano, S. S.; Wada, A. Optical control
of two-photon excitation efficiency of alpha-perylene crystal by pulse shaping. J. Chem.
Phys. 2004, 121, 6386.
13. Otake, I.; Kano, S. S.; Wada, A. Pulse shaping effect on two-photon excitation
efficiency of α-perylene crystals and perylene in chloroform solution. J. Chem. Phys.
2006, 124, 014501.
14. Vogt, G.; Krampert, G.; Niklaus, P.; Nurenberger, P.; Gerber, G. Optimal Control of
Photoisomerization. Phys. Rev. Lett. 2005, 94, 068305.
15. Hoki, K.; Brumer, P. Mechanisms in Adaptive Feedback Control: Photoisomerization
in a Liquid. Phys. Rev. Lett. 2005, 95, 168305.
16. Dietzek, B.; Bruggemann, B.; Pascher, T.; Yartsev, A. Mechanisms of Molecular
Response in the Optimal Control of Photoisomerization. Phys. Rev. Lett. 2006, 97,
Halder, A.: Optical control of excited state dynamics
19
258301.
17. Snee, P. T.; Yang, A.; Kotz, K. T.; Payne, C. K.; Harris, C. B. Ultrafast Infrared Studies
of the Reaction Mechanism of Silicon−Hydrogen Bond Activation by η5-CpV(CO)4. J.
Phys. Chem. A. 1999, 103, 10426.
18. Rybak, L.; Amitay, Z.; Amaran, S.; Kosloff, R.; Tomza, M.; Moszynski, R.; Koch, C.
P. Femtosecond coherent control of thermal photoassociation of magnesium atoms.
Faraday Discuss. 2011, 153, 383.
19. Feringa, B. L. Molecular Switches, Wiley-VCH, Weinheim, 2001.
20. Kuroda, D. G.; Singh, C. P.; Peng, Z.; Kleiman, V. D. Mapping Excited-State Dynamics
by Coherent Control of a Dendrimer’s Photoemission Efficiency. Science 2009, 326,
263.
21. Prokhorenko, V. I.; Nagy, A. M.; Waschuk, S. A.; Brown, L. S.; Birge, R. R.; Miller,
R. J. D. Coherent Control of Ratinal Isomerisation in Bacteriorhodopsin. Science 2006,
313, 1257.
22. Abe, M.; Ohtsuki, Y.; Fujimura, Y.; Domcke, W. Optimal control of ultrafast cis-trans
photoisomerization of retinal in rhodopsin via a conical intersection. Journal of
Chemical Physics 2005, 123, 144508.
23. Bassett, L. C.; Heremans, F. J.; Christle, D. J.; Yale, C. G.; Burkard, G.; Buckley, B.
B.; Awschalom, D. D. Ultrafast optical control of orbital and spin dynamics in a solid-
state defect. Science 2014, 345, 1333.
24. Tsubouchi, M.; Momose, T. Pulse shaping and its characterization of mid-infrared
femtosecond pulses: Toward coherent control of molecules in the ground electronic
states. Optics. Comm. 3757.
25. Mocker, H. W.; Collins, R. J. Mode Competition and Self-Locking Effects in a Q-
Switched Ruby Laser. Appl. Phys. Lett. 1965, 7, 270.
26. Misawa, K.; Kobayashi, T. Wave-packet dynamics in a cyanine dye molecule excited
with femtosecond chirped pulses. J. Chem. Phys. 2000, 113, 7546.
27. Horikoshi, K.; Misawa, K.; Lang, R. Rapid motion capture of mode-specific quantum
wave packets selectively generated by phase-controlled optical pulses. J. Chem. Phys.
2007, 127, 054104.
28. Hauera, J.; Skenderovica, H.; Kompa, K.-L.; Motzkus, M. Enhancement of Raman
modes by coherent control in β-carotene. Chem. Phys. Lett. 2006, 421, 523.
29. Fouassier, J.-P.; Lougnot, D.-J.; Faure, J. Excited states and laser properties of
Prajnan O Sadhona ……., Vol. 2, 2015
20
tricarbocyanine dyes. Optics Comm. 1976, 18, 263.
30. Fouassier, J.-P.; Lougnot, D.-J.; Faure, J. Photoisomerization processes in the IR-140
laser dye. Optics Comm. 1977, 23, 393.
31. Sahyun, M. R. V.; Serpone, N. Photophysical processes of polymethine dyes. An
absorption, emission, and optoacoustic study on 3,3'-diethylthiadicarbocyanine iodide.
J. Phys. Chem. A 1997,101, 9877.
32. Kovalenko, S. A.; Ernsting, N. P.; Ruthmann, J. Femtosecond Stokes shift in styryl dyes:
Solvation or intramolecular relaxation. J. Chem. Phys. 1997, 106, 3504.
33. Lee, S.-H.; Lee, J.-H.; Joo, T. Deuterium isotope effect on the solvation dynamics of a
dye molecule in methanol and acetonitrile. J. Chem. Phys. 1999, 110, 10969.
34. Beard, M. C.; Lotshaw, W. T.; Korter, T. M.; Heilweil, E. J.; McMorrow, D.
Comparative OHD-RIKES and THz-TDS Probes of Ultrafast Structural Dynamics in
Molecular Liquids. J. Phys. Chem. A. 2004, 108, 9348.
35. Vij, J. K.; Hufnagel, F. Dielectric relaxation and libration spectroscopy of some aliphatic
ketones and their molecular behavior. J. Phys. Chem. 1991, 95, 6142.
36. Oka, A.; Tominaga, K. Terahertz spectroscopy of polar solute molecules in non-polar
solvents. J. Non-Crystalline Solids 2006, 352, 4606.
37. Hunt, N. T.; Meech, S. R. Solvent dependence of low frequency vibrational modes: an
ultrafast optical Kerr effect study of diphenylmethane. Chem. Phys. Lett. 2003, 378,
195.
38. Chiang, W. Y.; Laane, J. Fluorescence spectra and torsional potential functions for
trans-stilbene in its S 0 and S 1(π,π*) electronic states. J. Chem. Phys. 1994, 100, 8755.
Tuhina, K..: Catalytic oxidation of alkanes……..
21
Research Article
Catalytic oxidation of alkanes and alcohols in presence of H2O2
using a polymer-anchored iron(III)-ferrocene complex
Kazi Tuhina
Assistant Professor, Department of Chemistry, B. S. College, Tangrakhali, Canning, South 24
Parganas, West Bengal, India
Correspondence should be addressed to Kazi Tuhina; [email protected]
Abstract
An eco-friendly, cheap and reusable polymer-anchored iron(III)-ferrocene Schiff base
catalyst has been designed for the efficient oxidation of alkanes and alcohols. Oxidation
reactions were done by using a greener oxidant 30% aqueous hydrogen peroxide in acetonitrile
medium at room temperature for alcohols and at 60oC for alkanes. Both the alkanes and
alcohols have been selectively oxidized to their corresponding aldehydes and ketones in
excellent yields. This catalyst has shown excellent catalytic activity, high selectivity and
recyclability. It was found that this catalyst can be reused up to six cycles without significant
loss of its activity.
Key Words: Alkane Oxidation, Alcohol Oxidation, Polymer Anchored, Iron Schiff Base
Catalyst, Hydrogen Peroxide
Introduction
Alkane oxidation is one of the basic reactions in industrial organic synthesis because
aldehydes and ketones are key intermediates for the manufacture of wide variety of valuable
products1. Therefore, alkane oxidation, under mild conditions, is a current challenge to modern
chemistry and the oxidation of the former by transition metal catalysts is a rather promising
approach1. However, the development and implementation of catalytic processes which
PS-Fe(III)-ferrocenecatalyst
60oC roomtemaprature
Prajnan O Sadhona ……., Vol. 2, 2015
22
eliminate the use of hazardous reactants and reduce the generation of waste, is an important
goal. The catalytic oxidation of inactivated C-H bonds of alkanes under mild conditions is
important in view of synthetic and industrial aspects2. Generally, catalytic oxidation of alkanes
under mild conditions is quite difficult, because of the lack of the reactivity in alkanes and of
their high C-H bond energy3,4. Conversion from alcohols to the corresponding aldehydes or
ketones is one of the most fundamental transformations in organic chemistry5. These include
(a) chromium- based reagents, such as Collins reagent (CrO3.Py2), PDC or PCC, (b) Swern
oxidation, Pfitzner–Moffatt oxidation, Parikh–Doering oxidation, (c) Oxidation by hypervalent
iodine compounds, (d) Ley-Oxidation and (e) catalytic TEMPO in the presence of excess
bleach (NaOCl)6. In these aforesaid processes, the reagents used are highly non eco-friendly
and poisonous in nature. Moreover these processes consume too much time and form unwanted
by-products. Cumbersome work-up procedures, use of toxic solvents and harsh reagents are
the main drawbacks of these processes which give rise to difficulties in the yield of
aldehyde/ketone from the corresponding alcohol. Hence, a search for other synthetic routes to
carbonyl compounds which would overcome at least some of the above confines is a matter of
present interest. That’s why, the selective oxidation of organic compounds by polymer-
anchored metal complexes is extensively studied to develop new synthetic strategies and
controlled oxidation of alcohol using a green oxidant is challenging work. In continuation of
our research interest in developing novel methodologies in organic synthesis using polymer
bound reagents, we became very much interested in investigating the oxidation of alcohols to
their corresponding aldehydes and ketones.
In the development of cost-effective and environmentally benign oxidation processes,
hydrogen-peroxide holds a prominent place among other oxidants7,9. Advantages of H2O2 are
low cost, high oxidation potential, eco-friendly nature and the formation of water as the by-
product. It is observed that H2O2 itself can’t afford the required high activation energy for
alkane oxidation. Therefore, a catalyst is required. Over the years, a wide range of catalysts
based on metals (-Ti, Mn, Fe, Cu, Ru etc.) have been designed for the in situ activation of H2O2
for the oxidation of alkanes and alcohols10-11.
Immobilization of active homogeneous catalysts on solid supports has attracted
enormous research interest because solid catalysts have the advantages of being easier to
recover and to recycle. Recently many studies are reported on the immobilization of Schiff
base and ligands on polymer supports12-14. The heterogeneous supports may be a range of
materials, including alumina15, amorphous silicates16, polymers17, zeolites18 and functionalized
Tuhina, K..: Catalytic oxidation of alkanes……..
23
mobil crystalline materials or functionalized MCM-4119. Application of a polymer supported
catalyst in oxidation reactions has been received attention in recent years due to their potential
advantages over the homogeneous ones20-22.
In this study, PS-ferrocene and PS-iron(III)–ferrocene complex was prepared and
applied for oxidation of alkanes and alcohols using 30% aqueous H2O2 as oxygen source. The
comparison between PS-ferrocene and PS-iron(III)-ferrocene complex was also studied in
catalytic oxidation of alkanes and alcohols. The recyclability experiments were performed to
see the catalytic activities.
Experimental
Materials:
Analytical grade reagents and freshly distilled solvents are used throughout the
experiment. Liquid substrates are pre-distilled and dried by appropriate molecular sieve.
Distillation and purification of the solvents and substrates are done by standard procedures23.
Chloromethylated polystyrene (5.5 mmol/g Cl), ferrocene-carboxaldehyde and other organic
substrates are supplied by Sigma-Aldrich chemicals Company, USA. Iron(III) chloride salt is
received from Merck and used without further purification.
Physical measurements:
Morphologies of the polymer-anchored ligand and complex is analyzed using a
scanning electron microscope (SEM)(ZEISS EVO40, England) equipped with EDX facility.
FT-IR spectra of the samples are recorded on a Perkin-Elmer FT-IR 783 spectrophotometer
using KBr pellets. Diffuse reflectance UV-Vis spectra are taken using a Shimadzu UV-2401
PC doubled beam spectrophotometer having an integrating sphere attachment for solid
samples. A Perkin-Elmer 2400 C elemental analyzer is used to collect microanalytical data (C,
H and N). The metal loading in the polymer is analyzed using a Varian AA240 atomic
absorption spectrophotometer (AAS).
Synthesis of catalyst:
The outline for the preparation of the polymer supported metal complex is given in
Scheme 1.
Prajnan O Sadhona ……., Vol. 2, 2015
24
Scheme 1: Synthesis of polymer supported metal complex
Synthesis of polymer-anchored Schiff base PS-ferrocene (4):
Polymer-anchored iron(III)–ferrocene complex can be prepared in two steps which is
shown in scheme 1. Polymer-anchored ligand (3) was prepared according to the literature24.
The chloromethylated polystyrene (1 g) is added to ethylenediamine (0.5 mL) in 20 mL THF
and then stirred for 48 h at room temperature. The light yellow coloured polymer beads are
filtered, washed thoroughly with methanol and dried in vacuum. Then this polymer supported
ligand (1 g) is kept in contact with 10 mL ethanol in a round bottom flask and to this, 10 mL
ethanolic solution of ferrocene-carboxaldehyde (1.177 g) is poured over a period of 45 min to
reflux for 24 h. Finally, grey coloured polymer supported Schiff base is filtered carefully,
washed with ethanol and dried in vacuum.
PS-ferrocene Schiff Base: C (%) = 66.89, H (%) = 6.58, N = 7.19, Fe (%) = 8.80.
Synthesis of immobilized catalyst:
PS-iron(III)-ferrocene is prepared by mixing 50 mL ethanolic solution of FeCl3 (0.892
g) with a PS-ferrocene (1.0 g) and the mixture is refluxed at 70 oC for 24 h in an oil bath under
inert atmosphere of argon. On cooling the solid polymer bound metal complex is filtered off,
washed several times by minimum amount of hot ethanol and dried under vacuum over
anhydrous CaCl2 to give the brown colour PS-iron(III)-ferrocene complex.
PS-iron(III)-ferrocene complex: C (%) = 60.11, H (%) = 6.09, N = 6.59, Fe (%) = 7.98.
CH2Cl +
Ethanol
THF
Ferrocene-
carboxaldehyde
room temparature
(1) (2) (3)
H2N
ferrocene
NH
NH2
NH2
NH N CHferroceneNH N CH
FeCl
Cl
ClH2O FeCl3,
Etanol, 70 oC(4)
(5)
70 OC
Tuhina, K..: Catalytic oxidation of alkanes……..
25
General experimental procedure for oxidation reaction of alkanes using H2O2 as oxidant:
In a 50 mL two necked round bottom flask a mixture of alkane (5.0 mmol), catalyst (50
mg) and 30% hydrogen peroxide (10 mmol) in acetonitrile (10 mL) is stirred at 60 oC for 10 h.
At the end of specified time, the contents are analyzed by Varian 3400 gas chromatograph
equipped with a 30 m CP-SIL8CB capillary column and a Flame Ionization Detector. Peak
position of various reaction products are compared and matched with the retention times of
authentic samples. Identity of the products is also confirmed by using an Agilent GC-MS.
General experimental procedure for oxidation reaction of alcohols using H2O2 as oxidant:
Catalytic reaction is carried out in a 50 mL two necked round bottom flask, which
charged with substrate (5 mmol), catalyst (50 mg), and 30% hydrogen peroxide (10 mmol) in
acetonitrile (10 mL) and stirred at room temperature for 10 h. The products are analyzed by
Varian 3400 gas chromatograph equipped with a 30 m CP-SIL8CB capillary column and a
Flame Ionization Detector. Identity of the products is also confirmed by using an Agilent GC-
MS.
Results and Discussion
Characterization of metal Schiff base complex:
Due to insolubilities of the polymer-anchored ligands and iron complex in all common
organic solvents, their characterizations are limited to their physicochemical properties,
elemental analysis, SEM, TGA, FT-IR, diffuse reflectance UV-vis and atomic absorption
spectroscopy which confirm the immobilization of iron onto polymer-anchored ligand.
Elemental analyses of the ligands and complex indicated the formulation of the complex.
Atomic absorption spectroscopy suggests 8.80% of iron in the PS-ferrocene (4) and 7.98% in
polymer-anchored iron(III)-ferrocene complex (5).
Scanning electron micrographs (SEM) and energy dispersive X-ray analyses (EDAX):
Field emission-scanning electron micrographs for polymer-anchored ferrocene
complex (4) and polymer-anchored iron(III)-ferrocene complex (5) are recorded to understand
the morphological changes which occurred on the polystyrene beads at various stages of the
synthesis.
The SEM images of the PS-ferrocene (A) and the PS-iron(III)-ferrocene (B) complex
are shown in Figure. 1.
Prajnan O Sadhona ……., Vol. 2, 2015
26
Figure 1: SEM images of polymer-anchored ligand (3) (A) and polymer-anchored iron(III)-
ferrocene complex (B)
The pure chloromethlated polystyrene bead has a smooth surface. After ligand (4)
formation and metal loading on polymer (5), a change in morphology of the polymer surface
is observed by SEM pictures. Also presence of metals along with oxygen and chlorine can be
further proved by energy dispersive spectroscopy analysis of X-rays (EDX) (Figure 2) which
suggests the immobilization of metal complex into the chloromethylated polystyrene bead.
Figure 2: EDX images of polymer-anchored ligand (3) (A) and polymer-anchored iron(III)-
ferrocene complex (B)
Tuhina, K..: Catalytic oxidation of alkanes……..
27
FT-IR spectral study:
The sharp C-Cl peak due to -CH2Cl group in polymer at 1264 cm-1 is found as weak
after loading of ethylenediamine on the support25. A strong broad band in the region from 3300-
3460 cm-1 in polymeric support is observed due to N-H (both secondary and primary amine)
stretching vibration. Polymer-anchored ligand (3) showed N-H bending vibration at 1640 cm-
1 (primary amine) and at 1491 cm-1 (secondary amine). Above IR data confirms the loading of
ethylenediamine on the polymer matrix. After condensation of polymer-anchored ligand (3)
with ferrocene-carboxaldehyde, polymer-anchored Schiff base, PS-ferrocene (4) has been
produced. PS-ferrocene exhibits a broad band around 1631 cm-1 due to the ν(C=N) stretching
vibration. The peaks at 827 and 1455 cm-1 related to C-H and C-C vibration of ferrocene appear
in PS-ferrocene which indicates that ferrocene has been loaded into the supports25. In polymer-
anchored iron(III)-ferrocene complex, the band respect to ν(C=N) shifted slightly towards
lower frequency. This suggests that the azomethine ‘N’ atom involved in coordination. In
polymer-anchored complex, new absorption bands at 520 and 312 cm-1 are assigned to ν(Fe-
N) and ν(Fe-Cl)25, respectively as shown in Figure 3.
Figure 3: FT-IR Spectra of Chloromethyl polystyrene (a), Polymer-anchored ligand (b), PS-
ferrocene (c) and polymer-anchored iron(III)-ferrocene complex (d)
TGA studies:
Thermal stability of complex is investigated using TGA at a heating rate of 10 oC/min
in air over a temperature range of 30-600 oC. TGA curve of polymer-anchored iron(III)-
ferrocene complex (Figure 4) shows that the mass loss is started at around 350 oC and this mass
loss is observed from 350 to 470 oC. So from the thermal stability, it concludes that polymer
supported metal complex degraded at considerably high temperature.
Prajnan O Sadhona ……., Vol. 2, 2015
28
Electronic spectral studies (DRS-UV spectroscopy):
The electronic spectrum (Figure 5) of the polymer supported metal complex is recorded
in diffuse reflectance spectrum mode as MgCO3/BaSO4 disc due to their solubility limitations
in common organic solvents. The diffuse reflectance UV–vis spectra of iron complex shows
three bands in the range 220-250 nm, 260-330 nm and 480-525 nm. The very low intensity
bands around 480-525 nm may be assigned to 6A1g →4A1g (G) and 6A1g →
4T2g (G) transition
in octahedral symmetry26. The other bands are due to LMCT transitions26.
Figure 4: Thermogravimetric weight loss
Plots for polymer supported
iron(III)-ferrocene Schiff base
complex
Figure 5: DRS-UV spectra of polymer-
anchored iron(III)-ferrocene
complex
Catalytic activity
Since polymer supported metal systems exhibit catalytic activity in a wide range of the
industrially important processes and have been extensively studied, we have decided to
investigate the catalytic activity of PS-ferrocene (4) and PS-iron(III)-ferrocene (5) complexes
in the oxidation of alkanes and alcohols in mild conditions. Selective oxidation of saturated
hydrocarbons under mild condition remains a challenging and attractive goal of contemporary
metal-complex catalysis27. Due to the high inertness of alkanes, their oxidations mainly undergo
in the presence of metal catalysts which require elevated temperature and pressure or strong
acidic medium3.
Effect of oxidants and solvents in toluene oxidation catalyzed by PS-iron(III)-ferrocene
complex (5):
To test the catalytic activities of this complex, oxidation of alkanes are examined at 60
oC. In search of optimal reaction conditions, the effects of various solvents and oxidants are
100 200 300 400 500 600
We
igh
t lo
ss (
%)
Temperature (0C)
200 300 400 500 600 700 800
Ab
so
rba
nce
(%
)
Wavelength (nm)
Tuhina, K..: Catalytic oxidation of alkanes……..
29
examined in the oxidation of toluene and the results are given in Table 1. The results showed
that acetonitrile is the best solvent (Table 1, run 5) and H2O2 is the best oxidant (Table 1, run
6) for the above oxidation reaction. It has been observed that amount of H2O2 played a crucial
role over oxidation reactions. Conversion increases when amount of H2O2 increases from 5
mmol to 10 mmol (Table 1, run 6) but there is no significant change in conversion when amount
changes to 15 mmol under the same reaction conditions (Table 1, run 7). With excess H2O2, the
yield of aldehyde decreases due to over oxidation of aldehyde to its corresponding acid (Table
1, run 8).
Under the optimized reaction conditions (as shown in Table 1), all the alkanes are
oxidized to their corresponding aldehydes or ketones, respectively with high yields. The results
are summarized in Table 2. Adamantane converts to 1-adamantanol with very high selectivity
(Table 2, entry 1). Toluene, 4-nitrotoluene, 4-chlorotoluene, cyclohexane are also oxidized
with good selectivity of aldehyde or ketones (Table 2, entries 2-4 and 12). Industrially
important ketones like acetophenone, propiophenone, benzophenone are synthesized with good
yields in the above oxidation process (Table 2, entries 5, 6 and 10). Position selective oxidation
has also been observed in case of n-octane, substituted cycloalkanes like phenylcyclohexane,
phenylcyclopentane, tetralin and indane (Table 2, entries 13-16). In all these cases 2 position
is selectively oxidized with high selectivity in presence of PS-iron(III)-ferrocene catalyst.
Table 1: Effect of different oxidants and solvents on oxidation of toluene with polymer-
anchored iron(III)-ferrocene complex
Entry Solvent Oxidant Benzaldehyde
Yield / Selectivitya (%)
1 CH3CN NaOCl 14/39
2 CH3CN NaIO4 26/43
3 CH3CN TBHP 23/57
4 CH3CN KHSO5 19/36
5b CH3CN H2O2 35/51
6c CH3CN H2O2 53/88
7d CH3CN H2O2 51/67
8e CH3CN H2O2 36/37
11 CH3CH2OH H2O2 33/59
12 i-PrOH H2O2 27/55
13 H2O H2O2 6/33
14f CH3CN H2O2 trace
Conditions: toluene (5 mmol); oxidant (10 mmol); CH3CN (10 mL); 50 mg catalyst at 60 oC
for 10 hr. (a Yield refers to GC & GC-MS analysis. b 5 mmol 30% H2O2 was used. c 10 mmol 30% H2O2 was
used. d 15 mmol 30% H2O2 was used. e 25 mmol 30% H2O2 was used. f Without any catalyst; 10 mmol 30%
H2O2 was used.)
Prajnan O Sadhona ……., Vol. 2, 2015
30
Comparison between PS-ferrocene (4) and PS-iron(III)-ferrocene (5):
The catalytic activity of PS-ferrocene with polymer-anchored iron(III)-ferrocene
complex is compared in the oxidation of alkanes and alcohols. Using PS-ferrocene as a catalyst
in oxidation of toluene, only 16% benzaldehyde is found while in case of polymer-anchored
iron(III)-ferrocene, it is 53% of same product in GC analysis under the same reaction
conditions (Scheme 2). It indicates that polymer-anchored iron(III)-ferrocene is more effective
in oxidation reaction than of PS-ferrocene. The same observation is found in case of other
alkanes and the results are summarized in Table 2.
Scheme 2: Oxidation product of toluene using PS-ferrocene and PS-iron(III)-ferrocene
Similar experiment is studied over benzyl alcohol. It gives only 29% benzaldehyde in
presence of PS-ferrocene where as using polymer-anchored iron(III)-ferrocene, we get 90%
same product in GC analysis remaining the reaction condition unchanged (Scheme 3). As
expected, the rest of the alcohols also oxidized more efficiently by PS-iron(III)-ferrocene than
by PS-ferrocene.
Scheme 3: Oxidation product of benzyl alcohol using PS-ferrocene and PS-iron(III)-ferrocene
Tuhina, K..: Catalytic oxidation of alkanes……..
31
Table 2: Oxidation of alkanes using 30% H2O2
Entry Alkane Product/s Yeild/Conversation(%)a,b
(Selectivity in %)
H2O2 conversion (%)
PS-ferrocene PS-
iron(III)-
ferrocene
PS-ferrocene PS-
iron(III)-
ferrocene
1
26/47
(55)
80/85
(94)
78 99
2
16/35
(46)
53/60
(88)
71 98
3
14/31
(45)
50/56
(89)
80 89
4
15/34
(44)
47/53
(89)
72 88
5
19/43
(44)
74/82
(90)
72
96
6
25/43
(58)
74/80
(93)
68
87
7
8
19/40
(47)
13/29
(45)
61/72
(85)
61/72
(85)
65
59
86
88
9
14/31
(45)
60/69
(87)
61 84
OH
CHO
O2N
CHO
O2N
CH3
Cl
CHO
Cl
O
O
Cl
MeO
Cl
O
MeO
O
O
Prajnan O Sadhona ……., Vol. 2, 2015
32
Conditions: alkanes (5 mmol); 30% aq H2O2 (10 mmol); CH3CN (10 mL); 50 mg catalyst at
60oC for 10 hr. (a Yield refers to GC & GC-MS analysis. b Products were identified by GC & GC-MS
analysis.)
Optimization conditions for alcohol oxidation catalyzed by PS-iron(III)-ferrocene complex
(5):
Oxidation of alcohol has its chemical and biological importance. It is often needed to
selectively oxidize the alcoholic part of a large organic moiety to its corresponding aldehyde
or ketone, without over oxidation to acid group28-30. Here acetonitrile is chosen as solvent and
hydrogen peroxide as an oxidant for benzyl alcohol oxidation in presence of the catalyst, which
has been treated as a probe reaction. In this study, benzyl alcohol selectively converted to
benzaldehyde with good yields in both 60oC and at room temperature. Not too much significant
change in yield is observed when the temperature drops down from 60oC to room temperature
10
27/52
(52)
76/83
(92)
71 96
11
9/16
(56)
37/45
(82)
52 69
12
11/18
(61)
41/49
(84)
54
68
13
22/35
(63)
67/77
(87)
63 84
14
19/31
(61)
63/70
(90)
63 82
15
28/47
(60)
77/88
(88)
83 97
16
30/52
(58)
81/93
(87)
87 99
O
C8H18
O
O
Ph Ph
O
PhPh
O
O
O
Tuhina, K..: Catalytic oxidation of alkanes……..
33
remaining the other conditions unchanged. So, room temperature is set as one of the optimized
conditions for the oxidation of alcohols as it is greener approach and under this condition all
the alcohols are converted to their corresponding aldehydes or ketones with very high yields.
Benzyl alcohol and its substituted derivatives show very good response in presence of
this catalytic system. The results are summarized in Table 3. Typical alcohol like 2,2-
dimethylpropiophenone is oxidized in high yields. Others industrially important ketones like
acetophenone, acetone, cyclohexanone etc. are obtained from the oxidation of alcohols by the
PS-iron(III)-ferrocene catalyst.
Table 3: Oxidation of alcohols using 30% H2O2
Entry Alcohol Product Yeild/Conversation(%)a,b H2O2 conversion (%)
PS-
ferrocene
PS-
iron(III)-
ferrocene
PS-
ferrocene
PS-
iron(III)-
ferrocene
1
29/43 90/94 78 99
2
25/40
89/92
72
99
3
26/37
87/91
68
98
4
31/47
88/92
80
99
5
27/42
86/94
72
99
6
19/39
82/87
70
94
7
21/40
68/74
71
87
OH O
HO
MeO
O
MeO
Cl
HO
Cl
O
NO2
HO
NO2
O
Br
HO
Br
O
HOH2C
Cl
OHC
Cl
OH O
Prajnan O Sadhona ……., Vol. 2, 2015
34
8
24/47
82/86
75
96
9
27/38
85/90
72
92
10
22/39
80/86
69
89
11
29/54
86/92
79
94
12
26/49
84/89
70
90
Conditions: alcohol (5 mmol); 30% aq H2O2 (10 mmol); CH3CN (10 mL); 50 mg catalyst at
room temparature for 10 hr. (a Yield refers to GC & GC-MS analysis. b Products were identified by GC
& GC-MS analysis).
Many efficient heterogeneous catalysts have been reported for the oxidation of alkanes
with 30% H2O231,32 (Table 4). Comparison of this catalyst with previously reported systems
reveals that the present system gives better conversion than other catalysts.
Table 4: Comparison with other reported catalysts
Catalyst Yield (%) Ref.
Cyclohexane Adamantane
Ps-Fe(III)-ferrocene 53 (ketone) 80 (alcohol) This study
LFeIII.SiO2 7.3 (ketone) - 32
aFe(salen)-POM - 66 (alcohol) 33
aReaction temperature 800C
OHO
O
OH
O
O
OH O
OH O
OH O
Tuhina, K..: Catalytic oxidation of alkanes……..
35
Stability and recycling of catalyst
To check the leaching of iron into the solution during the reaction, oxidation of toluene
is carried out under the optimum reaction conditions. The reaction is stopped after the reaction
proceeds 2 h. The catalyst is separated from the reaction mixture by filtration and the
conversion is determined. The separated filtrate is allowed to react for another 2 h under the
same reaction conditions, but no further increment in conversion is observed in gas
chromatographic analyses. The UV-vis spectroscopy is also used to determine the stability of
the heterogeneous catalyst. The UV-vis spectra of the reaction solution, at the first run, do not
show any absorption peaks characteristic of iron metal, indicating that the leaching of iron does
not take place during the course of the oxidation reaction. These results suggest that the catalyst
is heterogeneous in nature.
The recyclability of the catalyst is important for the catalysis reaction. The reusability
of polymer-anchored iron(III)-ferrocene is investigated in the oxidation of toluene and benzyl
alcohol (Figure 6). Catalyst is separated by filtration after the first catalytic run, washed with
solvent and dried under vacuum then subjected to the second run under the same reaction
conditions. The catalytic run is repeated with further addition of substrates under optimum
reaction conditions and the nature and yield of the final products are comparable to that of the
original one. It is found that the catalytic activity or selectivity does not change significantly
after six consecutive runs.
Figure 6: Recycling efficiency of the catalyst, PS-iron(III)-ferrocene in the oxidation of
toluene and benzyl alcohol
1 2 3 4 5 640
50
60
70
80
90
Yie
ld(%
)
Run
Toluene oxidation Benzyl alcohol oxidation
Prajnan O Sadhona ……., Vol. 2, 2015
36
Conclusions
In this study, a polymer-anchored iron(III)-ferrocene complex (5) is synthesized and
used as a heterogeneous catalyst for oxidation of alkanes and alcohols with H2O2 as an oxygen
source. The catalyst shows high catalytic activity and selectivity over corresponding aldehydes
and ketones. The catalytic activity between PS-ferrocene (4) and PS-iron(III)-ferrocene (5) is
compared. Interestingly, loading of iron(III) chloride to the PS-ferrocene complex increases
the catalytic activity remarkably during oxidation of alkanes and alcohols under mild
conditions. This catalyst is stable at high temperature, inexpensive and mostly easy to prepare.
The reusability of this catalyst is high and can be reused six times without significant decrease
in its initial activity.
Acknowledgements
I thank Dr. S. M. Islam, University of Kalyani for providing various supports for this
research work. Financial support from UGC through Minor research Project grant is gratefully
acknowledged.
References
1. (a) Grinstaff, M. W., Hill, M. G., Labinger, J. A., Gray, H. B. Mechanism of catalytic
oxygenation of alkanes by halogenated iron porphyrins. Science 1994, 264, 1311. (b)
White, M. C. Adding Aliphatic C-H Bond Oxidations to Synthesis, Science 2012, 335,
807.
2. Gormisky, P. E., White, M. C. Synthetic Versatility in C–H Oxidation: A Rapid
Approach to Differentiated Diols and Pyrans from Simple Olefins, Journal of the
American Chemical Society 2011, 133, 12584.
3. Sheldon, R.A., Kochi, J.K. Metal Catalyzed Oxidation of Organic Compounds.
Academic Press, New York, 1981.
4. Shilov, A.E., Shul’pin, G.B. Activation of C−H Bonds by Metal Complexes. Chem.
Rev. 1997, 97, 2879.
5. Hudlicky, M. Oxidations in Organic Chemistry (American Chemical Society),
Washington D.C., 1990.
6. Ley, S. V., Norman, J., Griffith, W. P., Marsden, S.P. Tetrapropylammonium
Perruthenate, Pr4N+RuO4
-, TPAP: A Catalytic Oxidant for Organic Synthesis. Synthesis
1994, 25, 639.
Tuhina, K..: Catalytic oxidation of alkanes……..
37
7. Hagen, J. Industrial Catalysis a practical Approach, 2nd edn., pp. 189-189 (Wiley-
Interscience, Weinheim, 2006.
8. Strukul, G., (Ed.), Catalytic Oxidations with Hydrogen Peroxide as Oxidant Klumer.
Academic Publishers, Dordrecht, Netherlands, 1992.
9. Muzart, J. Pd-mediated epoxidation of olefins. J. Mol. Catal. A: Chem. 2007, 276, 62.
10. Mizuno, N., Yamaguchi, K., Kamata, K. Coord. Epoxidation of olefins with hydrogen
peroxide catalyzed by polyoxometalates, Chem. Rev. 2005, 249, 1944.
11. Chen, M. S., White, M. C., Chen, M. S., White, M. C. A Predictably Selective Aliphatic
C-H Oxidation Reaction for Complex Molecule Synthesis. Science 2007, 318, 783.
12. A-E-Aziz A. et al. (Eds.) Chapt. 1 of Macromolecules Containing Metal and Metal-like
Elements, Vol. 1, A Half Century of Metal- and Metalloid-Containing Polymers
(Wiley-Interscience, Vol. 1), 2003.
13. A-E-Aziz A., Manners I. (Eds.) Chapter 1,"Organometalic Polymers: The Early Days",
C. U. Pittman, Jr. and C. E. Carraher, Jr. in Frontiers in Transition Metal-Containing
Polymers, Wiley Interscience, 2007.
14. A-E-Aziz A. S., Harvey P. (Ed.), “Organometallic and Coordination Clusters and
Polymers: Syntheses and Applications” Macromolecular Symposia. Wiley, Weinheim,
2004.
15. Dapurkar, S.E , Sakthivel, A., Selvam, P., Liquid Phase Oxidation via Heterogeneous
Catalysis: Organic Synthesis and Industrial Application, New J. Chem. 2003, 27, 1184.
16. Handzlik, J., Ogonowski, J., Stoch, J., Mikołajczyk, M. Comparison of metathesis
activity of catalysts prepared by anchoring of MoO2(acac)2 on various supports. Catal
Lett. 2005, 101, 65.
17. Mureseanu, M., Parvulescu, V., Ene, A. R., Cioatera, A. N, Pasatoiu, T.D., Andruh, M.
A. new example of coexistence of two different complexes in one crystal:
[{CuII(acac)(phen)}2CoII(NCS)4]·2[CuII(acac)(phen)(NCS)]. J. Mater. Sci. 2009, 44,
6795.
18. Lei, Z. Selective oxidation of cyclohexene catalyzed by polymer-bound ruthenium–
2,2′-bipyridine complexes. React. Func. Polym. 2000, 43, 139.
19. Salvati-Niasari, M. Synthesis, catalytic oxidation and antimicrobial activity of
copper(II) Schiff base complex. Inorg. Chem. Comnmun. 2005, 8, 174.
20. Nishiura, M., Hou, Z. Novel Polymerization Catalysts and Hydride Clusters from Rare
Earth Metal Dialkyls. Nature Chemistry 2010, 2, 257.
Prajnan O Sadhona ……., Vol. 2, 2015
38
21. Valodkar, V. B., Tembe, G. L., Ravindranathan, M., Rama, H. S. Catalyticoxidation of
alkanes and alkenes by polymer-anchored amino acid-rutheniumcomplexes. J. Mol.
Catal. A: Chem. 2004, 223, 31.
22. Maurya, M. R., Arya, A., Adao, P., Pesso, J. C. Oxidation of p-chlorotoluene and
cyclohexene catalysed by polymer-anchored oxovanadium(IV) and coper(I) complexes
of amino acid derived tridentate ligands. Appl. Catal. A: Gen. 2008, 351, 239.
23. Vogel, A. I. Text book of practical organic chemistry - Quantitative Analysis, 5th edn.,
Longman: London, 1998.
24. Burri, D. R., Shaikh, I. R., Choi, K. M., Park, S. E. Facile heterogenization of
homogeneous ferrocene catalyst on SBA-15 and its hydroxylation activity. Catal.
Comm. 2007, 8, 731.
25. Gnanasoundari, V. B., Natarajan, K. Use of a New Polymer Anchored Cu(II) Azo
Complex Catalyst for the Efficient Liquid Phase Oxidation Reactions Trans. Met.
Chem. 2004, 29, 511.
26. Antony, R., Tembe, G. L., Ravindranathan, M., Ram, R. N. Synthesis and catalytic
activity of Fe(III) anchored to a polystyrene-Schiff base support, J. Mol. Catal. A:
Chem. 2001, 171, 159.
27. Smith, J. R. L., Iamamoto, Y., Vinhado, F. S. Biomimetic oxidation studies of
monensin A catalyzed by metalloporphyrins: Identification of hydroxyl derivative
product by electrospray tandem mass spectrometr. J. Mol. Catal. A: Chem. 2006, 252,
23.
28. Ruiter, G. de J.S. Costa, Lappalainen, K., Roubeau, O., Gamez, P., Reedijk, J. A new
complex based on chelate copper coordination with divacant polyanion ligand [γ-
PW10O36]. Inorg. Chem. Commun. 2008, 11, 787.
29. Periana, R. A., Mironov, O., Taube, D., Bhalla, G., Jones, C. J. Catalytic, Oxidative
Condensation of CH4 to CH3COOH in One Step via CH Activation. Science 2003, 301,
814.
30. Kirillova, M. V., Kozlov, Y. N., Shul'pina, L. S., Lyakin, O. Y., Kirillov, A. M., ETalsi,
E. P., Pombeiro, A. J. L., Shul'pin, G. B. Remarkably fast oxidation of alkanes by
hydrogen peroxide catalyzed by a tetracopper(II) triethanolaminate complex:
promoting effects of acid co-catalyst and water, kinetic and mechanistic features, J.
Catal. 2009, 268, 26.
Tuhina, K..: Catalytic oxidation of alkanes……..
39
31. Bilis, G., Christoforidis, K. C., Deligiannakis, Y., Louloudi, M. Hydrocarbon oxidation
by homogeneous and heterogeneous non-heme iron(III) catalysts with H2O2, Catal.
Today 2010, 157, 101.
32. Mirkhani, V., Moghadam, M., Tangestaninejad, S., Mohammadpoor-Baltork, I.,
Rasouli, N. Oxidation of alkanes with hydrogen peroxide catalyzed by Schiff base
complexes covalently anchored to polyoxometalate. Catal. Commun. 2008, 9, 2171.
Prajnan O Sadhona ……., Vol. 2, 2015
40
Research Article
Terahertz time domain spectroscopy studies in liquids and
solutions
Partha Dutta
Assistant Professor, Department of Chemistry, Maharaja Manindra Chandra College,
20, Ramkanto Bose Street, Kolkata – 700003, West Bengal, India
Correspondence should be addressed to Partha Dutta; [email protected]
Abstract
Understanding solvent motion and solvent–solute interaction in solution is a central
issue to elucidate solvent effects on chemical reactions and various relaxation processes in
liquids and solutions. The intermolecular interaction often shows its characteristic frequencies
in the region below 100 cm-1. In this low-frequency region various motions such as librational
motion, intermolecular vibration, collective motions of associated liquids and reorientational
relaxation have specific spectral components. Terahertz time-domain spectroscopy (THz-TDS)
has been proven to be a powerful technique for studying these spectral components in low
frequency regions. Different theoretical and analytical models may be used to get an insight of
the experimental THz-TDS data.
Key Words: Liquids, Solutions, Dynamics, Low Frequency, Terahertz time-domain
spectroscopy
Introduction
The THz regime is sandwiched between the microwaves and the infrared, bridging the
gap between electronics and optics. For a long time, the THz regime was also referred to as the
“THz gap” as neither optical nor microwave devices could fully conquer this shadowy domain
with its many hidden scientific treasures.1, 2 Little commercial emphasis was placed on the
development of THz systems as the available sources, such as synchrotrons, backward wave
oscillators, Smith-Purcell emitters or free electron lasers, were very costly components.
But in the past two decades dramatic progress has been made in the generation and
detection techniques of freely propagating THz radiation based on femtosecond pulsed laser
which is cheaper compared to the previously mentioned instruments.1 Because the pulse
Dutta, P.: Terahertz time domain spectroscopy ……..
41
duration of the THz radiation is in a sub-picosecond time region, it is possible to measure the
electric field of the radiation by coherent detection methods, which consequently allows to
conduct THz-TDS. The refractive index and extinction coefficient of a medium can be easily
obtained by measuring the phase and amplitude of the THz radiation. This technique is a type
of transmission spectroscopy and is suitable for studying the properties of the liquids and also
the characteristics of solute molecules in solutions since the contribution of the solvent can be
subtracted from the spectrum of the solution.2 Thus, THz-TDS is an attractive method for the
detection of low frequency spectra and also for studying dynamics with time scales of sub-
picoseconds and picoseconds of various kinds of material, such as liquids and mixtures of
liquids,3,4 solutions of organic solvents,2,5-8 biological molecules,9-11 and ionic liquids and their
mixtures with other solvents12-13.
There has been considerable interest in both the experimental and theoretical
investigation of the low-frequency motion associated with molecules and molecular aggregates
in condensed phases. Vibrational motions with resonance frequencies in the terahertz (THz; 1
THz = 1012 Hz) frequency range are characterized by weaker potential forces and/or larger
reduced masses, which are in sharp contrast to vibrations localized within a molecule with
resonance frequencies in the mid infrared (IR) region.14-15 In this article the special emphasis
will be given to discuss the studies of condensed phases such as solutions and liquids namely
water and methanol by THz-TDS technique.
Discussion
(a) Liquids:
Hydrogen bonding liquids such as water and methanol form characteristic network
structures, which dynamically fluctuate by making and breaking hydrogen bonds. The structural
fluctuation of the network has a great influence on chemical reactions and relaxation processes.1,16
Water shows a tremendous absorption in the low frequency domain. Water can form a
three-dimensional network structure. Low frequency spectra of water show two distinct regions in
the low frequency region, one around 60 cm-1 and other around 170 cm-1.16 The lower region may
be attributed to the frustrated translations due to the local structure around a given molecule
that produces the so-called cage effect. The librational motion of the molecules in a cage may
affect this kind of band. On the contrary, the frequency band around 170 cm-1 is absent in the
spectral densities of non-associated liquids, which is consistent with the attribution of this band
to the stretching of hydrogen-bonded molecules.16 Thus for the liquid water, the low frequency
band should not be associated with the existence of hydrogen bonding. The experimental
Prajnan O Sadhona ……., Vol. 2, 2015
42
observations have been supported by different theoretical interpretations reported earlier
also.17-18 The low-frequency dynamics of water is also characterized by a distribution of the time
scale of dynamics. Actually in water there are different kinds of hydrogen bonding networks with
different sizes and conformation. Each network has its own characteristic time scale of dynamics.1
Methanol is the simplest alcohol, and its hydrogen bond dynamics have been investigated
by femtosecond laser spectroscopy such as time-dependent fluorescence Stokes shift and OKE
spectroscopy. It has been observed that the deuterations on the hydroxyl and methyl groups have
an influence on the solvation dynamics19 and the OKE response of methanol.20 The deuteration
affects the hydrogen bond bending motion and libration, which have characteristic frequencies in
the THz range between 10 and 100 cm-1. In recent years, dielectric properties of liquids have been
investigated in this frequency range by THz-TDS. The low-frequency spectra of normal and
deuterated methanols (CH3OH, CH3OD, CD3OH, and CD3OD) by THz-TDS to get an insight into
the dynamics of the hydrogen bond network of methanol.4 The spectra of the four methanols show
two components around 20 cm-1 and 60 cm-1 which were assigned to an overdamped mode and an
underdamped oscillation, respectively. Around 20 cm -1 the absorption intensities of all the
methanols are nearly the same, while around 60 cm-1 the absorption intensity of CH3OH is a little
greater than those of deuterated methanols. This observation is due to the isotope effect on the
change of the dipole moment along the coordinate of the normal mode. The hydrogen bonding
networks of the shorter chain alcohol may be well developed compared to those of the longer chain
alcohol. THz-TDS investigations also show that the shorter and longer alcohols roughly correspond
to the biopolymers and amino acids, respectively.
(b) Solutions:
Dielectric relaxation of solvents in solution in the MHz and GHz regions are often
dominated by the orientational motion of the molecules, which can be described by several
theoretical models, including the Debye theory.3 The Debye relaxation time characterizes the time
scale of the orientational motion, which depends on the bulk viscosity of the system, which is in
turn described by the Stokes-Einstein-Debye (SED) equation. The dynamics of the interaction in
the solution are often characterized by time scales ranging between sub-picoseconds to tens of
picoseconds, which correspond to the THz and GHz spectral ranges in the frequency domain,
respectively. The high frequency (THz > GHz) response might be due to intermolecular vibration,
librational motion, structural fluctuation of the hydrogen bonding network, as well as
intramolecular large-amplitude motion. The high frequency dielectric response is especially
important for understanding the ultrafast component in the solvation dynamics.1 It has often been
observed that the initial response of the solvation dynamics takes place in less than 100 fs, which
Dutta, P.: Terahertz time domain spectroscopy ……..
43
is due to the inertial motion of the solvent molecules.1 In order to understand this ultrafast response
of the solvent on a quantitative level, it is necessary to obtain accurate data regarding the dielectric
response in the THz region .THz-TDS study actually provides the accurate experimental data in
this region.
For a particular non-polar aromatic solute and polar solvent interacting system, there is a
characteristic peak frequency of absorption in the THz region detected by THz-TDS. The peak
wavenumber shifts towards higher wavenumbers with an increase in the chain length of the alkane
solvent in the solution. The peak around 10-20 cm-1 is possibly due to a librational motion in the
solution. Smith and Meech interpreted this high-frequency component to be a result of the
“librational motion in a cage model”.21 In a simplified expression, librational frequency is given by
(k/Ieff)1/2 where k is the force constant of the harmonic potential surface, and Ieff is the effective
moment of inertia of a molecule. Since the force constant depends on the strength of the
intermolecular interactions, the librational frequency is expected to decrease with increasing
dilution. The peak frequency is around 10–20 cm-1, which may be considered as a frequency at an
infinite dilution limit. Thus, the shift in the peak frequency of ΔOD could be explained according
to the above relation. It is reasonable to assume that Ieff is independent of the type of solvent, which
suggests that the peak frequency shift is due to a change in force constant caused by changing the
solvent.
The low frequency spectra can be analyzed by different models.2, 5-6 The following equation
(Eq.1) fits the experimental data (Figure 1) very well in the THz region considering the analytical
model equation (Eq. 2)
, ]020~2exp
~exp1~Re[)~()~(
i
solute
k
solvent
i
solute
k
solute
k
solute
tttcidt
kThcNn
(1)
, expexpexpexp
)0()(
2
43
2
322
21
21
21
11
2
tatatta
t solutesolute
(2)
where ∆ ~~ n is the change of the product spectra of the refractive index and the absorption
coefficient between solution and solvent; Nsolute is the number of solute molecules,
0solutesolute t is time correlation function of solute in solution, ai -amplitude of the time
components, 1 denotes the reorientational relaxation time, 2 is the angular velocity
Prajnan O Sadhona ……., Vol. 2, 2015
44
correlation time including the inertial effect, 3 is related to the mean time between collisions
and 4 corresponds to the additional high frequency component with a Gaussian function.
Figure 1: Spectral fitting for the difference product spectra ~~ n with Eq. (1) for the
TCF of the solute dipole moment. (Eq. 2)
Hydrogen bonds (HB) play an important role in structural formation of biological
macromolecules. Since the strength of HB is between those of covalent bonds and van der
Waals forces, it also results in structural flexibility. To deepen understanding on hydrogen
bond, there have been efforts to study intermolecular vibrations of dimers in the liquid phases.8
In solutions, the interaction with surrounding solvent molecules affect the intermolecular
interaction and structural stability of the dimer.
Conclusion
The accurate experimental findings in the low frequency region by THz–TDS study
along with theoretical models having proper physical meanings help to understand different
kind of motions like librational motion, intermolecular vibration, collective motions of
associated liquids and reorientational relaxation etc. in the condensed phases. Numerous
applications for THz–TDS exist and many industrial branches can benefit from its unique
capabilities in future.
References
1. Schmuttenmaer, C. A. Exploring dynamics in the far-infrared with terahertz
spectroscopy. Chem. Rev. 2004, 104, 1759.
2. Dutta, P.; Tominaga, K. Obtaining Low Frequency Spectra of Acetone Dissolved in
Cyclohexane by Terahertz Time-Domain Spectroscopy. J. Phys. Chem. A 2009, 113,
8235.
~~ n
Dutta, P.: Terahertz time domain spectroscopy ……..
45
3. Rønne, C.; Thrane, L.; Astrand, P.-O.; Wallqvist, A.; Mikkelsen, K.V.;
Keiding, S.R. Investigation of the temperature dependence of dielectric relaxation in
liquid water by THz reflection spectroscopy and molecular dynamics simulation. J.
Chem. Phys. 1997, 107, 5319.
4. Kambara, O.; Kawaguchi, S.; Dutta, P.; Ponseca Jr., C. S.; Ikeshima,K.; Yamaguchi,
S.; Hirai, S.; Banno, M.; Naito, S.; Tominaga, K. Low-Frequency Dynamics in
Condensed Phases Studied by Terahertz Radiation Spectroscopy. Proceedings of
International Symposium on Terahertz between Japan and Sweden, TMU Symp. Ser.,
2008, 1, 44.
5. Dutta, P.; Tominaga, K. Dependence of low frequency spectra on solute and solvent in
solutions studied by terahertz time-domain spectroscopy. Mol. Phys. 2009, 107, 1845.
6. Dutta, P.; Tominaga, K. Terahertz Time-Domain Spectroscopic Study of the Low-
Frequency Spectra of Nitrobenzene in Alkanes. J. Mol. Liq. 2009, 147, 45.
7. Oka, A.; Tominaga, K. Terahertz spectroscopy of polar solute molecules in non-polar
solvents. J. Non-Crystalline Solids 2006, 352, 4606.
8. Yamaguchi, S.; Tominaga, K.; Saito, S. Intermolecular vibrational mode of the benzoic
acid dimer in solution observed by terahertz time-domain spectroscopy. Phys. Chem.
Chem. Phys. 2011, 13, 14742.
9. Rungsawang, R.; Ueno, Y.; Tomita, I.; Ajito, K. Angle-dependent terahertz time-
domain spectroscopy of amino acid single crystals. J. Phys. Chem. B 2006, 110, 21259.
10. Xu, J.; Plaxco, K.; Allen, S. J. Collective dynamics of lysozyme in water: terahertz
absorption spectroscopy and comparison with theory. J. Phys. Chem. B 2006, 110,
24255.
11. Markelez, A. G.; Knab, J. R.; Chen, J. Y.; He, Y. Protein dynamical transition in
terahertz dielectric response. Chem. Phys. Lett. 2007, 442, 413.
12. Koeberg, M.; Wu, C.-C.; Kim, D.; Bonn, M. THz dielectric relaxation of ionic
liquid:water mixtures.Chem. Phys. Lett. 2007, 439, 60.
13. Chakraborty, A.; Inagaki, T.; Banno, M.; Mochida, T.; Tominaga, K. Low-frequency
spectra of metallocenium ionic liquids studied by terahertz time-domain spectroscopy.
J. Phys. Chem. A. 2011, 115, 1313.
14. Möller, K. D.; Rothschild, W. G. Far-infrared Spectroscopy, Wiley, New York, 1971.
15. Yarwood, J. Ed. Spectroscopy and Structure of Molecular Complex, Plenum Press,
London, 1973.
Prajnan O Sadhona ……., Vol. 2, 2015
46
16. Padro, J. A.; Marti, J. An interpretation of the low-frequency spectrum of liquid water.
J. Chem.Phys.2003, 118, 452.
17. Ohmine, I.; Tanaka, H. Fluctuation, relaxations, and hydration in liquid water.
Hydrogen-bond rearrangement dynamics. Chem. Rev. 1993, 93, 2545.
18. Sutmann, G.; Vallauri, R. Hydrogen bonded clusters in the liquid phase: I. Analysis of
the velocity correlation function of water triplets. J. Phys. Condens. Matter 1998, 10,
9231.
19. Shirota, H.; Pal, H.; Tominaga, K.; Yoshihara, K. Deuterium isotope effect of
salvation dynamics in methanol: CH3OH, CH3OD, CD3OH, and CD3OD. J. Phys. Chem.
1996, 100, 14575.
20. Shirota, H.; Yoshihara, K. ; Smith, N. A. ; Lin, S.; Meech, S. R. Deuterium isotope
effects on ultrafast polarisability anisotropy relaxation in methanol Chem. Phys. Lett.
1997, 281, 27.
21. Smith, N. A.; Meech, S. R. J. Phys. Chem. A 2000, 104, 4223.
Ghosh, P.: Discussion on reciprocal graph ……..
47
Research Article
Discussion on reciprocal graph from graph theoretical point of
view
Piyali Ghosh
Department of Chemistry, M.U.C. Women’s College, Burdwan - 713104, West Bengal, India
Correspondence should be addressed to Piyali Ghosh; [email protected]
Abstract
Over the few years graph theory has been one of the most rapidly growing areas of
mathematics. Very basic ideas of graph theory with its introduction as well as its few
applications in the field of Chemistry are discussed here. In the field of graph theory reciprocal
graphs are very important because of their distinct characteristics. Eigenspectral properties of
such graphs are mainly focused here.
Key Words: Graph theory; Reciprocal graphs; Eigenspectral properties.
Introduction
Graph theoretical ideas were first introduced in the year of 1736 when Leonhard Euler
presented his solution of Königsberg bridge problem1-6. In the year of 1857 Aurther Cayley
used graphical concept of tree while he was trying to enumerate the number of isomers of
alkanes, CnH2n+21-7. Four colours problem first presented by A. F. Möbius became well known
after Cayley published it in the first volume of the Proceedings of the Royal Geographical
Society in 18791-5,8. Till date, the four colours conjecture so far is a famous unsolved problem
in graph theory, although an enormous amount of research has been done in this field. The first
mention of a graph was not before 1878. J.J. Sylvester first introduced the terminology ‘graph’
in the year of 18789. From the time of terminology ‘graph’ was used in mathematics and also
in the field of chemistry to represent the molecular structural formulas. The essence and beauty
of graph theory attracted mathematicians thus a great deal of research has been done and is
being done in this area. By now graph theory exists as a separate field in Mathematics. Huge
numbers of books on mathematical graph theory have been appeared. Oystein Ore, Robin
J.Wilson and Frank Harary have a great contribution to this field2,10,11. Although graph theory
is a discipline of pure mathematics, it is applied in theoretical chemistry along with its
Prajnan O Sadhona ……., Vol. 2, 2015
48
considerable applications12-16. A great deal of research has been done and is being done in this
area. Graph theoretical ideas are applied in quantum mechanical molecular orbital theory.
Determination of eigenspectral properties17-20 and correlations with some important physico-
chemical properties are interesting research area to the theoretical chemists. Quantitative
Structure-Activity Relationship (QSAR) and Quantitative Structure-Property Relationship
(QSPR) studies are the active areas of chemical research21-25. Topological indices are the graph
invariants and are enormously used in these studies particularly for searching molecular
databases, selecting compounds for drug screening, drug designing, predicting molecular
properties and modeling drug receptor sites. New molecular descriptors are introduced to
explore the structure-dependent properties of molecules with important physico-chemical
properties.
Finite Graphs and Infinite Graphs:
A graph 𝐺 = (𝑣, 𝐸) consists of a set of objects 1 2 3( , , ,...)v v v v called vertices and
another set1 2 3 ( , , .......)E e e e whose elements are called edges. A graph is represented by a
diagram, in which each vertex is represented by dot and each edge is represented by segment
of line joining its end vertices.
A finite graph and infinite graph are shown in Figure 1a and Figure 1b respectively. In
Figure-1(a) v1, v2, v3 and v4 are the vertices and e1, e2, e3, e4, e5, e6 and e7 are the edges of the
graph. Here e6 is called loop because its initial and final vertices are the same i.e. v4. An infinite
graph consists of infinite number of edges and vertices as in Figure 1b. Crystal lattice is an
example of infinite graph.
(a) (b)
Figure 1: (a) A finite graph (b) An infinite graph
e4
v1
e2
e1
e5
e6
v4
v2 v3
e7
Ghosh, P.: Discussion on reciprocal graph ……..
49
Reciprocal graphs and its some properties:
Different types of graphs are considered depending on the number of vertices or edges,
mode of connection among the vertices or edges or the value of their eigenspectra etc.
Reciprocal graphs are very special type of graphs. Reciprocal graphs26-29 are those whose
eigenvalues occur in the form of reciprocal pair ( /1, ) with palendromic characteristic
polynomials. Four-vertex reciprocal graphs of linear chain, cycle and star are shown in Figure
2.
Figure 2: 4-vertex reciprocal graphs of linear chain, cyclic and star graphs
Characteristic polynomial of reciprocal graphs
Characteristic polynomial of any graph can be written as follows.
1 2 2
0 1 2 2 1... 0 (1)n n n
n n na x a x a x a x a x a
Here .),1,...(2,1,0, nniai are called characteristic polynomial coefficients and x
represents the eigenvalue of the graph. If x be the eigenvalue for a reciprocal graph according
to the definition x/1 will be another eigenvalue so equation can also be written in the form
given below
1 2 2
0 1 2 2 1(1/ ) (1/ ) (1/ ) ... (1/ ) (1/ ) 0 (2)n n n
n n na x a x a x a x a x a
Multiplying equation (2) by nx we get
2 2 1
0 1 2 2 1... 0 (3)n n n
n n na a x a x a x a x a x
Comparing equation (1) and equation (3) we can write
,...,, 22110 nnn aaaaaa so on.
Prajnan O Sadhona ……., Vol. 2, 2015
50
Characteristics polynomials of reciprocal graphs with such special relation among its co-
efficients are called palendromic type of characteristics polynomial.
A method28 for construction of characteristics polynomial (CP) coefficients of three
classes of reciprocal graphs (linear, cyclic and star graphs) has been developed. In this method
working formula was expressed in matrix product form.
Cardinalities of reciprocal graphs:
Two vertices are said to be ‘independent’ if they are not connected by an edge. The
number of sets of such independent is called “cardinality”. Mandal and et.al.30 studied on the
cardinalities of three classes of reciprocal graphs (linear, cyclic and star graphs). Recurrence
as well as analytical formula was derived. These relations were applied to calculate the bond
orders of some conjugated molecules.
Topological indices of reciprocal graphs:
Topological indices are graph invariants are used for Quantitative Structure-Activity
Relationship (QSAR) and Quantitative Structure-Property Relationship (QSPR) studies. Two
important topological indices namely Hosoya indices and Wiener indices of three classes of
reciprocal graphs (linear, cyclic and star graphs) are extensively studied31. Mandal et.al.32
farther studied to express these two topological indices for reciprocal graphs in terms of number
of pendant vertices.
Conclusion
Reciprocal graphs are not all hypothetical; some represents real molecules which are
fairly synthesized. Reciprocal graph of the type cyclic graph with six base vertices attached
with six pendant vertices represents hexamethylene cyclohexane molecule was already
synthesized. When pendant vertices of reciprocal graphs are replaced by hetero atoms
particularly sulphur, oxygen or nitrogen it has been conjectured that their electrical
conductivity should be very high. These reciprocal graphs are very important to theoretical
chemists because of their reciprocal and also self-complementary eigenvalues.
References
1. Theory of graphs, American Mathematical Society, Providence, R. I., 1962.
2. Ore, O, Graphs and Their Uses, Random House, Inc., New York, 1963.
Ghosh, P.: Discussion on reciprocal graph ……..
51
3. Berge, C., The Theory of Graphs and Its Applications, John Wiley and Sons, Inc., New
York, 1962.
4. Wilson, R. J., Introduction to graph theory, Longman Group Ltd., London, 1978.
5. Harary, F, Graph Theory, Addison- Wesley, Reading, Mass, 1969.
6. Euler, L, ‘Solutio Ptoblematis ad Geometriam Situs Pertinantis’, Academimae
Petropolitanae (St. Petersburg Academy), 1736, 8, 128.
7. Cayley, A, The Algebra of Organic Synthesis: Green Metrics, Design Strategy, Route
Selection and Optimization. Philos. Mag. 1857, 13, 172.
8. Cayley, A, Graph Theory Applications Philos. Mag. 1874, 67, 444.
9. Sylvester, J. J., The Emergence of the American Mathematical Research Community.
Amer. J. Math. 1878, 1, 64.
10. Wilson, R. J., Introduction to graph theory, Longman Group Ltd., London, 1978.
11. Harary, F., Graph Theory, Addison- Wesley, Reading, Mass 1969.
12. Coulson, C. A. and Rushbrooke, G. S., Proc. Camb. Philos. Soc. 1940, 36, 193.
13. Rutherford, D. E. Proc. R. Soc. Edinb. 1946, A62, 229.
14. Coulson, C. A. Hückel theory for organic chemists. Proc. Camb. Philos. Soc. 1950, 46,
202.
15. Coulson, C. A. and Streitwieser. A. Jr., Dictionary of -electron calculations,
Pergamon Press, Oxford, 1965.
16. Sachs, H., Chemical Group Theory: Techniques and Applications, Publ. Math.
(Debrecen). 1963, 11, 119.
17. Trinajstić, N, Chemical Graph Theory, CRC Press, Boca Raton, FL, 1992.
18. Balaban A. T., (Eds.), Chemical Applications of Graph Theory, Academic Press,
London, 1976.
19. Gutman, I. and Polansky, O. E., Mathematical Concepts in Organic Chemistry,
Springer-Verlag, New York, 1986.
20. Dias, J. R., Molecular Orbital Calculations Using Chemical Graph Theory, Springer-
Verlag, New York, 1993.
21. Randić, M., Artificial Intelligence in Chemistry: Structure Elucidation and Simulation
of Organic Reactions, J. Am. Chem. Soc. 1975, 97, 6609.
22. Hosoya, H. Symmetry: Unifying Human Understanding, Bull. Chem. Soc. Japan. 1971,
44, 2332.
Prajnan O Sadhona ……., Vol. 2, 2015
52
23. Merrifield, R. E., and Simmons, H. E., Topological Methods in Chemistry, Wiley, New
York, 1989.
24. Klein, D. J. and Randić, M. (Eds.), Mathematical Chemistry, VCH, Weinheim, 1990.
25. Balaban, A. T., (Eds.), Topological Indices and Related Descriptors in QSAR and
QSPR, Gordon and Breach Science Publishers, The Netherlands, 1999.
26. Sarkar, J and Mukherjee, A. K, Graphs with reciprocal pairs of eigenvalues, Mol. Phys.,
1997, 90, 903.
27. Mandal, B, Datta. K, Mukherjee, A. K. and Banerjee, M., A Pascal’s triangle-like
approach for the determination of characteristic polynomial coefficients of reciprocal
graphs Mol. Phys. 1999, 96, 1609.
28. Mandal, B, Mukherjee, A. K. and Banerjee, M, Cardinalities of reciprocal graphs, Int.
J. Quant. Chem. 2005, 101, 119.
29. Tiwary, A. S., Mandal, B. and Mukherjee, A. K. Construction and studies of a new class
of reciprocal trees: interknitting of the Pascal's triangle, Mol. Phys. 2008, 106, 1813.
30. Mandal, B, Mukherjee, A. K. and Banerjee, M., Cardinalities of reciprocal graphs, Int.
J. Quant. Chem. 2004, 99, 119.
31. Mandal, B., Mukherjee, A. K, Banerjee, M, Wiener and Hosoya indices of reciprocal
graphs Mol. Phys. 2005, 103, 2665.
Pal, P. et al.: Studies on the anti-inflammatory effect …….
53
Research Article
Studies on the anti-inflammatory effect of leishmanial lipid in vitro
and in vivo
Prajnamoy Pal1, Subhadip Das2, Nabanita Chatterjee2, Dipayan Bose2
and Krishna Das Saha2
1Associate Professor, Department of Chemistry, Fakir Chand College, Diamond Harbour -
743331, South 24 Parganas, West Bengal, India
2Cancer Biology and Inflammatory Disorder Division, CSIR-Indian Institute of Chemical
Biology, 4 Raja S. C. Mullick Road, Kolkata - 700032, West Bengal, India
Correspondence should be addressed to Krishna Das Saha; [email protected]
Abstract
Inflammation is a key process of many diseases. This may occur as a result of gram-
negative bacteria sepsis including Escherichia coli infection that gives rise to excessive
production of inflammatory mediators and causes severe tissue injuries. We have reported
earlier that the lipid of attenuated Leishmania donovani suppresses the inflammatory responses
in arthritis patients. Using heat killed E. coli and LPS stimulated macrophages, we have now
investigated the effect of leishmanial total lipid (LTL) isolated from Leishmania donovani
(MHO/IN/1978/UR6) for amelioration of the inflammatory mediators and transcriptional
factor. To evaluate the in vivo effect, E. coli induced murine sepsis model was used focusing
on the changes in different inflammatory mediator levels and pathophysiology. Significant
improvement was observed in the mortality of endotoxemic mice. The carrageenan and
formalin-induced paw edema thicknesses were found to be reduced significantly with
thetreatment of LTL in time dependent manner. These findings indicate that LTL may prove
to be a potential anti-inflammatory agent and provide protection against gram-negative
bacterial toxin.
Key Words: Leishmania, Lipid, Bacteria, Toxin, Macrophage
Introduction
Endotoxins are constituents of the outer membrane of Gram-negative bacteria.
Bacterial endotoxin is a major candidate for the inflammatory reactions. Endotoxins are agents
Prajnan O Sadhona ……., Vol. 2, 2015
54
of pathogenicity of Gram-negative bacteria, implicated in the development of Gram-negative
shock. Endotoxins are lipopolysaccharides (LPS). In many cases of Gram-negative bacteria,
LPS are found to consist of three covalently-linked regions, the lipid A, the core
oligosaccharide and the O-specific polysaccharide. All three parts of the LPS molecule are
immunogenic, eliciting the formation of antibodies interacting specifically with distinct
epitopes in the respective region. There are a large spectrum of toxic biological activities that
are found to be expressed by purified LPS or isolated free lipid A. These activities are not
direct effects of the LPS molecule but are induced indirectly by endogenous mediators that are
produced following interaction of endotoxin with LPS-sensitive cells. Macrophages are cells
mediating the toxic activities of LPS and tumour necrosis factor alpha (TNF-) is a primary
mediator of the lethal action of endotoxin.
It has been well-established that endotoxins are mitogenic for B-cells and function as
polyclonal B-cell activators. They also mediate cell activation of macrophages and activate
the complement cascade. They act as a physiological stimulus for the synthesis of pro-
inflammatory cytokines such as TNF, IL1, IL6, IL8 and non-protein mediators, which in turn,
are responsible for most pathophysiological consequences of a bacterial infection1. Endotoxic
shock is a complex phenomenon resulting from systemic release of inflammatory mediators.
Endotoxin interacts with inflammatory cells, platelets, and vascular endothelium. Cytokines,
such as tumor necrosis factor and interleukins, and lipid mediators (platelet activating factor,
thromboxane, prostacylin, leukotrienes) are released. These primary mediators act
synergistically to cause many of the harmful effects associated with endotoxemia. Multiple
secondary mediators are released in response to the primary mediators, compounding the
damage. The end result is the species-specific clinical syndrome recognized as endotoxemia2.
Treatment of endotoxemia is difficult because of the numerous mediators involved in the body's
response to endotoxin. There are three possible approaches in treating endotoxemia. The
interaction of endotoxin with target cells can be blocked by inducing tolerance, decreasing
plasma endotoxin concentrations, or interfering with endotoxin binding. Once endotoxin has
interacted with target cells, endogenous mediators can be blocked with a huge variety of drugs.
The effects of corticosteroids, cyclooxygenase blockers, leukotriene blockers, platelet
activating factor blockers, tumor necrosis factor blockers, oxygen radical scavengers, opiate
antagonists, antihistamines and calcium channel blockers are detailed. Supportive care of the
endotoxemic patient continues to be a critical aspect of treatment3. There are reports that
immune response of host cells is significantly modified by leishmanial lipids. They impair
Pal, P. et al.: Studies on the anti-inflammatory effect …….
55
inflammatory cytokines and NO production by stimulated macrophages4,5. L. donovani-
induced immunosuppression and alteration of host cell signaling is mediated by ceramide, a
pleiotropic second messenger playing an important role in regulation of several kinases,
including mitogen-activated protein kinase and phosphatases6,7. Recently, we have shown that
a sphingolipid-rich lipid fraction isolated from an attenuated Leishmania donovani
promastigote (MHO/IN/1978/UR6) nduces apoptosis when administered exogenously in
mouse melanoma (B16F10) and human melanoma (A375) cells8. In 2008, our findings shown
that leishmanial lipid has strong anti-inflammatory and apoptosis-inducing effects on SFMCs
from patients with RA and that apoptosis occurs via the mitochondrial pathway9. Leishmanial
lipid is a strong immunosuppressor of host cells. Inhibition of the inflammatory responses of
synovial cells through induction of apoptosis is one of the main targets of therapeutic
intervention in rheumatoid arthritis (RA). This study was undertaken to examine the
antiinflammatory and apoptosis-inducing effects of leishmanial lipid on adherent synovial fluid
mononuclear cells (SFMCs) in patients with RA. Leishmanial lipid inhibited the release of
tumor necrosis factor, interleukin-1, and NO in the culture, decreased their cytosolic protein
levels, and decreased NF- B p65 levels in SFMCs, in a dose-dependent manner. It had the
reverse effect on interleukin-10 levels. Leishmanial lipid-induced apoptosis involved the
activation of caspase 3, caspase 9, and Bax, the release of cytochrome c, the alteration of
mitochondrial membrane potential, and the down-regulation of Bcl-2.
These results suggest that leishmanial lipid has strong anti-inflammatory and
apoptosis-inducing effects on SFMCs from patients with RA, and that apoptosis occurs via the
mitochondrial pathway9. Septic shock remains the one of the leading causes of death in hospital
patients. Barely more than 50% of the patients with severe sepsis survive their hospital
admission. The incidence of sepsis is increasing year by year10. Septic shock is a serious
medical condition caused by decreased tissue perfusion and oxygen delivery as a result of
infection and sepsis, though the microbe may be systemic or localized to a particular site. It
can cause multiple organ dysfunction syndrome (formerly known as multiple organ failure)
and death. Its most common victims are children, immunocompromised individuals, and the
elderly, as their immune systems cannot deal with the infection as effectively as those of
healthy adults. The mortality rate from septic shock is approximately 50 percent11.
According to the US CDC, septic shock is the 13th leading cause of death in the United
States, and the #1 cause of deaths in intensive care units. There has been an increase in the rate
of septic shock deaths in recent decades, which is attributed to an increase in invasive medical
devices and procedures, increases in immunocompromised patients, and an overall increase in
Prajnan O Sadhona ……., Vol. 2, 2015
56
elderly patients. Tertiary care centers (such as hospice care facilities) have 2-4 times the rate
of bacteremia than primary care centers, 75% of which are nosocomial infections. The
mortality rate from sepsis is approximately 40% in adults, and 25% in children12. Treatment
primarily consists of a) Oxygen administration and airway support, b) Volume resuscitation,
c) Early antibiotic administration, d) Rapid source identification and control, e) Support of
major organ dysfunction.
All medications may cause side effects. Antibiotics can cause allergic reactions,
stomach upset, and other side effects. Other effects depend on the medicines used. A ventilator
can rarely cause a new infection or lung damage. Surgery can be complicated by bleeding,
infection, an allergic reaction to the anaesthetic or even death. Vasopressin and terlipressin are
thus last resort therapies in septic shock states that are refractory to fluid expansion and
catecholamines. However, current data in humans remain modest, and properly powered,
randomized controlled trials with survival as the primary endpoint are required before these
drugs can be recommended for more widespread use. So the fact which can be accentuated
unhesitatingly that there is a genuine demand for an effective preparation with therapeutic
potentiality in treating sepsis. As mentioned earlier, the outcomes of our previous
investigations has prompted us to enthusiastically speculate that the findings of our present
study as proposed will definitely be an effective march ahead to satisfy that serious ongoing
demand.
Our present study is designed to explore the anti-inflammatory role of leishmanial lipid
in LPS stimulated macrophage responses. Monocytes-macrophages are the first line of defence
against infections and varieties of immunomodulatory diseases.
Lipids from Leishmania spp. are known to have immunosuppressive role on immune
systems. Monocyte-macrophages are the host for Leishmania where it grows and multiply.
Leishmanial lipid impairs the signalling system and the immune responses of macrophages. So
the lipids from leishmania are expected to be immunosuppressive against inflammation.
Materials and Methods
Isolation of Lipid from Leishmania donovani Promastigote Cells:
Leishmania strain UR6 (MHO/IN/1978/UR6) will be grown in Ray’s modified medium
and subcultured at 72-hour intervals. Cells will be collected and washed in phosphate buffered
saline (PBS). For extraction of lipids from Leishmania promastigote cells standard methods
like Bligh and Dyer method will be followed. The total lipid thus obtained from the lower
organic phase after evaporation to dryness at - 40°C will be stored at 4°C in vacuum desiccators
Pal, P. et al.: Studies on the anti-inflammatory effect …….
57
until used. Since lipids are usually not soluble in aqueous medium, leishmanial lipid will then
be dissolved in solvents like DMSO, the required amount of leishmanial lipid will be
emulsified by sonication in RPMI 1640 containing 10% FBS. The emulsion and will then be
used for subsequent experiments13.
Thin-Layer Chromatography (TLC):
The leishmanial lipid was dissolved in 2:1 (v/v) chloroform-methanol. TLC was
performed in chloroform-methanol-water (90:10:1, v/v) and lipid spots were visualized using
iodine spray14.
Isolation of mouse peritoneal macrophages:
Macrophages will be obtained from peritoneal cavities of BALB/c mice by washing
with 5 ml of Hanks balanced salt solution (HBSS). The pooled cells will be washed and re-
suspended in HBSS. Approximately 2.5 x 106 cells will be cultivated in sterile cell culture
plate in HBSS supplemented with 10% calf serum. After 2 to 4 h of incubation at 37°C in a
moist atmosphere of 5% CO2, non-adhering cells on each plate will be removed by intensive
rinsing with phosphate-buffered saline (PBS). The number of adherent cells on each plate will
be determined by counting 10 to 15 microscopic fields and multiplying the arithmetic means
of the counts by the ratio of plate area to the area of the microscopic field15.
Cytokine assays in vitro:
To determine the effect of LTL on the production of cytokines from LPS-stimulated
cells, RAW 264.7 or macrophages cells were plated onto 24-well plates (1×106 cells/well),
pretreated in the presence or absence of LTL for 1 h, and then stimulated with LPS for time
dependent manner at 37°C in a 5% CO2 incubator. At each time point, cell-free supernatants
were collected and the concentrations of cytokines TNF-α, IL-1ß and IL-6 were measured by
sandwich ELISA, using commercially available assay kit from GE Healthcare Bio-Sciences
(NJ,USA) according to the manufacturer's instructions.
Extraction of nuclear proteins and assay of NF-kB p65:
RAW 264.7 cells were plated onto 24-well plates (1×106 cells/well), pretreated in the
presence or absence of LTL for 1 h, and stimulated with LPS (1μg/mL) for 12 h. After
centrifugation, it was resuspended in 400 μL of ice cold hypotonic buffer for 10 min, vortexed,
and centrifuged at 15,000 g at 4°C to get the supernatant containing nuclear protein. Aliquots
of this were added to incubation wells precoated with the NF-kB p65 DNA-binding consensus
sequence, and the translocated p65 subunits present in the nuclear lysate were assayed
according to the recommendations of the manufacturer of the NF-kB assay kit (Cayman,
Michigan, USA).
Prajnan O Sadhona ……., Vol. 2, 2015
58
NO assay:
RAW 264.7 cells (1×106 cells/well) were plated onto 24-well plates, pretreated with the
indicated concentrations of LTL for 1 h, and subjected to stimulation with of LPS for time
dependent manner. The sample supernatants were mixed with equal volumes of Griess reagent
(1% sulfanilamide in 5% phosphoric acid and 0.1% naphthylethylenediamine dihydrochloride)
and then incubated at room temperature for 10 min. The absorbance was measured at 540 nm
on a microplate reader. Nitrite concentration was determined using a dilution of sodium nitrite
as a standard. Concentration values were determined for two wells of each sample and the
experiment was performed in triplicate.
Carrageenan-induced paw edema:
Animals were divided into four groups (n=10). In all groups, inflammation was induced
by single sub-plantar injection of 0.02 mL of freshly prepared 1% carrageenan in normal saline.
The group treated with carrageenan alone served as control. Three groups received LTL, i.p. at
different time point, 30 min before the carrageenan injection. The paw thickness was measured
using vernier calipers and the difference between paw volumes was calculated every hour after
carrageenan injection16.
Formalin-induced paw edema:
The experiment was the same as described before for carrageenan induced paw edema
except that a single dose of 0.02 mL of formalin (2%) was used as the inflammation inducer17.
Results
Morphological changes of macrophage cells Fig.1 presents the cell morphology of the
macrophage RAW 264.7 cells under Leishmanial total lipid (LTL) treatment in the presence or
absence of LPS (1μg/ml). The cells were monitored under optical microscopy (200X) and
pictures were captured after 24h of treatment. In the LPS-unstimulated cells, the cell
morphology generally showed round in form whereas LPS-activated RAW 264.7 cells had
changed to an irregular form with accelerated spreading and forming pseudopodia. Leishmanial
total lipid (LTL) reduced the level of cell spreading and pseudopodia formation by suppressing
cell differentiation.
Pal, P. et al.: Studies on the anti-inflammatory effect …….
59
Figure 1: Inhibition of LPS-induced cell morphological changes in RAW cells by the
Leishmanial total lipid (LTL)
Effects of Leishmanial total lipid (LTL) on the expressions of inflammatory mediators in
LPS stimulated macrophage cells:
To determine whether Leishmanial total lipid (LTL) modulates the production of pro-
inflammatory cytokines, the productions of TNF-α, IL-1β, IL-6, IL-10 and NO were examined
using ELISA methods in the LPS stimulated RAW 264.7 macrophage supernatants. These
compounds alone had no significant effect on the secretions of those inflammatory mediators
in the normal RAW264.7 cells (not stimulated with LPS) (data not shown). The treatment with
different concentrations of LTL resulted in an inhibition of the LPS-induced (1µg/µl) IL-1β
(Fig. 2A) TNF-α (Fig. 2B), IL-6 (Fig. 2C) and NO (Fig. 2F) secretions. With 30ug/ml of LTL
a remarkable >50% inhibition compared to control set were noticed in each cases.
Simultaneous release of another cytokines, IL-10 was also observed expectedly with more than
50% increase in production with 30ug/ml of LTL (compared to control) (Fig. 2D). The result
of Western Blot analysis convincingly corroborated the above findings as shown in Figure 2E.
These observed effects were not due to the cytotoxicity of LTL, showed no impairment of the
cell viability in concentration ranges used in this study, compared with the cells treated with
LPS alone.
A B
Prajnan O Sadhona ……., Vol. 2, 2015
60
Figure 2: Effects of Leishmanial total lipid (LTL) on the expressions of inflammatory
mediators in LPS stimulated macrophage cells
Effects of Leishmanial total lipid (LTL) on the expressions of inflammatory mediators in
E. coli K-13 stimulated macrophage cells:
To determine whether Leishmanial total lipid (LTL) modulates the production of pro-
inflammatory cytokines, the productions of TNF-α, IL-1β, IL-6, IL-10 and NO were examined
using ELISA methods in the E. coli K-13 stimulated RAW 264.7 macrophage supernatants just
in the similar manner as above. The findings are equally impressive as are delineated in Fig.
3A-3E below. Here also, our LTL responded in a remarkably effective manner as the figures
described.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 10 30 50 100 200
LTL in µg/ml
IL-1
0 (n
g/m
l)
0
20
40
60
80
100
120
140
0 10 30 50 100 200
LTL in mg/ml
NO
(m
M)
C D
F E
Pal, P. et al.: Studies on the anti-inflammatory effect …….
61
[A] [B]
[D]
[C] [D]
[E]
Figure 3: Effects of Leishmanial total lipid (LTL) on the expressions of inflammatory
mediators in E. coli K-13 stimulated RAW 264.7 macrophage cells
0
0.5
1
1.5
2
2.5
0 10 30 50 100 200
LTL in ug/ml
IL-1
β (
ng
/ml)
0
0.5
1
1.5
2
2.5
3
3.5
4
0 10 30 50 100 200
LTL in ug/ml
TN
F-α
in (n
g/m
l)
0
0.5
1
1.5
2
2.5
3
0 10 30 50 100 200
LTL in ug/ml
IL-6
in( n
g/m
l)
0
50
100
150
200
0 10 30 50 100 200
LTL in ug/ml
NO
in
uM
0
0.5
1
1.5
2
0 10 30 50 100 200
LTL in ug/ml
IL-1
0 (
ng
/ml)
Prajnan O Sadhona ……., Vol. 2, 2015
62
Effects of Leishmanial total lipid (LTL) on the expressions of inflammatory mediators in
LPS stimulated serum and peritoneum macrophages of mice:
Next we have conducted our experiments on in vivo mouse model to see whether our
LTL is equally effective in in vivo system. To determine whether Leishmanial total lipid
(LTL) modulates the production of pro-inflammatory cytokines, the productions of TNF-α, IL-
1β, IL-6, IL-10 and NO were examined using ELISA methods in both serum and peritoneum
macrophages of mice pre-treated with LTL and then with sublethal doses of LPS. Here also
very much encouraging results were found as can be clearly observed from Fig. 4A-4E. The
animals were separately pre-treated with 3mg/kg wt and 6mg/kg wt of LTL followed by a
sublethal dose of LPS (2.5mg/kg wt). After first 24 hours of study inhibitory effect of LTL on
the production of inflammatory mediators, like TNF-α, IL-1β, IL-6 and NO, were distinctly
noticed and were recorded in a remarkable fashion after 36 hours of study (compared to only
LPS-treated control). For IL-6 production, the inhibitory effect of LTL even after 24 hours
was significant (Fig. 4A-4D). Also, in complete agreement of our expectation the production
of anti-inflammatory cytokine IL-10 was conspicuously manifested after first 24 hour of study
and after 36 hours the increase in production was really noteworthy (Fig. 4E). Fig. 4F
significantly delineates the efficacy of LTL on survival rate of mouse treated with LPS
compared to treatment with LPS alone as control.
Effects of Leishmanial total lipid (LTL) on the expressions of inflammatory mediators in
E. coli K-13 stimulated serum and peritoneum macrophages of mice:
Next, we have conducted similar experiments with on in vivo mouse model by using
sublethal doses of E. coli K-13 strain to determine whether Leishmanial total lipid (LTL)
modulates the production of pro-inflammatory cytokines, the productions of TNF-α, IL-1β, IL-
6, IL-10 and NO were examined using ELISA methods in both serum and peritoneum
macrophages of mice. The findings are equally impressive as are delineated in Fig. 5A-5E
below. Here also, our LTL responded in a remarkably effective manner as the figures
described. Fig. 5F significantly delineates the efficacy of LTL on survival rate of mouse treated
with E. coli K-13 compared to treatment with E. coli K-13 alone as control.
Pal, P. et al.: Studies on the anti-inflammatory effect …….
63
Figure 4: Level of cytokines in serum and peritoneum macrophages of mice pre-treated with
lipid and then with sub-lethal doses of LPS for 24 hrs.
[A]
Prajnan O Sadhona ……., Vol. 2, 2015
64
[B]
[C]
Figure 5: Effects of Leishmanial total lipid (LTL) on the expressions of inflammatory
mediators in E. coli K-13 stimulated serum and peritoneum macrophages of mice
[D]
Pal, P. et al.: Studies on the anti-inflammatory effect …….
65
[E]
[F]
Figure 5 (contd.): Effects of Leishmanial total lipid (LTL) on the expressions of inflammatory
mediators in E. coli K-13 stimulated serum and peritoneum macrophages
of mice
LTL inhibits NF-𝜅B expression:
LPS induced inflammatory stress is known to cause activation of the transcriptional
factor NF-𝜅B and the subsequent release of inflammatory mediatorswith signalling cascade.
Thus, we examined if LTL inhibited the levels of NF-𝜅B p65 expression in 100ug/ml manner.
Immunocytochemistry studies provided evidence that the expression level of NF-𝜅Bp65
subunit is lowered in LPS stimulated macrophage cells in the presence of LTL. NF-𝜅B
activation represents a paradigm for controlling the function of different regulatory proteins
via phosphorylation based on the cascade series including proteolytic degradation of I𝜅B
followed by TLR4-CD14 signalling pathway.
Prajnan O Sadhona ……., Vol. 2, 2015
66
Figure 6: Immunofluorescence expression measured by confocal laser scanning microscopy
(magnification 600x)
Carregeenan induced paw edema
Leishmanial total lipid (LTL) was administered intra-peritoneally at the doses of 200µg,
500µg and 2mg/kg of body weight. These LTL at different doses inhibited carregeenan-
induced rat paw edema up to 4hrs observations. Moreover, the anti-inflammatory effect was
more significant at the dose of 2mg/kg than dose of the 200µg, 500µg/kg LTL as compared
with control (carregeenan induced group) (Figure 7).
Figure 7: Effect of Leishmanial total lipid (LTL) at different doses on local effect of carrageenan
induced rat paw edema (mean±SEM; n=6)
DIC DAPI NF-ĸBp65 Merge
Pal, P. et al.: Studies on the anti-inflammatory effect …….
67
Formalin induced paw edema
Leishmanial total lipid (LTL) was administered intraperitoneally at the concentration
of 200µg, 500µg and 2mg/kg of body weight. These LTL at different doses inhibited formalin-
induced rat paw edema up to 4 hrs observation. Moreover the anti-inflammatory effect was
more significant at the dose of 2mg/kg than dose of the 200µg, 500µg/kg LTL as compared
with control (formalin induced group) (Figure 8).
Figure 8: Effect of Leishmanial total lipid (LTL) at different doses on local effect of formalin-
induced rat paw edema (mean±SEM; n=6)
Discussion
Our previous study has found that a novel compound, lipid isolated from Leishmanial
donovani inhibitory effects on endotoxin induced TNF- and IL-6 release in mouse
macrophages. In the present study, we demonstrated anti-inflammatory activities of this
compound both in vitro and in vivo, using LPS-stimulated Raw 264.7 cells and several mouse
models of topical inflammation respectively.
Macrophages play important roles in a host’s immune defense system during infection
as well as in the processes of disease development18. Activation of macrophages by stimuli,
such as LPS, increases the production of numerous inflammatory mediators, including various
cytokines and nitric oxide (NO)10. Excessive production of NO has been widely reported to be
associated with inflammatory response19,20. First, we showed that LTL has inhibitory activity
upon morphological features Raw 264.7 cells (Figure 1B). Our next experiments provided the
evidence that LTL significantly inhibits TNF-α, IL-1β, IL-6, IL-10 and NO in macrophages
cells. (Figure 2). Strong inhibition of inflammatory cytokine release has been ascribed to
down-regulation of NF-kB dependent gene transcription21,22. The validation of experiment was
0
0.2
0.4
0.6
0.8
1
1.2
1st 2nd 3rd 4th
Time (hour)
Paw
volu
me
(ml)
Control
Formalin
Formalin+LTL (200ug)
Formalin+LTL (500ug)
Formalin+LTL(2mg/kg)
Prajnan O Sadhona ……., Vol. 2, 2015
68
done with E.coli also and we found the similar result. The significant reduction was found in
inflammatory mediators and cytokines level with the LTL in stimulated macrophages. Next,
we designed to study the effect of LTL in murine serum and macrophages also in presence of
LSP or E.coli. LTL subdued the level of TNF-α, IL-1β, IL-6, IL-10 in vivo system also in time
dependent manner with simultaneous improvement of the survival rate of the endotoxin or
E.coli challenged mice.
Besides the endotoxin and etiologic agents, some chemicals also induce the production
of inflammation. During tissue inflammation, there is normally vasodilatation and transient
increases in capillary permeability producing extravasation of plasma proteins and tissue
oedema. Generally, these reactions are linked to painful perception or hyperalgesic
sensitization23-25. Our results shown in Figure 6 indicate that pre-treatment with LTL reduced
the inflammatory pain, and also reduced the paw oedema induced by carrageenan and formalin
model. These inflammatory models are mainly related to local production of bradykinin and
prostaglandins, such as PGE2 and leukotrienes, both derivatives of arachidonic acid. These
prostanoids bind the prostaglandin sub-type receptors, triggering the inflammatory and
hyperalgesic pathway in tissues. Bradykinin receptor B1 is associated with a metabotropic
signaling pathway producing vasodilatation, an increase of vascular permeability, and an
increase of eicosanoids production, such as PGE2 and NO produced by PGES, COX-2 and
NO. On the other hand, B1 receptors promote pain stimuli and inflammation by the NF-k
pathway25. In another inflammatory model of formalin-caused pain and oedema, the pain in the
first phase is induced through formalin directly stimulating C-fibers, whereas in the second
phase, the pain is generated by the release of several inflammatory mediators26,27. The higher
inhibitory rate of LTL in phase II than phase I also partly indicated that the main mechanism
of LTL activity is to inhibit the production of inflammatory mediators.
This study represents that leishmanial total lipid (LTL) exerts anti-inflammatory
responses via regulating the inflammatory factors in vitro and in vivo. Thus, LTL reduces
mortality rate of gram-negative bacteria induced septic mice.
Acknowledgments
The authors convey their sincere thanks to Dr. Subires Bhattacharyya, Principal, Fakir
Chand College, Diamond Harbour, and to Prof. (Dr.) Siddhartha Roy, Director, CSIR-Indian
Institute of Chemical Biology, Jadavpur, Kolkata, for their relentless support to carry out the
Pal, P. et al.: Studies on the anti-inflammatory effect …….
69
present work. Financial assistance from University Grants Commission, Govt. of India, to the
first author is also duly acknowledged.
References
1. Rietschel, E. T. et. al., Bacterial endotoxin : chemical constitution, biological
recognition, host response and immunological detoxification. Curr. Topics in
Microbiol. and Immunol. 1996, 216, 39-81.
2. Hardie, E. M., Kruse-Elliott, K. Endotoxic shock. Part I : A review of causes. J. Vet.
Intern. Med. 1990, 4, 258-66.
3. Elizabeth, M., Hardie, D. V. M., Kris Kruse-Elliott, D. V. M. Endotoxic Shock Part II:
A review of treatment. J. Vet. Intern. Med. 1990, 4, 306-314.
4. Hatzigeorgiou, D. E., Geng, J, Zhu, B, Zhang, Y, Liu, K., Rom, W. N., et. al.
Lipophosphoglycan from Leishmania suppresses agonist-induced Interleukin1β gene
expression in human monocytes via a unique promoter sequence. Proc. Natl. Acad. Sci.
USA 1996, 93, 14708–14713.
5. Proudfoot, L., O’Donnell, C. A., Liew, F. Y. Glycoinositolphospholipids of Leishmania
major inhibit nitric oxide synthesis and reduce leishmanicidal activity in murine
macrophages. Eur. J. Immunol. 1995, 25, 745–750.
6. Ghosh, S., Bhattacharyya, S., Das, S., Raha, S., Maulik, N., Das, K. D., Roy, S.,
Majumdar, S. Generation of ceramide in murine macrophages infected with
Leishmania donovani alters macro phage signaling events and aids intracellular
parasitic survival. Mol. Cell. Biochem. 2001, 223, 47–60.
7. Ghosh, S., Bhattacharyya, S., Sirkar, M., Sa, G. S., Das, T., Majumdar, D., Roy, S.,
Majumdar, S. Leishmania donovani suppresses activated protein 1 and NF-B in host
macrophages via ceramide generation: involvement of extracellular signal-regulated
kinase. Infect. Immun. 2002, 70, 6828–6838.
8. Ratha, J., Majumdar, K. N., Mandal, S. K., Bera, R., Sarkar, C., Saha, B., et. al. A
sphingolipid rich lipid fraction isolated from attenuated Leishmania donovani
promastigote induces apoptosis in mouse and human melanoma cells in vitro. Mol. Cell.
Biochem. 2006, 290, 113–123.
9. Majumdar, K. N., Banerjee, A., Ratha, J., Mandal, M., Sarkar, R. N., Das Saha, K.
Leishmanial lipid suppresses TNF, IL1β, and nitric oxide production by adherent
synovial fluid mononuclear cells in rheumatoid arthritis patients and induces apoptosis
Prajnan O Sadhona ……., Vol. 2, 2015
70
through the mitochondrial-mediated pathway. Arthritis and Rheumatism 2008, 58,
696–706.
10. Qureshi, K., Rajah, A. Septic shock : a review article. Brit. J. Med. Practitioners 2008,
1, 7-12.
11. Kumar, V., Abbas, A. K., Fausto, N., Mitchell, R. N. Robbins Basic Pathology (8th ed.).
Saunders Elsevier. 2007, pp. 102-103. ISBN 978-1-4160-2973-1
12. Huether, S. E., McCance, K. L. Understanding Pathophysiology. 4th Edition, 2008.
13. Bligh, E. G., Dyer, W. J. A rapid method of total lipid extraction and purification.
Canadian Journal of Biochemistry and Physiology 1959, 37(8), 911–917.
14. Majumdar, K. N., Banerjee, A., Ratha, J., Mandal, M., Sarkar, R. N., Saha, K. D.
Leishmanial lipid suppresses tumor necrosis factor α, interleukin-1, and nitric oxide
production by adherent synovial fluid mononuclear cells in rheumatoid arthritis patients
and induces apoptosis through the mitochondrial mediated pathway. Arthritis and
Rheumatism 2008, 58(3), 696–706.
15. Heinzel, F. P. The role of IFN- in the pathology of experimental endotoxemia. J
Immunol. 1990, 145(9), 2920–2924.
16. Chattopadhyay, D., Arunachalam, G., Mandal, A. B., Sur, T. K., Mandal, S. C.,
Bhattacharya, S. K. Antimicrobial and anti-inflammatory activity of folklore: Mallotus
peltatus leaf extract. J. Ethnopharmacol. 2002, 82, 229–237.
17. Ajith, T. A., Janardhanan, K. K. Antioxidant and anti-inflammatory activities of
methanol extract of Phellinus rimosus. Indian J. Exp. Biol. 2001, 39, 1166–1169.
18. Duffield, J. S. Macrophages and immunologic inflammation of the kidney. Semin. Nephrol.
2010, 30, 234–254.
19. Edirisinghe, I., Yang, S. R., Yao, H., Rajendrasozhan, S., Caito, S., et al. VEGFR-2
inhibition augments cigarette smoke-induced oxidative stress and inflammatory
responses leading to endothelial dysfunction. FASEB J. 2008, 22, 2297–2310.
20. Panayiotou, C. M., Baliga, R., Stidwill, R., Taylor, V., Singer, M., et al. Resistance to
endotoxic shock in mice lacking natriuretic peptide receptor-A. Br. J. Pharmacol. 2010,
160, 2045–2054.
21. Chiu, J., Khan, Z. A., Farhangkhoee, H., Chakrabarti, S. Curcumin prevents diabetes-
associated abnormalities in the kidneys by inhibiting p300 and nuclear factor-.
Nutrition 2009, 25, 964–972.
Pal, P. et al.: Studies on the anti-inflammatory effect …….
71
22. Ghosh, S. S., Massey, H. D., Krieg, R, Fazelbhoy, Z. A., Ghosh, S., et al. Curcumin
ameliorates renal failure in 5/6 nephrectomized rats: role of inflammation. Am. J.
Physiol. Renal. Physiol. 2009, 296, F1146–1157.
23. Paulino, N., Rodrigues, N. C., Pardi, P. C., Suarez, J. A., dos Santos, R. P., et al.
Evaluation of anti-inflammatory effect of synthetic 1,5-bis(4-acetoxy-3-
methoxyphenyl)- 1,4-pentadien-3-one, HB2. Bioorg. Med. Chem. 2009, 17, 4290–
4295.
24. Garrido-Suarez, B. B., Garrido, G., Marquez, L., Martinez, I., Hernandez, I., et al. Pre-
emptive anti-hyperalgesic effect of electroacupuncture in carrageenan- induced
inflammation: role of nitric oxide. Brain Res. Bull. 2009, 79, 339–344.
25. Lee, C. H., Shieh, D. C., Tzeng, C. Y., Chen, C. P., Wang, S. P., et al. Bradykinininduced
IL-6 expression through bradykinin B2 receptor, phospholipase C, protein kinase
Cdelta and NF- pathway in human synovial fibroblasts. Mol. Immunol. 2008, 45,
3693–3702.
26. Correa, C. R., Calixto, J. B. Evidence for participation of B1 and B2 kinin receptors in
formalin-induced nociceptive response in the mouse. Br. J. Pharmacol. 1993, 110, 193–
198.
27. Liu, X. J., White, T. D., Sawynok, J. Involvement of primary sensory afferents,
postganglionic sympathetic nerves and mast cells in the formalin-evoked peripheral
release of adenosine. Eur. J. Pharmacol. 2001, 429, 147–155.
Prajnan O Sadhona ……., Vol. 2, 2015
72
Research Article
Studies on the effect of biofertilizer comprising of the plant growth
promoting rhizobacteria Bacillus thuringiensis on different types of
plants
Sandip Bandopadhyay1 and Swati Roy Gangopadhyay2
1Assistant Professor, Post Graduate Dept. of Microbiology, Bidhan Nagar College, EB-2, Salt
Lake, Kolkata-700064, West Bengal, India.
2Assistant Professor, Post Graduate Dept. of Microbiology, Barrackpore Rastraguru
Surendranath College, 85, Middle Road, Barrackpore, Kolkata -700120, West Bengal, India.
Correspondence should be addressed to Swati Roy Gangopadhyay; [email protected]
Abstract
We have isolated and characterized phosphate solubilizing and heavy metal resistant
Bacillus thuringiensis from the local agricultural fields of district North 24 Parganas of West
Bengal. This organism exhibits different plant growth promoting characteristics and can
promote plant growth when applied as biofertilizer on different non-leguminous plants like
Amaranthus viridis, Capsicum annuum, Ocimum tenuiflorum, and Abelmoschus esculentus.
The application of biofertilizer resulted in high catalase and peroxidase activity in leaves;
increased seed germination rate, increased sugar and phosphate content in leaves as well as in
fruits and dry weight of A. esculentus plants. Although the application of the strain as
biofertilizer caused an increase in total protein content in Abelmoschus leaves, no newly
synthesized protein was detected on SDS-PAGE.
Key Words: Bacillus thuringiensis, Abelmoschus esculentus, Peroxidase, SDS-PAGE
Introduction
Plant growth promoting effects of PGPR in pot as well as in fields were exhaustively
studied for last three decades. It was reported that particular strains of Pseudomonas
fluorescens, Bacillus megaterium and Azotobacter vinelandii accelerated the rate of growth of
different wild plants in pots under greenhouse condition1. Wu et al. reported the combined effect
of nitrogen fixing, phosphate and potassium solubilizing fungi in the growth of maize under
greenhouse condition2. Effect of Pseudomonas putida and Trichoderma attoviride in growth
Bandopadhyay, S. et al.: Studies on the effect of biofertilizer …….
73
stimulation of tomato plants under greenhouse state was reported earlier by the researchers3.
They reported that inoculation of those microorganisms not only stimulates in the increase of
root length and shoot length, but accelerates in the secretion of IAA also. Combined effect of
different PGPR belongs to the genus Azospirillum, Azotobacter, Mesorhizobium, Pseudomonas
Bacillus and Stretomyces was also previously reported4,5,6. Their study showed that dry matter
accumulation in different plant organs was significantly affected by inoculation with PGPR in
the flowering stage. Inoculation of PGPR also affected on grain yield and biomass
concentration. Grain yield was almost doubled in inoculated plants in comparison to control
plants. Total protein content of the grains collected from the inoculated plants also showed a
sharp increase than control. They also reported that the effect of microorganisms enhanced
shoot height, canopy width, and total number of fruits, fruit weight, and fruit length of plants
under pot as well as field condition.
Inoculation of PGPR also helped to increase the length of lateral roots and root diameter
as well as root hair formation7,8,9,10. It was observed that introduction PGPR in pot not only
increased initial root hair formation, but also increased plant height, leaf area, total leaf
chlorophyll content and total dry matter accumulation. They also concluded that bacterial
inoculants significantly increased seed germination rate. The rate of seed germination was
calculated by Vigor Index [VI = (mean root length + mean shoot length) x % of seed
germination]. A. brasilense was reported to be the strain with highest VI, followed by P. putida
(strain R-168)11. Cyanobacteria like Anabaena, Scytonema, Oscillatoria and Lyugbya was also
reported to act as good inoculants in pot culture12. Inoculation of cyanobacterial culture induced
an appreciable increase of chlorophyll-a, chlorophyll-b and total chlorophyll content in leaves.
It also increased root and shoot length as well as enhances plant catalase and peroxidase activity
significantly. Growth promoting effects of PGPR in different non-leguminous plants were also
reported by various researchers in last one decade13,14,15,16.
Microbial activities of PGPR in pot condition were measured by various ways. Total
microbial activity in soil may be measured by Dehydrogenase activity or Electron Transfer
System (ETS) activity assay1,17,18,19. The method involved reduction of Triphenyl Tetrazolium
Chloride (TTC) by microbial dehydrogensae to Tri Phenyl Formazan (TPF). The colour of TPF
was measured at 485 nm in spectrophotometer. Carbon di-oxide evolution method was also
reported as a method of choice for determining the activity of aerobic microorganisms in soil,
in which the amount of oxidized carbon (i.e., CO2) produced due to respiration, was
measured20,21,22,23. The amount of CO2 released is an indication of the metabolic activities as
well as the interaction of soil microflora with environmental factor. The carbon assimilated in
Prajnan O Sadhona ……., Vol. 2, 2015
74
microbial cell is burned aerobically to produce CO2. Therefore, high rate of CO2 production by
soil microflora indicates high metabolic activity of the microorganisms.
We have already isolated and purified phosphate solubilizing and heavy metal resistant
rhizobacteria from the soil sample of the agricultural field from Kalyani Road (Koirapur), North
24 Parganas. Biochemical characterization followed by molecular characterization by 16S
rDNA analysis detected the particular strain as Bacillus thuringiensis. (Gene Bank entry:
DQ286337)24. This strain was shown to be the potent phosphate solubilizer, phytohormone
producer like IAA and have been found to promote the plant growth when applied to different
vegetable and medicinal plants24. Moreover our preliminary studies also revealed that the
isolated Bt can resist the toxicity of some heavy metals like Hg+2, Zn+2, Ni+2 and Pb+2 and Cd+2
24 and our recent studies revealed that the resistance to lead toxicity may be due to bio
accumulation of lead by metallothionein protein30. Thus the relevance of such multi beneficial
bacterial isolate may be in its application in agricultural field as bio fertilizer and in
bioremediation in heavy metal contaminated soil.
Materials and Methods
The culture:
The PGPR strain Bacillus thuringiensis A5-BRSC was isolated from the agricultural
field of West Bengal, India and identified by 16S rDNA analysis (Gene Bank entry: DQ286337)
. The strain exhibited high phosphate solubilizing and phytohormone (Indole Acetic Acid)
producing activity as evident in our previous study24.
Preparation of pots & Cultivation of seeds:
Surface soil from a bare ground area of local field was collected and sieved to discard
small pieces of bricks and stones. About 5 kg of loamy soil (pH- 7.2) was taken in each pot.
Seeds of Amaranthus viridis (non leguminous), a common vegetable for the people of West
Bengal; Ocimum tenuiflorum, Capsicum annuum and Abelmoschus esculentus were eventually
distributed on the surface of each of the three sets of pots. 20 pots were selected for each of the
three different sets of plants.
Preparation & application of biofertilizer:
Culture of A5-BRSC with a cell density of 2x106 CFU/ml was mixed with sterile
charcoal (1:1, w/v). Charcoal mediated culture applied to the soil of the pot as biofertilizer after
7 days of sowing of seeds. One set of un-inoculated pots for each of the three plants served as
Bandopadhyay, S. et al.: Studies on the effect of biofertilizer …….
75
control. All inoculated and un-inoculated control pots were maintained in a sunny area and
water was sprayed regularly.
Measurement of growth parameters:
Growth of the plants (shoot height), leaf area, number of leaves, flowering time and
length and weight of fruits were monitored for 75 days in every 15 days of interval. Fresh weight
and dry weight of the plants was taken after 75 days.
Estimation of microbial activity in pot:
Total activity of aerobic microorganisms in both test and control pots were determined
by carbon di-oxide evolution method and soil dehydrogenase assay. In every 15 days interval
60 g of soil from each of the pots were withdrawn and divided into two parts. One part of the
soil was taken for carbon di-oxide evolution assay and another part was taken for soil
dehydrogenase assay. 50 g of moistened soil from each pot was collected in 1 l conical flask
and a test tube with 15 ml of 0.5 N NaOH was kept inside the flask. The flask was incubated
for 24 hours at 30ºC by sealing it with parafilm. An empty conical flask (without soil) was
treated in the same manner as ‘negative control’. Next day the residual NaOH was titrated with
standard 0.5 N HCl in both negative control and soiled pots. The amount of carbon dioxide
evolved was calculated by using the formula: (M.W. of carbon / M.W. of CO2) x volume of
HCl consumed.
For soil dehydrogenase assay 6 g of air-dried soil was thoroughly mixed with 0.6 g of
CaCO3 and the mixture was taken in sterile culture tube. 2.5 ml of sterile water and 1 ml of 3
% aqueous Triphenyl Tetrazolium Chloride (TTC), the substrate, was added to the tube. One
tube without soil was treated in the same manner acted as control. All the tubes were incubated
for 24 hours at 30ºC. After 24 hours 10 ml of 100 % methanol was added to each tube and the
content was filtered through Wattman filter paper no.5 and the filtrate was collected. The
absorbance of the filtrate was recorded in spectrophotometer at 485 nm. The amount of product
formed was estimated from the standard curve of the product Tri Phenyl Formazan (TPF).
Activity of 1 ml of the enzyme was expressed as µmoles of product formed/min under the
specified assay condition.
Determination of chlorophyll content:
1 g of youngest fully expanded leaf from each plant was put in 50 ml of 96% methanol.
The mixture was homogenized at 2000 rpm for 1 minute. Homogenate was filtered through
Prajnan O Sadhona ……., Vol. 2, 2015
76
two-layered cheese cloth filter. The filtrate was centrifuged at 2500 x g for 10 minutes.
Absorbance was taken to determine the amount of chlorophyll a, chlorophyll b and total
carotene present in leaf extract.
Determination of total soluble sugar content in leaves and fruits:
1 g each of leaf and fruit samples of Abelmoschus plant were crushed separately in
mortar-pestle and homogenized with 10 ml of double distilled water. The homogenate was
centrifuged at 10,000 x g for 10 minutes at room temperature. Pellet was discarded and soluble
sugar content of the supernatant was estimated by adding Anthron reagent. After incubation of
20 minutes at room temperature the absorbance was measured at 620 nm. Amount of soluble
sugar present in per g of fruit and leaf was determined from the standard curve of glucose.
Estimation of phosphate content in leaf and fruit:
To determine the phosphorus content of the leaf and fruit sample of Abelmoschus plant,
10 g of sample was digested by dry ash method. The sample was taken in silica crucible and
heated in low flame of Bunsen burner till the contents get charred. The content was transferred
in muffle furnace and heated it at 5500C till it completed turned into white ash. The ash was
mixed with 5 ml of dilute HCl and 5 ml of distilled water and warmed it in boiling water bath.
The solution was filtered with Whatman filter paper no. 40 and clear filtrate was used as
phosphate source. Finally phosphate content was estimated spectrophotometrically by L-
ascorbic acid method25.
Catalase and Peroxidase enzyme activity of leaves:
Catalase and peroxidase activity of the leaves were estimated by following the protocol
described by Chance and Maehly26 with slight modification. 0.5 g of leaf samples were crushed
in pre-chilled mortar-pestle and homogenized with 10 ml of 0.1 M cold phosphate buffer, pH:
7.0. The homogenate was centrifuged at 17000 x g for 10 minutes at 4ºC (Plastocraft, India)
and diluted 5 times with 0.1M phosphate buffer, pH: 7.0. This diluted sample was taken as
enzyme source for biochemical studies. To determine catalase activity 1 ml of this leaf extract
was mixed with 2 ml of 0.1 M phosphate buffer, pH: 7.0 and 2 ml of 100 µmoles of H2O2 at 30
ºC for 1 minute. The reaction was terminated by adding 2 ml of 6N H2SO4. Residual H2O2 was
titrated against 0.01 N KMNO4. A control set was treated in the same manner where the
enzymatic reaction was stopped at zero time. One unit of catalase activity was expressed as the
amount of enzyme that breaks down 1 µmole of H2O2 /min under the specified assay condition.
Bandopadhyay, S. et al.: Studies on the effect of biofertilizer …….
77
For peroxidase assay 1 ml of 10-times diluted leaf extract was mixed with 1 ml 0.1 M
phosphate buffer, pH: 7.0, 1 ml of 50 µmoles of pyrogallol (the chromogen) and 2 ml of 100
µmoles of H2O2. Reaction mixture was incubated at 30ºC for 5 minutes and the reaction was
stopped by adding 2 ml of 6N H2SO4. The colour of purpurogallin formed was measured by
taking absorbance at 420 nm. The amount of purpurogallin released was determined from the
standard curve. Activity of 1 ml of the enzyme was expressed as µmoles of product formed/min
under the specified assay condition.
Extraction of leaf protein:
50 g of Abelmoschus leaf samples were crushed in pre-chilled mortar-pestle and
homogenized with 100 ml of 50 mM cold Tris-HCl buffer, pH: 7.5. The homogenate was
centrifuged at 20,000 x g for 15 minutes at 4ºC. The pellet was discarded and the supernatant
was taken. Solid ammonium sulphate was slowly added to the supernatant with a continuous
stirring by magnetic stirrer to obtain 60% saturation. The solution was kept at 40C for overnight.
Precipitated protein by ammonium sulphate fractionation was centrifuged at 15000 x g for 30
minutes at 4ºC. It was resuspended in 50mM cold Tris-HCl buffer of pH 7.5 and dialyzed
against the same buffer for overnight. Dialyzed protein was concentrated through lyophilization
and protein concentration was determined by Lowry method. Fifty μg of leaf protein was loaded
for SDS-PAGE27.
Result and Discussion
Inoculation of biofertilizer B. thuringiensis (A5-BRSC) in each pots, containing the
seeds of Amaranthus viridis, Capsicum annuum (Chili), Abelmoschus esculentus (Ladies’
finger) and Ocimum tenuiflorum (Tulsi: medicinal plant) showed that germinated seeds
remarkably grow higher than that of un-inoculated pot in 45 days (Figure 1a-1d). Induction of
growth of experimental plants in presence of the inoculum in pot is clearly evident from the
data shown in Figure 2. Growth rate of tulsi plant was fastest among the test plant sets; although
it had shown no significant shoot length increase after 75 days. Inflorescence of Ocimum and
Amaranthus occurred after 32 and 21 days of inoculation respectively. Fluorination and fruit
formation in Capsicum and Abelmoschus plants were observed after 24 and 38 days as well as
22 and 30 days of inoculation respectively.
Application of Bacillus thuringiensis (A5-BRSC) culture resulted in significant
enhancement in seed germination as well as Vigor index in Abelmoschus esculentus. Seed
germination was observed in both types of pots after 72 hours of sowing. About 31.1% more
Prajnan O Sadhona ……., Vol. 2, 2015
78
seeds (out of 95 seeds in both inoculated and un-inoculated pots) were germinated in inoculated
pots within 7 days in comparison to control pots. Initial root length (after 2 days of seed
germination) in inoculated pots were 15 times higher than un-inoculated pots, but no significant
difference was observed in initial shoot length of both test and control pots. Vigor index was
calculated as 432 and 170 for inoculated and un-inoculated pots respectively. Significant
difference in shoot length as well as in leaf area was observed after 20 day onwards of seed
germination (Table 1).
Figure 1: Comparative growth of (a) Amaranthus viridis and (b) Abelmoschus esculentus (c)
Capsicum annuum and (d) Abelmoschus esculentus in pot after applying Bacillus
thuringiensis (A5-BRSC) culture against control
[Note: In the Figure 1(d), Right pot (test) contained biofertilizer and left pot
contained no biofertilizer]
Bandopadhyay, S. et al.: Studies on the effect of biofertilizer …….
79
Figure 2: Shoot height in cm of different experimental plants in pot culture study [It should be
noted that no significant increase of shoot height was recorded in case of Tulsi
(Ocimum tenuiflorum) and Amaranthus plant at 75 days]
In 60 days old matured plants, almost 1.5 times longer shoot length and 1.13 times thicker
average shoot diameter, 1.2 times longer root length and 1.6 times larger leaf area was recorded.
Experimental plants showed greater number of lateral roots than control plants (Table 1).
Gholami et al. (2009) reported similar effect of PGPR inoculation on germination, seedling
growth and maize yield in both pot cultures as well as under field conditions. Similar result was
reported by other researchers4,6,12.
Application of inoculum increased significant amount of total fresh weight and dry
weight of plants, increased protein content as well as catalase and peroxidase enzyme activities;
but total chlorophyll content did not varied significantly in comparison to un-inoculated pots
with Abelmoschus esculentus plant (Table 1).
Prajnan O Sadhona ……., Vol. 2, 2015
80
Table 1: Comparative biochemical and morphological analysis of Abelmoschus esculentus
plant in both inoculated and un-inoculated pots (Note: All biochemical tests were
done by taking young leaves of 14 days old plant and for other tests 60 days old
matured plants were used)
Same pattern of result was also described by Mia et al9. Their study with tissue cultured banana
plant in pot condition revealed that introduction PGPR in pot not only increased initial root hair
formation, but also increased plant height, leaf area, total leaf chlorophyll content and total dry
matter accumulation. Growth promoting effects of PGPR in different non-leguminous plants
were also reported by other scientists13,14,15,16. There was no significant difference in flowering
and fruiting time (24 days and 32 days after seed germination respectively) in plants treated
with biofertilizer as compared to the control plants, but inoculated plants showed rapid increase
in fruit length and width as well as fruit weight in comparison to control plants. However,
biofertilizer treated other test plants also showed similar type of results. Results of biofertilizer
treated Ocimum tenuiflorum plants against controls were shown in Table 2.
Bandopadhyay, S. et al.: Studies on the effect of biofertilizer …….
81
Table 2: Effect of biofertilizer on growth characteristics of Tulsi (Ocimum tenuiflorum)
seedlings at 60 days after inoculation
Proteins were extracted from the leaf of Abelmoschus esculentus and analysed on SDS
PAGE, to determine whether any different protein was expressed in culture treated plants. The
result (Figure 3) showed no such new protein synthesis was triggered in inoculated plants in
comparison to controls.
Figure 3: SDS-PAGE analysis of leaf proteins of Abelmoschus esculentus plant from
inoculated and un-inoculated pots as well as field samples [Lane 1: Molecular
weight marker; lane 2: leaf protein from inoculated pot; lane 3: leaf protein from
un-inoculated pot; Note that no such detectable changes in all protein profiles]
Prajnan O Sadhona ……., Vol. 2, 2015
82
Both soil dehydrogenase assay and carbon di-oxide evolution assay showed that
microbial activities were much higher in inoculated pots than control pots. The result of
dehydrogenase assay implied that after the addition of PGPR B. thuringiensis A5-BRSC culture
in pots the microbial activity had a sharp rise on first 30 days and then gradually the activity
become lowered, where as in control pots microbial activities remained more or less same for
total period of 60 days. Similarly, high metabolic activity of aerobic soil microbes in inoculated
pots were indicated by higher amount of carbon di-oxide evolution in first 30 days, where as
almost similar CO2 evolution rate was observed in control pots during total study period of 60
days (Figure 4). Almost similar pattern of result in soil dehydrogenase activity and CO2
evolution rate was reported earlier1,22,28,29.
Figure 4: Measurement of microbial activities in inoculated pots of Abelmoschus esculentus,
compared to control pots by soil dehydrogenase assay and CO2 evolution method
Our study revealed that Bacillus thuringiensis (A5-BRSC, Gene Bank entry:
DQ286337) can promote the growth of various plants including Amaranthus viridis (notey),
Capsicum annuum (chili) and Ocimum tenuiflorum (tulsi) . Growth accelerating activity of this
strain is also evident in Abelmoschus esculentus (Ladies’ finger or vindi or okra) plant under
both pot and field condition. (Field data not shown). All the plants used in the present study are
non-legumious and hence none of these plants can act as natural habitat for nodulating nitrogen
fixing bacteria. Therefore the plant growth promoting effect was due to the association and
interaction of the rhizobacteria with the plants. As the potent phosphate solubilizer24, it helped
an easy and better uptake of soluble phosphate by plants which were also evident from the
Bandopadhyay, S. et al.: Studies on the effect of biofertilizer …….
83
increased phosphorous content in test plant. Moreover, all the plants chosen for this study are
either herb or shrub and annual according to their life span so that they can be easily handled in
pots. Inoculation of Bacillus thuringiensis (A5-BRSC) in pots as culture broth or in the form of
carrier based biofertilizer increased the root length, shoot height and diameter, leaf area, fruit
weight, fresh weight and dry weight of plants. Total amount of soluble sugar and protein in
leaves and fruits, protein content of seeds, catalase and peroxidase activities of leaves are
significantly increased in Abelmoschus esculentus plant during pot study, although leaf
chlorophyll content is not significantly higher in test plants, in comparison to control, The strain
Bacillus thuringiensis (A5-BRSC) shows better shelf life at 40C when it is immobilized with
charcoal as carrier compared to Indian peat (low grade coal) (data not shown). Moreover, due
to more water containing capacity, neutral pH and easy availability makes charcoal most
suitable carrier for biofertilizer in this study.
Acknowledgement
This investigation was financially supported by University Grants Commission (UGC).
References
1. Jeon, J. S., Lee, S. S., Kim, H. Y., Ahn, T.S., Song, H. G. Plant growth promotion in
soil by some inoculated microorganisms. J. Microbiol., 2003, 41(4), 271-276.
2. Wu, S. C., Cao, Z. H., Li, Z. G., Cheung, K. C., Wong, M. H. Effects of biofertilizer
containing N-fixer, P and K solubilizers and AM fungi on maize growth: a greenhouse
trial. Geoderma, 2005, 125, 155–166.
3. Gravel, V., Antoun, H., Tweddell R. J. Growth stimulation and fruit yield improvement
of greenhouse tomato plants by inoculation with Pseudomonas putida or Trichoderma
atroviride: Possible role of indole acetic acid (IAA). Soil. Biol. Biochem., 2007, 39,
1968–1977.
4. Datta, M., Palit, R., Sengupta, C., Pandit, M. K., Banerjee S. Plant growth promoting
rhizobacteria enhance growth and yield of chilli (Capsicum annuum L.) under field
conditions. Australian Journal of Crop Science, 2011, 5(5), 531-536.
5. Fallik, E., Sarig, S., Okon, Y. Morphology and physiology of plant roots associated with
Azospirillum. In: Azospirillum / Plant Associations., ed: Okon, Y. pp.77-85, CRC Press,
Florida, 1994.
6. Rokhzadi, A., Asgharzadeh, A., Darvish, F., Mohammadi, G.N., Majidi, E. Influence of
plant growth-promoting Rhizobacteria on dry matter accumulation and yield of
Prajnan O Sadhona ……., Vol. 2, 2015
84
chickpea (Cicer arietinum L.) under field conditions. American-Eurasian J. Agric.
Environ. Sci., 2008, 3(2), 253-257.
7. Hadas. R., Okon. Y. Effect of A. brasilense on root morphology and respiration in
tomato seedlings, Biol. Fertil. Soils, 1987, 5, 241-247.
8. Hartmann, A., Singh, M., Klingmuller, W. Isolation and characterization of
Azospirillum mutants excreting high amounts of indoleacetic acid, Can. J. Microbiol.,
1983, 29, 916-923.
9. Mia, M. A. B., Shamsuddin, Z. H., Wahab, Z., Marziah, M. Effect of plant growth
promoting rhizobacterial (PGPR) inoculation on growth and nitrogen incorporation of
tissue-cultured Musa plantlets under nitrogen-free hydroponics condition. Australian
Journal of Crop Science, 2010, 4(2), 85-90.
10. Okon, Y. Azospirillum as a potential inoculant for agriculture. Trends Biotechnol., 1985,
3, 223-228.
11. Gholami, A., Shahsavani, S., Nezarat S. The Effect of Plant Growth Promoting
Rhizobacteria (PGPR) on Germination, Seedling Growth and Yield of Maize. World
Academy of Science, Engineering and Technology, 2009, 49, 19-24.
12. Das, P. K. Effect of cyanobacterial biofertilizer on growth and biochemical
characteristics of Vinca rosea Linn. Res. J. Biotechnol., 2010, 5(4), 76-79.
13. Bashan Y. Inoculants of plant growth promoting rhizobacteria for use in agriculture.
Biotechnol. Adv., 1998, 16, 729-770.
14. Bashan, Y., Holguin, G., de-Bashan, L. E. Azospirillum- plant relationships:
physiological, molecular, agricultural, and environmental advances, Can. J. Microbiol.,
2004, 50, 521–577.
15. Mia, M. A. B., Shamsuddin, Z. H., Zakaria, W., Marziah, M. High-yielding and quality
banana production through plant growth promoting rhizobacterial inoculation. Fruits,
2005, 60, 179-185.
16. Shaukat, K., Affrasayab, S., Hasnain S. Growth responses of Triticum aestivum to plant
growth promoting rhizobacteria used as a biofertilizer. Res. J. Microbiol., 2006, 1(4),
330-338.
17. Lenhard, G. The dehydrogenase activity in soil as a measure of the activity of soil
Microorganisms, Z. Pflanzenernaehr. Dueng. Bodenkd., 1956, 73, 1-11.
18. Klein, D. A., Loh, T. C., Goulding, R. L. A rapid procedure to evaluate the
dehydrogenase activity of soils low in organic matter. Soil Bid. Biochem. 1971, 3, 385-
387.
Bandopadhyay, S. et al.: Studies on the effect of biofertilizer …….
85
19. Bayer, L., Wachendrof, C., Elsner, D.C., Knabe R. Suitability of dehydrogenase activity
assay as an index of soil biological activity. Biol. Fertil. Sci., 1993, 16, 52-56.
20. Anderson, J. P. E. Soil respiration, In Methods of soil analysis, part 2, 2nd edn., Page, A.
L., Miller, R. H., Keeney D. R. (ed.), 837–871, Madison, Wisc.: ASA and SSSA, 1982.
21. Staben, M. L., Bezdicek, D. F., Smith, J. L., Fauci, M. F. Assessment of soil quality in
conversation reserve program and wheat–fallow soils. Soil Sci. Soc. Am. J., 1997, 61,
124–130.
22. Haney, R. L., Brinton, W. H., Evans, E. Estimating Soil Carbon, Nitrogen, and
Phosphorus Mineralization from Short-Term Carbon Dioxide Respiration. Commun.
Soil Sci. Plant Anal., 2008, 39, 2706–2720.
23. Ghany, A. E., Bouthaina, F., Arafa Rhawhia, A. M., Rahmany, E., Tomader, A., Shazly
E., Morsy, M. Effect of Some Soil Microorganisms on Soil Properties and Wheat
Production under North Sinai Conditions. J. Appl. Sci. Res., 2010, 4(5), 559-579.
24. Bandopadhyay, S., Pal, S., Gangopadhyay S. R., Isolation and characterization of plant
growth promoting Bacillus thuringiensis from agricultural soil of West Bengal, Res. J.
Biotechnol., 2011, 6(2), 9-13.
25. Clesceri, L. S., Greenberg, A. E., Eaton, A. D. Standard methods for the examination of
water and wastewater, 20th ed. APHA-AWWA-WEF, Washington DC, 1998.
26. Chance, B, Maehly, A. C. Assay of catalase and peroxidase, Methods Enzymol., 1955,
2, 764-775.
27. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature, 1970, 277, 680-685.
28. Fierer, N., Schimel J. P. A proposed mechanism for the pulse in carbon dioxide
production commonly observed following the rapid rewetting of a dry soil. Soil Sci.
Soc. Am. J, 2003, 67, 798–805.
29. Wongkaew, P., Homkratoke T. Enhancement of soil microbial metabolic activity in
tomato field plots by chitin application. As. J. Food Ag-Ind. Special Issue, 2009, 325-
335.
30. Saha, A., Gangopadhyay, S. R. Screening of lead resistant colonies of plant growth
promoting rhizobacteria Bacillus thuringiensis and insight into the mechanism of lead
resistance, Proceedings of UGC- sponsored National Seminar on Frontiers of
Microbiology: Prospects and Challenges. Ramkrishna Mission Vidyamandir, Belur
Math, pp 29-36.
Prajnan O Sadhona ……., Vol. 2, 2015
86
Review Article
Synthesis and binding pattern of Calix[4]Pyrrole compounds
Debabrata Pal
Assistant Professor, Department of Chemistry, Sreegopal Banerjee College, Bagati, Magra,
Hooghly, Pin – 712148, West Bengal, India.
Correspondence should be addressed to Debabrata Pal; [email protected]
Abstract
In this short review, focus has been on the improvisations in the synthetic procedures
of the Calix[4] pyrrole type compounds, since their first discovery in the year 1886 and
subsequent discussion on their binding pattern towards different chemical species. Calix[4]
pyrrole, which is an analogue of hetero calixarenes, is the representative of a group of
macrocycles. It has an ability to bind neutral and anionic species. The selectivity towards guest
species emerges from the energetics and conformational features of this compound. Different
structural modifications and fuctionalizations have been incorporated in the parent Calix[4]
pyrrole moiety in order to achieve better selectivity.
Key Words: Calix[4]Pyrrole, Synthesis, Binding, Selectivity, Energetics
Introduction
Since the discovery of Calix[4] pyrrole by Baeyer in the year 1886,1 this compound
finds an outstanding passage of potential application in diversified dimensions of chemistry
and related areas. In the nineties of the past decade, about a hundred years after the discovery,
Floriani and coworkers initiate remarkable growth of interest by their extensive work on the
metabolism and synthetic chemistry of deprotonated calixpyrroles.2 Pioneering work by Sessler
and his group triggers the opening of a new domain on these group of compounds,3,4 which
encompasses an area of developing new methodologies, strategies aiming at elaborate
elucidation of functionality, characterization and overall to tune the approach towards
multidisciplinary applications. The calix[4]pyrroles are a most venerable class of materials.
Calixpyrroles are previously considered as Porphyrinogens, the macrocyclic species composed
of four pyrrole rings linked in the α-position via sp3-hybridized carbon atoms. Porphyrinogens
which carry meso-hydrogen atoms are prone to oxidation to the corresponding porphyrin. On
the other hand, fully meso-substituted porphyrinogens are generally not only stable crystalline
Pal, D.: Synthesis and binding pattern of …….
87
materials but also readily obtainable. While the term porphyrinogen is now widely accepted in
the literature, octaalkylporphyrinogens are not bona fide precursors of the porphyrins and
might, therefore, be better considered as being calixpyrroles. Such a renaming, which has
precedent in the chemistry of other heterocyclic ring systems, would allow the analogy to
calixarenes to be more fully stressed. Interestingly, while functionalized calixarenes have been
shown to be capable of binding anions, unmodified calixarene frameworks show no affinity for
anionic guests. In the case of the simple octaalkyl functionalized Calix[4] pyrrole, it is found
that both fluoride and chloride anion are bound in the solid state and that these two anions, and
to lesser extent dihydrogen phosphate, are bound in dichloromethane solution.3 Shortly
thereafter, it is discovered that calixpyrroles bind neutral substrates, though weakly.4 Since
then, much of research have been directed towards the development of systems whose inherent
anion binding properties are modified relative to the "parent" Calix[4] pyrrole. Following the
course of time, different systems such as calix[n]pyrroles,5-8 calixbipyrrole,9,10 strapped
calixpyrrole,11-13 calixpyrrole dimer,14 pentapyrrolic calix[4]pyrrole,15 and cryptand-like
calixpyrrole16,17 have been reported. Calix[4]pyrrole adopts four major conformations as in the
case of calix[4]arene. While 1,3-alternate conformation is found to be preferred in the absence
of a guest, as determined by theory and X-ray method, the cone conformation is observed to
be dominant in the presence of an anionic guest by the formation of hydrogen bonding between
pyrrolic NH and an anion.3 Apart from conventional binding with anionic and neutral guests,
calix[4]pyrrole has recently been reported to function as ditopic ion-pair receptor18 and also to
act as mediators for the selective transport of an ion pair (CsCl) through model phospholipid
bilayers.19 Interaction of calix[4]pyrrole with fullerenes has also been explored recently, where
large binding constants has been reported, indicative of strong binding between these species.20.
In the context of this review article, the major focus will be on different synthetic pathways,
the binding phenomenon and the diversified field of applications.
Synthesis
Baeyer originally synthesized meso-octamethylcalix[4]pyrrole 1 as a white crystalline
material by condensing pyrrole and acetone in the presence of hydrochloric acid.1The reaction
has been refined over the following century to a stage where 90% yields of the macrocycle are
readily obtainable without the need for any form of templation or for high dilution conditions.
Subsequent to the work of Baeyer, Dennstedt and Zimmemann also studied this reaction, using
‘chlorzink’ as the acid catalyst.21,22,23 In the 1950s, Rothemund and Gage reported improved
yields by using methanesulfonic acid as the acid catalyst.24 Protic acids (HCl, H2SO4), organic
Prajnan O Sadhona ……., Vol. 2, 2015
88
acids (CH3SO3H) and Lewis acids (BBr3 and BF3) have also been used in the synthesis of
calix[4]pyrroles.25-28 In recent past, M. Radhakishan et al have reported a novel, shape-
selective, zeolite-catalyzed synthesis of calix[4]pyrroles by refluxing an equimolar ratio of
pyrrole and ketone in dichloromethane over Al-MCM-41,29,30 and Co-MCM-41 is found to
give maximum yields.31 In continuation of their work, efficient evaluation of porphyrins and
calix[4]pyrroles by in situ synthesis from pyrrole with aromatic aldehydes and ketones,
respectively, over zeolite based molecular sieve catalysts as sorbents in thin layer
chromatography (TLC) are accomplished in one step with microwave heating.32 A facile,
highly efficient and eco-friendly protocol for the synthesis of calix[4]pyrroles in excellent
yields is reported in the meantime ,where simple recrystallization techniques instead of tedious
column chromatography, has been employed for compound purification yielding better results
and leading to multi gram scale synthesis.33 Another approach, rather a green chemistry
approach for the synthesis of calix[4]pyrroles and N-confused calix[4]pyrroles in moderate to
excellent yields is done by the reaction of dialkyl or cycloalkyl ketones with pyrrole catalyzed
by reusable Amberlyst™-15 under eco-friendly conditions.34
Binding phenomenon
Calix[4]pyrroles are effective and selective anion binding agents both in solution and
in the solid state. The first ever binding studies have been carried out with meso-octamethyl
calix[4]pyrrole 1 and tetraspirocyclohexylcalix[4]pyrrole 2,3 the structures of 1 and 2 are
provided in Figure 1. The binding occurs via the interaction of the anion and the pyrrolic H
atoms through formation of H bonding.35 The same reasoning can be well attributed during
complexation with neutral guests like alcohols, amides etc where the electronegative atoms in
the guest molecules are employed in H-bonding. Another point, to be evoked, is the inherent
flexibility of calix[4]pyrrole moity,36 which significantly prompts towards a geometrically
favourable local alignment of the core to incorporate the guest in the cavity.37
Figure 1: Structure of meso-octamethyl calix[4]pyrrole[1] and
tetraspirocyclohexylcalix[4]pyrrole[2]
Pal, D.: Synthesis and binding pattern of …….
89
Crystals of the tetrabutylammonium chloride complex of calixpyrrole 1 were obtained
by slow evaporation of a dichloromethane solution containing an excess of the chloride salt.
Crystals of the tetrabutylammonium fluoride complex of 2 were obtained using a similar
procedure.3 X-ray crystal analysis revealed that in both cases the calix[4]pyrrole ligand adopts
a conelike conformation such that the four NH protons can hydrogen bond to the halide anion.
While these two structures are thus grossly similar, in the case of the chloride complex, the
nitrogen-to-anion distances are in the range of 3.264(7)-3.331(7) Å, while for the
corresponding fluoride complex they are 2.790(2) Å. (the four pyrrole groups are equivalent
by symmetry). As a result, in these two complexes the chloride and fluoride anions reside
2.319(3) and 1.499(3) Å above the N4 root-mean-square planes of calixpyrroles 1 and 2,
respectively. Thus, fluoride anion appears to be more tightly bound, at least in the solid state.
Analyses of the solution phase anion-binding properties of calix[4]pyrroles 1 and 2 have been
made using 1H NMR titrations carried out in dichloromethane-d2. The values of the binding
constants are given in table 1 and 2 , for anionic and neutral guests respectively, which reveals
that both compounds 1 and 2 are not only effective 1:1 anion-binding agents in solution, they
are also selective ones; they show a marked preference for F- relative to other putative anionic
guests (Viz. Cl-, Br-, I-, H2PO4-, and HSO4
-). Thus, the anion-binding behavior displayed by 1
and 2 appears to be a direct consequence of having available for hydrogen bonding a
polypyrrolic macrocycle of appropriate size and shape. From this viewpoint it is anticipated
that 1 would modify its shape to accommodate different neutral substrates, provided that these
can act as hydrogen-bonding acceptors. In accord with this expectation, the calixpyrrole unit
in 1.•2(MeOH) is found to assume a 1,3-alternate conformation in the solid state, as judged
from single-crystal X-ray diffraction analysis.4 Single molecules of the alcohol lie above and
below the macrocycle, each one held in place by hydrogen bonds to two pyrrolic NH groups.
Further evidence that methanol is bound to 1, rather than merely occupying space in the lattice,
is given by the inward tilt of the pyrroles of 1.•2(MeOH).Complex 1•2(DMF) [DMF = N,N-
dimethylformamide] has also been studied by X-ray crystallography. As in the methanol
adduct, individual symmetry-equivalent guests are found above and below the host, but in this
case each amide is hydrogen bonded to adjacent pyrroles. The conformation of the calixpyrrole
is therefore 1,2-alternate. Structurally characterized examples of 1,2-alternate calix[4]arenes
are scarce, and 1•2(DMF) is the first calixpyrrole in this conformation to be unambiguously
characterized. The unsaturated portion of the amide lies 3.363(3) Å above the plane of a third
pyrrole ring (the planar twist angle between these two moieties is 7.1(1)°), leading to the
proposal that a π - π stacking interaction helps to stabilize 1•2(DMF). From the results of table.
Prajnan O Sadhona ……., Vol. 2, 2015
90
2 it can be said that Calixpyrrole 1 have a measurable affinity for all of the studied substrates
except nitromethane, which is a notoriously poor hydrogen-bond acceptor. A trend is evident
across both the alcohol and amide series, in which the association constants uniformly decrease
with increasing bulk near the substrate oxygen atom. Methanol is therefore bound most
strongly among the alcohols, with Ka=12.7±1.0 M-1, the two primary alcohols cluster about
Ka~10 M-1, and the secondary alcohols fall near Ka ~ 7 M-1. For the amides, the Ka values are
sensitive to changes in substituents on the nitrogen atom and on the carbonyl carbon. Thus,
receptor 1 has a higher affinity for a secondary formamide (N-formylglycine ethyl ester) than
for a tertiary one (DMF) and forms a more robust complex with DMF than with N,N -
dimethylacetamide. The low Ka value of 2.2 ± 0.1 M-1recorded for 1,1,3,3-tetramethylurea
indicates that the presence of two sets of N-bound methyl groups severely hinders approach of
the amide oxygen atom o the calixpyrrole pseudocavity.
Table 1: Stability constants for 1 and 2 with Anionic Substrates in CD2Cl2 at 298K
Stability constant (M-1)
anion added Calix[4]pyrrole 1 Calix[4]pyrrole 2
fluoride 17170(±900) 3600(±395)
Chloride 350(±5.5) 117(±4.0)
Bromide 10(±0.5)
Iodide <10
Dihydrogen phosphate 97(±3.9) <10
Hydrogen sulfate <10
Pal, D.: Synthesis and binding pattern of …….
91
Table 2. Association constants for 1 with Neutral Substrates
substrate added Ka(M-1) substrate added Ka(M
-1)
methanol 12.7 ± 1.0 N,N-dimethylformamide 11.3 ± 0.8
ethanol 10.7 ± 0.7 N,N-dimethylacetamide 9.0 ± 0.9
benzyl alcohol 9.7 ± 0.7 1,1,3,3-tetramethylurea 2.2 ± 0.1
isopropyl alcohol 7.0 ± 0.4 dimethyl sulfoxide 16.2 ± 1.1
sec-butanol 6.2 ± 0.4 1,2-dimethylimidazole 5.4 ± 0.3
N-formylglycine ethyl
ester
13.3 ± 1.0 acetone 2.2 ± 0.2
However, the selectivity among the halides is lost with increasing solvent polarity and
thus necessitates the idea of enthalpy-driven interaction to play the major part in the binding
process.38,39 This view, though seems to be rational concerning the nature of host and guest in
the present system, is not quite conceivable if cummulative enthalpy of all processes happening
simultaneously in solution is taken into consideration rather than the partial event of the isolated
host-guest interaction, e.g., in free space. The results evolve out of Isothermal titration
calorimetry (ITC) of calix[4]pyrrole 1 with anionic guests clearly reimburse the fact that in
condensed phases selectivity in its thermodynamic sense with competing guests is not a
function of the host structure alone but is heavily dependent on the actual solvent used and the
fluoride specificity in dichloromethane or in the gas phase, too, is compromised and eventually
vanishes totally in more polar solvents such as acetonitrile or DMSO.40 Since calorimetry
determines the integral heat response of all reactions happening simultaneously in solution it
can furnish a detailed mirror image of the molecular events only when the system concerned
are ideal in thermodynamic sense without the necessity of making adjustment for competing
simultaneous events that run within the same time domain of the measurement process. This
situation is quite demanding and practically not attainable considering host-guest interaction in
polar solvents and thus the calorimetric study cannot be rendered as a true thermodynamic
preview of the interaction process rather it govern most attempts of host design grossly.
Retorting to the actual thermodynamic scenario of the intricate host –guest interaction of
calix[4]pyrrole 1 with the anions, a nice study by the group of Angela F. Danil de Namor,
which is claimed to be the first detailed thermodynamic study on the calix[4]pyrrole-anion
complexation process, ascertains the selective recognition of the halides by 1 in polar solvents
Prajnan O Sadhona ……., Vol. 2, 2015
92
like acetonitrile and N,N-dimethylformamide.41 Their approach is quite explanatory in
consideration of the fact that host-guest associations in polar solvents are prone to enthalpy-
entropy compensation42 and the finest of the study is that the selective behavior of the ligand
for the anions have been analyzed in terms of the solvation properties of reactants and products
in the solvents which is justifiable on the results of 1H NMR, conductance measurements, and
titration calorimetry experiments and.thus a relationship between the stability constants and the
chemical shift changes of the pyrrole protons has been established redirecting a supportive
consequences towards selective recognition of anions by 1, the notion that has been quite
outweighed, rather questioned by the previous Isothermal titration calorimetry study.
Following the track of advancement, sole attention was centered on the anion binding
aspect in all respect, the role of the counter-cation in the complexation process remained largely
unrevealed, until manifested by the fact that replacement of the quaternary ammonium by the
{potassium_cryptand[222]} counter-cation of the guest anion viz fluoride, chloride, bromide
and dihydrogen phosphate influences the magnitude of the experimental enthalpy by up to
20%, as reported in the Isothermal titration calorimetry studies of the complexation process
with 1 in acetonitrile medium .40 This reflects the role of the counter-cation not to be that of
an innocent observer, which is already boosted from the results of solid state structural analyses
of the complexes formed from a range of presumably similar chloride anion salts, showing the
cations of different size, to interact differently with the calixpyrrole bowl.43 Various X-ray
crystal structures with variation in the counter cation clearly show differing degrees of
encapsulation of the cation into the anion-induced calixpyrrole cup-shaped cavity in the solid
state. This substantiates the fact that ion pairing, and hence counter cation-derived effects,
could be important under at least some solution-phase conditions. This particular aspect was
treated explicitly by Sessler and collaborators.44 They focus entirely on the solvation and ion-
pairing effect and investigated the variation of solvents and the counter-cation in studying the
complexation of 1 with chloride ions by 1H NMR and ITC method together with
crystallographic studies. The results show that in solvents like nitromethane, acetonitrile and
DMSO, the effect of countercation is reasonably nil, whereas this is much pronounced in
1,2,dichloroethane and even more in dichloromethane, reflecting the substantial effect of
charge density and size of the counter cation on the stability of the ion pair that would form as
the result of the cation interacting with the electron-rich walls of the calix[4]pyrrole “bowl”.
These effects are anticipated to be masked to a greater extent in a more highly solvating solvent
and hence would not necessarily be reflected in observable changes in the calculated affinity
constant for anion binding, Ka. The effect of the counter cation in the binding process is
Pal, D.: Synthesis and binding pattern of …….
93
subsequently demonstrated by solution binding studies of calix[4]pyrroles with anion (added
as tetraalkylammonium salts) investigated using UV–vis spectroscopic techniques.45 The
observation of red-shift of absorption maximum band of 1 in EtOH in the presence of the
tetramethylammonium (TMA+) or tetraethylammonium (TEA+) salts is indicative of the fact
that calix[4]pyrrole receptors linking anionic species through multiple hydrogen bonding
interactions are capable of using the periphery electron-rich “walls” for selectively binding
electron-deficient tetraalkylammonium cation subunits by cation-π charge–transfer interaction
and concomitantly the stability of the calix[4]pyrrole–anion complex is strongly dependent on
the nature of the cation. The meso-alkyl groups of the calix[4]pyrrole, the affinity for the anion
subunits and the structure of tetraalkylammonium cations have considerable effects on the
formation of cation-π charge–transfer interaction. Taking in concert, it can be said that the apart
from the host-designing, the association process is influenced by the solvent and the counter
cation, although any apparent correlation between the observed binding behavior and the
solvent parameters viz. permittivity, refractive index (polarizability), dielectric constant,
donicity, or acceptor strength etc. are yet to be established. The selective recognition of anions
by calix[4]pyrroles is substantiated by a recent study on the binding of the macrocycle with
fluoro, chloro, bromo, iodo and sulphato anions generated from the corresponding normal-
TBAsalts, investigated by electrospray ionization mass spectrometry (ESI-MS) in
dichloromethane–acetonitrile in negative ion mode.46 The binding strength is found to be
inversely proportional to the size of anion, which is previously interpreted based on the
thermodynamic studies referred earlier.41 The association constants of calix[4]pyrroles and
anions obtained from electronic transition studies were in good agreement with that observed
from 1H NMR titration studies.
Applications
The most important application of the calixpyrrole moiety is perceived by utilizing its
ability to bind with both anionic and neutral guest species and also by their potency to act as
ion-pair receptors. In order to achieve responses with discernible specificity, different redox or
fluorescent moieties are incorporated in the parent calixpyrrole skeleton to render the system
effective towards discrimination between different guests. Recently a new calix[4]pyrrole-
based, dual functional, chemodosimetric sensor is reported and studied as a cyanide selective
indicator; where complete color bleaching is observed even in the co-presence of an excess of
other anions.47 This is quite a land mark in the development of sensors, concerning the toxicity
Prajnan O Sadhona ……., Vol. 2, 2015
94
of cyanide ions. The calixpyrrole is effective in facilitating ion transportation across
membranes. The calixpyrrole moiety also finds application in the field of capillary
electrophoresis48 calixpyrrole and calixpyrrole based materials also find use in fabrication of
electrodes and electroanalytical sensors49, promoter50 and catalysts51 in different reactions, as
well as in the preparation of nanofilms52 and selective ion carriers53. The domain of application
seems to be increasing in a faster way and thus turning this easy to make simple macrocycle
namely calixpyrrole, a very important molecule for the future days to come.
References
1. Baeyer, A. Ueber ein Condensationsproduct von Pyrrol mit Aceton. Ber. Dtsch. Chem.
Ges. 1886, 19, 2184.
2. Jacoby, D.; Floriani, C.; C.-Villa, A.; Rizzoli, C. The π and σ bonding modes of meso-
octaethylporphyrinogen to transition metals: the X-ray structure of a meso-
octaethylporphyrinogen–zirconiuml (IV) complex and of the parent meso-
octaethylporphyrinogen ligand. J. Chem. Soc., Chem. Commun. 1991, 790.
3. Gale, P. A.; Sessler, J. L.; Král, V.; Lynch, V. Calix[4]pyrroles: Old Yet New Anion
Binding Agents. J. Am. Chem. Soc. 1996, 118, 5140.
4. Allen, W. E.; Gale, P. A.; Brown, C. T.; Lynch, V. M.; Sessler, J. L. Binding of Neutral
Substrates by Calix[4]pyrroles. J. Am. Chem. Soc. 1996, 118, 12471.
5. Turner, B.; Botoshansky, M.; Eichen, Y. Extended Calixpyrroles: meso-Substituted
Calix[6]pyrroles. Angew. Chem., Int. Ed. 1998, 37, 2475.
6. Cafeo, G.; Kohnke, F. H.; la Torre, G. L.; White, A. J. P.; Williams, D. J. From Large
Furan-Based Calixarenes to Calixpyrroles and Calix[n]furan[m]pyrroles: Synthesis and
Structures. Angew. Chem., Int. Ed. 2000, 39, 1496.
7. Cafeo, G.; Kohnke, F. H.; la Torre, G. L.; White, A. J. P.; Williams, D. J. The
Complexation of Halide Ions by a Calix[6]pyrrole. Chem. Commun. 2000, 1207.
8. Sessler, J. L.; Anzenbacher, P., Jr.; Shriver, J. A.; Jurisíková, K.; Miyaji, H.; Lynch, V.
M.; Marquez, M. Direct Synthesis of Expanded Fluorinated Calix[n]pyrroles:
Decafluorocalix[5]pyrrole and Hexadecafluorocalix[8]pyrrole. J. Am. Chem. Soc.
2000, 122, 12061.
9. Sessler, J. L.; An, D.; Cho, W.-S.; Lynch, V. Calix[n]bipyrroles: Synthesis,
Characterization, and Anion Binding Studies, Angew. Chem. Int. Ed. 2003, 42, 2278.
Pal, D.: Synthesis and binding pattern of …….
95
10. Sessler, J. L.; An, D.; Cho, W. S.; Lynch, V. Calix[2]bipyrrole[2]furan and
Calix[2]bipyrrole[2]thiophene: New Pyrrolic Receptors Exhibiting a Preference for
Carboxylate Anions, J. Am. Chem. Soc. 2003, 125, 13646.
11. Yoon, D. W.; Hwang, H.; Lee, C. H. Synthesis of a strapped calix[4]pyrrole: structure
and anion binding properties. Angew. Chem., Int. Ed. 2002, 41, 1757.
12. Lee, C.-H.; Na, H.-K.; Yoon, D.-W.; Won, D.-H.; Cho, W.-S.; Lynch, V. M.; Shevchuk,
S. V.; Sessler, J. L. Single Side Strapping. A New Approach to Fine Tuning the Anion
Recognition Properties of Calix[4]pyrroles. J. Am. Chem. Soc. 2003, 125, 7301.
13. Lee, C. H.; Lee, J.-S.; Na, H.-K.; Yoon, D.-W.; Miyaji, H.; Cho, W.-S.; Sessler, J. L.
Cis and transStrapped calix[4]pyrroles Bearing Phthalimide Linkers; Synthesis and
Anion Binding Properties. J. Org. Chem. 2005, 70, 2067.
14. Sato, W.; Miyaji, H.; Sessler, J. L. Calix[4]pyrrole Dimers Bearing Rigid
Spacers. Towards the Synthesis of Cooperative Anion Binding Agents. Tetrahedron
Lett. 2000, 41, 6731.
15. Warriner, C. N.; Gale, P. A.; Light, M. E.; Hursthouse, M. B. Pentapyrrolic
calix[4]pyrrole. Chem. Commun. 2003, 1810.
16. Gale, P. A.; Genge, J. W.; Král, V.; McKervey, M. A.; Sessler, J. L.; Walker, A. First
Synthesis of an Expanded Calixpyrrole. Tetrahedron Lett. 1997, 38, 8443.
17. Bucher, C.; Zimmerman, R. S.; Lynch, V.; Sessler, J. L. First Cryptand-like
Calixpyrrole. Synthesis, X-ray Structure and Anion binding Properties of a
Bicyclic[3,3,3]nonapyrrole. J. Am. Chem. Soc. 2001, 123, 9716.
18. Custelcean, R.; Moyer, B. A.; Sessler, J. L.; Cho, W.-S.; Gross, D.; Bates, G. W.;
Brooks, S. J.; Light, M. E.; Gale, P. A. Calix[4]pyrrole : An Old yet New Ion-Pair
Receptor. Angew. Chem. Int. Ed. 2005, 44, 2537.
19. Tong, C. C.; Quesada, R.; Sessler, J. L.; Gale, P. A. Meso-
Octamethylcalix[4]pyrrole: an old yet new transmembrane ion-pair transporter.
ChemComm. 2008, 6321.
20. Pal, D.; Goswami, D.; Nayak, S. K.; Chattopadhyay, S.; Bhattacharya, S. Spectroscopic
and Theoretical Insights into the Origin of Fullerene-Calix[4]pyrrole Interaction. J.
Phys. Chem. A, 2010, 114, 6776.
21. Dennstedt, M.; Zimmerman, J. Ueber die Einwirkung des Paraldehyds auf das Pyrrol.
Chem. Ber. Dtsch. Ges. 1886, 19, 2189.
22. Dennstedt, M.; Zimmerman, J. Ueber die Einwirkung des Acetons auf das Pyrrol.
Chem. Ber. Dtsch. Ges. 1887, 20, 850.
Prajnan O Sadhona ……., Vol. 2, 2015
96
23. Dennstedt, M. Ueber die Einwirkung des Acetons auf das Pyrrol. Chem. Ber. Dtsch.
Ges., 1890, 23, 1370.
24. Rothemund P.; Gage, C. L. Concerning the Structure of “Acetonepyrrole”. J. Am.
Chem. Soc. 1955, 77, 3340.
25. Shao, S.; Wang, A.; Yang, M.; Jiang, S.; Xianda, Y. Synthesis of meso-aryl-substituted
calix[4]pyrroles. Synth. Commun. 2001, 31, 1421.
26. Chen, Q.; Wang, T.; Zhang, Y.; Wang, Q.; Ma, J. Doubly n-confused calix[4]pyrrole
prepared by rational synthesis. Synth. Commun. 2002, 32, 1051.
27. Anzenbacher, P. Jr.; Jursíková, K.; Lynch, V. M.; Gale, P. A.; Sessler, J. L.
Calix[4]pyrroles Containing Deep Cavities and Fixed Walls. Synthesis, Structural
Studies and Anion Binding Properties of the Isomeric Products Derived from the
Condensation of p-Hydroxyacetophenone and Pyrrole. J. Am. Chem. Soc. 1999, 121,
11020.
28. Schmidtchen, F. P.; Berger, M. Artificial Organic Host Molecules for Anions. Chem.
Rev. 1997, 97, 1609.
29. Kishan, M. R.; Srinivas, N.; Raghavan, K. V.; Kulkarni, S. J.; J. Sarma, A. R. P.;
Vairamani, M. A novel, shape-selective, zeolite-catalyzed synthesis of calix(4)pyrroles.
Chem. Commun. 2001, 2226.
30. Kishan, M. R.; Rani, V. R.; Murty, M. R. V. S.; Devi, P. S.; Kulkarni, S. J.; Raghavan,
K. V. Synthesis of calixpyrroles and porphyrins over molecular sieve catalysts. J. Mol.
Catalysis. A: Chemical. 2004, 223, 263.
31. Kishan, M. R.; Rani, V. R.; Kulkarni, S. J.; Raghavan, K. V. A new environmentally
friendly method for the synthesis of calix [4] pyrroles over molecular sieve catalysts.
J. Mol. Catalysis. A: Chemical. 2005, 237, 155.
32. Kishan, M. R.; Rani, V. R.; Devi, M. R. P. S.; Kulkarni, S. J.; Raghavan, K. V. A
novel zeolite based stationary phases for in situ synthesis and evaluation of porphyrins
and calix [4] pyrroles. J. Mol. Catalysis. A: Chemical. 2007, 269, 30.
33. Dey, S.; Pal, K.; Sarkar, S. An Efficient and Eco-friendly Protocol to Synthesize
Calix[4]pyrroles. Tetrahedron. Lett. 2006, 47, 5851.
34. Chauhan, S. M. S.; Garg, B.; Bisht, T. Syntheses of Calix[4]Pyrroles by Amberlyst-
15 Catalyzed Cyclocondensations of Pyrrole with Selected Ketones. Molecules. 2007,
12, 2458.
35. Sessler, J. L.; Andrievsky, A.; Gale, P. A.; Lynch, V. Anion Binding: Self-Assembly
of Polypyrrolic Macrocycles. Angew. Chem. Int. Ed. 1996, 35, 2782.
Pal, D.: Synthesis and binding pattern of …….
97
36. Floriani, C. The porphyrinogen-porphyrin relationship: the discovery of artificial
porphyrins. Chem. Commun. 1996, 1257.
37. Gale, P. A.; Sessler, J. L.; Král, V. Calixpyrroles. Chem. Commun. 1998, 1.
38. Anzenbacher, P., Jr.; Try, A. C.; Miyaji, H.; Jurisíková, K.; Lynch, V. M.; Marquez,
M.; Sessler, J. L. Fluorinated Calix[4]pyrrole and Dipyrrolylquinoxaline. Neutral Anion
Receptors with Augmented Affinites and Enhanced Selectivities, J. Am. Chem. Soc.
2000, 122, 10268.
39. Camiolo, S.; Gale, P. A.; Fluoride recognition in “Super-extended cavity”
calix[4]pyrrole. Chem. Commun. 2000, 1129.
40. Schmidtchen, F. P. Surprises in the Energetics of Host−Guest Anion Binding to
Calix[4]pyrrole. Org. Lett. 2002, 4, 431.
41. de Namor, A. F. D.; Shehab, M. Selective Complexation of Anions (Chloride, Bromide,
Iodide) by a Calix[4]pyrrole. J. Phys. Chem. B. 2003, 107, 6462.
42. Dunitz, J. D. Win some, lose some: enthalpy-entropy compensation in weak
intermolecular interactions. Chem. Biol. 1995, 2, 709.
43. Blas, J. R.; Marquez, M.; Sessler, J. L.; Luque, F. J.; Orozco, M. Theoretical Study of
Anion Binding to Calix[4]pyrrole: The Effect of Solvent, Fluorine Substitution,
Cosolute and Water Traces. J. Am. Chem. Soc. 2002, 124, 12796.
44. Sessler, J. L.; Gross, D. E.; Cho, W.-S.; Lynch, V. M.; Schmidtchen, F. P.; Bates, G.
W.; Light, M. E.; Gale, P. A. Calix[4]pyrrole as a Chloride Anion Receptor: Solvent
and Counter-Cation Effects, J. Am. Chem. Soc. 2006, 128, 12281.
45. Liu, K.; Xu, J.; Sun, Y.; Guo, Y.; Jiang, S.; Shao, S. Spectroscopic study on anion
recognition properties of calix[4]pyrroles: effects of tetraalkylammonium cations.
Spectrochim. Acta A. 2008, 69, 1201.
46. Giri, N. G.; Chauhan, S. M. S.; Spectroscopic and spectrometric studies of anion
recognition with calix[4]pyrroles in different reaction conditions. Spectrochim. Acta A.
2009, 74, 297.
47. Hong, S. J.; Yoo, J.; Kim, S. H.; Kim, J. S.; Yoon, J.; Leem, C. H. β-Vinyl substituted
calix[4]pyrrole as a selective ratiometric sensor for cyanide anion. Chem. Commun.
2009, 189.
48. Ma, H.; Luo, M.; Shao, S.; Liu, X.; Jiang, S. Cation effects in the separation of
calix[4]pyrroles by nonaqueous capillary electrophoresis with tetraalkylammonium
chloride salts as background electrolytes. J. Chromatography A. 2008, 1192, 180.
Prajnan O Sadhona ……., Vol. 2, 2015
98
49. Yang, W.; Yin, Z.; Wang, C.-H.; Huang, C.; He, J.; Zhu, X.; Cheng, J.-P. New redox
anion receptors based on calix[4]pyrrole bearing ferrocene amide. Tetrahedron. 2008,
64, 9244.
50. Martínez-García, H.; Morales, D.; Pérez, J.; Coady, D. J.; Bielawski, C. W.; Gross, D.
E.; Cuesta, L.; Marquez, M.; Sessler, J. L. Calix[4]pyrrole as a promoter of the CuCl-
catalyzed reaction of styrene and Chloramine-T. Organometallics. 2007, 26, 6511.
51. Cafeo, G.; De Rosa, M.; Kohnke, F. H.; Neri, P.; Soriente, A.; Valenti, L. Efficient
organocatalysis with a calix[4]pyrrole derivative. Tetrahedron Lett. 2008, 49, 153.
52. Taner, B.; Özcan, E.; Üstündağ, Z.; Keskin, S.; Solak, A. O.; Ekşi, H. Fabrication of
calix[4]pyrrole nanofilms at the glassy carbon surface and their characterization by
spectroscopic, optic and electrochemical methods. Thin Solid Films. 2010, 519, 289.
53. Amiri, A. A.; Safavi, A.; Hasaninejad, A. R.; Shrghi, H.; Shamsipur, M. Highly
selective transport of silver ion through a supported liquid membrane using
calix[4]pyrroles as suitable ion carriers. J. Membrane Science. 2008, 325, 295.
Sen Sarma, M.: Cytotoxic activity of organotin(IV) complexes …….
99
Review Article
Cytotoxic activity of organotin(IV) complexes - a short review
Moumita Sen Sarma
Assistant Professor, Department of Chemistry, Fakir Chand College, Diamond Harbour –
743331, West Bengal, India.
Correspondence should be addressed to Moumita Sen Sarma; [email protected]
Abstract
Over the recent decades, investigations on organotin(IV) complexes have received
considerable attention due to the structural diversity they exhibit and their diversified non-
biological and biological applications. Organotins are also finding important place as effective
antitumour agents. This review briefly discusses the cytotoxic activity of various organotin
compounds against different cancer cell lines.
Key Words: Organotin (IV) complexes, Anti-tumour activity, Cytotoxic activity
Introduction
The relationship between metal ions and cancer are both intriguing and controversial.
Since its discovery by Furst in 19631, metal chelates continue to play an important role in the
cure and cause of malignancy. In addition to this, the discovery of the antitumour activities of
titanocene dichloride2-4 and certain diorganotin derivatives5,6 stimulated much interest in the
research of organometallic compounds as antitumour agents.
The introduction of cisplatin as the first inorganic compound approved for clinical use
in 1978 produced a great impetus in cancer treatment. Cisplatin is used to treat various types
of cancers like small cell lung cancer, ovarian cancer, lymphomas, bladder cancer, cervical
cancer and germ cell tumours7. Unfortunately, the use of cisplatin was also identified to be
associated with number of side effects such as nephrotoxicity, neurotoxicity, ototoxicity,
myelotoxicity, hemolytic anemia and several disadvantages such as the emergence of
cisplatin resistance. Hence, cisplatin combination chemotherapy (which now includes
carboplatin and oxalipatin, which are also platinum derivative drugs) was consequently being
introduced8. In addition to the organometallic complexes of Pt, other organometallic
compounds derived from Sn, Au, Ti, Cu, Pd and Ru have also emerged as potent antitumour
Prajnan O Sadhona ……., Vol. 2, 2015
100
agents. Recent studies on the antitumour activities of organotins have shown that low doses
of organotins can induce cell death and have better or similar potential when compared with
other drugs which are in clinical use9.
Antitumour activity of organotin(IV) complexes
Although the first organotin(IV) compound was tested for its antitumour activity10 in
1929, no systematic study was undertaken afterwards and as a result only 1509 organotin
compounds have been tested in various tumour systems by the end of 198111. Organotin
compounds with coordination number greater than four are significant for their large spectrum
of biological activity12,13 including antifungal, antibacterial, antimicrobial, antinematicidal,
antiinsecticidal, antiherbicidal and anti-inflammatory activities and also interesting structures.
Along with this their potent antitumour activity has led many researchers to investigate them
as effective antitumour agents and a large body of literature is now available14-29.
Brown firstly reported30 the restraint effect of triphenyltin acetate for the growth of
anticancer cells. It has been demonstrated that certain triphenyltin benzoates exhibit
exceptionally high in vitro activity31 against MCF-7 and WiDr cell lines. Some novel
triphenyltin carboxylates32 also revealed interesting low ID values compared to the clinically
established antitumour drugs.
Di-n-butyltin, di-t-butyltin and diphenyltin 2, 6-pyridine carboxylates33-35 were found
to be more active than cisplatin against MCF-7, a mammary tumour, and WiDr, a colon
carcinoma. The anhydrous bis(dicylohexylammonium)bis(2,6-pyridine dicarboxy- lato)
dibutyl stannate is reported to display higher in vitro antitumour activity than those of
carboplatin and cisplatin36.
Bis{3-methoxysalicylato[di(n-butyl)]tin(IV)}oxide, obtained by the condensation of
Bu2SnO and 3-methoxysalicylic acid , exhibited higher antitumour activities in vitro against
two tumour cell lines , MCF-7 and WiDr than those of the 5-Me and 4-Meo analogues37.
Gielen et al. synthesized and screened38 a series of organotin compounds against MCF-
7 and WiDr and found that the complexes exhibited very promising antitumour activities. Di-
n-butyltin bis (2, 5-dihydroxybenzoate) was found to be as active as cisplatin in vitro against
murine Colon 26 carcinoma.
Saxena and Tandon39 screened a series of di-n-butyltin complexes of Schiff bases
derived from S-substituted dithiocarbazates and p-fluoroaniline for their activity against P-388
Sen Sarma, M.: Cytotoxic activity of organotin(IV) complexes …….
101
Lymphoyte Leukaemia and suggested that the presence of highly electronegative groups can
greatly enhance the activity.
The dibutyltin complexes of the general formula Bu2SnL (where L= dianion of
tridentate Schiff bases derived from the condensation of 2-hydroxy-1-naphthaldehyde or acetyl
acetone with Gly, L- -Ala, DL- Val, DL- 4-aminobutyric acid, L-Met, L-Leu and PhGly) have
exhibited higher antitumour activities than those observed for cisplatin and carboplatin40.
Barbieri et al.41 reported the antitumour activity of diorganotin(IV) complexes with
adenine (R2SnAd2) and glcylglycine(R2SnGly-Gly) against the P-388 lymphocyte leukaemia
in mice. The (R2SnGly-Gly) were active against leukaemia in very small doses. They suggested
the antileukaemic activity of R2SnGly-Gly depends upon the peculiar structure and bonding of
the solids.
Ruisi et al. have tried to interpret the action of R2Sn(IV) glycylglycinates (R= Me, n-
Bu, n-Oct and Ph) on a molecular basis. Of the various tumours studied the Bu2Sn(Gly-Gly)
was only active against P-388 leukaemia. On the basis of solution studies using various
spectroscopic techniques, it is likely that solvated species in aqueous solution or a mixture of
water and organic solvent and unsolvated species (mainly organic solvent) are present in
equilibrium and contribute to passage of alkyltin complexes across the cell membrane which
in turn produces the biological activity42. A series of organotin dipeptide compounds and a
number of diorganotin halides and pseudohalides [SnR2X2L2] (L= amino ligand e.g., py, bpy,
phen, en) have displayed modest antitumour activity43.
Many di-n-butyl, tri-n-butyl and triphenyltin complexes with hydroxyaryl- carboxylic,
ketocarboxylic, fluoroarylcarboxylic acids and many other oxygen and nitrogen containing
derivatives of carboxylic acids display high antitumour activities44-48.
Cardarelli et al. investigated the antitumour activity of a number of organotin
compounds, namely, tri-butyltin fluoride, dibutyltin dichloride.2-2’-bipyridyl, 1,10-
phenanthroline.dibutyltin complex and dibutyltin histidine by administering these to cancerous
mice in drinking water and found that the tumour growth rates were significantly reduced49.
Studies on the tin content in various body organs led Cardarelli et al.50 to hypothesize that
soluble organotin compounds of various types introduced into the body are concentrated in the
thymus gland. The tin in the thymus is then processed into one or more biochemical that acts
as anti-carcinogens. The isolation and evaluation of thymic extracts reasonably pointed to the
unknown tin-bearing anti-oncogenic biochemicals of steroid nature. On the basis of their
Prajnan O Sadhona ……., Vol. 2, 2015
102
hypothesis, Cardarelli et al.51 patented several organotin compounds of steroids which show
marked antitumour activity.
The synthesis and in vitro screening of even more water soluble organotin compounds,
containing a polyoxaalkyl moiety linked to tin either by a carbon-tin or by a carbon-oxygen
bond is one of the latest development by Gielen and his coworkers52,53.
The compounds Ph2Sn(OH)Cl, (Et2SnO)n and ClMe2SnOSnMe2Cl as well as their
octahedral adducts R2SnX2L2 (where R is alkyl or aryl, X is halide or thiocyanate and L2 are
nitrogen atoms of the bidentate ligands 1,10-phenanthroline, 2-2’-bipyridyl and 2-
aminomethylpyridine) have been determined to be active against P-388 Lymphocytic
Leukaemia in mice54. The mechanism of antitumour action of these drugs proposed was the
preferred transportation of the complex species into the tumour cells, which would be attacked
by hydrolyzed R2Sn(IV)2+ moieties.
Casas et al. reported that [SnMe2(PyTSC)(OAc)].HOAc, [SnMe2(DAPTSC)],
[SnPh2(DAPTSC)].2DMF where H2DAPTSC= 2,6-diacetylpyridinebis(thiosemicarbazo- ne)
all suppress proliferation of FLC . DMSO-induced differentiation of FLC was slightly
suppressed by [SnMe2(DAPTSC)] and was unaffected by [SnPh2(DAPTSC)].2DMF and
[SnMe2(PyTSC)(OAc)].HOAc55.
S. G. Teoh and coworkers reported that bis(acetonethiosemicarbazone-S) dichloro
diphenyltin(IV), SnPh2Cl2(atsc)2 exhibited significant cytotoxicity against human colon
adenocarcinoma, breast adenocarcinoma, hepatocellular carcinoma and acute lymphoblastic
leukaemia56.
In aqueous solution, most of the organotin complexes undergo dissociation, aquation
and hydrolysis reactions. For diorganotin complexes, mono-, di- and poly-nuclear
dialkyltin(IV) species co-exist in the aqueous solution, depending on pH. Moreover dialkyl
dihydroxo tin(IV) species prefer to be five- or six- coordinated by binding weakly to one or
two water molecules57. R2Sn bond seems rather stable in aqueous solution, but the triorganotins
will release one alkyl group to give active R2Sn species, the half-life of this process has been
shown to be between 3 and 6 days58. The R2Sn(IV)2+ has been suggested as the active species
for the organotin anticancer agents59.
Atassi assumed that water-soluble organotin(IV) compounds are probably more active
than complexes soluble only in organic solvents. More recent results on the antitumour activity
of the organotin complexes on the tumour cell lines seem to point to a necessary balance
between solubility and lipophilicity in order to optimize their efficacy48,60,61. Attempts to
Sen Sarma, M.: Cytotoxic activity of organotin(IV) complexes …….
103
improve the bioavailability of the organotin(IV) cations by the formation of water soluble
complexes62 or by their inclusion into -cyclodextrin63 have also been reported.
Organotin carboxylated derivatives have shown high cytotoxic activity against various
cell lines of human origin. Three new tri-n-butyltin derivatives from 4-oxo-4-
(arylamino)butanoic acids were tested for their in vitro anti-proliferative effect on HeLa, CaSki
and ViBo cell lines. All of the compounds showed potency against all three strains and null or
low cytotoxic activity (necrotic) as well. The most potent of the derivatives as an anti-
proliferative agent against the three cell lines was tributylstannyl 4-oxo-4-[(3-trifluoromethyl-
4-nitrophen-1-yl)amino]butanoate, exhibiting an IC50 value of 0.43 μM against the HeLa cell
line64.
The biological activity of organotin(IV) complexes with pyridoxal thiosemicarbazone
(PLTSC) [SnR2(PLTSC-2H)] (R=Me, Et, Bu, Ph) were also evaluated65. With the exception
of the Me complex, the other three suppressed Friend erythroleukaemia cells (FLC)
proliferation with the lowest thresholds for the latter two compounds.
Nine diorganotin(IV) compounds of Schiff bases derived from salicylaldehyde /
substituted salicylaldehyde and thiosemicarbazide were synthesized. Their cytotoxicity was
also investigated against several human cancer cell lines66. The results obtained indicated that
amongst the different organotins selected for the study, Ph2SnL1was found to possess the
highest potency against most of the cell lines studied followed by n-Bu2SnL1 and Ph2SnL2
respectively. Me2SnL2 appeared to be the least potent.
The results further suggested that all the compounds selected for the study were not
equally effective against the various cancer cell lines. This difference in the degree of
selectivity could, presumably, be either due to differential binding potential of the compounds
or metabolic activities of the cell lines which helps in overcoming the cytotoxic effects of the
organotins. The formulae of the ligands and the abbreviations of the complexes used for this
study are presented in Scheme 1.
L1: X = H; L2: X = Br
1: Me2SnL1; 2: n-Bu2SnL1; 3: Ph2SnL1; 4: Me2SnL2.H2O; 6: Ph2SnL2
Scheme 1: The formulae of the ligands and the abbreviations of the complexes66
Prajnan O Sadhona ……., Vol. 2, 2015
104
Figure 1: Crystal structure of [Ph2SnL1] (3)66
Five organotin complexes of terpyridine derivatives were synthesized and
characterized. The mononuclear Sn(IV) complexes were six-coordinated adopting a distorted
octahedral geometry. A brief outline of the fluorescence spectra and the in vitro cytotoxicity
of Sn(IV) complexes were reported67.
Figure 2: Inhibitory effect of dibutyltin complex of salicylaldehyde thiosemicarbazone (n-
Bu2SnL1) on different Human cancer cell lines. Adriamycin (ADR) has been used
as positive control66
A new arenetelluronic triorganotin ester, namely (Me3Sn)4[o-Me-PhTe(μ-O)(OH)O2)]2
has been prepared by the reaction of o-tolyltelluronic acid and Me3SnCl in the presence of
potassium hydroxide. Cytotoxic assessments showed that the complex can induce apoptotic
cell death via accumulation of reactive oxygen species, ROS, collapse of the matrix
0
20
40
60
80
100
120
Colo205 Hop62 MCF7 PC3 SiHa ZR-75-1 A-2780
Human cancer cell lines
% I
nh
ibit
ion
10 ug/ml20 ug/ml40 ug/ml80 ug/ml
Sen Sarma, M.: Cytotoxic activity of organotin(IV) complexes …….
105
metalloproteinase, MMP and activating caspase-3, a caspase protein (cysteine-aspartic acid
protease). The results indicated that ROS is crucial to the cytotoxicity induced by the
complex68.
Figure 3: Crystal structure of (Me3Sn)4[o-Me-PhTe(μ-O)(OH)O2)]268
Very recently a review by Hadjikakou et al focusses on the antiproliferative and anti-
tumour activity of organotin compounds69.
Three main factors which have been found to play an important role in determining the
structure-activity relationship for organotin(IV) derivatives (L)xRnSnX4-n are:
the nature of the organic group R
the nature of halide or pseudohalide X
the nature of donor ligand
Examination of the structures of Sn(IV) compounds containing a N-donor atom and
tested for antitumour activity revealed that in the active tin complexes the average Sn-N bond
lengths were greater than 239 pm, whereas inactive complexes had Sn-N bond lengths < 239
pm, which implies that predissociation of the ligand may be an important step in the mode of
action of these complexes , while the coordinated ligand may favour transport of the active
species to the site of action in the cells, where they are released by hydrolysis70.
Also, a comparison of the structures of the active and inactive compounds suggest71
that in all the active compounds there is
The availability of coordination positions at Sn
The occurrence of relatively stable ligand-Sn bonds, Sn-N and Sn-S
Low hydrophilic decomposition of these bonds
Prajnan O Sadhona ……., Vol. 2, 2015
106
With regard to the data published on all the Sn(IV) derivatives , it can be concluded
that R2Sn(IV)2+ compounds generally exhibit higher antitumour activity than those of the
corresponding mono-, tri- and tetra- organotin(IV) or the inorganic Sn(IV) derivatives, and
within the diorganotin series , the highest activity is exerted by the Et2Sn(IV)2+ and Ph2Sn(IV)2+
complexes. Also, it has been established that the R2Sn(IV)2+ compounds which exhibit
maximum antitumour activity combined with low mammalian toxicity are adducts of the type
R2SnX2L2 (X=halogen, pseudohalogen, L= O- or N- donor ligand)70.
Conclusion
From the foregoing description of the antitumour properties of organotin(IV)
complexes it is clear that such complexes are likely to find applications in medicine. Organotins
offer the potential of reduced cytotoxicity, non-cross resistance and a different spectrum of
activity compared to conventional chemotherapeutic agents such as platinum-containing
compounds72-74. The exact mechanism of action of organotins on controlling tumour growth is
not well known75. It has been reported that organotins induce tumour cell death in a dose
dependent manner75-78. At higher concentrations, these compounds induce degenerative
changes indicative of necrosis accompanied by random DNA breakdown. Electron microscopic
studies revealed that organotin-induced cytotoxicity to be associated with cell shrinkage,
chromatin condensation, fragmentation of DNA, and development of apoptotic bodies79. It has
been suggested that organotins, like other DNA binding drugs, induce cellular stress which
activates the tumour suppressor p53 leading to apoptotic death79. The involvement of zinc and
calcium dependent endonucleases has also been suggested in cytotoxicity induced by
organotins80.
DNA is believed to be the main cellular target for most of the metal anticancer agents.
The anticancer nature is the coordination of metal ions with DNA molecules, causing DNA
damage in cancer cells, blocking the division of the cancer cells and resulting in cell death. The
aqueous interactions of solvated di- and tri- organotin (IV) species R2Sn(IV) and R3Sn(IV)
(R=Me, Et, n-Bu, n-Oct, Ph in ethanol solution) with native DNA(calf thymus) have been
investigated by 119Sn Mössbauer spectroscopy at pH between 5 and 7.481-84. The addition of
ethanolic organotins [R2SnCl2(EtOH)2 or R3SnCl(EtOH)] to DNA yielded solid products, the
119Sn Mössbauer spectra suggested that the tin was coordinated by the phosphodiester groups
of the nucleotides. While the water soluble hydrolyzed dimethyltin(IV) species did not show
any interaction with native DNA81,84.
Sen Sarma, M.: Cytotoxic activity of organotin(IV) complexes …….
107
Yang et.al.85 studied the binding modes of (CH3)2SnCl2, (C2H5)2SnCl2,
(C2H5)2SnCl2(phen) with DNA and nucleotides in aqueous solution by using modern
techniques. The results show that all the above compounds exhibit high affinity to the
phosphate group of the nucleotides. The interactions of (CH3)2SnCl2 and (C2H5)2SnCl2 with
calf thymus DNA suggested that organotins(aqueous) binded to DNA via only the phosphate
group. In addition, the relationship between DNA binding property of metal anticancer
complex and their anticancer activity was discussed in the review and a hypothesis named
‘Two-Pole Complementary Principle’ was also put forward and some criterias were suggested
as well.
Recently, a review by Tabassum et al focusses on chemical and biotechnological
developments in organotin cancer chemotherapy [86]. This review provides a substantial
knowledge on the action mode of organotins in cancer chemotherapy. The coordinating mode
of organotin compounds towards DNA and cancer cells is discussed. Most of the organotins
tested are DNA-targeted and mitotic, the action mode occurring via a gene-mediated pathway.
These potential anti-cancer drugs are actually being studied widely, and whilst they are
efficacious and perhaps curative against a select number of neoplasias, suffer from a variety of
deficiencies, notably severe systemic toxicity and a tendency to elicit drug resistance.
Acknowledgement
The author is grateful to her research guide Prof. (Dr.) Abhijit Roy, Retired Professor,
Department of Chemistry, North Bengal University, for introducing and teaching her the facets
of organotin chemistry during her doctoral works. She is also obliged to the authority of Fakir
Chand College for providing necessary infrastructure and to all her colleagues for their constant
support and encouragement.
References
1. Furst, A. Chemistry of Chelation in Cancer, Thomas Springfield, New York, 1963, 111.
2. Köpf, H.; Köpf-Maier, P. Titanocene dichloride--the first metallocene with
cancerostatic activity. Angew. Chem. Int. Ed. Engl. 1979, 18, 477.
3. Köpf- Maier, P.; Köpf, H. Vanadocene Dichloride - Another Antitumor Agent from
the Metallocene Series. Z. Naturforsch. 1979, 34b, 805.
4. Köpf- Maier, P.; Leitner, M.;Voigtlander, R.; Köpf, H. Molybdocene Dichloride as an
Antitumor Agent. Z. Naturforsch. 1979, 34c, 1174.
Prajnan O Sadhona ……., Vol. 2, 2015
108
5. Crowe, A. J.; Smith, P. J.; Atassi, G. Investigations into the antitumour activity of
organotin compounds. I. Diorganotin dihalide and di-pseudohalide complexes. Chem.
Biol. Interact. 1980, 32, 171.
6. Crowe, A. J.; Smith, P. J.; Atassi, G. Investigations into the antitumour activity of
organotin compounds. 2. Diorganotin dihalide and dipseudohalide complexes. Inorg.
Chim. Acta. 1984, 93, 179.
7. Florea, A.M.; Busselberg, D. Cisplatin as an anti-tumor drug: Cellular mechanism of
activity, drug resistance and induced side effects. Cancers. 2011, 3, 1351.
8. Stordal, B.; Davey, M. Understanding cisplatin resistance using cellular models.
IUBMB Life. 2007, 59, 696.
9. Alama, A.; Tasso, B.; Novelli, F.; Sparatore, F. Organometallic compounds in
oncology: implications of novel organotins as antitumor agents. Drug Discovery
Today. 2009, 14, 500.
10. Krause, E. An effective treatment of experimental mouse-cancers using organo -lead
compounds (also some organotins). Ber. 1929, 62, 135.
11. Narayanan, V.L.; in Reinhoudt, D.N.; Connors, T.A.; Pinedo, H.M.; van de Poll,
K.W.(Eds.). Structure-activity relationship of anti-tumour agents, Martinus Nijhoff,
The Hague. 1983, 5.
12. Caruso, F.; Gianini, M.; Giuliani, A.M.; Rivarola, E. Synthesis and spectroscopic
studies (Mo¨ssbauer, IR and NMR) of [R2SnCl2bipym] (R = butyl or phenyl) and the
crystal and molecular structure of [Ph2SnCl2bipym]. J. Organomet. Chem. 1994, 466,
69 and references therein.
13. Nath, M.; Saini, P.K. Chemistry and applications of organotin(IV) complexes of Schiff
bases. Dalton Trans. 2011, 40, 7077.
14. Crowe, A.J. In metal Complexes in Cancer Chemotherapy, Keppler, B.K. (Ed.), VCH:
Weinhem, 1993, 369-379 and references cited therein.
15. Gielen, M. Review: Organotin compounds and their therapeutic potential: a report from
the Organometallic Chemistry Department of the Free University of Brussels, Appl.
Organomet. Chem. 2002, 16, 481 and references cited therein.
16. Cagnoli, M.; Alama, A.; Barbieri, R.; Novelli, F.; Bruzzo, C.; Sparatore, F. Synthesis
and biological activity of gold and tin compounds in ovarian cancer cells. Anticancer
Drugs. 1998, 9, 603.
Sen Sarma, M.: Cytotoxic activity of organotin(IV) complexes …….
109
17. Nagy, L.; Mehner, H.; Christy, A; Sletten, E; Edelmann, F; Anderson, F.Q. Preparation
and structural studies on organotin(IV) complexes with flavonoids. J. Radioanal. Nucl.
Chem. 1998, 227, 89.
18. Ng, S.W.; Hook, J. M.; Gielen, M. Solid-state 119Sn NMR and antitumor activity of
bis[1,3-bis(3-oxapentamethylenecarbamoylthioacetato)-1,1,3,3-tetrabutyl-1,3-
distannoxare], and the crystal structure of its bis-ethanol solvate. Appl. Organomet.
Chem. 2000, 14, 1.
19. Jancso, A.; Nagy, L.; Moldrheim, E.; Sletten, E. Potentiometric and spectroscopic
evidence for co-ordination of dimethyltin(IV) to phosphate groups of DNA fragments
and related ligands. J. Chem. Soc., Dalton Trans. 1999, 1587.
20. Atkinson, A.; Rodriguez, M.D.; Shewmaker, T.E.; Walmsley, J.A. Synthesis and
characterization of compounds of di- and tributyltin chloride with adenine and guanine
mononucleotides. Inorg. Chim. Acta. 1999, 285(1), 60.
21. Buza’s, N.; Gajda, T.; Nagy, L.; Kuzmann, E.;Vertes, A.; Burger, K. Unusual
coordination behavior of D-fructose towards dimethyltin(IV): metal-promoted
deprotonation of alcoholic OH groups in aqueous solutions of low pH. Inorg. Chim.
Acta. 1998, 274, 167.
22. Willem, R.; Bouhdid, A.; Mahieu, B.; Ghys, L.; Biesemans, M.;Tiekink, E.R.T.; de
Vos, D.; Gielen, M. Synthesis, characterization and in vitro antitumour activity of
triphenyl- and tri-n-butyltin benzoates, phenylacetates and cinnamates. J. Organomet.
Chem. 1997, 531, 151.
23. Barbieri, R.; Ruisi, G.; Atassi, G. The antitumor activity and the structure of
bis(adeninato-N9) (diphenyltin(IV). J. Inorg. Biochem. 1991, 41, 25.
24. Barbieri, R. QSAR approach to understand the antitumour activity of organotins. Inorg.
Chim. Acta. 1992, 191, 253.
25. Atassi, G. Antitumour and toxic effects of Silicon, Germanium, Tin and Lead. Rev. Si
Ge Sn Pb Compd. 1985, 8, 219.
26. Blunden, S.J.; Cusack, P.A.; Hill, R. The Industrial Use of Tin Compounds, Royal
Society of Chemistry, London, 1985.
27. Dalil, H.; Biesemans, M.; Willem, R.; Gielen, M. Tetrabutylbis (2,3:4,6-di-
isopropylidene-2-keto-L-gulonato)distannoxane. Main Group Met. Chem. 1998, 21,
741.
Prajnan O Sadhona ……., Vol. 2, 2015
110
28. Boualam, M.; Biesemans, M.; Menuier-Piret, J.; Willem, R.; Gielen, M. In vitro
antitumour activities of a novel 2:3 condensation product of salicylaldoxime with di-n-
butyltin(IV) oxide. Appl. Organomet. Chem. 1992, 6, 197.
29. Basu Baul, T. S.; Dutta, S.; Rivarola, E.; Scopelliti, M.; Choudhuri, S. Synthesis,
characterization of diorganotin(IV) complexes of N-(2-hydroxyarylidene)amino-acetic
acid and antitumour screening in vivo in Ehrlich ascites carcinoma cells. Appl.
Organomet. Chem. 2001, 15, 947.
30. Brown, N.M. Tin-based Antitumour Drugs. 1990, 69.
31. Gielen, M.; Willem, R.; Biesemans, M.; Boulam, M.; El Khloufi, A.; de Vos, D.
Exceptionally high in vitro antitumor activity of substituted triphenyltin benzoates
including salicylates against a human mammary tumor, MCF-7, and a colon carcinoma,
WiDr. Appl. Organomet. Chem. 1992, 6, 287.
32. Gielen, M.; El Khloufi, A.; Biesemans, M., Bouhdid, A.; de Vos, D.;Mahieu, B.;
Willem, R. Synthesis, Characterization and High In Vitro Antitumour Activity of Novel
Triphenyltin Carboxylates. Metal-Based Drugs. 1994, 1, 305.
33. Gielen, M.; Joosen, E.; Mancilla, T.; Jurkschat, K.; Willem, R.; Roobol, C.; Bernheim,
J.; Atassi, G.; Huber, F.; Hoffmann, Z.E.; Preut, H.; Mahieu, B. Main Group Met.
Chem. 1987, 10, 147.
34. Gielen, M.; Joosen, E.; Mancilla, T.; Jurkschat, K.; Willem, R.; Roobol, C.; Bernheim,
J. in Nicolini, M. (Ed.) , Platinum and other Metal Coordination Compounds in Cancer
Chemotherapy, Martinus Nijhoff, Dordrecht, 1988, 662-665.
35. Willem,R.; Biesemans, M.; Bouâlam, M.; Delmotte, A.; El Khloufi, A.; Gielen, M.
Tetraethylammonium (diorgano)halogeno-(2, 6-pyridinedicarboxylato)stannates:
Synthesis, characterization and in vitro antitumour activity. Appl. Organomet. Chem.
1993, 7, 311.
36. Ng, S.W.; Kumar Das, V.G.; Holecek, J.; Lycka, A.; Gielen, M.; Drew, M.G.B.
Organostannate derivatives of dicyclohexylammonium hydrogen 2,6-
pyridinedicarboxylate: solution/solid-state 13C,119Sn NMR and in vitro antitumour
activity of bis(dicyclohexylammonium) bis(2,6-pyridinedi carboxylato) dibutyl
stannate, and the crystal structure of its monohydrate. Appl. Organomet. Chem. 1997,
11, 39.
37. Gielen, M.; Lelieveld, P.; de Vos, D.; Willem, R. in : Gielen, M. (Ed.), Metal Based
Antitumour Drugs , Freund , Tel Aviv, 1992, Vol. 2, 29-54.
38. Gielen, M. Tin -based antitumour drugs. Coord. Chem. Rev. 1996, 151, 41.
Sen Sarma, M.: Cytotoxic activity of organotin(IV) complexes …….
111
39. Saxena, A.; Tandon, J.P. Antitumor activity of some diorganotin and tin(IV) complexes
of Schiff bases. Cancer Lett. 1983, 19, 73.
40. Nath, M.; Yadav, R.; Gielen, M.; Dalil, H.; de Vos, D.; Eng, G. Synthesis, characteristic
spectral studies and in vitro antimicrobial and antitumour activities of organotin(IV)
complexes of Schiff bases derived from amino-acids. Appl. Organomet. Chem. 1997,
11, 727.
41. Barbieri, R.; Pellerito, L.; Ruisi, G.; Lo Giudice, M.T.; Huber, F.; Atassi, G. The
antitumour activity of diorganotin(IV) complexes with adenine and glycylglycine.
Inorg. Chim. Acta. 1982, 66, L 39.
42. Ruisi, G.; Silvestri, A.; Lo Giudice, M.T.; Barbieri, R.; Atassi, G.; Huber, F.; Graetz,
K.; Lamartina, L. The antitumor activity of di-n-butyltin(IV) glycylglycinate, and the
correlation with the structure of dialkyltin(IV) glycylglycinates in solution. J. Inorg.
Biochem. 1985, 25, 229.
43. Huber, F.; Barbieri, R. in Keppler, B. (Ed.) Metal Complexes in Cancer Chemotherapy,
VCH, Weinhem, 1993, 351.
44. Tao, J.; Xiao, W.; Yang, Q. Structural chemistry of organotin ferrocenecarboxylic
esters II. The crystal structure of dibutyltin ferrocenecarboxylate oxide. J. Organomet.
Chem. 1997, 531, 223.
45. Bonire, J.J.; Fricker, S.P. The in vitro antitumour profile of some 1,2-
diaminocyclohexane organotin complexes. J. Inorg. Biochem. 2001, 83, 217.
46. Gielen, M.; Handlir, K.; Hollein, M.; de Vos, D. Synthesis, Characterization and
Antitumour Activity of Some Butyltin(IV) Cysteaminates and N,N-
Dimethylcysteaminates. Metal-Based Drugs. 2000, 7, 233.
47. Li, J.; Ma,Y.; Liu, R.; Li, J. Synthesis, antitumor activity and crystal structure of the
triphenyltin derivative of exo-7-oxa-bicyclo[2,2,1]hept-5-ene-3-N-p-tolylamide-2-acid.
Appl. Organomet. Chem. 2001, 15, 227.
48. de Vos, D.; Willem, R.; Gielen, M.; van Wingerden, K.E.; Nooter, K. The Development
of Novel Organotin Anti-Tumor Drugs: Structure and Activity. Metal-Based Drugs.
1998, 5, 179.
49. Cardarelli, N.F.; Quitter, B.M.; Allen, A.; Dobbins, E.; Libby, E.P.; Hager, P.;
Sherman, L.R. Organotin implications in anticarcinogenesis. Background and Thymus
involvement. Aust. J. Expl. Biol. Med. Sci. 1984, 62, 199.
Prajnan O Sadhona ……., Vol. 2, 2015
112
50. Cardarelli, N.F.; Cardarelli, B.M.; Libby, E.P.; Dobbins, E. Organotin implications in
anticarcinogenesis. Effects of several organotinson tumour growth rate in mice. Aust.
J. Expl. Biol. Med. Sci. 1984, 62, 209.
51. Cardarelli, N.F.; Kanakkant, S.V. Tin steroids and their use as antineoplastic agents.
U.S. Patent 45, 41,956, 1984.
52. Gielen, M. Review: Organotin compounds and their therapeutic potential: a report from
the Organometallic Chemistry Department of the Free University of Brussels. Appl.
Organomet. Chem. 2002, 16, 481.
53. Kemmer, M.; Miesemans, M.; Gielen, M.; Tiekink, E.R.T.; Willem, R. Synthesis and
structural characterization of (4,7-dioxaoctyl)phenyldichlorostannane and triphenyltin
compounds containing various polyoxaalkyl moieties. J. Organomet. Chem. 2001, 634,
55.
54. Crowe, A.J.; Smith, P.J.; Atassi, G. Investigations into the antitumour activity of
organotin compounds. I. Diorganotin dihalide and di-pseudohalide complexes. Chem.
Biol. Interactions. 1980, 32, 171 and references therein.
55. Casas, J. S.; Garcia-Tasende, M. S.; Mössmer, C. M.; Rodriguez-Argüelles, M.C.;
Sánchez, A.; Sordo, J.; Vázquez- López, A.; Pinelli, S.; Lunghi, P.; Albertini, R.
Synthesis, structure, and spectroscopic properties of acetato (dimethyl) (pyridine-2-
carbaldehydethiosemicarbazonato)tin(IV) acetic acid solvate, [SnMe2
(PyTSC)(OAc)].HOAc. Comparison of its biological activity with that of some
structurally related diorganotin(IV) bis(thiosemicarbazonates). J. Inorg. Biochem.
1996, 62, 41.
56. Teoh, S.-G.; Ang, S.-H.; Teo, S.-B.; Fun, H.-K.; Khew, K. -L.; Ong, C.-W. Synthesis,
crystal structure and biological activity of bis(acetonethiosemicarbazone-S)
dichlorodiphenyltin(IV). J. Chem. Soc., Dalton Trans. 1997, 465.
57. Nataume, T.; Aizawa, S.; Hatano, K.; Funahashi, S. Hydrolysis, polymerisation, and
structure of dimethyltin (IV) in aqueous solution. Molecular structure of the polymer
[(SnMe2)2(OH)3]ClO4. J. Chem. Soc., Dalton Trans. 1994, 2749.
58. Iwai, H.; Manabe, M.; Ono,T.; Wada, M. Distribution, bio-transformation and
biological half-life of tri-, di-, and mono-butyltin in rats. J. Toxicol. Sci. 1979, 4, 285.
59. Crowe, A.J.; in: Fricker, S.P. (Ed.) Metal Compounds in Cancer Therapy, Chapman
and Hall, London, 1994, pp. 147.
60. Kemmer, M.; Gielen , M.; Biesemans, M.; de Vos, D.; Willem, R. Synthesis,
Characterization and In Vitro Antitumour Activity of Di-n-Butyl, Tri-n-Butyl and
Sen Sarma, M.: Cytotoxic activity of organotin(IV) complexes …….
113
Triphenyltin 3,6-Dioxaheptanoates and 3,6,9-Trioxadecanoates. Metal-Based Drugs.
1998, 5, 189.
61. Tiekink, E.R.T.; Gielen, M.; Bouhdid, A.;Willem, R.; Bregadze, V.I.; Ermanson, L.V.;
Glazun, S.A. X-Ray Structure and In Vitro Anti-Tumoural Activity of the Dimeric
Bis[(2-Phenyl-1,2-Dicarba-Closo-Dodecaborane-1-Carboxylato)-Di-n-Butyltin]
Oxide. Metal-Based Drugs. 1997, 4, 75.
62. Gielen, M. in: Cardinelli, N.F. (Ed.), Tin as a Vital Nutrient, CRC Press Boca Raton,
Fl, 1985.
63. Szejth, J. Cyclodextrins and Their Inclusion Complexes. Akademiai Kiado, Budapest,
1982.
64. Rojas-Oviedo, I.; Camacho-Camacho, C.; Sánchez-Sánchez, L.; Cárdenas, J.; López-
Muñoz, H.; Eugenio-Robledo, H.; Velázquez, I.; Toscano, R.A. Synthesis and
characterization of tributyltin derivatives from 4-oxo-4-(arylamino)butanoic acids and
their in vitro biological activity against cervical cancer cell lines. Appl. Organomet.
Chem. 2014, 28, 884.
65. Casas, J. S.; Rodriguez-Argüelles, M. C.; Russo, U.; Sánchez, A.; Sordo, J.; Vázquez
- López, A.; Pinelli, S.; Lunghi, P.; Bonati, A.; Albertini, R. Diorganotin(IV)
complexes of pyridoxal thiosemicarbazone: Synthesis, spectroscopic properties and
biological activity. J. Inorg. Biochem. 1998, 69, 283.
66. Sen Sarma, M.; Mazumder, S.; Ghosh, D.; Roy, A.; Duthie, A.; Tiekink, E.R.T.;
Synthesis, spectroscopic characterization and biocidal activity of some diorganotin(IV)
complexes of salicylaldehydethiosemicarbazones and related ligands. Molecular and
supramolecular structures of [R2Sn(OArCH N N CSNH2)], where R = Me, Ph
and Ar = C6H4, C6H3(5-Br) and C6H3(5-Cl), and of [Me2Sn{OC6H3(5-Br)CH N
N CSNH2}]·OH2. Appl. Organomet. Chem. 2007, 21, 890.
67. Shi, P.F.; Jiang, Q.; Duan, H.-C.; Wang, D.-Q. Synthesis, characterization and
cytotoxicity of fluorescent organotin complexes of terpyridine derivatives. Chinese
Chemical Letters. 2014, 25, 586.
68. Ma, C.; Cheng, S.; Hu, Z.; Li, Q.; Zhang, R.; Zhang, S. Synthesis and characterization
of a novel o-tolyltelluronic trimethyltin ester and its cytotoxic assessment in vitro.
Dalton Trans. 2014, 43, 671.
69. Hadjikakou, S.K.; Hadjiliadis, N. Antiproliferative and anti-tumor activity of organotin
compounds. Coord. Chem. Rev. 2009, 253, 235.
Prajnan O Sadhona ……., Vol. 2, 2015
114
70. Pellerito, L.; Nagy, L. Organotin(IV)n+ complexes formed with biologically active
ligands: equilibrium and structural studies, and some biological aspects. Coord. Chem.
Rev. 2002, 224, 111.
71. Nath, M. Organotin(IV) complexes of amino acids and peptides. Coord. Chem. Rev.
2001, 215, 99.
72. Moebus, V.J.; Stein, R.; Kieback, D.G.; Runnebaum, I.B.; Sass, G.; Kreienberg, R.
Antitumor activity of new organometallic compounds in human ovarian cancer cell
lines and comparison to platin derivatives. Anticancer Res. 1997 , 17, 815.
73. Winship, K.A. Toxicity of tin and its compounds. Adverse Drug React. Acute Poisoning
Rev. 1988, 7, 19.
74. Barbieri, F.; Viale, M.; Sparatore, F.; Schettini, G.; Favre, A.; Bruzzo, C.; Novelli,
F.; Alama, A. Antitumor activity of a new orally active organotin compound: a
preliminary study in murine tumor models. Anti-Cancer Drugs. 2002, 13, 599.
75. Gielen, M.; Tiekink, E.R.T. in: Gielen, M.; Tiekink, E.R.T. (Eds.) Metallotherapeutic
Drugs and Metal-Based Diagnostic Agents: The Use of Metals in Medicine, Wiley:
Chichester, 2005, Chapter 22, 421.
76. Gielen, M.; Tiekink, E.R.T. in: Gielen, M.; Tiekink, E.R.T. (Eds.) Metallotherapeutic
Drugs and Metal-Based Diagnostic Agents: The Use of Metals in Medicine, Wiley:
Chichester, 2005, Chapter 22, 421.
77. Barbieri, F.; Viale, M.; Sparatore, F.; Favre, A.; Cagnoli, M.; Bruzzo, C.; Novelli, F.;
Alama, A. Cytotoxicity in vitro and preliminary antitumor activity in vivo of a novel
organotin compound. Anticancer Res. 2000, 20, 977.
78. Chou, R. H.; Huang, H. Restoration of p53 tumor suppressor pathway in human cervical
carcinoma cells by sodium arsenite. Biochem. Biophys. Res. Commun. 2002, 26, 298.
79. Singh, H. L.; Varshney, A. K. Synthetic, Structural, and Biochemical Studies of
Organotin(IV) With Schiff Bases Having Nitrogen and Sulphur Donor Ligands
Bioinorg. Chem. Appl. 2006, 2006: 23245.
80. Cima, F.; Ballarin, L. Appl. Organomet. Chem. 1999, 13, 697.
81. Barbieri, R.; Silvestri, A. The interaction of native DNA with dimethyltin(IV) species.
J. Inorg. Biochem. 1991, 41, 31.
82. Barbieri, R.; Silvestri, A. Giuliani, A.M.; Piro, V.; Disimone, A.F.; Madonia, G.
Organotin compounds and deoxyribonucleic acid J. Chem. Soc., Dalton Trans. 1992,
585.
Sen Sarma, M.: Cytotoxic activity of organotin(IV) complexes …….
115
83. Piro, V.; Disimone, A. F.; Madonia, A. G.; Silvestri, A.; Giuliani, A. M.; Ruisi, G.;
Barbieri, R. The interaction of organotins with native DNA, Appl. Organomet. Chem.
1992, 6, 537.
84. Barbieri, R.; Ruisi, G.; Silvestri, A.; Giuliani, A. M.; Bardieri, A.; Spina, G.; Pieralli,
F.; Delgiallo, F. Dynamics of tin nuclei in alkyltin(IV)–deoxyribonucleic acid
condensates by variable-temperature tin-119 Mössbauer spectroscopy. J. Chem. Soc.,
Dalton Trans. 1995, 467.
85. Yang, P.; Guo, M. Interactions of organometallic anticancer agents with nucleotides
and DNA. Coord. Chem. Rev. 1999, 185-186, 189.
86. Tabassum, S.; Pettinari, C. Chemical and biotechnological developments in organotin
cancer chemotherapy. J. Organomet. Chem. 2006, 691, 1761.
Prajnan O Sadhona ……., Vol. 2, 2015
116
Review Article
Quantum Dots and it method of preparations - revisited
Rana Karmakar
Assistant Professor, Fakir Chand College, Diamond Harbour - 743331, South 24 Parganas,
West Bengal, India.
Correspondence should be addressed to Rana Karmakar; [email protected]
Abstracts
Quantum dots are inorganic nanocrystal semiconductors that behave exceptionally well
as fluorophores and find ample applications in electronics, chemical and bio-medical
researches and mass production. From the last two decades lot of efforts have been employed
to invent a newer method for its preparation that will be beneficial over the conventional one.
In this review articles emphasis has been given on the meticulous discussion of traditional
methods as well as current development in preparation of modern quantum dots. Synthetic
processes based on ultrasonic or microwave irradiation, using microorganism bio-template,
cluster-seed method and different green non-toxic techniques are highlighted.
Key words: Quantum Dots, Lithography, Reactive ion etching, Molecular beam epitaxy,
Micro emulsion, Solvothermal synthesis, Cluster Seed method, CVD, DNA-functionalized
quantum dots.
Introduction
In the last two decades scientists and researchers have shown enormous interest on the
nanostructured materials as it shows some characteristic properties that are intermediate
between the bulk and molecular levels.1 Quantum dots are typically belong in nano dimension
and their unique physical properties make them a suitable candidates for observing different
biological processes such as single molecule binding in living cells using fluorescence
microscopy, which shows its potential in neuroscience research.2 They are photo stable and
many of them shows a wide absorption band however, their precise emission wavelength are
helpful in biological imaging processes. Quantum dots are useful tools in the monitoring of
cancer cells, produces inexpensive white LEDs, photovoltaic solar cells. In future it may be
used in fabrics, polymer matrices and even in various defence applications.
Karmakar, R.: Quantum Dots and it method …….
117
Generally a crystalline solid is called a nanostructure when it has a particle size less
than 100 nm. When the fabrications are in two dimensions, it is terminated as thin films or
quantum wells, one dimensional are quantum wires and zero dimensional are called quantum
dots. Quantum dots generally consist of a semiconductor core of less than 30 nm, coated by a
shell (e.g. ZnS) and an organic cap (e.g. tri-n-octyl phosphine oxide – TOPO) or an inorganic
layer (a material having larger band gap) that enhance the solubility in aqueous buffer
solutions.3 (Fig. 1) DNA passivated quantum dots are also reported that are stable and nontoxic
to biological systems4,5
Fig. 1 Typical schematic diagram of Quantum dots.6
In quantum dot, limited number of electrons from group II–VI or III–V elements results
in discrete quantized energies in the density of states for non-aggregated zero dimensional
structures. The size of quantum dots are comparable to the wavelength of the electron therefore
the energy difference between two consecutive levels exceed the multiplied values of
Boltzmann constant, (k) and absolute temperature, (T). This results a quantum confinement
effect that impose a restriction of the mobility of electron and holes in that nanocrystal.
Specifically, its excitons are confined in all three spatial dimensions. The electronic properties
of these materials are closely related to the size and shape of the individual crystal and are
intermediate between those of bulk semiconductors and a single molecules.7,8 Quantum dots
are sometimes called as ‘artificial atoms’ where these quantized energy level gap can be
precisely modulated by varying the size.9 Quantum dots are not new, it was discovered in a
glass matrix by a Russian solid state Physicist, Alexei I. Ekimov and also in colloidal solutions
of CdS crystallites by Louis E. Brus10 in 1983. The term "quantum dot" was coined by Mark
A. Reed in 1988.11
Prajnan O Sadhona ……., Vol. 2, 2015
118
Quantum dots shows wide and continuous absorption spectra in presence of white light
whereas the few nanosecond delayed emission occur at a different wavelength region
characteristics of the material used.12,13 The size and band gap are inversely related in quantum
dots. The fluorescent studies indicate that the emission frequencies increase as the size of the
quantum dot decreases, resulting a red to blue shift in the electromagnetic spectra. Excitation
and emission of the quantum dot are therefore highly tunable. (Fig. 2)
Fig. 2. A typical fluorescence spectra of different size CdSe quantum dots (A), and illustration
of the relative particle sizes (B). From left to right, the particle diameters are 2.1 nm,
2.5 nm, 2.9 nm, 4.7 nm, and 7.5 nm14
As the size of the crystals can be controlled during synthesis, the conductive properties
can also be carefully controlled. Several excellent review articles15-22, interesting papers5,23-34
and edited books35-39 have appeared addressing the detailed physiochemical properties,
synthesis and applications in electronics, optoelectronics, fluorescence spectroscopy &
imaging, sensor for biomedical purpose and also its toxicity as a clinical products. This review
articles presents in-depth view of the conventional literature methods15 as well as focused on
the current techniques for preparation of modern stable and non-toxic quantum dots citing
recent examples.
Karmakar, R.: Quantum Dots and it method …….
119
Synthesis of quantum dots
The synthesis of quantum dots can be achieved mainly by two techniques, one is called
as top-down approach and another is bottom-up approach.
1) Top-Down Synthesis Processes
In this process, quantum dots of size ~ 30 nm are to be formed from bulk semiconductor
materials with the help of electron beam lithography, focused ion or laser beams, reactive-ion
or wet chemical etching techniques. To obtain quantum dots of specific packing geometries
with particular shapes and sizes a series of experiments on quantum confinement effect are
required however, these fabrication processes suffers from structural defects and incorporation
of various impurities during its long synthetic pathways.
1.1 Electron beam lithography.
In this approach a focused beam of electrons is scanned to draw custom designed shapes
on a surface covered with an electron sensitive film called a resist. The resist material normally
consist of a polymeric compound. If the resist consists of a long chain polymer with high
molecular weight (~105 units) it is called as negative tone whereas short chain polymeric resist
are known as positive tone. In case of negative tone resist exposure to electron beam induces
further polymerization of the chain length but for positive resist a reduction in the chain length
occur. The electron beam changes the solubility of the resist enabling selective removal of
either the exposed or covered regions of the resist by immersing it in a solvent that is termed
as developing process. The purpose is to create very small structures in the resist that can
subsequently be transferred to the substrate material, often by etching. The resolution of this
electron beam process not only depend on the minimum width of source electron beam but it
also depends on the development process. The source of electron beams are usually hot
lanthanum hexaboride for lower-resolution systems, whereas, higher-resolution instrument
uses field electron emission sources, such as heated W/ZrO2 for lower energy spread and
enhanced brightness. The primary advantage of electron-beam lithography is that it offers a
high degree of flexibility in the design of nanostructured systems (quantum dots, wires or rings)
with sub 10 nm resolution.
Prajnan O Sadhona ……., Vol. 2, 2015
120
Fig. 3 A schematic representation of a six stage electron beam lithography process.
1.2 Focused ion beam (FIB) techniques40
Focused ion beam (FIB) technique is used in semiconductor manufacturing process
from the last two decades. This process closely resembles to a scanning electron microscope
(SEM) however, FIB uses a finely focused beam of ions that can be operated at low or high
beam currents for imaging or site specific sputtering/milling respectively whereas a beam of
electrons is used in SEM. The size, shape and inter-particle distance of the quantum dots
depend on the size of the ion beam but a minimum beam diameter of 8 – 20 nm has been
reported for both lab and commercial systems, allowing etching of quantum dots to dimensions
of less than 100 nm.41 The FIB technique can also be used to selectively deposit material from
a precursor gas with a resolution of ~100 nm.
Most FIB instruments are using liquid-metal ion sources like gallium ion sources.
Sometimes elemental gold and iridium are also used. In a gallium liquid metal ion sources,
gallium metal is placed in contact with a tungsten needle and heated gallium wets the tungsten
and flows to the tip of the needle where the opposing forces of surface tension and electric field
form the gallium into a point shaped tip called a Taylor cone. The tip radius of this cone is
extremely small (~2 nm). The huge electric field at this small tip (> 1 x 108 volts/cm) causes
ionization and field emission of the gallium atoms. Source ions are then generally accelerated
to an energy of 1–50 keV, and focused onto the sample by electrostatic lenses. A modern FIB
can deliver tens of nanoamperes of current to a sample, or can image the sample with a spot
size on the order of a few nanometers. However, by using expensive instruments this process
is producing low yield and leaves residual surface damage.
1.3 Etching techniques
Etching process are well known and plays a very important role in these nanofabrication
processes. In dry etching, a reactive gas species is taken in an etching chamber and with the
Karmakar, R.: Quantum Dots and it method …….
121
help of a controlled radio frequency voltage a plasma is formed that breaks down the gas
molecules to more reactive fragments. These high kinetic energy species strike the surface and
form a volatile reaction product to etch a patterned sample. When the energetic species are
ions, this etching process is called reactive ion etching (RIE). With a masking pattern, selective
etching of the substrate is achieved. Fabrication of GaAs /AlGaAs quantum structures as small
as 40 nm has also been reported using RIE with a mixture of boron trichloride and argon. This
RIE process has been used to produce close-packed arrays for testing of lasing in quantum dot
semiconductors. Close packed arrays of ZnTe quantum dots with inter dot distance of 180 nm
to 360 nm were produced by RIE using CH4 and H2.42
2) Bottom-up Approach
The bottom up approach is of two types a) wet-chemical and b) vapour-phase methods.
Wet-chemical method deals with reactions in solution phase such as sol-gel process,
microemulsion, hot-solution decomposition etc. whereas in vapour-phase method, molecular
beam epitaxy (MBE), sputtering, liquid metal ion sources, physical or chemical vapour
deposition are get importance.
2.1 Wet-Chemical Methods
It is the conventional precipitation methods from a single solution or mixture of
solutions with careful control of different parameters. The quantum dots of a particular size
and shape can be prepared by tuning temperature of the reaction media, electrostatic double
layer thickness and ratios of anionic to cationic species. Use of stabilizers or miceller solution,
varying concentrations of precursors with different solvents also have pronounced effect in
these process. The precipitation process involves both nucleation and limited growth of
nanoparticles. Nucleation may be homogeneous, heterogeneous or secondary where
homogeneous nucleation occurs when solute atoms or molecules combine and reach a critical
size without the assistance of a pre-existing solid interface. A general discussion of different
wet chemical processes are given below.
2.1.1 Sol-Gel Process
This process is a combination of three main steps, which are hydrolysis, condensation
i.e. sol formation followed by growth i.e. formation gel. In a typical technique, a sol, here it is
the nanoparticles dispersed in a solvent by Brownian motion, is prepared using a metal
precursor (generally alkoxides, and metal chloride) in an acidic or basic medium. The metal
Prajnan O Sadhona ……., Vol. 2, 2015
122
precursor hydrolyses in this medium and condenses to form a sol, followed by polymerization
to form a networked elastic solid (gel). Formation of a metal oxide involves connecting the
metal centers with oxo (M-O-M) or hydroxo (M-OH-M) bridges, therefore generating metal-
oxo or metal-hydroxo polymers in solution. Thus, the sol evolves toward the formation of a
gel-like diphasic system containing both a liquid and solid phase whose morphologies range
from discrete particles to continuous polymer networks. After that the remaining solvents are
removed from it resulting a significant amount of shrinkage and densification. The rate at which
the solvent can be removed is ultimately determined by the distribution of porosity in the gel.
Afterward, a thermal treatment, or firing process, is often necessary for further
polycondensation and enhance mechanical properties and structural stability via final sintering,
densification, and grain growth. The precursor sol can either be deposited by dip-coating or
spin-coating on a substrate to form a film, cast into a suitable container with the desired shape
or used to synthesize microspheres, or nanospheres. For example, ZnO Quantum dots have
been prepared by mixing solutions of Zn-acetate in alcohol and sodium hydroxide, followed
by control aging in air.43 Park et.al. have prepared a ZnO thin films, fabricated by a low-
temperature sol–gel conversion method and used as n-type electron-transporting layers in
depleted-heterojunction quantum dot solar cells.44 Several research articles/chapter has been
published where sol-gel procedure is used to synthesis nano structures and quantum dots.45-48
The main advantages of this process are low temperature requirements, cost-effective and
suitable for scale-up but it suffers from non-precise size distribution and a high concentration
of defects.
2.1.2 Microemulsion Process
Similar to Sol-Gel process, this processes can also be carried out at room temperature.
One can tune the size of quantum dots by changing the molar ratio of surfactant to water and
also have a narrower distribution of size as compared to the sol gel process. Although low yield
and incorporation of impurities and defects are main disadvantages of this method. The reverse
micelle process is popular for synthesizing quantum dots, where nano meter water droplets
dispersed in n-alkane solutions can be achieved using surfactants, like aerosol OT (AOT), cetyl
trimethyl-ammonium bromide (CTAB), sodium dodecyl sulphate (SDS) or triton-X. As these
surfactants have both hydrophilic and hydrophobic groups on opposite ends, numerous tiny
droplets called micelles are formed in the continuous oil medium. These micelles are
thermodynamically stable and can act as ‘nanoreactors’. Mixing of vigorous stirred micellar
solutions leads to a continuous exchange of reactants due to dynamic collisions. Growth of the
Karmakar, R.: Quantum Dots and it method …….
123
resultant Quantum dots is limited by the size of the micelle which is controlled by the molar
ratio of water and surfactant. The reverse micelle technique has been used to prepare II-VI core
and core/shell Quantum dots, such as CdS49,50, CdS:Mn/ZnS51,52 and IV-VI Quantum dots53
and carbon quantum dots using hydride reducing agents.54 Darbandi et.al. has reported a
straightforward method for incorporating single quantum dots (QDs) into spherical silica
nanoparticles by means of a one-step synthesis using cyclohexane as the oil phase and
synperonic NP-5 as the surfactant.55 A recent study shows the effect of ultrasonic irradiation
on the preparation of CdS quantum dots with a hexagonal phase in microemulsion.56
2.1.3 Hot-Solution Decomposition Process
This method involves pyrolysis of organometallic compound at high temperature and a
detailed discussion was first given by Bawendi and co-workers in 1993.57 A typical procedure
involves initial degassing and drying of a coordinating solvent like tri-octyl-phosphine oxide
(TOPO) at ~300ºC temperature under vacuum in a three-necked round flask. A moisture free
mixture of Cd-precursor and tri-n-octyl-phosphine selenide is prepared and injected with
vigorous stirring into the flask at that temperature, which induces homogeneous nucleation to
form quantum dots, with the subsequent growth through ‘Ostwald ripening’ being relatively
slow. In Ostwald ripening, the higher free energy of smaller quantum dots makes them lose
mass to large size that results a slow increase of the size of quantum dots at the reaction
temperature of ~250ºC. The coordinating TOPO solvent (purity ~90%) stabilizes the quantum
dot dispersion, improves the passivation of the surface, and provides an adsorption barrier to
slow the growth of the quantum dots. Aliquots may be removed from the flask at regular
intervals during the first few hours and the optical absorption edge used to achieve a desired
particle size. This method has been extensively used to synthesize II-VI, IV-VI and III-V
quantum dots. The size, shape, and control of the overall reaction depends not only on process
parameters like reaction time, temperature, precursors, solvents and coordinating agents but
also on the purity of the coordinating solvent, such as TOPO. A detailed & systematic
discussion with various precursor and process parameters are well described by Bera et.al. in
their review article.15 This method provides sufficient thermal energy to anneal defects and
results in mono dispersed quantum dots. Also a series of quantum dot sizes can be prepared
from the same precursor bath by modulating the temperature. But this process suffer for its
higher costs due to the use of high temperature, toxicity of some of the organometallic
precursors, and generally poor dispersions in water.
Prajnan O Sadhona ……., Vol. 2, 2015
124
2.2 Vapour-Phase Methods
In vapour-phase methods layers of quantum dots are grown in an atom-by-atom process
without any patterning instead by hetero-epitaxial growth of highly strained materials.58-60 In
general, the layered materials grow as a uniform, often epitaxial layer (Frank-van der Merwe
mode–FvdM),61 initially as a smooth layer sometimes followed by nucleation and growth of
small islands (Volmer-Weber mode–VW),62 or as small (quantum dots) islands directly on the
substrate (Stranski-Krastonow mode–SK).63 Depending on the interfacial/surface energies and
lattice mismatch (i.e. lattice strain) one of these growth modes is observed. For example, In the
case of substrates with an overlayer with a large lattice mismatch and appropriately small
surface and interface energies, initial growth of the overlayer occurs through by a layer-by-
layer FvdM growth. However, when the film is thick consisting of few monolayers, to induce
a large strain energy the system lowers its total free energy by breaking the film into isolated
islands or quantum dots (i.e., the VW mode). SK growth is observed with an overlayer material
that has a good lattice match with the substrate, but the substrate surface energy (σ1) is less
than the sum of the interfacial energy between the substrate and overlayer (γ12) and the
overlayer surface energy (σ2), i.e., when σ1 < σ2+γ12.64 Although, self-assembling of quantum
dots using vapour-phase methods is effective in producing quantum dots arrays without
template, fluctuation in size of quantum dots often results in inhomogeneous optoelectronic
properties.
2.2.1 Molecular beam epitaxy (MBE)
Molecular beam epitaxy (MBE), is one of the technique, widely used for growing III-
V semiconductors65-68 and II-VI semiconductors crystals.69,70 In solid-source MBE, ultra-pure
elements e.g. gallium and arsenic, are heated in separate quasi-Knudsen effusion cells until
they begin to slowly sublime. The gaseous elements then condense on the wafer, where they
may react with each other to form gallium arsenide quantum dots. MBE has lower throughput
than other forms of epitaxy. Combination of solid and gases (e.g., AsH3, PH3 or metal-organics
such as tri-methyl gallium or tri-ethyl gallium) are also used as source. During operation,
reflection high energy electron diffraction (RHEED) is often used for monitoring the growth
of the crystal layers. A computer controls shutters in front of each furnace, allowing precise
control of the thickness of each layer, down to a single layer of atoms. MBE has been mainly
used to self-assemble of quantum dots from using the large lattice mismatch e.g., InAs on GaAs
has a 7% mismatch and leads to SK growth, as discussed above.
Karmakar, R.: Quantum Dots and it method …….
125
2.2.2 Physical vapour deposition (PVD)
In physical vapour deposition (PVD) process material is vaporised from a solid or liquid
source in the form of atom or molecules and transported in the form of a vapour through a
vacuum or low pressure gas i.e. plasma to the substrate where condensation takes place. Most
commonly used PVD technique in industrial application is sputtering, where the surface of the
sputtering target is bombard with gaseous ion under high voltage acceleration. The
atom/molecules ejected from the target is due to momentum transferred from the incoming
particle. Vacuum deposition, arc vapour deposition, ion plating and pulsed laser ablation are
also used in PVD process.71,72 Preparation of Nb2O5, CdSe/CdTe quantum dots and other
nanocrystals using PVD technique are already reported.73-75
2.2.3 Chemical vapour deposition (CVD)
In CVD a chemical reaction transform the atom/molecules in the gas phase, known as
‘precursors’ into solid film or powder to deposit on the substrate at elevated temperature. This
process generally accompanied by volatile reaction by products and unused precursors. CVD
are of various type, such as vapour phase epitaxi (VPE) when CVD is used to deposit single
crystal film, metal organic CVD (MOCVD) when precursors are metal-organic species, plasma
enhanced CVD (PECVD) when a plasma is used to enhance the reaction and if the
decomposition is carried out at low pressure it is called low pressure CVD (LPCVD). Similarly
atmospheric pressure CVD (APCVD), photochemical vapour deposition (PCVD), laser
chemical vapour deposition (LCVD) are also well known.71,72 Kim et.al synthesized ZnSe/ZnS
quantum dots with the SK growth mode using a metal-organic chemical vapour deposition
(MOCVD) technique in an atomic layer epitaxy (ALE) mode.76 Dhawan et.al prepared self-
assembled InAs quantum dots on germanium substrate using metal organic CVD (MOCVD)
technique and investigated the effects of growth temperature and InAs coverage on the size,
density, and height of quantum dots.77 Jinsup Lee et.al reported the size controlled fabrication
of uniform graphene quantum dots using self-assembled block copolymer (BCP) as an etch
mask on graphene films.78 Modern research have been employed to direct synthesis of
graphene quantum dots in large scale79 and also to investigate the growth dynamics and
recrystallization of 2D material graphene under a mobile hot-wire assisted CVD Process.80
Prajnan O Sadhona ……., Vol. 2, 2015
126
2) Other Synthetic Processes
3.1 Using Ultrasonic or Microwave irradiation
By passing ultrasonic or microwaves through a mixture of precursors in water
preparation of quantum dots of size range 1–5 nm have been reported since 2000.56,81,82 . Such
acoustic cavitation generates a localized hotspot through adiabatic compression within the gas
inside the collapsing bubble, resulting growth of quantum dots. Zhu et.al. have prepared ZnSe
nanoparticles of about 3 nm in size where zinc acetate were dispersed in a solution and seleno-
urea was added and sonicated for an hour under an argon atmosphere.81 The temperature of the
solution increased to 80ºC during the time required to produce quantum dots.
3.2 Hydrothermal synthesis
Hydrothermal synthesis methods83,84 or similar synthesis process85 have been used to
produce quantum dots. These are crystallization of inorganic salts from aqueous solution,
controlled by pressure and temperature. The solubility of inorganic compounds typically
decreases as the temperature and/or pressure is lowered, leading to crystalline precipitates. By
changing pressure, temperature, reaction and aging time and reactants, different shapes and
sizes of the quantum dots can be achieved. Several other wet-chemical processes for
synthesizing quantum dots86,87 were also reported in literatures. Passing H2S gas through
precursors has also been used to prepare sulfide-based quantum dots.88,89
3.3 Solvothermal synthesis
Solvothermal synthetic method is similar to the hydrothermal route, where the chemical
reaction is conducted in a stainless steel autoclave, but the only difference being that the
precursor solution is usually non-aqueous in the former. Solvothermal synthesis has been used
to prepare different morphologies of nanostructured TiO290 and carbon nanosheets (graphene)
in a bottom-up approach using ethanol and sodium, which are reacted to give an intermediate
solid that is then pyrolized, yielding a fused array of graphene sheets that are dispersed by mild
sonication.91 Du et.al reported synthesis of ultra-small CuInSe2 quantum dots with diameters
below the exciton Bohr radius 10.6 nm by a solvothermal method. The synthesis was conducted
in oleylamine without any organometallic precursors.92
3.4 From microorganism bio-template
Karmakar, R.: Quantum Dots and it method …….
127
Bio-templates from microbial origin are become a new source for designing and
fabricating intricate, high surface area structures that possess potent applications in
nanotechnology based on a bottom up approach. Microbial bio-templates are natural product
having quick growth and multiplication along with controlled structure and size, so it is cost
effective for preparing nano materials compared to the conventional lithographic techniques.
It has previously been shown that genetically engineered viruses can recognize specific
semiconductor surfaces through the method of selection by combinatorial phage display.93 It
was also observed that liquid crystalline structures of wild-type viruses (Fd, M13, and TMV)
are adjustable by controlling the solution concentrations, solution ionic strength, and the
external magnetic field applied to the solutions. This specific recognition properties of the virus
can be used to organize inorganic nano crystals, forming ordered arrays over the length scale
defined by liquid crystal formation. Using this information, Lee et al. were able to create self-
assembled, highly oriented, self-supporting films from a phage and ZnS precursor solution.
This system allowed them to vary both the length of bacteriophage and the type of inorganic
material through genetic modification and selection. In the year 2002, they have reported using
genetically engineered M13 bacteriophage viruses to create quantum dot bio-composite
structures.94 A review article addresses the recent advancement in the synthesis of nano/micro
structures using bio-templates obtained from various types of microorganisms like bacteria,
fungi, algae and virus, and have highlighted its possible applications.95
3.5 Electrochemical assembly
Highly ordered arrays of quantum dots may also be self-assembled by electrochemical
techniques. A template is created by causing an ionic reaction at an electrolyte-metal interface
which results in the spontaneous assembly of nanostructures, including quantum dots, onto the
metal which is then used as a mask for mesa-etching these nanostructures on a chosen substrate.
This process have several advantages like, it is relatively cheap and does not causes radiation
damage to nanostructures unlike beam lithography, have high yield so eligible for mass
production and structures can be delineated on non-planar substrates. Bandyopadhyay et.al
described two electrochemical self-assembly processes for producing highly ordered quasi-
periodic arrays of quantum dots on a surface96 and nonlinear optical properties of self-
assembled CdS quantum dots.97 Raman spectroscopic study of electrochemically self-
assembled quasiperiodic arrays of CdS quantum dots into a porous anodized alumina film was
also reported in the year 2000.98
3.6 Cluster-Seed method
Prajnan O Sadhona ……., Vol. 2, 2015
128
This method was developed by the Nigel Pickett, the founder of a company named
Nanoco, Inc., Manchester, Great Britain to produce large quantities of quantum dots in a single
batch. It was stipulated that clusters of Cd10Se4(SPh)16 seed the growth of CdSe quantum dots
and the number of quantum dots produced in a batch process is the same as the number of
clusters added.99 Further research employed by “The Snee Group” at the University of Illinois,
Chicago by synthesizing CdSe nanocrystals in the presence of an increasing number of clusters
and found that the number of clusters and nanocrystals synthesized are linearly correlated to
each other. Also, the use of a large number of clusters results in the formation of small quantum
dots due to competition of larger number of dots for less precursors and that no quantum dots
are synthesized if the clusters are absent. This group also reported a method for synthesizing a
single CdSe quantum dot doped with four copper ions using [Na(H2O)3]2[Cu4(SPh)6] as a
cluster seed.100 The synthesis is a derivative of the cluster-seed method, whereby
organometallic clusters act as nucleation centers for quantum dots.
3.7 Green methods
3.7.1 Heavy metal free quantum dots
Due to enhanced environmental awareness, restriction or ban is imposed on the use of
heavy metals in many household goods so that most cadmium based quantum dots are unusable
for consumer-goods applications. For commercial viability, a range of cadmium-free quantum
dots such as InP/ZnS and CuInS/ZnS has been developed that shows bright emissions in the
visible and near infra-red region of the spectrum and have similar optical properties to those of
CdSe quantum dots.
A green method for the synthesis of hydrophilic CuInS2 and CuInS2–ZnS colloidal
quantum dots was developed employing N,N-dimethylformamide (DMF) as a solvent. To
synthesize hydrophilic CuInS2 quantum dots, the sulphur source H2S gas, was generated in situ
from the reaction between FeS and H2SO4. Short chain thiols were applied as ligands, reactivity
controllers and sulphur sources for the growth of the ZnS shell. The growth of CuInS2 quantum
dots experiences a typical Ostwald ripening process, and it should be controlled in 45 min due
to the pyrolysis of the ligands at ~130°C temperature.29 Recently, Wang et.al synthesized
CuInS2 quantum-dot sensitized TiO2 photoanodes with In2S3 buffer layer were in situ prepared
via chemical bath deposition of In2S3, where the Cd-free In2S3 layer then reacted with
TiO2/CuxS which employed a facile SILAR process to deposit CuxS quantum dots on TiO2
film, followed by a covering process with ZnS layer. Polysulfide electrolyte and Cu2S on FTO
Karmakar, R.: Quantum Dots and it method …….
129
glass counter electrode were used to provide higher photovoltaic performance of the
constructed devices.101 Environmentally benign cadmium free quantum dot Light-Emitting
Diodes (QLED) is also prepared that enabled by the direct formation of excitons within
InP@ZnSeS quantum dots.102
3.7.2 DNA-functionalization of quantum dots
Quantum dots can behave as a fluorophore and becoming an exceptionally good
candidates to draw pictures on the cellular and sub-cellular levels as its size directly reflects
the wavelength of emitted light. But common CdSe-ZnS quantum dots are not the best choice
for this purpose as Cd is toxic to biological cells. To make biocompatibility, surface
modification of quantum dots becomes another pathway in addition to replace Cd from it. After
a surface modification has been made to limit toxicity, the particle can be further coated with
a hydrogel or bioconjugate layer to selectively bind to DNA, which may then be used for
cellular or molecular level detection.103 In a hydrogel encapsulation, the ZnSe shell surface
may be charged to bind to the hydrophobic interior of a micelle, which then allows the
hydrophilic exterior to remain in contact with biological systems. However, in bioconjugation
two covalently bonded biomolecules can form a protective layer around the quantum dot.
Hydrophobic bioconjugation inhibits degradation of the quantum dot structure within the
body.104 Xiaohu Gio et.al used TOPO as a coordinating ligand, and a tri-block polymer
consisting of two hydrophobic and one hydrophilic segment, all with hydrophobic hydrocarbon
side chains. The strong hydrophobic interactions between the TOPO and polymer hydrocarbon
allow the two layers to “bond” to one another, forming a hydrophobic protection structure. This
structure resists quantum dot optical properties in a wide range of pH (1-14), salt conditions
(0.01-1.0M), and even after 1.0M HCl treatment.105
Pong, B.K. et.al developed another synthetic strategy to efficiently functionalise
CdSe/ZnS quantum dots with 3-mercaptopropionic acid that exhibit good colloidal stability
and quantum yield. With this surface functionalized by carboxylic groups, amine modified
DNA can be coupled to the quantum dots surface via the formation of an amide linkage. The
coupling is promoted by1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-
hydroxysuccinimide (NHS). They found that alkaline medium (pH 9.5) was optimal for
hybridisability of the immobilised DNA.106
Ionic strength, pH of the solution and DNA/nanoparticle ratio have major influence on
the chemical conjugation process and structural and optical stability of carboxyl-functionalized
Prajnan O Sadhona ……., Vol. 2, 2015
130
quantum dots functionalized with amino-modified DNA. A neutral pH of 7 allows enough
protons from the carboxyl group to facilitate covalent bonding of amino modified DNA, but
not much protons to destabilize the colloids. By carefully adjusting the other variables at pH 7,
up to 20 DNA strands can be conjugated per quantum dot.5
3.7.3 Ionic liquid (IL) assisted quantum dots
Ionic liquids are well known for their environment friendly green nature and used as an
alternative reaction media by substituting conventional volatile organic solvents for couple of
years. Robina Shadid et.al reported a new method using ionic liquid as Microwave absorbing
solvent for the synthesis of ZnS quantum dots in 2012.107,108 They have used 1-butyl-3-
methylimidazolium tetrafluoroborate, [BMIM][BF4] ionic liquid with inorganic precursor and
trihexyl(tetradecyl)phosphonium chloride [P6,6,6,14][Cl], trihexyl(tetradecyl)phosphonium
bis(trifluoromethanesulfonyl) amide [P6,6,6,14][TSFA] and also [BMIM][BF4] ionic liquids
where organic precursors are utilized. In first case, equal volume of ZnCl2 and sulfur powder
were used as inorganic precursor and they are mixed with [BMIM][BF4] at ~2500C under MW
irradiation with constant stirring for 1 to 10 minutes. Subsequently, ethanol was added to
precipitate the quantum dots from the cooled solution. The product was then centrifuged and
re-dispersed in ethanol.
In second system, equal volumes of Zn-oleate and TOP-S stock solution was added as
organic precursor to 10 mL of ionic liquid. The solution was Microwave irradiated at ~250ºC
for reaction times ranging from 5 minutes to 1 hour under continuous stirring. Subsequently,
ethanol was added to precipitate the quantum dots from the cooled solution. The product was
then centrifuged and re-dispersed in different solvents.
A facile and efficient approach for preparation of ionic liquid functionalized silica-
capped CdTe quantum dots (CdTe/SiO2/IL) was proposed by Li et.al., where imidazolium-
based ionic liquid N-3-(3-trimethoxysilylpropyl)-3-methyl imidazolium chloride was
introduced. It was anchored on the surface of silica-capped CdTe quantum dots by the sol–gel
technique, which played the role of a recognition element due to the covalent coordination
binding between the heme group of hemoprotein and the imidazolium cation in the ionic liquid.
This CdTe/SiO2/IL exhibited high adsorption capacity and good specificity toward
hemoproteins and also was successfully applied in the fluorescence detection of hemoprotein
in biological fluid.109
Choi et.al reported a new synthetic route to prepare a water soluble CdTe quantum dots
by using dithiol-functionalized ionic liquids (dTFILs). The dTFILs were designed to have
Karmakar, R.: Quantum Dots and it method …….
131
dithiol and vinylimidazolium functional groups and used as a ligand molecule of CdTe
quantum dot to utilize the bidendate chelate interaction afforded by the dithiol groups of
dTFILs. The photoluminescence quantum yield of dTFIL-capped CdTe quantum dots reached
up to ∼40%, and their luminescent property was maintained for 8 weeks, suggesting an
improved stability in water phase.110
Samanta et.al used 1-butyl-3-methylimidazolium hexafluorophosphate ionic liquid
instead of aqueous solvents to synthesize CdTe quantum dots and studied the hole transfer
process from this photoexcited quantum dots to sulfide anion. This ionic liquid capped CdTe
quantum dots show enhanced stability compared to conventional aqueous environment in
presence of sulfide electrolyte.111 Improved luminescence properties of the CdTe quantum
dots of different sizes in a large number of thiol-capped imidazolium ionic liquids are reported
and the enhanced stability of the quantum dots toward molecular oxygen is depicted to the
capping agent and the ionic liquids, because of low solubility of oxygen in these viscous
media.112 In another study influence of ligand and solvent on light-induced modulation of the
emission behavior of the quantum dots has been investigated for CdTe quantum dots capped
with hexadecylamine, mercaptopropionic acid, and 1-(1-undecanethiol)-3-methyl imidazolium
bromide in [bmim][PF6] ionic liquid, CHCl3, and H2O using steady state and time-resolved
fluorescence and fluorescence correlation spectroscopy techniques.113
The emission spectra of ionic liquid passivated CdSe/ZnS quantum dots dispersed in
POC copolymer shows a wide spectrum from blue to orange wavelengths due to the
combination of colors emitted by the polymer and quantum dots, respectively.114 Chao and
coworker used a hydrophilic ionic liquid, 1-ethyl-3-methylimidazolium dicyanamide, as a
medium for the synthesis of highly luminescent CdTe nanocrystals capped with thioglycolic
acid. The synthesis was performed with low-cost inorganic salts as precursors and continued
for 8 hours at 130°C, similar to nanocrystal preparation in an aqueous medium. The
photoluminescence quantum yield of the CdTe quantum dots prepared in ionic liquid medium
was found significantly higher than that prepared in water. Also, the quenching effect of Hg²⁺
on the fluorescence signals in ionic liquid medium was distinctively unique compared to other
interfering ions, such as Pb²⁺, Fe³⁺, Co²⁺, Ni²⁺, Ag⁺, and Cu²⁺. Thus this ionic liquid assisted
nanocrystals can potentially be used to develop a probe system for the determination of Hg²⁺
in physiological samples.115
Prajnan O Sadhona ……., Vol. 2, 2015
132
4) Bulk-manufacturing procedure
Conventional small scale production uses a process called "high temperature dual
injection". This is a reproducible production method that can be applied to a wide range of
quantum dot sizes and compositions. But it is quite difficult to separate nanoparticle nucleation
and growth via a high temperature dual injection method for III-V quantum dots, as the nature
of bonding is more covalent than that in II-VI materials. Also with increasing reaction scales
rapid injection of large solution volumes into one another results in temperature differentials
and culminates in wide particle size distributions.
A reproducible method for creating larger quantities of consistent, high-quality
quantum dots involves producing nanoparticles from chemical precursors in the presence of a
molecular cluster compound under conditions whereby the integrity of the molecular cluster is
maintained and acts as a prefabricated seed template. Individual molecules of a cluster
compound act as a seed or nucleation point upon which nanoparticle growth can be initiated.
In this way, a high temperature nucleation step can be avoided to initiate nanoparticle growth
because suitable nucleation sites are already provided in the system by the molecular
clusters.116 Particle growth is maintained by the periodic addition of precursors at moderate
temperatures until the desired particle size is reached.100
In the year 2011, Quantum Materials Corporation and the Access2Flow consortium of
the Netherlands have jointly developed a new microreactor and software controlled continuous
flow process which can mass produce tetrapod cadmium selenide quantum dots including
heavy metal free (cadmium free) quantum dots and other biologically inert materials. This new
quantum dot production process replaces batch synthesis and has potential for high
improvement in both yield and conversion. Tetrapod Quantum Dots are used in a variety of
emerging applications including solid state lighting, QLED displays and nanobio
applications.117 Recently the fabrication and characterization of large-scale graphene quantum
dots grown on hexagonal boron nitride substrates with different layers and similar size of island
diameters is reported by chemical vapor deposition (CVD) technique.79
Perspective
From the past few years the current trends in science and research are directed to
nanotechnology and its application in electronics and bioscience focusing drug delivery in
human cells and organ. Fastening of nano materials in drug delivery also demands a nontoxic
nature that was absent in previous CdS quantum dots. The modern lighting, electronic gadgets
and digital display systems uses energy efficient LED resulting from bright quantum dots. All
Karmakar, R.: Quantum Dots and it method …….
133
these demands for a synthetic route of nano materials through an easy and cost effective
manufacturing process in a commercial scale. In this review both the traditional top down and
bottom up approaches along with various current techniques are highlighted. Other than III-V
and II-VI quantum dots nowadays carbon based graphene quantum dots become a promising
one. Heavy metal free and DNA functionalized quantum dots already proved their candidature
as a nature friendly non-toxic agent in a biological cells. Room temperature ionic liquids that
are famous for their environment friendly green nature, are also used in preparation of different
quantum dots in replacement of conventional volatile organic solvents. This technique shows
enhanced luminescence properties and even become a tool for sensing a typical metal ion in
low concentration. Tetrapod quantum dots are now popular with software controlled
continuous flow process that also have large scale commercial application. With the fast
progress in research in nonoscience, the literatures are being flooded with new techniques and
potent applications in a very short period. This review article addresses some of the current
updates so that the recent advancement in this field can be easily tracked.
References
1. Brus, L. E. Chemistry and Physics of Semiconductor Nanocrystals[Online early
access]2007.
2. Pathak, S.; Cao, E.; Davidson, M. C.; Jin, S.; Silva, G. A. Quantum Dot Applications
to Neuroscience: New Tools for Probing Neurons and Glia. J. Neurosci. 2006, 26, 1893-
1895.
3. Ghasemi, Y.; Peymani, P.; Afifi, S. Quantum dot: magic nanoparticle for imaging,
detection and targeting. Acta. Biomed. 2009, 80, 156-165.
4. Ma, N.; Yang, J.; Stewart, K. M.; Kelley, S. O. DNA-passivated CdS nanocrystals:
Luminescence, bioimaging, and toxicity profiles. Langmuir 2007, 23, 12783-12787.
5. Sun, D.; Gang, O. DNA-Functionalized Quantum Dots: Fabrication, Structural, and
Physicochemical Properties. Langmuir 2013, 29, 7038-7046.
6. Shashkov, E. V.; Everts, M.; Galanzha, E. I.; Zharov, V. P. Quantum Dots as
Multimodal Photoacoustic and Photothermal Contrast Agents. Nano Letters 2008, 8,
3953-3958.
7. Norris, D. J. Measurement and Assignment of the Size-Dependent Optical Spectrum in
Cadmium Selenide (CdSe) Quantum Dots. 1995.
Prajnan O Sadhona ……., Vol. 2, 2015
134
8. Murray, C. B.; Kagan, C. R.; Bawendi, M. G. Synthesis and Characterization of
Monodisperse Nanocrystals and close-packed Nanocrystal Assemblies. Annual Review
of Materials Research 2000, 30, 545-610.
9. Klimov, V. L. Spectral and dynamical properties of multiexcitons in semiconductor
nanocrystals. Annu. Rev. Phys. Chem. 2007, 58, 635-673.
10. Rossetti, R.; Nakahara, S.; Brus, L. E. Quantum size effects in the redox potentials,
resonance Raman spectra, and electronic spectra of CdS crystallites in aqueous solution.
J. Chem. Phys. 1983, 79, 1086-1088.
11. Reed, M. A.; Randall, J. N.; Aggarwal, R. J.; Matyi, R. J.; Moore, T. M.; Wetsel, A. E.
Observation of discrete electronic states in a zero-dimensional semiconductor
nanostructure. Phys. Rev. Lett. 1988, 60, 535–537.
12. Deb, P.; Bhattacharyya, A.; Ghosh, S. K.; Ray, R.; Lahiri, A. Excellent biocompatibility
of semiconductor quantum dots encased in multifunctional poly(N-
isopropylacrylamide) nanoreservoirs and nuclear specific labeling of growing neurons.
Appl. Phys. Lett. 2011, 98, 103702–103703.
13. Bakalova, R.; Ohba, H.; Zhelev, Z. Quantum dots as photosensitizers? Nat. Biotechnol.
2004, 22, 1360-1361.
14. Smith, A. M.; Nie, S. Chemical analysis and cellular imaging with quantum dots.
Analyst 2004, 129, 672-677.
15. Bera, D.; Qian, L.; Tseng, T. K.; Holloway, P. H. Quantum Dots and Their Multimodal
Applications: A Review. Materials 2010, 3, 2260-2345.
16. Dhar, R.; Deepika. Synthesis and Current Applications of Quantum Dots: A review.
Nanoscience and Nanotechnology: An International Journal 2014, 4, 32-38.
17. Mishra, S.; Panda, B.; Rout, S. S. An Elaboration of Quantum Dots and Its Applications.
International Journal of Advances in Engineering & Technology 2012, 5, 141-145.
18. Petryayeva, E.; Algar, W. R.; Medintz, I. L. Quantum dots in bioanalysis: a review of
applications across various platforms for fluorescence spectroscopy and imaging. Appl
Spectrosc. 2013, 67, 215-252.
19. Valizadeh, A.; Mikaeili, H.; Samiei, M.; Farkhani, S. M.; Zarghami, N.; Kouhi, M.;
Akbarzadeh, A.; Davaran, S. Quantum dots: synthesis, bioapplications, and toxicity.
Nanoscale Research Letters 2012, 7, 480-493.
20. Cheng, X.; Lowe, S. B.; Reece, P. J.; Gooding, J. J. Colloidal silicon quantum dots:
from preparation to the modification of self-assembled monolayers (SAMs) for bio-
applications. Chem. Soc. Rev. 2014, 43, 2680-2700.
Karmakar, R.: Quantum Dots and it method …….
135
21. Pisanic II, T. R.; Zhang, Y.; Wang, T. H. Quantum dots in diagnostics and detection:
principles and paradigms. Analyst 2014, 139, 2968-2981.
22. Lim, S. Y.; Shen, W.; Gao, Z. Carbon quantum dots and their applications. Chem. Soc.
Rev. 2014.
23. Mulpur, P.; Tanu Mimani Rattan, T.; Kamisetti, V. One-Step Synthesis of Colloidal
Quantum Dots of Iron Selenide Exhibiting Narrow Range Fluorescence in the Green
Region. Journal of Nanoscience 2013, 2013, 5.
24. Thuy, U. T. D.; Chi, T. T. K.; Nga, P. T.; Nghia, N. D.; Khang, D. D.; Liem, N. Q.
CdTe and CdSe quantum dots: synthesis, characterizations and applications in
agriculture. Adv. Nat. Sci.: Nanosci. Nanotechnol. 2012, 3, 043001-043011.
25. Novak, S.; Scarpantonio, L.; Novak, J.; Dai Prè, M.; Martucci, A.; Musgraves, J. D.;
McClenaghan, N. D.; Richardson, K. Incorporation of luminescent CdSe/ZnS core-
shell quantum dots and PbS quantum dots into solution-derived chalcogenide glass
films. Opt. Mater. Express 2013, 3, 729-738.
26. Zhang, H.; Li, Y.; Liu, X.; Liu, P.; Wang, Y.; An, T.; Yang, H.; Jing, D.; Zhao, H.
Determination of Iodide via Direct Fluorescence Quenching at Nitrogen-Doped Carbon
Quantum Dot Fluorophores. Environ. Sci. Technol. Lett. 2014, 1, 87-91.
27. Hoshino, K.; Gopal, A.; Glaz, M. S.; Vanden Bout, D. A.; Zhang, X. Nanoscale
fluorescence imaging with quantum dot near-field electroluminescence. Appl. Phys.
Lett. 2012, 101, 043118.
28. Bowers, M. J.; McBride, J. R.; Rosenthal, S. J. White-Light Emission from Magic-
Sized Cadmium Selenide Nanocrystals. J. Am. Chem. Soc. 2005, 127, 15378-15379.
29. Zhang, J.; Sun, W.; Yin, L.; Miao, X.; Zhang, D. One-pot synthesis of hydrophilic
CuInS2 and CuInS2-ZnS colloidal quantum dots. J. Mater. Chem. C 2014, 2, 4812-
4817.
30. Bottrill, M.; Green, M. Some aspects of quantum dot toxicity. Chem. Commun. 2011,
47, 7039-7050.
31. Tyrakowski, C. M.; Snee, P. T. A primer on the synthesis, water-solubilization, and
functionalization of quantum dots, their use as biological sensing agents, and present
status. Phys. Chem. Chem. Phys. 2014, 16, 837-855.
32. Corredor, E.; Testillano, P. S.; Coronado, M. J.; González-Melendi, P.; Fernández-
Pacheco, R.; Marquina, C. Nanoparticle penetration and transport in living pumpkin
plants: In situ subcellular identification. BMC. Plant Biol. 2009, 9, 45.
Prajnan O Sadhona ……., Vol. 2, 2015
136
33. Santos, A. R.; Miguel, A. S.; Tomaz, L.; Malhó., R.; Christopher. The impact of
CdSe/ZnS quantum dots in cells of Medicago sativa in suspension culture. J.
Nanobiotechnology. 2010, 8, 24.
34. Wu, Y. L.; Lim, C. S.; Fu, S.; Tok, A. I. Y.; Lau, H. M.; Boey, F. Y. C. Water-soluble
quantum dots for biomedical applications. Biochem. Biophys. Res. Commun. 2006, 348,
781-786.
35. Luminescent Materials and Applications Kitai, A., Ed.; John Wiley & Sons Ltd.: The
Atrium, Southern Gate, ChiChester, West Sessex, England, 2008.
36. Nanostructured Materials and Nanotechnology; Nalwa, H. S., Ed.; Academic Press:
San Diego, California, USA, 2002.
37. Bandyopadhyay, S.; Nalwa, H. S.: Quantum Dots and Nanowires. American Scientific
Publishers, 2003.
38. Bera, D.; Qian, L.; Holloway, P. H.: Phosphor Quantum Dots. In Luminescent Materials
and Applications; Kitai, A., Ed.; John WIley & Sons, Ltd: Chichester, UK., 2008.
39. Bera, D.; Qian, L.; Holloway, P. H.: Semiconducting Quantum Dots for Bioimaging.
In Drug Delivery Nanoparticles Formulation and Characterization; 1st ed.; Pathak, Y.,
Thassu, D., Eds.; CRC Press: UK, 2009; pp 349-366.
40. Focused Ion Beam - Wikipedia. Http://en.Wikipedia.Org/wiki/focused_ion_beam.
41. Chason, E.; Picraux, S. T.; Poate, J. M.; Borland, J. O.; Current, M. I.; delaRubia, T.
D.; Eaglesham, D. J.; Holland, O. W.; Law, M. E.; Magee, C. W.; Mayer, J. W.;
Melngailis, J.; Tasch, A. F. Ion beams in silicon processing and characterization. J.
Appl. Phys. 1997, 81, 6513-6561.
42. Tsutsui, K.; Hu, E. L.; Wilkinson, C. D. W. Reactive Ion Etched II-VI Quantum Dots:
Dependence of Etched Profile on Pattern Geometry. Jpn. J. Appl. Phys. 1993, 32, 6233-
6236.
43. Bang, J.; Yang, H.; Holloway, P. H. Enhanced and stable green emission of ZnO
nanoparticles by surface segregation of Mg. Nanotechnology 2006, 17, 973-978.
44. Park, H. Y.; Ryu, I.; Kim, J.; Jeong, S.; Yim, S.; Jang, S. Y. PbS Quantum Dot Solar
Cells Integrated with Sol–Gel-Derived ZnO as an n-Type Charge-Selective Layer. J.
Phys. Chem. C. 2014, 118, 17374-17382.
45. Mashford, B.; Baldauf, J.; Nguyen, T. L.; Funston, A. M.; Mulvaney, P. Synthesis of
quantum dot doped chalcogenide glasses via sol-gel processing. J. Appl. Phys. 2011,
109, -.
Karmakar, R.: Quantum Dots and it method …….
137
46. Spanhel, L.; Schmidt, H.; Uhrig, A.; Klingshirn , C. Inorganic-Organic Sol-Gel
Processing of Semiconductor Quantum Dots and some Preliminary Self-Diffraction
Studies on CdS-PbS. . MRS Online Proceedings 1992, 272, 53.
47. Selvan, S. T.: Fabrication of Inorganic Nanocomposites Using Self-Assembly and Sol-
Gel Processing. In Nanoscale Materials; Liz-Marzán, L. M., Kamat, P. V., Eds.;
Springer US, 2003; pp 247-272.
48. Korala, L.; Wang, Z.; Liu, Y.; Maldonado, S.; Brock, S. L. Uniform Thin Films of CdSe
and CdSe(ZnS) Core(Shell) Quantum Dots by Sol–Gel Assembly: Enabling
Photoelectrochemical Characterization and Electronic Applications. ACS Nano 2013,
7, 1215-1223.
49. Colvin, V. L.; Goldstein, A. N.; Alivisatos, A. P. Semiconductor nanocrystals
covalently bound to metal-surfaces with self-assembled monolayers. J. Am. Chem. Soc.
1992, 114, 5221-5230.
50. Lemke, K.; Koetz, J. Polycation-Capped CdS Quantum Dots Synthesized in Reverse
Microemulsions. Journal of Nanomaterials 2012, 2012, 10.
51. Yang, H.; Holloway, P. H. Enhanced photoluminescence from CdS:Mn/ZnS core/shell
quantum dots. Appl. Phys. Lett. 2003, 82, 1965-1967.
52. Yang, H.; Holloway, P. H.; Cunningham, G.; Schanze, K. S. CdS:Mn nanocrystals
passivated by ZnS: Synthesis and luminescent properties. J. Chem. Phys. 2004, 121,
10233-10240.
53. Ogawa, S.; Hu, K.; Fan, F. R. F.; Bard, A. J. Photoelectrochemistry of films of quantum
size lead sulfide particles incorporated in self-assembled monolayers on gold. J. Phys.
Chem. B 1997, 101, 5707-5711.
54. Linehan, K.; Doyle, H. Size controlled synthesis of carbon quantum dots using hydride
reducing agents. J. Mater. Chem. C 2014, 2, 6025-6031.
55. Darbandi, M.; Thomann, R.; Nann, T. Single Quantum Dots in Silica Spheres by
Microemulsion Synthesis. Chemistry of Materials 2005, 17, 5720-5725.
56. Entezari, M. H.; Ghows, N. Micro-emulsion under ultrasound facilitates the fast
synthesis of quantum dots of CdS at low temperature. Ultrasonics Sonochemistry 2011,
18, 127-134.
57. Murray, C. B.; Norris, D. J.; Bawendi, M. G. Synthesis and characterization of nearly
monodisperse CdE (E = sulfur, selenium, tellurium) semiconductor nanocrystallites. J.
Am. Chem. Soc. 1993, 115, 8706-8715.
Prajnan O Sadhona ……., Vol. 2, 2015
138
58. Leonardi, K.; Selke, H.; Heinke, H.; Ohkawa, K.; Hommel, D.; Gindele, F.; Woggon,
U. Formation of self-assembling II–VI semiconductor nanostructures during migration
enhanced epitaxy. J. Cryst. Growth 1998, 184-185, 259-263.
59. Kurtz, E.; Shen, J.; Schmidt, M.; Grun, M.; Hong, S. K.; Litvinov, D.; Gerthsen, D.;
Oka, T.; Yao, T.; Klingshirn, C. Formation and properties of self-organized II–VI
quantum islands. Thin Solid Films 2000, 367, 68-74.
60. Swihart, M. T. Vapor-phase synthesis of nanoparticles. Curr. Opin. Colloid Interface
Sci. 2003, 8, 127-133.
61. Frank, F. C.; van der Merwe, J. H. One-Dimensional Dislocations. I. Static Theory.
Proc. Roy. Soc. London A 1949, 198, 205-216.
62. Volmer, M.; Weber, A. Z. Phys. Chem. 1926, 119, 277.
63. Stranski, I. N.; Krastanow, V. L. Self-assembled quantum dots. Akad. Wiss. Lit. Mainz
Math.-Natur. KI. IIb 1939, 146, 797.
64. Eaglesham, D. J.; Cerullo, M. Dislocation-free Stranski-Krastanow growth of Ge on
Si(100). Phys. Rev. Lett. 1990, 64, 1943-1946.
65. Leonard, D.; Krishnamurthy, M.; Reaves, C. M.; Denbaars, S. P.; Petroff, P. M. Direct
formation of quantum‐sized dots from uniform coherent islands of InGaAs on GaAs
surfaces. Appl. Phys. Lett. 1993, 63.
66. Trevisi, G.; Seravalli, L.; Frigeri, P.; Bocchi, C.; Grillo, V.; Nasi, L.; Suárez, I.; Rivas,
D.; Muñoz-Matutano, G.; Martínez-Pastor, J. MBE growth and properties of low-
density InAs/GaAs quantum dot structures. Crystal Research and Technology 2011, 46,
801-804.
67. Ma, Y.; Huang, S.; Zeng, C.; Zhou, T.; Zhong, Z.; Zhou, T.; Fan, Y.; Yang, X.; Xia, J.;
Jiang, Z. Towards controllable growth of self-assembled SiGe single and double
quantum dot nanostructures. Nanoscale 2014, 6, 3941-3948.
68. Seravalli, L.; Trevisi, G.; Frigeri, P. Design and growth of metamorphic InAs/InGaAs
quantum dots for single photon emission in the telecom window. CrystEngComm 2012,
14, 6833-6838.
69. Xin, S. H.; Wang, P. D.; Yin, A.; Kim, C.; Dobrowolska, M.; Merz, J. L.; Furdyna, J.
K. Formation of self‐assembling CdSe quantum dots on ZnSe by molecular beam
epitaxy. Appl. Phys. Lett. 1996, 69, 3884-3886.
70. Nakamura, S.; Kitamura, K.; Umeya, H.; Jia, A.; Kobayashi, M.; Yoshikawa, A.;
Shimotomai, M.; Nakamura, S.; Takahashi, K. Bright electroluminescence from CdS
quantum dot LED structures. Electron. Lett. 1998, 34, 2435-2436.
Karmakar, R.: Quantum Dots and it method …….
139
71. Comini, E.: One and two dimentional metal oxide nano structures for chemical sensing.
In Semiconductor Gas Sensors; Jaaniso, R., Tan, O. K., Eds.; Woodhead Publising
Limited, 2013; pp 299-310.
72. Mattox, D. M.: Hand book of Physical Vapor Deposition (PVD) Processing; 2nd ed.;
Elsevier Inc.: USA, 2010. pp. 1-24.
73. Burda, C.; Chen, X. B.; Narayanan, R.; El-Sayed, M. A. Chemistry and Properties of
Nanocrystals of Different Shapes. Chem. Rev. 2005, 105, 1025-1102.
74. Dhawan, S.; Dhawan, T.; Vedeshwar, A. G. Growth of Nb2O5 quantum dots by physical
vapor deposition. Matlet. 2014, 126, 32-35.
75. Kumar, M. M. D.; Devadason, S.; Rajesh, S. Formation of CdSe/CdTe quantum dots in
multilayer thin films using PVD method. AIP Conference Proceedings 2012, 1451,
176-178.
76. Kim, Y. G.; Joh, Y. S.; Song, J. H.; Baek, K. S.; Chang, S. K.; Sim, E. D. Temperature-
dependent photoluminescence of ZnSe/ZnS quantum dots fabricated under the
Stranski–Krastanov mode. Appl. Phys. Lett. 2003, 83, 2656-2658.
77. Dhawan, T.; Renu Tyagi, R.; Bag, R. K.; Singh, M.; Mohan, P.; Haldar, T.;
Murlidharan, R.; Tandon, R. P. Growth of InAs Quantum Dots on Germanium
Substrate Using Metal Organic Chemical Vapor Deposition Technique. Nanoscale Res.
Lett. 2010, 5, 31-37.
78. Lee, J. Y.; Kim, K.; Park, W. I.; Kim, B. H.; Park, J. H.; Kim, T. H.; Bong, S.; Kim, C.
H.; Chae, G.; Jun, M.; Hwang, Y.; Jung, Y. S.; Jeon, S. Uniform Graphene Quantum
Dots Patterned from Self-Assembled Silica Nanodots. Nano Letters 2012, 12, 6078-
6083.
79. Ding, X. Direct synthesis of graphene quantum dots on hexagonal boron nitride
substrate. J. Mater. Chem. C 2014, 2, 3717-3722.
80. Lee, J.; Baek, J.; Ryu, G. H.; Lee, M. J.; Oh, S.; Hong, S. K.; Kim, B. H.; Lee, S. H.;
Cho, B. J.; Lee, Z.; Jeon, S. High-Angle Tilt Boundary Graphene Domain
Recrystallized from Mobile Hot-Wire-Assisted Chemical Vapor Deposition System.
Nano. Lett. 2014, 14, 4352-4359.
81. Zhu, J. J.; Koltypin, Y.; Gedanken, A. General Sonochemical Method for the
Preparation of Nanophasic Selenides: Synthesis of ZnSe Nanoparticles. Chem. Mater.
2000, 12, 73-78.
Prajnan O Sadhona ……., Vol. 2, 2015
140
82. Qian, H. F.; Li, L.; Ren, J. One-step and rapid synthesis of high quality alloyed quantum
dots (CdSe–CdS) in aqueous phase by microwave irradiation with controllable
temperature. Mater. Res. Bull. 2005, 40, 1726-1736.
83. Wang, L. C.; Chen, L. Y.; Luo, T.; Qian, Y. T. A hydrothermal method to prepare the
spherical ZnS and flower-like CdS microcrystallites. Mater. Lett. 2006, 60, 3627-3630.
84. Yang, H.; Yin, W.; Zhao, H.; Yang, R.; Song, Y. A complexant-assisted hydrothermal
procedure for growing well-dispersed InP nanocrystals. J. Phys. Chem. Solids 2008, 69,
1017-1022.
85. Xie, Y.; Qian, Y. T.; Wang, W. Z.; Zhang, S. Y.; Zhang, Y. H. A Benzene-Thermal
Synthetic Route to Nanocrystalline GaN Science 1996, 272, 1926-1927.
86. Rogach, A. L.; Katsikas, L.; Kornowski, A.; Su, D. S.; Eychmuller, A.; Weller, H.
Synthesis and Characterization of Thiol-Stabilized CdTe Nanocrystals. Ber. Bunsenges.
Phys. Chem. 1996, 100, 1772-1778.
87. O'Connor, E.; O'Riordan, A.; Doyle, H.; Moynihan, S.; Cuddihy, A.; Redmond, G.
Near-infrared electroluminescent devices based on colloidal HgTe quantum dot arrays.
Appl. Phys. Lett. 2005, 86, 201114.
88. Sankaran, V.; Cummins, C. C.; Schrock, R. R.; Cohen, R. E.; Silbey, R. J. Small lead
sulfide (PbS) clusters prepared via ROMP block copolymer technology. J. Am. Chem.
Soc. 1990, 112, 6858–6859.
89. Winiarz, J. G.; Zhang, L. M.; Park, J.; Prasad, P. N. Inorganic: Organic Hybrid
Nanocomposites for Photorefractivity at Communication Wavelengths. J. Phys. Chem.
B 2002, 106, 967–970.
90. Xie, R. C.; Shang, J. K. Morphological control in solvothermal synthesis of titanium
oxide. J Mater Sci 2007, 42, 6583-6589.
91. Mohammad, C.; Pall, T.; Stride, J. A. Gram-scale production of graphene based on
solvothermal synthesis and sonication. Nat. Nano. 2009, 4, 30-33.
92. Du, C. F.; You, T.; Jiang, L.; Yang, S. Q.; Zou, K.; Han, K. L.; Deng, W. Q. Controllable
synthesis of ultrasmall CuInSe2 quantum dots for photovoltaic application. RSC Adv.
2014, 4, 33855-33860.
93. Whaley, S. R.; English, D. S.; Hu, E. L.; Barbara, P. F.; Belcher, A. M. Selection of
peptides with semiconductor binding specificity for directed nanocrystal assembly.
Nature 2000, 405, 665-668.
94. Lee, S. W.; Mao, C.; Flynn, C. E.; Belcher, A. M. Ordering of Quantum Dots Using
Genetically Engineered Viruses. Science 2002, 296, 892-895.
Karmakar, R.: Quantum Dots and it method …….
141
95. Selvakumar, R.; Seethalakshmi, N.; Thavamani, P.; Naidu, R.; Megharaj, M. Recent
advances in the synthesis of inorganic nano/microstructures using microbial
biotemplates and their applications. RSC Adv. 2014, 4, 52156-52169.
96. Bandyopadhyay, S.; Miller, A. E.; Chang, H. C.; Banerjee, G.; Yuzhakov, V.; Yue, D.
F.; Ricker, R. E.; Jones, S.; Eastman, J. A.; Baugher, E.; Chandrasekhar, M.
Electrochemically assembled quasi-periodic quantum dot arrays. Nanotechnology
1996, 7, 360.
97. Bandyopadhyay, S.; Menon, L.; Kouklin, N.; Zeng, H.; Sellmyer, D. J.
Electrochemically self-assembled quantum dot arrays. Journal of Electronic Materials
1999, 28, 515-519.
98. Balandin, A.; Wang, K. L.; Kouklin, N.; Bandyopadhyay, S. Raman spectroscopy of
electrochemically self-assembled CdS quantum dots. Appl. Phys. Lett. 2000, 76, 137-
139.
99. Pickett, N. Controlled preparation of nanoparticle materials U.S. Patent 7,867,556 B2.
100. Jawaid, A. M.; Chattopadhyay, S.; Wink, D. J.; Page, L. E.; Snee, P. T. Cluster-Seeded
Synthesis of Doped CdSe:Cu4 Quantum Dots. ACS Nano 2013, 7, 3190-3197.
101.Wang, Y. Q.; Rui, Y. C.; Zhang, Q. H.; Li, Y. G.; Wang, H. Z. A Facile in Situ Synthesis
Route for CuInS2 Quantum-Dots/In2S3 Co-Sensitized Photoanodes with High
Photoelectric Performance. ACS Appl. Mater. Interfaces. 2013, 5, 11858-11864.
102.Lim, J.; Park, M.; Bae, W. K.; Lee, D.; Lee, S.; Lee, C.; Char, K. Highly Efficient
Cadmium-Free Quantum Dot Light-Emitting Diodes Enabled by the Direct Formation
of Excitons within InP@ZnSeS Quantum Dots. ACS Nano 2013, 7, 9019-9026.
103.Murphy, C. J.; Brauns, E. B.; Gearheart, L. Quantum Dots as Inorganic DNA-Binding
Proteins. MRS Proceedings 1996, 452, 452–597.
104.DNA-Functionalized Quantum Dots. http://en.wikipedia.org/w/index.php?title=DNA-
Functionalized_Quantum_Dots&oldid=629611362 2014).
105.Gao, X.; Cui, Y.; Levenson, R. M.; Chung, L. W. K.; Nie, S. In vivo cancer targeting
and imaging with semiconductor quantum dots. Nat. Biotech. 2004, 22, 969-976.
106.Pong, B. K.; Trout, B. L.; Lee, J. Y. Preparation of DNA-functionalised CdSe/ZnS
Quantum Dots. Chemical and Pharmaceutical Engineering 2007.
107.Shahid, R.; Gorlov, M.; El-Sayed, R.; Toprak, M. S.; Sugunan, A.; Kloo, L.;
Muhammed, M. Microwave assisted synthesis of ZnS quantum dots using ionic liquids.
Mater. Lett. 2012, 89, 316-319.
Prajnan O Sadhona ……., Vol. 2, 2015
142
108.Shahid, R. Green Chemical Synthesis of II‐VI Semiconductor Quantum Dots. Royal
Institute of Technology (KTH), 2012.
109.Li, D. Y.; Wang, Y. Z.; Zhao, X. L.; He, X. W.; Li, W. Y.; Zhang, Y. K. Facile synthesis
of ionic liquid functionalized silica-capped CdTe quantum dots for selective recognition
and detection of hemoproteins. J. Mater. Chem. B 2014, 2, 5659-5665.
110.Choi, S. Y.; Shim, J. P.; Kim, D. S.; Kim, T. Y.; Suh, K. S. Aqueous Synthesis of CdTe
Quantum Dot Using Dithiol-Functionalized Ionic Liquid. Journal of Nanomaterials
2012, 2012, 6.
111.Sekhar, M. C.; Santhosh, K.; Praveen Kumar, J.; Mondal, N.; Soumya, S.; Samanta, A.
CdTe Quantum Dots in Ionic Liquid: Stability and Hole Scavenging in the Presence of
a Sulfide Salt. J. Phys. Chem. C 2014, 118, 18481-18487.
112.Santhosh, K.; Samanta, A. Exploring the CdTe Quantum Dots in Ionic Liquids by
Employing a Luminescent Hybrid of the Two. J. Phys. Chem. C 2012, 116, 20643-
20650.
113.Patra, S.; Samanta, A. Effect of Capping Agent and Medium on Light-Induced
Variation of the Luminescence Properties of CdTe Quantum Dots: A Study Based on
Fluorescence Correlation Spectroscopy, Steady State and Time-Resolved Fluorescence
Techniques. J. Phys. Chem. C 2014, 118, 18187-18196.
114.Borriello, C.; Concilio, S.; Minarini, C.; Piotto, S.; Di Luccio, T. Optical properties of
ionic liquid passivated CdSe/ZnS quantum dots dispersed in POC copolymer. Polymer
Composites 2013, 34, 1471-1476.
115.Chao, M. R.; Chang, Y. Z.; Chen, J. L. Hydrophilic ionic liquid-passivated CdTe
quantum dots for mercury ion detection. Biosens Bioelectron. 2013, 42, 397-402.
116.Pickett, N. L.; Masala, O.; Harris, J. Commercial Volumes of Quantum Dots:
Controlled Nanoscale Synthesis and Micron-Scale Applications. Material Matters
2008, 3, 24-26.
117.Scottsdale, A.: Quantum dot mass production breakthrough achieved..... Quantum
Materials Corporation: USA, 15 Sept, 2011.
Kar, S.: p300/CBP proteins: A HAT Trick …….
143
Review Article
p300/CBP proteins: A HAT Trick to determine cell fate
Sanchari Kar
Assistant professor, Department of Chemistry, Bangabasi College, 19 Rajkumar Chakraborty
Sarani, Kolkata - 700009, West Bengal, India
Correspondence should be addressed to Sanchari Kar; [email protected]
Abstract
p300 and CBP are highly related nuclear proteins, which act as a transcriptional
coactivator in response to various extracellular and intracellular stresses. p300/CBP responds
as a histone acetyltransferase (HAT) that regulates gene expression by acetylating histones and
other transcription factors. Dysregulation of p300/CBP HAT activity contributes to various
diseases including cancer. p300/CBP also delivers essential functions in virtually all known
cellular programs, including the decision to grow, to differentiate, or to commit suicide by
apoptosis. Other proteins, including the p53 tumour suppressor, are targets for acetylation by
p300/CBP. Active research is currently on to understand fundamental processes involved in
transcriptional control of p300/CBP.
Key Words: Transcription, Signal transduction, Chromatin, p300/CBP, Acetyl transferase
Introduction
All cellular activity, namely, cell development, division and differentiation, that are
required for the proper development of any organism is controlled by the genetic information
coded in the DNA and is commonly known as the gene expression. The first step in this gene
expression is known as transcription, which is the elaborate process that cells use to copy from
the DNA (into units of RNA replica) the necessary information of a particular gene that
generates a particular protein and also the regulatory mechanism that controls the generation
of such protein as may be required for the cellular activity. Two important proteins that are
central to our discussion in this article, namely, p300 and CREB binding protein (CBP), are
ubiquitous and critical regulators of transcription in the cells of higher eukaryotes like human1.
Their action is said to be as a coactivator, in the sense that they generally interact with
transcription factors and assist in the activation of the transcription from the target gene and is
a major area of research.
Prajnan O Sadhona ……., Vol. 2, 2015
144
p300 was originally identified using protein-interaction assays with adenoviral E1A
oncoprotein2. This protein has been implicated in a number of diverse biological functions
including proliferation, cell cycle regulation, apoptosis (the process of programmed cell death),
differentiation (creating different types of cells required to produce different organs) and DNA
damage response1,3. It is highly homologous to CBP with 63% homology at the level of amino
acids (that are building blocks of any protein). The two proteins have a number of common
roles in physiological processes, although it is increasingly clear that they serve several distinct
and non-overlapping functions as well. CBP and p300 function primarily as transcription
cofactors for a number of nuclear proteins. These include oncoproteins (a protein coded by
oncogene that has the potential to cause cancer) as well as tumor suppressor proteins such as
p532,4. Recent evidence indicates that p300/CBP genes are altered in various human tumors5.
Transcriptional coactivation is mediated by CBP/p300 acting as a bridge linking DNA-binding
transcription factors to the basal transcriptional machinery. Furthermore, p300/CBP proteins
are endowed with histone acetyltransferase (HAT) activity6,7, which transfers an acetyl group
to the e-amino group of a lysine residue, and the acetylation level of chromatin has been
established to be a key mechanism in regulation of transcription8. Although it has not been
formally proven that p300/CBP HAT activity is directly involved in chromatin remodeling, a
growing body of evidences suggests that other targets including transcription factors and the
transcription apparatus [9] are also regulated by acetylation, thereby highlighting the potential
importance of the HAT activity in p300/CBP functions.
Genes
The p300/CBP genes are conserved in a variety of multi cellular organism, from worms
to human. The human CBP locus is located in a region on chromosome 1610 which shows
extensive homology to a region on chromosome 22, where p300 resides2. Apart from
p300/CBP, these two regions may contain eight other pairs of paralogous genes11.
Structure and functions
The p300/CBP proteins share several conserved regions, which constitute most of the
known functional domains in the proteins5 that determines its cellular functions: (1) the
bromodomain, which is responsible for HAT activity; (2) three cystine-histidine (CH)-rich
domains (CH1, CH2 and CH3); (3) a KIX domain; and (4) ADA2-homology domain (see Fig.
1). The CH1, CH3 and the KIX domains are likely to be important in mediating protein-protein
interaction and a number of cellular and viral proteins bind to these regions. The HAT domain
Kar, S.: p300/CBP proteins: A HAT Trick …….
145
resides in the central region of the protein and provides a scaffold for assembly of multi-
component transcription coactivator complexes as illustrated in the next section.
Although p300 and CBP share extensive homology, genetic and molecular analyses
suggest that they perform not only overlapping but also unique functions. Most sequence
specific transcription factors can be coactivated by either p300 or CBP.
p300/CBP proteins play very important role in the cell cycle regulation as is evident
from the fact that these two proteins are targets for adenovirus E1A oncoproteins in order to
destroy the normal cellular regulatory mechanism, which in turn promote cancerous growth.
Studies of the interaction between E1A and p300/CBP support such a role in the control of
DNA synthesis and S-phase progression12. Cell proliferation and growth are also influenced by
p300/CBP activity12. Another important role of p300/CBP is to regulate stability of p53, a
tumor suppressor protein13 as discussed later on.
Transcriptional regulation by p300/CBP
Transcriptional regulation is the means by which a cell regulates the conversion of
DNA to RNA (transcription), thereby organizing gene activity. Both RNA and DNA are
nucleic acids, which use base pairs of nucleotides as a complementary language. It is
orchestrated by transcription factors (proteins that control which genes are to be turned on or
off in the genome) and other proteins working in concert to finely tune the amount of RNA
being produced through a variety of mechanisms.
It is now widely accepted that p300/CBP proteins are versatile and perhaps general
transcriptional regulator14. Current evidence suggests that they act through variety of
mechanisms as elucidated below in Fig. 2.
(a) A bridge to the transcriptional machinery:
The initiation of transcription by RNA polymerase II enzyme requires sequence-
specific promoter binding transcription factors as well as the basal transcription machinery (a
Figure 1: Schematic Representation of p300 Domains
Prajnan O Sadhona ……., Vol. 2, 2015
146
central component of general transcription). Unless transcription factors can directly interact
with the basal transcription machinery, other protein must act as bridges that connect them to
the basal machinery. p300/CBP is known to interact both with a wide variety of transcription
factors and with components of basal transcriptional machinery. Therefore, in one model,
p300/CBP provides such a bridge.
(b) A scaffold for the assembly of multi-protein complexes:
p300/CBP proteins might nucleate the assembly of diverse cofactor proteins into multi-
component coactivator complexes15. By providing a scaffold for the assembly of transcription
cofactors, it might increase the relative concentration of these factors in the local transcription
environment and thereby facilitate protein-protein and protein-DNA interactions.
Figure 2: Cartoons showing mechanisms of transcriptional activation by p300/CBP
Adapted from Chan et al.14
Acetylation dependent transcription regulation:
Acetylation of multiple sites in a core histone tails is associated with transcriptional
activity. Hypo-acetylation generally correlates with transcriptional repression, and hyper-
Basal
machi
nery
TF
p300
(a) A Bridge: Here 300/CBP proteins
connect sequence specific
transcription factors (TF) to the
transcription apparatus
NAP TF
p300
(b) A Scaffold: p300/CBP acts as a
protein scaffold for the assembly of
multi component complexes that
confer transcriptional activation.
Examples of possible components,
such as HAT, JMY and NAP are
indicated.
HAT JMY
TF
(c) A HAT: the intrinsic HAT activity
of p300/CBP or HATs assembled in
multicomponent complexes target
chromatin and TF to facilitate a
transcriptional response.
p300
A
c
A
c
Kar, S.: p300/CBP proteins: A HAT Trick …….
147
acetylation correlates with transcriptional activation8. It is now known that the p300/CBP HAT
activity targets nucleosomal histones directly and regulates transcription by chromatin
remodeling. Various studies indicate that non-chromosomal proteins may also be target of
acetylation, suggesting that p300/CBP HAT acts upon transcription factors, or the basal
transcription apparatus, to influence transcription. A host of transcription factors, like p53 have
been shown to be acetylated and, in almost all cases, acetylation enhances their DNA-binding
activity.
p300/CBP/p53 interaction and regulation of the p53 response
As a specific example, we discuss how p300/CBP play as coactivator of p53, another
protein which is known as the cellular gate keeper16. The tumor suppressor protein p53 is
essential for the prevention of cancer development and often referred to. About 50% of human
cancers have either lost p53 gene or encoded a mutated inactive protein. In normal cells
concentration of p53 is controlled by Mdm2 through a negative feedback loop maintaining a
very low level17. But as the cell is subjected to any oncogenic stress, p53 level goes up and
starts functioning as a transcription activator. The first step in transcriptional activation is
believed to be facilitated by the ability of p53 to simultaneously bind the specific DNA
sequences and recruit CBP/p300 to the p53 responsive promoters. CBP/p300 recruitment
brings RNA polymerase II to the target promoters increasing the rate of pre-initiation complex
assembly18. CBP/p300 also facilitates transcriptional activation through nucleosome and
transcription factor acetylation. The co-activators have been shown to directly acetylate lysine
residues present within the amino terminal tails of the four core histones19. Acetylation appears
to increase the accessibility of the nucleosomal DNA to transcription factor binding, a critical
step in gene activation20.
p300/CBP genes and disease
In general, mutation in p300/CBP genes has been observed in a number of human
tumours3 which, together with the properties discussed earlier, suggests that p300/CBP proteins
possess classical tumour suppressor-like activity. Mutations in CBP, and to a lesser extent
p300, are the cause of Rubinstein-Taybi syndrome, which is characterized by severe mental
retardation. These mutations result in the loss of one copy of the gene in each cell, which
reduces the amount of CBP or p300 protein by half. Some mutations lead to the production of
a very short, nonfunctional version of the CBP or p300 protein, while others prevent one copy
of the gene from making any protein at all. Although researchers do not know how a reduction
Prajnan O Sadhona ……., Vol. 2, 2015
148
in the amount of CBP or p300 protein leads to the specific features of Rubinstein-Taybi
syndrome, it is clear that the loss of one copy of the CBP or p300 gene disrupts normal
development5. Defects in CBP HAT activity appear to cause problems in long-term
memory formation21.
Furthermore, bi-allelic somatic mutations in the p300 gene have been observed in
gastric, colon and breast cancers, and mutations in p300 that result in truncated proteins were
detected in primary tumors and tumor cell lines [21]. Dysregulation of the transcriptional
functions of CBP/p300 is associated with rare chromosomal translocations that are associated
with acute myeloid leukemia1 and other types of cancer, thus it has been recognized as a
potential anti-cancer drug target.
These observations suggest that in some circumstances p300 behaves as a classical
tumor suppressor gene. The p300/CBP genes are involved in various chromosomal
translocation events during hematological malignancy and may contribute to aberrant growth
control possibly through a gain of function mutation.
Conclusion
It has become increasingly clear that p300/CBP proteins are versatile transcriptional
co-activators that can influence different physiological processes, including cell growth,
proliferation and differentiation. They are likely to participate in DNA replication, cell cycle
checkpoints, the response to hypoxia, cell adhesion control and the interplay between distinct
signal transduction pathways. Indeed, p300/CBP proteins are biologically multifunctional.
However, many key questions still remain. For example, the HAT activity of p300/CBP
undoubtedly plays a crucial role in its physiological function, and many cellular proteins are
known to be acetylated. The mechanism by which HAT activity is directly involved in
nucleosome remodeling is yet to be elucidated. There is again much to be learnt about the
structural mechanisms of p300/CBP involving multivalent and dynamic interactions with
binding partners, which may pave new avenues for anti-cancer drug development.
References
1. Goodman, R. H.; Smolik, S. CBP/p300 in cell growth, transformation, and
development. Genes Dev. 2000, 14, 1553–1577.
2. Eckner, R.; Ewen, M. E.; Newsome, D.; Gerdes, M.; DeCaprio, J. A.; Lawrence, J. B.;
Livingston, D. M. Molecular cloning and functional analysis of the adenovirus E1A-
Kar, S.: p300/CBP proteins: A HAT Trick …….
149
associated 300-kD protein (p300) reveals a protein with properties of a transcriptional
adaptor. Genes. Dev. 1994, 8, 869–884.
3. Giles, R. H.; Peters, D. J.; Breuning, M. H. Conjunction dysfunction: CBP/p300 in
human disease. Trends Genet. 1998, 14, 178–183.
4. Bannister, A. J; Kouzarides, T. CBP-induced stimulation of c-Fos activity is abrogated
by E1A. EMBO J. 1995, 14, 4758–4762.
5. Petrij, F.; Giles, R. H.; Dauwerse, H. G.; Saris, J. J.; Hennekam, R. C.; Masuno, M.;
Tommerup, N.; van Ommen, G. J.; Goodman, R. H.; Peters D. J. Rubinstein-Taybi
syndrome caused by mutations in the transcriptional co-activator CBP. Nature 1995,
376, 348-351.
6. Bannister, A. J.; Kouzarides, T. The CBP co-activator is a histone acetyltransferase.
Nature.1996, 384, 641-643.
7. Ogryzko, V. V.; Schilltz, R. L.; Russanova, V.; Howard, B. H.; Nakatani, T. The
transcriptional coactivators p300 and CBP are histone acetyltransferases. Cell 1996, 87,
1107-1112.
8. Brownell, J. E.; Allis, C. D. Special HATs for special occasions: linking histone
acetylation to chromatin assembly and gene activation. Curr. Opin. Genel. Dev. 1996,
6, 176-184.
9. Gu, W.; Roeder, R. G. p53 C-Terminal Domain. Cell 1997b, 90, 596-606.
10. Chen, X.; Korenberg, J. R. Localization of human CREBBP (CREB binding protein)
to 16p13.3 by fluorescence in situ hybridization. Cytogenet. Cell. Genet. 1995, 71, 56-
57.
11. Giles, R. H.; Dauwerse, H. G.; Van Ommen, G. B.; Breuning, M. H. Do human
chromosomal bands 16p13 and 22q11-13 share ancestral origins? Am. J. Hum. Genet.
1997, 63, 1240-1242.
12. Stein, R. W.; Corrigan, M.; Yaciul, P.; Whelan, J.; Moran, Analysis of E1A-mediated
growth regulation functions: binding of the 300-kilodalton cellular product correlates
with E1A enhancer repression function and DNA synthesis-inducing activity. E. J.
Virol. 1990, 64, 4421-4427.
13. Grossman, S. R.; Perez, M.; Kung, A. L.; Joseph, M.; Mansur, C.; Xiao, Z. X.; Kumar,
S.; Howley, P. M.; Livingston, D. M. p300/MDM2 complexes participate in MDM2-
mediated p53 degradation. Mol. Cell. 1998, 2, 405–415.
14. Chan, H. P. La Thangue, N. B. p300/CBP proteins: HATs for transcriptional bridges
and scaffolds. J. Cell. Sci. 2001, 114, 2363-2373.
Prajnan O Sadhona ……., Vol. 2, 2015
150
15. Xu, L.; Glass, C. K.; Rosenfeld, M. G. Coactivator and corepressor complexes in
nuclear receptor function. Curr. Opin. Genet. Dev. 1999, 9, 140-147.
16. Lane, D. P. Cancer. p53, guardian of the genome. Nature 1992, 358, 15-16.
17. Marine, C. J.; Jochemsen, A. G. Mdmx as an essential regulator of p53 activity.
Biochem. Biophys. Res. Commun. 2005, 331, 750-760.
18. Yie, J.; Senger, K.; Thanos, D. Mechanism by which the IFN-beta enhanceosome
activates transcription. Proc. Natl. Acad. Sci. U. S. A. 1990, 96, 13108–13113.
19. Schiltz, R. L.; Mizzen, C. A.; Vassilev, A.; Cook, R. G.; Allis, C. D.; Nakatani,
Overlapping but distinct patterns of histone acetylation by the human coactivators p300
and PCAF within nucleosomal substrates. Y. J. Biol. Chem. 1999, 274, 1189–1192.
20. Lee, D. Y.; Hayes, J. J.; Pruss, D.; Wolffe, A. P. A positive role for histone acetylation
in transcription factor access to nucleosomal DNA. Cell 1993, 72, 73–84.
21. Gayther, S. A.; Batley, S. J.; Linger, L.; Bannister, A.; Thorpe, K.; Chin,S.; Daigo, Y.;
Russell, P.; Wilson, A.; SowJoy, H. M. Mutations truncating the EP300 acetylase in
human cancers. Nat. Genet. 2000, 24, 300-304.
Chaudhuri, S.: Vaccination and autism …….
151
Review Article
Vaccination and autism: a new perspective
Sanjukta Chaudhuri
Assistant Professor, Department of Zoology, Fakir Chand College, Diamond Harbour -
743331, West Bengal, India
Correspondence should be addressed to Sanjukta Chaudhuri; [email protected]
Abstract
Autism Spectrum Disorder (ASD), a rare neurodegenerative disease, affecting 1 in 100
people worldwide, 1 in 68 in US, and is often referred as the hidden epidemic. India has over
10 million autistic people. The number is steadily increasing each year, and in US alone 20%
rise was recorded in the last two years. Several factors including genetic, environmental, and
psychological, seem to be involved, and an individual’s genetic make-up susceptible to any
external stimuli may lead to autism. Several reports indicated that MMR (Measles, Mumps,
and Rubella) vaccine may act as a trigger for autism. In MMR, the rubella vaccine is actually
a live-attenuated RNA virus. Studies indicated that the live-attenuated RNA virus may become
virulent as RNA viruses with reverse transcriptase have a higher mutation rate and can cause
viral attack. The aim of this review is to find out a possible link between the rubella vaccine
and ASD.
Key Words: Autism, Vaccine, Rubella, Immunity.
Introduction
In an overpopulated and developing country like India, importance of a disease or
disorder largely depends on its outcome, for example life-threatening diseases are more
important as the value of human life depends on the quantity rather than the quality. This is
evident from the research funding, public awareness and government policies on health care.
There is often a pronounced misconception about some diseases and disorders, which
deteriorate the quality of lives of individuals and their families. Mental disorders specifically
fall into this category. Autism Spectrum Disorders (ASD) or Autism is a group of
developmental disorders that pose threat to the society because it affects the children, the future
of any society, and thus called as a ‘curse of civilization’ or hidden epidemic. In modern days
ASD is spreading like cancer.
Prajnan O Sadhona ……., Vol. 2, 2015
152
Autism Spectrum Disorder or ASD is the collective term for neurodevelopmental
disorders where a person has impairment in social interaction, communication, with limited
activities and interest.1 It includes autistic disorder, pervasive developmental disorder (PDD),
Apergers syndrome, Rett syndrome etc.2 There is a growing concern about ASD in developed
countries, including US, UK, Canada and Australia as the incidence of ASD is alarmingly
increasing day by day. In US 1 out of 68 children are diagnosed with ASD in 2013; which was
1 in 110 in 2010; while in India over 10 million people are affected.3
Types and cause of ASD:
ASD is broadly classified into innate and acquired form. When an individual is autistic
right from infancy is called innate; while in acquired autism the individual develops ASD traits
later in his life, now known as regressive autism.4 As ASD has a strong genetic background,
every ASD can be treated as innate. Till date the cause of ASD is unknown. It is primarily an
autoimmune disorder/disease where considerable neurodegeneration results in impairment of
social and verbal communication. However, some risk factors may induce ASD in genetically
predisposed individuals.5
Risk factors and remedy:
Available internet database of any widely used search engine will blame almost
everything and nothing to be the cause of ASD. Some valid findings have marked certain risk
factors that might induce or trigger ASD. Of these, maternal infection (viral and bacterial),
fragile-X, severe fungal diarrhoea, vaccination are important risk factors for ASD.6 Till date
there is no remedy or medicine that can be used for curing ASD. Since a multitude of factors
are responsible for this disorder, there cannot be one single solution to correct it. Medicines,
however, can reduce certain behavioural conditions. Psychological therapies, early
intervention, behaviour therapy, special education etc may help the autistic individual to some
extent.7
Rubella or German measles is an air borne viral disease that may cause the congenital
rubella syndrome (CRS) in infants. Infection during pregnancy, especially within the first 20
weeks, by Rubella virus can be serious and may lead to the birth of child with CRS8. German
measles caused by the rubella virus can occur in school going children also.8
Chaudhuri, S.: Vaccination and autism …….
153
Symptoms and CRS:
The symptoms of German measles are similar to flu, and the most common symptom
is the appearance of rash, followed by fever, swollen glands, joint pains, headache and
conjunctivitis9; while the CRS causes severe damage to the brain. Extensive degenerative
changes in leptomeningeal and intrinsic blood vessels of cerebrum, deep white matter and gray
nuclei occur due to necrosis. Retarded myelinisation also occurred in some infants.10 Rubella
causes significant loss of executive functioning in children at school going age.
Vaccines and types:
Immunization or vaccination is an effective way to reduce or control many infectious
diseases. Vaccines are primarily of three types: (i) antigenic preparation of live attenuated
strains of disease producing organisms, e.g. measles, mumps, rubella, typhoid; (ii) killed
preparations like anthrax, cholera, hepatitis-A; and (iii) antitoxin or toxoid preparations like
tetanus and diphtheria.
Constituents of Rubella vaccine:
Rubella vaccine is a live attenuated vaccine which is often administered as a
combination vaccine with measles and mumps, known as MMR. This vaccine is reported to
contain a mercurious compound thimerosal, but FDA recently stopped the use of this
compound as a constituent.11
Risks of live attenuated viral vaccine:
The live attenuated viruses are known to retain the ability to replicate within the host
body without causing any illness. This method of immunization is very effective in building
up immunity against many life threatening infectious diseases. However, recently a group of
researchers have expressed concern over the fact that attenuated viruses, particularly RNA
viruses are able to cause virulence in host body after administration of the vaccine; as RNA
virus can undergo antigenic change and become infectious, particularly in immune-
compromised people and infants.12
Immunity and Autism
ASD is a condition often with dysfunctional immune system and the affected
individuals often show abnormal immune responses.13 The autistic individuals have low levels
of immunoglobulins (Ig), and it was evident that higher severity of the disorder correlates with
Prajnan O Sadhona ……., Vol. 2, 2015
154
lower level of immunoglobulins.14 A recent study15 on the plasma of 99 children, aged between
5 and 10 years, with autism showed significantly lower levels of eight cytokines, compared
with that of 40 unrelated healthy siblings without AD, under the same conditions. Three of the
cytokines are known to be involved with hematopoiesis and five with attraction of T-cells,
natural killer cells and monocytes. This necessitate further study to confirm immunological
role in hematopoiesis and antibody production in the children with AD along with the linking
genes that encode immune related proteins and cytokines for their impact on critical periods of
brain development and function15.
Autism and Rubella
Rubella is a major contributor of multisensory impairment (MSI), and in autistic people
MSI is a common occurrence. Many children have shown complete or partial autism when
infected with rubella.16
Discussion
From the analysis of the available data so far, it can be concluded that ASD individuals
have low levels of several cytokines and immunoglobulins that makes them prone to infections.
On the other hand, the vaccines although immunize against many diseases, including the live
attenuated rubella in MMR, may increase the virulence of the live virus in host body by
antigenic alterations. So, when a child is genetically vulnerable to ASD, MMR or rubella
vaccine can cause infection, since, the child would be immunocompromised. Hence, it is
pertinent that the immunoglobulin levels of infants should be checked before administering the
vaccines like MMR, and the children with lower levels of Ig should further be tested for ASD.
As time ticks by, the current data from Centre for Disease Control, US, shows more than 1%
of the world population is affected by some form of autism17, hence, it is of grave concern and
a massive awareness and research drive is to be initiated at the very basic level.
References
1. American Psychiatric Association. Diagnostic and statistical manual of mental
disorders. 4th ed., text revision. Washington, DC: American Psychiatric Association;
2000.
2. WHO. The ICD-10 classification of mental and behavioral disorders: diagnostic criteria
for research. Geneva: World Health Organization; 1993.
3. www.autismweb.com.
Chaudhuri, S.: Vaccination and autism …….
155
4. www.autismresearch.com
5. O’Roak, B.J.; State, M.W. Autism genetics: strategies, challenges, and opportunities.
Autism Res. 2008, 14–17.
6. Autism and Developmental Disabilities Monitoring Network Surveillance Year 2002
Principal Investigators; Centers for Disease Control and Prevention. Prevalence of
autism spectrum disorders--autism and developmental disabilities monitoring network,
14 sites, United States, 2002. MMWR Surveill Summ 2007, 56, 12-28.
7. Ospina, M.B.; Krebs, S.J.; Clark, B. et al. Behavioral and developmental interventions
for autism spectrum disorder: a clinical systematic review. PLoS ONE 2008, 3, e3755.
8. Neighbors, M.; Tannehill-Jones, R. Childhood diseases and disorders. Human
diseases (3rd ed.). Clifton Park, New York: Delmar, Cengage Learning. 2010, 457-
79. ISBN 978-1-4354-2751-8.
9. Edlich, R.F.; Winters, K.L.; Long, W.B.; Gubler, K.D. Rubella and congenital rubella
(German measles). J. Long Term Eff. Med. Implants 2005, 15, 319-28.
10. Rorke, L.B.; Spiro, A. J. Cerebral lesions in congenital rubella syndrome. The J
Pediatrics. 1967, 70, 243-255.
11. Online database of US-FDA.
12. Yao, K.; Wang, Z. Reverse genetics for live attenuated virus vaccine development.
Cancer Immunotherapy. 2008, 2, 57-62.
13. Meldrum, S. J.; Strunk, T.; Currie, A.; Prescott, S. L.; Simmer, K.; Whitehouse, A. J.
O. Autism spectrum disorder in children born preterm—role of exposure to perinatal
inflammation. Frontiers. Neurosc. 2013, 7, 1-10.
14. Goines P.; Van der Water, J. The immune system’s role in the biology of autism. Curr.
Opin. Neurol. 2010, 23, 111-117.
15. Manzardo, A.M.; Henkhaus, R.; Dhillon, S.; Butler M.G. Plasma cytokine levels in
children with autistic disorder and unrelated siblings. International J Developmental
Neuroscience, 2012, 30(2), 121-127.
16. Chess, S.; Korn, S.; Fernandez, P. Psychiatric Disorders of Children with Congenital
Rubella. Brunner/Mazel, New York. 1971.
17. www.cdc.gov/ncbddd/autism, 2014
Prajnan O Sadhona ……., Vol. 2, 2015
156
Review Article
Mechanism of action of antimicrobial peptides and proteins
Suranjana Chattopadhyay
Assistant Professor, Department of Chemistry, Maharaja Manindra Chandra College, Kolkata
- 700003, West Bengal, India
Correspondence should be addressed to Suranjana Chattopadhyay; [email protected]
Abstract
A wide variety of organisms produce antimicrobial peptides as part of their first line of
defense. They are typically relatively short, positively charged, amphiphilic, and have been
isolated from single-celled microorganisms to insects, other invertebrates, plants, amphibians,
birds, fish, and mammals, including humans. To date, hundreds of such peptides have been
identified indicating their importance in the innate immune system. The direct antimicrobial
activity is largely evident in dilute media, and direct microbe killing is almost certainly
prevented by physiological conditions, including high monovalent or moderate divalent cation
concentrations, host proteases, polyvalent anions, and low local peptide concentrations.
Conversely these peptides are important effector molecules of the innate immune system. They
are able to enhance phagocytosis, stimulate prostaglandin release, neutralize the septic effects
of LPS (lipopolysaccharides), and promote recruitment and accumulation of various immune
cells at inflammatory sites.
Key Words: Antimicrobial, Peptides, Amphiphilic, Bilayer, Immunomodulatory
Introduction
Antimicrobial peptides are short (12 to 100 amino acids), predominantly cationic (net
charge of +2 to +9), amphiphilic, and naturally occurring in a large number of sources1. The
expression of these antimicrobial peptides can be constitutive or can be inducible by infectious
and/or inflammatory stimuli, such as pro inflammatory cytokines, bacteria, or bacterial
molecules that induce innate immunity, e.g., lipo-polysaccharides (LPS)2.
Conversely these peptides are important effector molecules of the innate immune
system3. They are able to enhance phagocytosis, stimulate prostaglandin release, neutralize the
septic effects of LPS, and promote recruitment and accumulation of various immune cells at
inflammatory sites. Peptides of mammalian origin have also been demonstrated to have an
Chattopadhyay, S.: Mechanism of action of antimicrobial …….
157
active role in the transition to the adaptive immune response by being chemotactic for human
monocytes and T cells and by exhibiting adjuvant and polarizing effects in influencing
dendritic cell development4. Although such peptides may have a direct effect on the microbe,
such as by damaging or destabilizing the bacterial, viral, or fungal membrane or acting on other
targets, they appear to be broadly involved in the orchestration of the innate immune and
inflammatory responses. Thus, they are increasingly being referred to as host defense peptides.
Despite their similar general physical properties, individual cationic peptides have very limited
sequence homologies and a wide range of secondary structures with at least four major themes.
The most prominent structures are amphiphilic peptides with two to four -strands,
amphipathic -helices, loop structures, and extended structures (Fig. 1).
Figure 1: The structural classes of antimicrobial peptides
Natural distribution of antimicrobial peptides
Antimicrobial peptides are a universal feature of the defense systems of virtually all
forms of life, with representatives found in organisms ranging from bacteria to plants and
invertebrate to vertebrate species, including mammals5. They form part of the ancient,
nonspecific innate immune system, which is the principal defense system for majority of living
organisms. Antimicrobial peptides produced by bacteria were among the first to be isolated
and characterized. While they do not protect against infection in the classical sense, they
contribute to survival of individual bacterial cells by killing other bacteria that might compete
for nutrients in the same environment. Bacterial antimicrobial peptides, also called
Prajnan O Sadhona ……., Vol. 2, 2015
158
bacteriocins, are thought to be produced by many or most bacteria. Mersacidin, a tetra cyclic
peptide that is produced by Bacillus species6, displays bactericidal activity against methicillin-
resistant Staphylococcus aureus that is comparable to that of vancomycin, an important drug,
but without the development of cross resistance. In plants, it is widely believed that
antimicrobial peptides play an important and fundamental role in defense against infection by
bacteria and fungi. So far, only peptides with a -sheet globular structure have been identified
in plants, with the two major and best-studied groups being thionins and defensins.
Invertebrates lack the adaptive immune system found in vertebrate species, they are reliant
solely upon their innate immune systems to counteract invading pathogens. Numerous
antimicrobial peptides have now been identified in invertebrates, and they are recognized as
playing a key role in protection from pathogenic organisms. Antimicrobial peptides are found
in the hemolymph (plasma and hemocytes), in phagocytic cells, and in certain epithelial cells
of invertebrates. Defensins are antimicrobial peptides that contain six cysteines. Defensins
form at least three structural groups whose evolutionary relationship is uncertain: the originally
described ‘classical’ defensins or the -defensins, the newly discovered -defensins and the
subsequently discovered insect defensins. The three defensin groups differ from each other in
the spacing and connectivity of their six cysteines residues.
Structure–function relationship of antimicrobial peptides
All of the peptides are highly cationic and hydrophobic. It is widely believed that they
act through nonspecific binding to biological membranes, even though the exact nature of these
interactions is presently unclear7. High-resolution nuclear magnetic resonance (NMR) has
contributed greatly to knowledge in this field, providing insight about peptide structure in
aqueous solution, in organic co-solvents, and in micellar systems8. Solid-state NMR can
provide additional information about peptide-membrane binding. Despite the different three-
dimensional structural motifs of the various classes, they all have similar amphiphilic surfaces
that are well-suited for membrane binding. Many antimicrobial peptides bind in a membrane-
parallel orientation, interacting only with one face of the bilayer9. This may be sufficient for
antimicrobial action. At higher concentrations, peptides and phospholipids translocate to form
multimeric transmembrane channels that seem to contribute to the peptide's hemolytic activity.
Although extremely variable in length, amino acid composition and secondary
structure, all peptides can adopt a distinct membrane-bound amphipathic conformation. Recent
studies demonstrate that they achieve their antimicrobial activity by disrupting various key
Chattopadhyay, S.: Mechanism of action of antimicrobial …….
159
cellular processes. Some peptides can even use multiple mechanisms. A molecular description
of functional modules in the cell is the focus of many high-throughput studies in the post-
genomic era10. A large portion of biomolecular interactions in virtually all cellular processes is
mediated by compact interaction modules, referred to as peptide motifs. Such motifs are
typically less than ten residues in length, occur within intrinsically disordered regions, and are
recognized and/or post-translationally modified by structured domains of the interacting
partner. In this review, we suggest that in spite of a million instances of peptide motifs in the
human proteome, an understanding of the key features of the secondary and tertiary structures
of the antimicrobial peptides and their effects on bactericidal and hemolytic activity can aid the
rational design of improved analogs for clinical use11.
A better understanding of the structure–activity relationships of AMPs might be
required to facilitate the rational design of novel antimicrobial agents.
Structural Requirements and mode of action for Antibacterial Peptides
As mentioned above, cationic antimicrobial peptides are generally categorized into four
structural classes, i.e., -helical, sheet, loop, or extended structures (Fig. 1); however, there
are many peptides that do not fit into this simplified classification scheme. One approach to
increase the antibacterial activity of cationic peptides has been to alter their flexible secondary
structures. Thus, preconditioning peptides to adopt structures related to their final membrane-
associated ones can occasionally be advantageous while giving rise to other assets such as
protease stability. Nonetheless, antibacterial peptides seem largely able to affect their
antimicrobial activity because of their amphipathicity or amphiphilicity and because of the
presence of regions within the folded structure with high concentrations of positively charged
residues12.
It was originally proposed that permeabilization of the bacterial cell membrane was the
sole mode of action of antibacterial peptides. There is an increasing body of evidence, however,
that indicates that some antimicrobial peptides exert their effects through alternative modes of
action. Regardless of their precise mode of action, the activities of antibacterial peptides are
almost universally dependent upon interaction with the bacterial cell membrane13. The first
step in this interaction is the initial attraction between the peptide and the target cell, which is
thought to occur through electrostatic bonding between the cationic peptide and negatively
charged components present on the outer bacterial envelope, such as phosphate groups within
the lipo-polysaccharides of gram-negative bacteria or lipoteichoic acids present on the surfaces
Prajnan O Sadhona ……., Vol. 2, 2015
160
of gram-positive bacteria. The events that occur at the membrane surface are the subject of
considerable debate, and several prominent models have been proposed (Fig. 2).
Figure 2: Different models to describe the antibacterial action of peptide
A mechanism, known as the aggregate model, with some similarity to the toroidal pore
model, has been proposed (Fig. 2A). In this model peptides reorient to span the membrane as
an aggregate with micelle-like complexes of peptides and lipids but the peptides adopt no
particular orientation. In the toroidal pore model (Fig. 2B), aggregates of peptides insert
themselves in an orientation perpendicular to the membrane to form a pore, with the membrane
also curving inward to form a hole with the head groups facing towards the center of the pore,
and the peptides line this hole. Examples of antimicrobial peptides that are proposed to form
this type of trans-membrane pore include the magainins, melittin, and LL-3714. In the barrel-
stave model (Fig. 2C); peptides reorient to become the “staves” in a “barrel”-shaped cluster
which orients perpendicular to the plane of the membrane. The hydrophobic regions of each
peptide in the cluster are associated with the lipid core, while the hydrophilic regions are facing
the lumen of the newly formed transmembrane pore. In contrast to the barrel-stave and toroidal
pore models, the carpet model proposes that aggregates of peptide align parallel to the lipid
bilayer, coating local areas in a carpet-like fashion (Fig. 2D). At sufficiently high concentration,
this is thought to have a detergent-like activity (causing patches of the membrane to break up
Chattopadhyay, S.: Mechanism of action of antimicrobial …….
161
into micelles), causing local disturbances in membrane stability which can lead to the formation
of holes in the membrane. Many cationic antimicrobial peptides will do this at high enough
concentrations due to their amphipathic character; however, there is very limited evidence
demonstrating that most peptides cause membrane dissolution at the minimal effective
concentrations in vivo (or, in many studied cases, in vitro; for example, Zhang et al.15. Alpha-
helical peptides such as derivatives of pleurocidin, a fish-derived antimicrobial peptide, and
dermaseptin, isolated from frog skin, cause inhibition of DNA and RNA synthesis at their MICs
without destabilizing the membrane of E. coli cells (Fig. 2E). Inhibition of nucleic acid
synthesis has also been demonstrated for antimicrobial peptides from different structural
classes, such as the sheet human defensin, HNP-116, and the extended-structure bovine
peptide indolicidin17 (Fig 2F). Inhibition of cellular enzymatic activity by proline-rich insect
antimicrobial peptides has also been observed. Pyrrhocidin enters the target cell and binds to
DnaK, a heat shock protein that is involved in chaperone-assisted protein folding. Specifically,
the peptide inhibits the ATPase activity of DnaK, preventing protein folding, which results in
the accumulation of misfolded proteins and cell death18 (Fig. 2G). Antimicrobial peptides can
also target the formation of structural components, such as the cell wall (Fig. 2I). It is likely
that the mode of action of individual peptides may vary according to the particular bacterial
target cell, the concentration at which they are assayed, and the physical properties of the
interacting membrane. It is also likely that in the context of infection, antimicrobial peptides
may use more than one mechanism of action, such as destabilization of the cell membrane
combined with inhibition of one or more intracellular targets. An example of multi target
mechanism is that of aminoglycoside modifying enzymes, which contain an anionic binding
pocket. It was recently demonstrated that cationic peptides of various structural classes can
bind and specifically inhibit the activity of these enzymes19 (Fig. 2H).
Each of these indicates a different type of intermediate that can lead to one of three
types of events: formation of a transient channel, micellarization or dissolution of the
membrane, or translocation across the membrane. As a result, the peptide can permeabilize the
membrane and/or translocate across the membrane and into the cytoplasm without causing
major membrane disruption. This high degree of complexity of the mechanism is almost
certainly the cause of the observation that it is extremely difficult to select cationic-peptide-
resistant mutants.
Prajnan O Sadhona ……., Vol. 2, 2015
162
Structural Requirements and mode of action for Antifungal Peptides
Viejo-Diaz et al.20 identified two novel human lactoferrin-derived peptides with
different anti-Candida activities but quite high sequence homology. Alignments of one of these
peptides with the sequence of brevinin-1Sa also indicated high homology. However, other
studies have shown that antifungal peptides vary substantially in sequence and structure.
Modification of ineffective antimicrobial peptides has revealed that relatively modest changes
often result in antifungal activity. For example, conjugation of undecanoic acid or palmitic acid
to magainin resulted in analogue peptides that had gained potent activity against both yeast and
opportunistic fungal infections.
Lehrer et al. demonstrated quite early that the rabbit -defensin NP-2 resulted in
permeabilization of C. albicans. Lee et al. used scanning electron microscopy observations to
demonstrate morphological changes in response to the potent permeabilizing peptides pig
myeloid antimicrobial peptide (PMAP-23) and melittin. They also presented data indicating
that indolicidin an antimicrobial peptide exerts its fungicidal activity by disrupting the structure
of the fungal cell membrane, in a salt-dependent and energy-independent fashion, via direct
interactions with the lipid bilayer. This contrasts to the situation for bacteria, in which
indolicidin, although membrane active appears to penetrate cells and act on macromolecular
synthesis. The mechanism of action of certain antifungal peptides is still a matter of debate.
The formation of reactive oxygen species has been suggested to be the crucial step in the
fungicidal mechanism of a number of antimicrobial peptides, including histatin 5 and
lactoferrin-derived peptides21. Conversely, Veerman et al. have concluded that reactive oxygen
species do not play a role in the histatin 5-mediated killing of C. albicans. Histatins from saliva
of humans and some other higher primates bind to a receptor on the fungal cell membrane and
enter the cytoplasm, where they target the mitochondrion. Thus, it is reasonable to hypothesize
that antimicrobial peptides may interact with mitochondria in a manner very similar to some of
their actions on bacteria, as suggested by the studies of Helmerhorst et al.
Structural Requirements and mode of action for Antiviral Peptides
Synthetic analogues of several naturally occurring antimicrobial peptides have been
made in an attempt to identify important structural features contributing to the antiviral activity.
Several groups have looked at the importance of charged and aromatic amino acids, since
antiviral peptides are often highly cationic and amphiphilic. They concluded that the presence
of hydrophobic and positively charged residues is critical but not sufficient for antiviral
Chattopadhyay, S.: Mechanism of action of antimicrobial …….
163
activity, and this may relate to different conformations adopted by these peptides in the context
of the native protein. Heparan sulfate is the most important glycosaminoglycan molecule with
respect to viral attachment22; consequently, blocking of heparan sulfate can reduce the viral
infection. One might hypothesize that antimicrobial peptides that interact with heparan sulfate
should be able to block many different viral infections. Human -defensin, LL-37, and
magainin have all been shown to interact with different glycosaminoglycan molecules.
Antimicrobial peptides might interact directly with specific viral receptors on the host cell,
influencing viral attachment, entry, or intracellular shuttling. The most obvious example of this
is the known ability of the polyphemusin analogue T22 to bind to the chemokine receptor
CXCR4, which serves as a co receptor for HIV-1 entry into T cells. Thus, it antagonizes that
subgroup of HIV strains which use this chemokine receptor but not those that use CCR5.
Antimicrobial peptide interactions with glycoproteins in the viral envelope have been proposed
to influence the viral entry process. defensin (retrocyclin-2) interacts with the HSV-2
glycoprotein B with high affinity, thus protecting the cells from HSV-2 infection.
Conclusion
Antimicrobial cationic host defense peptides have been demonstrated to have activity
towards a wide spectrum of infectious bacterial, viral, fungal, and parasitic pathogens. Their
activities differ both within and between distinct structural peptide classes, although all the
peptides appear to be able to adopt some sort of cationic and amphipathic structure. Their
modes of action are strongly dependent on experimental conditions and demonstrate the
tremendous ability of peptides to exert a diverse range of antimicrobial effects. The leukocyte-
attracting activity of many mammalian CAMPs (cyclic antimicrobial peptides) requires
specific receptor interactions, and only some amino acids can be exchanged without losing the
immunomodulatory functions of CAMPs23. It remains an open question, in many instances,
which of these two roles is more important for a given peptide.
Antibiotics are usually designed to function at low concentrations through a defined
high-affinity antimicrobial target—a set of circumstances that makes it comparatively easy for
bacteria to develop resistance. By contrast, CAMPs are released precisely at infected sites and
are usually active at much higher concentrations than antibiotics. It is possible that such low-
affinity interactions with universal non- protein targets are not favourable for the development
of bacterial resistance. In other words, it can be argued that the evolution of the innate host
defense systems has favoured the design of ‘dirty’ antimicrobials that disturb many biological
Prajnan O Sadhona ……., Vol. 2, 2015
164
functions with low potency rather than blocking a specific high-affinity target. Such properties
enable the host to control a much wider range of potential pathogens without creating the
selection pressure for high-level resistance that is observed with potent, high-affinity
antibiotics. Diverse immunomodulatory activities of such host defense peptides are the most
recent characterized property and will provide an additional stimulus to consideration of these
molecules as a new class of therapeutic agents24.
References
1. Brogden, K. A. Antimicrobial peptides: pore formers or metabolic inhibitors in
bacteria. Nature Reviews Microbiology. 2005, 3(3), 238–250.
2. Campos, M. A., Vargas, M. A., Regueiro, V., Llompart, C. M., Alberti, S., Bengoechea,
J. A. Capsule Polysaccharide Mediates Bacterial Resistance to Antimicrobial
Peptides. Infection and Immunity. 2004, 72 (12), 7107–7114.
3. Matsuzaki, K. Control of cell selectivity of antimicrobial peptides. Biochimica et
Biophysica Acta – Biomembranes. 2008, 1788 (8), 1687–92.
4. Davidson, D. J., Currie, A. J., Reid, G. S., Bowdish, D. M., MacDonald, K. L., Ma, R.
C., Hancock, R. E., Speert, D. P. The cationic antimicrobial peptide LL-37 modulates
dendritic cell differentiation and dendritic cell-induced T cell polarization. J. Immunol.
2004, 172(2), 1146-56.
5. Arenas, G., Marshall, S. H. Antimicrobial peptides: A natural alternative to chemical
antibiotics and a potential for applied biotechnology. Electronic Journal of
Biotechnology 2003, 6(3).
6. Herzner, A. M., Dischinger, J., Szekat, C., Yakéléba, A., Jansen, A., Sahl, H.G., Piel,
J., Bierbaum, G., Reinartz, R. Expression of the Lantibiotic Mersacidin in Bacillus
amyloliquefaciens FZB42.. PLOS ONE 2011, 6(7), e22389.
7. Rokitskaya, T. I. et al. Indolicidin action on membrane permeability: carrier
mechanism versus pore formation. Biochim. Biophys. Acta. 2011, 1808, 91–97.
8. Chan, D. I. et al. Human macrophage inflammatory protein 3 alpha: protein and peptide
nuclear magnetic resonance solution structures, dimerization, dynamics, and anti-
infective properties. Antimicrob. Agents Chemother. 2008, 52, 883–894.
9. Holt, A., Kilian, J. A. Orientation and dynamics of transmembrane peptides: the power
of simple models. Eur. Biophys. J. 2010, 39, 609–621.
10. Blondelle, S. E., Lohner, K. Optimization and high-throughput screening of
antimicrobial peptides. Curr. Pharm. Des. 2010, 16, 3204–3211.
Chattopadhyay, S.: Mechanism of action of antimicrobial …….
165
11. Williams, K. J., Bax, R. P. Challenges in developing new antibacterial drugs.
Curr. Opin. Investig. Drugs. 2009, 10, 157–163.
12. Wheaten, S. A., Ablan, F. D. O., Spaller, B. L., Tieu, J. M., Almeida, P. F.
Translocation of Cationic Amphipathic Peptides across the Membranes of Pure
Phospholipid Giant Vesicles. J. Am. Chem. Soc. 2013, 135 (44), 16517–16525.
13. Melo, M. N., Castanho, M. A. The mechanism of action of antimicrobial peptides:
lipid vesicles vs. Bacteria. Front. Immunol. 2012, 3, 236.
14. Bucki, R., Pastore, J. J., Randhawa, P., Vegners, R., Weiner, D. J., Janmey, P. A.
Antibacterial Activities of Rhodamine B-Conjugated Gelsolin-Derived Peptides
Compared to Those of the Antimicrobial Peptides Cathelicidin LL37, Magainin II, and
Melittin. Antimicrob. Agents Chemother. 2004, 48(5), 1526-1533.
15. Zhang, L.; Rozek, A.; Hancock, R. E. Interaction of cationic antimicrobial peptides
with model membranes. J. Biol. Chem. 2001, 276(3), 5714-35722.
16. Varkey, J.; Nagaraj, R. Antibacterial activity of human neutrophil defensin HNP-1
analogs without cysteines. Antimicrob agents Chemother. 2005, 49(11), 4561-6.
17. Hsu, C. H.; Chen, C.; Jou, M. L.; Lee, A. Y.; Lin, Y. C.; Yu, Y. P.; Huang, W. T.; Wu,
S. H. Structural and DNA-binding studies on the bovine antimicrobial peptide,
indolicidin: evidence for multiple conformations involved in binding to membranes and
DNA. Nucleic Acid Res. 2005, 33(13), 4053-64.
18. Kragol, G.; Lovas, S.; Varadi, G.; Condie, B. A.; Hoffmann, R.; Otvos, L. J. The
Antibacterial Peptide Pyrrhocoricin Inhibits the ATPase Actions of DnaK and Prevents
Chaperone-Assisted Protein Folding. Biochemistry. 2001, 40, 3016-3026.
19. Muller, A.; Ulm, H.; Reder-Christ, K.; Sahl, H. G.; Schneider, T. Interaction of type A
lantibiotics with undecaprenol-bound cell envelope precursors. Microb. Drug
Resist. 2012, 18, 261–270.
20. Viejo-Dı´az, M.; Andre´s, M. T.; Fierro, J. F. Different Anti-Candida Activities of Two
Human Lactoferrin-Derived Peptides, Lfpep and Kaliocin-1. Antimicrobial Agents and
Chemotherapy 2005, 2583–2588.
21. Van der Does, A. M.; Hensbergen, P. J.; Bogaards, S. J.; Cansoy, M.; Deelder, A.
M.; Van Leeuwen, H. C.; Drijfhout, J. W.; Van Dissel, J. T.; Nibbering, P.H. The human
lactoferrin-derived peptide hLF1-11 exerts immunomodulatory effects by specific
inhibition of myeloperoxidase activity. J. Immunol. 2012, 188(10), 5012-9.
22. Sarrazin, S.; Lamanna, W. C.; Esko, J. D. Heparan Sulfate Proteoglycans. Cold Spring
Harb. Perspect. Biol. 2011, 3(7).
Prajnan O Sadhona ……., Vol. 2, 2015
166
23. Nijnik, A.; Hancock, R.E. Host defence peptides: antimicrobial and immunomodulatory
activity and potential applications for tackling antibiotic-resistant infections. Emerg.
Health Threats J. 2009, 2.
24. Vale, N.; Aguiar, L.; Gomes, P. Antimicrobial peptides: a new class of antimalarial
drugs? Front. Pharmacol. 2014, 5, 275.
Ghosh, S.: Some synthetic and structural aspects …….
167
Review Article
Some synthetic and structural aspects of chromium nitrosyl
complexes
Swapna Ghosh
Associate Professor, Department of Chemistry, Sreegopal Banerjee College, Bagati, Hooghly-
712148, West Bengal, India
Correspondence should be addressed to Swapna Ghosh; [email protected]
Abstract
Much attention has been recently paid to nitrosyl complexes in the fields of physiology
as well as in coordination chemistry, since the physiological effects of NO comes mostly from
the presence of metal ions, metal-proteins or metal-enzymes. Considerable progress has also
been made toward the understanding of the redox non-innocence of the nitrosyl ligand. In
particular, the electronic structure of linear metal nitrosyls has proven far more complicated
than the traditional ‘NO+’ description given to these species. Chromium (VI) reacts with
hydroxylamine in acidic, neutral, or alkaline aqueous solutions, yielding nitrosyl complexes of
the type (Cr-NO). In the presence of excess hydroxylamine and chelating ligands, several
complexes have been isolated and characterized by X-ray crystallography.
Key Words: Reductive nitrosylation, Hydroxylamine hydrochloride, Chromium(VI), Nitric
Oxide, Cis/Trans-dinitrosyl
Introduction
Previously, Nitric oxide (NO) thought to be a poisonous, pungent-smelling gas: an
unpleasant and dangerous product of the oxidation of ammonia and of incomplete combustion
of gasoline in motor vehicle exhausts. However, the present studies indicate that NO is one of
the most important physiological regulators,1 playing a key role in signal transduction and
cytotoxicity. The fascinating coordination chemistry of NO has drawn considerable attention
of coordination chemists, since much of the biochemistry of NO involves metal nitrosyl
complexes. Metal nitrosyls can also be seen as useful delivery agents of nitric oxide and, in
particular, hold promise for the photochemical delivery of NO to biological targets. The
transition metal nitrosyl complexes have attracted increasing attention because of their intrinsic
Prajnan O Sadhona ……., Vol. 2, 2015
168
chemical interest, specially the electron transfer properties2, catalytic uses in organic synthesis3
via carbon-nitrogen bond formation4, and their potentiality in pollution control5.
Considering the above facts a brief account of the nitrosyl complexes of chromium
synthesized mainly by reductive nitrosylation methods is being present in the present
communication. Although, special attention has been given to the complexes synthesized by
the reaction of hydroxylamine hydrochloride on higher valent chromium substrates, the nitrosyl
complexes obtained by some other ways are also described. There exist several reviews on
metal nitrosyls6 but no unified description of the bonding in metal nitrosyl complexes which
adequately accounts for all their known structural, physical, and chemical properties has yet
been provided.
Background of Nitrosyl (NO) ligands:
Nitric oxide is a stable free radical, having an unpaired electron in this molecule resides
in a π* molecular orbital. This electronic configuration explains the high reactivity of the NO
molecule, in particular the ease of oxidation to the nitrosonium ion (NO+), the probability of
reduction to the nitroxide ion, NO-, the facile attack by oxygen leading to formation of NO2.
NO is isoelectronic with the dioxygen monocation (O2+), and NO+ is isoelectronic with CO and
CN-, while NO- is isoelectronic with O2, having a triplet ground state. NO can be an effective
probe of metalloenzyme structure (geometrical and electronic) and function, where a
spectroscopic examination of the resting or oxygenated enzyme is difficult or impossible
because of instability. The nitrosonium ion has been isolated as a series of stable salts, and is a
useful synthetic and oxidizing agent. However, NO+ in all likelihood has an extremely short
independent life in biological media, although metal complexes may function as transport
agents. The independent chemistry of reduced nitric oxide (NO-) is currently minimal, although
the anion formally plays a significant role in binding with transition metals, as is reported later.
The nitric oxide molecule is redox-active in solution, a most important property which has a
major influence on the chemistry of its transition metal complexes. The redox potential for the
reversible process NO to NO+ is strongly solvent dependent, and in water is also pH-
dependent7. The bond length of free NO is 1.154 Å, lying between that of a double (1.18 Å)
and a triple (1.06 Å) bond. Convention regards this bond length as equivalent to a bond order
of 2.5, (Fig. 1,2,3)8. Oxidation to NO+ causes the bond distance to contract to 1.06 Å,
equivalent to bond order 3. Reduction of NO to NO- leads, concomitantly, to an increase in
bond length (1.26 Å) because of further population of the π* orbital.9 The bond length changes
discussed above are reflected in the IR stretching frequencies of these simple diatomic species:
Ghosh, S.: Some synthetic and structural aspects …….
169
NO decreasing with increasing charge, from 2377 (NO+) through 1875 (NO) to 1470 cm-1 (NO-
).10 Electron spin resonance studies indicate that ca. 60% of the spin density is concentrated on
the N atom of neutral nitric oxide.8
Figure 3: Molecular orbitals involved in d-*
Bonding between metal and NO
Late transition metal nitrosyl complexes, in particular, feature complicated electronic
structures, and often exhibit ambiguous oxidation state assignments for the nitrosyl ligand11.
The root of this ambiguity can be ascribed to the highly covalent nature of the M(NO) bond12.
Traditionally, the NO stretch, as determined by IR spectroscopy, was used to differentiate
between the two resonance extremes NO+ and NO– 13; however, significant spectral overlap
between these forms makes a definitive assignment based on the NO stretch alone a challenge.
Likewise, the M–N–O bond angle, as determined by X-ray crystallography, is not a good
predictor of the NO electronic structure, as the M–N–O bond angle also overlaps between
resonance forms. Accordingly, in recent years a combination of techniques, including IR and
Mössbauer spectroscopies, X-ray crystallography, and computational methods, have been used
to confidently determine the applicable resonance form of a metal nitrosyl complexes.
Synthetic aspect of chromium nitrosyl complexes
The bright green complex, K3[Cr(NO) (CN)5].H2O the first known14 chromium
nitrosyl complex was reported by Griffith et al.14 by the action of NH2OH.HCl and KCN on
CrO3 in a basic solution. The compound reportedly shows υNO at 1625 cm-1, and the magnetic
moment (µeff) of 1.87 B.M. Subsequently, another group15 prepared it from K3[Cr(CN)6]
Figure 2: Valence bond representation of
metal-nitrosyl bonding involving
NO+ ion
Figure 1: Valence bond and other
representations of NO
Prajnan O Sadhona ……., Vol. 2, 2015
170
Scheme: Representation of the three binding modes of metal nitrosyls
using NH2OH.HCl & the complex was structurally characterized. Later on, the compound
was also isolated by Bhattacharyya et. al.16 at a higher yield by synthesizing it from CrO42-,
KCN and excess of NH2OH.HC1 in an alkaline medium. Polarographic reduction of a 0.1 M
[Cr(CN)5NO]3- solution resulted a blue solution which when treated with air-free ethanol
precipitated K4[ Cr(CN)5NO].2H20 17 as a blue solid (υNO at 1515 cm-1 and υCN at 2020 cm-1).
Bhattacharyya et. al. also reported18 several five coordinate cyano-nitrosyl complexes having
the composition [ Cr(NO)(CN)4]2-, [Cr(NO) (CN)3H2O]- and [Cr(NO)(CN)2(L-L)] (where L-L
= bipy or phen) using excess of NH2OH.HCl as nitrosylating agent.
In 1978, Muller and his coworkers19 reported that NH2OH.HCl can reductively
nitrosylate CrO42- in a slightly acidic medium in the presence of SCN- ion. In that work they
obtained a limited yield (because they employed, insufficient amount of NH2OH.HCl) of a
paramagnetic (µeff = 2.23B.M.) hexacoordinate anionic thiocyanatonitrosyl complex19 (Ph4P)3
[Cr(NO) (NCS)5], containing {Cr(NO)}5 moiety.
Bhattacharyya et al.20,21 discovered that the use of an excess of NH2OH.HCl
dramatically improves the smoothness of the reaction and almost a quantitative yield of the
product could be obtained. Besides, (Ph4P)3[Cr(NO)(X)5], [Cr(NO)(X)2(L-L)] and
Cr(NO)(dtc)2 (where X = NCS- or N3- and L-L = bipy or phen and dtc = diethyl
dithiocarbamate anion). Bhattacharyya et. al. also observed that while the reductive
nitrosylation of CrO42-, with NH2OH.HCl in the presence of NCS-, requires a slightly acidic
medium, such reaction in the presence of N3- requires an alkaline medium, though the
composition of the respective products, [Cr(NO)X5]3- ( X = NCS or N3) is identical. The
υNO bands for all those above compounds were reported to appear around 1640-1705 cm-1. It
Ghosh, S.: Some synthetic and structural aspects …….
171
was also observed by Bhattacharyya et al 20,21 that the changes in different parameters of the
experimental course did not drag the reaction down to Cr(NO)+ or to dinitrosylation.
The reductive nitrosylation reaction of [Cr(H2O)6]2+ with NO2
-/H+ to afford
[Cr(NO)(H2O)5]2+ was reported by Ardon et al. 22. Mori et al.23 carried out the same reactions
with the subsequent addition of NH3 and reportedly isolated several salts of [Cr(NO)(NH3)5]2+.
The detailed kinetic study of these reactions was reported by Takenaka and his coworkers24.
The tetranitrosyl complex of chromium, Cr(NO)4, which is isoelectronic with Ni(NO)4, was
prepared by passing a slow stream of NO through a photolyzed solution of Cr(CO)6 in
pentane25. The compound was characterized by Raman and I.R. spectroscopic studies.
The diamagnetic yellow crystalline complex [Cr(NO)(Cl)(das)2] [das = O-phenylene-
-bis(dimethylarsine)] was synthesized26 by the reduction of [Cr(NO)(Cl)(das)2]ClO4, with
dithionite (S2O42-). A series of low spin four coordinate diamagnetic chromium nitrosyl
complexes viz., [Cr(NO)(NPr2i)3], [Cr(NO)(2,6-dimethylpiperidide)3], [Cr(NO)(OBut)3] and
[Cr(NO)(OPri)3] were isolated by Bradley et al.27 The NO stretching frequencies of those
complex were observed at 1641, 1673, 1707 and 1720 cm-1 respectively. Dinitrosyl chromium
complexes are relatively less known. Some diamagnetic dinitrosyl complexes were reported.
[Cr(NO)2{OP(Ph3)3}2X2]28 (X = Cl, Br, I), and [Cr(NO)2{CH3CN)4][PF6]
29 were isolated
as diamagnetic complexes, but, for the first compound υNO was obtained at 1847 and 1714
cm-1 conforming to the cis arrangement of the two nitrosyl groups whereas the second one
showed only one υNO band indicating a trans arrangement between the two nitrosyl groups a
very rare occurance in nitrosyl chemistry. Reaction of second compound with [As(dtc)3] and
[Na2S2C2(CN)2]/Ph4AsCl yielded [Cr(NO)2(dtc)2] and (Ph4As)2[Cr(NO)2(S2C2(CN)2)2]
respectively. Both the compounds were found to be diamagnetic and υNO was obtained at 1775
and 1678 cm-1. Carlin et. al.30 had also synthesized the same compound [Cr(NO)2(dtc)2] as
diamagnetic maroon crystals in a different experimental method which was reported to show
the υNO bands at 1785 and 1660 cm-1.
The formally Cr(I) nitrosyl compounds (NO+ formalism) possess {Cr(NO)}5 moiety
and are always of low-spin type. The complexes of the type [Cr(NO)L5]2+ (where L = H2O or
NH3) were extensively studied by EPR spectrophotometer.
Several nitrosyl complexes of Cr were synthesized by Pandey et. al.31 using RNO2 (R=
Me, Et, Pr, Bu) as nitrosylating agent. The nitrosyls were characterized on the basis of I.R.
data, magnetic and conductance measurements and elemental analyses.
Although, Maurya et al.32 reported lots of derivatives by conducting substitution
reaction on K3[Cr(NO)(CN)5] with different heterocyclic or aromatic bases, the products were
Prajnan O Sadhona ……., Vol. 2, 2015
172
formulated as [Cr(NO)(CN)2L2], but the work was rather naively reported and seemed to be
superfluous and requires authentication by detailed analytical and physicochemical evidences.
The X-ray single crystal structures of [Cr(NO)(NH3)5]Cl2, [Cr(NO)(NH3)5]Cl(ClO4),
and [Cr(NO)(NH3)5] (ClO4)2 complexes have been reported33-36 and found that the interatomic
distances and angles within the complex cations change very little with the change of the
counter anions, while the distances between the O (nitrosyl) and H (ammonia in adjacent
complex cations) atoms increase clearly in the order of [Cr(NO)(NH3)5]Cl2 <
[Cr(NO)(NH3)5]Cl(ClO4) < [Cr(NO)(NH3)5] (ClO4)2. It seemed that the bulky perchlorate
anions separated the complex cations widely in [Cr(NO)(NH3)5](ClO4)2, while the small
chloride anions were not large enough to separate them in [Cr(NO)(NH3)5]Cl2. To clarify the
reason for the color change of the nitrosyl compounds, two additional compounds,
[Cr(NO)(NH3)5](PF6)2 and [Cr(NO)(NH3)5]Cl(PF6), were prepared, and their the X-ray
structures were also determined.
Table 1: Peak positions of reflection spectra: wavenumbers of N-O stretching vibration
(cm-1) in the IR spectra and colour of the crystal
Figure 4: ORTEP view of [Cr(NO)(NH3)5]2+
Complex N-O stretching
wavenumber (cm-1)
Colour of
crystal
[Cr(NO)(NH3)5](PF6)2 1693 Red
[Cr(NO)(NH3)5]Cl2 1683 Red-orange
[Cr(NO)(NH3)5]Cl(ClO4) 1710 Brown
[Cr(NO)(NH3)5]Cl(PF6) 1713 Brown
[Cr(NO)(NH3)5](ClO4)2 1728 Green
Ghosh, S.: Some synthetic and structural aspects …….
173
The difference in colour in the solid states was attributed to a rise in energy of the
excited level π* NO caused by a donor–accepter interaction with anion acting as donor.
Interatomic distances between the oxygen atom (nitrosyl) and counter anion are in the range of
3.1–4.7 Å among [Cr(NO)(NH3)5]Cl2, [Cr(NO)(NH3)5]Cl(ClO4), [Cr(NO)(NH3)5]Cl(PF6), and
[Cr(NO)(NH3)5] (ClO4)2, where no donor–accepter interaction can be seen between the oxygen
atom (nitrosyl) and counter anion, since the interatomic distances are longer than the sum of
the van der Waals radius of each atom. The case of [Cr(NO)(NH3)5](PF6)2 is exceptional: the
interatomic distances (2.743(7) Å) between O (nitrosyl) and F (PF6 - anion) are shorter than the
sum (2.99 Å) of the vander Waals radius of each atom, and some donor–accepter interaction
may exist between O (NO) of [Cr(NO)(NH3)5] and F (PF6 -). The irregularity may be due to
the donor–acceptor interaction suggested by Mori and co-workers.
Table 2: Comparison of selected interatomic distances (Å) and angles (°)
Compound [Cr(NO)
(NH3)5](PF6)2
[Cr(NO)
(NH3)5]Cl2
[Cr(NO)
(NH3)5]Cl(ClO4)
[Cr(NO)
(NH3)5]ClPF6
[Cr(NO)
(NH3)5](ClO4)2
NO 1.156(7) 1.169(9) 1.18(1) 1.179(18) 1.181(7)
Cr-N(NO) 1.700(6) 1.692(7) 1.71(1) 1.684(14) 1.677(6)
Cr-NH3
(trans) 2.177(6) 2.113(7) 2.139(9) 2.165(13) 2.140(5)
Cr–NH3
(cis) 2.092(3)
2.089(5)
2.104(4) 2.104(6) 2.097(4)
Cr–N–O 180 180 180 180 179.9(6)
Figure 5: Molecular structure of the
cation in the complex
[Cr(dmso)5(NO)]2+
Figure 6: Molecular structure of the
cation in the complex
Cr(NO)(NiPr2)(CH2SiMe3)2
Prajnan O Sadhona ……., Vol. 2, 2015
174
Biological activity of nitrosyl complexes
It is now well established that nitric oxide plays fundamental roles in biochemical
processes, including cardiovascular control, neuronal signaling and as an agent for defense
mechanisms against microorganisms and tumors. It has been demonstrated that NO is involved
as a mediator in one tumor-induced angiogenic process, which is a key step in the formation of
metastasis. Both NO and O2 are stable paramagnetic gases with neutral charge and one-
electron reduction to HNO/NO− or O2−, respectively, results in the formation of an anion. In
aqueous conditions, the formed anion has a pKa associated with the equilibrium of protonation
of the anion. For superoxide, a pKa of 6.8 has been measured, while the pKa of NO− has been
calculated to be around 11.4.
The reactions of NO with heme are of great biological significance. The first known
physiological target of NO was the soluble guanylate cyclase (sGC). NO binds to the ferrous
heme in sGC and releases the heme-ligating histidine, resulting in a heme Fe2+– NO complex
formation. This reaction triggers a change in heme geometry and a subsequent conformational
change of the protein to an enzymatically active form37. Therefore, hemoglobin in erythrocytes
could be one of the most important elements in the biological transformation of NO donors and
NO transportation inside the organism. Moreover, NO can react not only with Hb SH-groups,
but with heme too, producing nitrosyl complexes (HbNO). HbNO was detected in human blood
plasma in ischemia/reoxygenation, tumor necrosis. Hemoglobin nitrosyl complexes are formed
when NO is associated with heme iron, and the iron atom is most often in the reduced state
(heme Fe2+- NO). The paramagnetic properties of this complex are due to the presence of an
unpaired electron that belongs to NO•. Both reduced (Hb(Fe2+)) and oxidized (Hb(Fe3+) –
metHb) forms of hemoglobin can interact with NO. The reaction of metHb with NO is
reversible, and the rates of the direct and reverse reactions are rather low. The reaction of Hb
(deoxyHb) with NO is diffusion limited and practically irreversible.
NO + metHb ↔ metHb_NO.
The biological properties of NO are generally attributed to its interaction with iron in
the heme groups of enzymes. However, NO also interacts with a wide range of other cellular
components, many of which do not contain heme. Enzymes that react with oxygen (e.g.
monooxygenases, dioxygenases) have the potential to make nitrosyl complexes as shown in
the case of lipoxygenase37.
To date, all reported HNO-transition-metal complexes have been obtained by insertion
or redox reactions of NO-related species. For example, the initial route to Mb-HNO was by
Cr(II) reduction of the nitrosyl adduct Mb-NO or {Mb-NO}7.
Ghosh, S.: Some synthetic and structural aspects …….
175
Mb-NO + Cr(II) + H+ Mb-HNO + Cr(III)
The possibility of usage of nitrosyl ruthenium complexes as novel antitumor agents
which might release cytotoxic NO within tumor cells, leading to cell death, Additionally, it
has been claimed that the activity of NAMI-A against disseminated tumors might be related
with NO metabolism in vivo.
As, the nitrosyl complexes have enormous biological implications. So, chromium
nitrosyl complexes can also be used for manipulation of many biological transformations.
Conclusion
Chromium forms a relatively wide range of nitrosyl complexes, and some of their
synthetic and structural properties have been discussed in this review, which helps to
understand and development of new organic methodologies. The reductive nitrosylation yields
chromium nitrosyl complexes with different counter anion which affects the colour of crystal
and stretching of N-O ligand in the complexes. However, there are several areas of chromium
nitrosyl chemistry that are deserving of further exploration.
Acknowledgment
S.G thanks UGC for financial support in the form of a Minor Research Project [SPSW-
044/09-10(ERO)]. S.G. is also thankful to Sreegopal Banerjee College for assistance.
References
1. Goodrich, L. E.; Lehnert,, N. J. The trans effect of nitroxyl (HNO) in ferrous heme
systems: Implications for soluble guanylate cyclase activation by HNO, Inorg.
Biochem. 2013, 118, 179.
2. Enemark , J. H.; Feltham, R. D. Stereochemical Control of Valence and Its Application
to the Reduction of Coordinated NO and N2, Proc. Natl. Acad. Sci. U.S.A. 1972, 69,
3534.
3. Zuech, E. A.; Huges, I. B.; Kubicek, D. H.; Kittleman, E. T. Homogeneous catalysts
for olefin disproportionations from nitrosyl molybdenum and tungsten compounds, J.
Am. Chem. Soc. 1970, 92, 528.
4. McCleverty, J. A. Reactions of nitric oxide coordinated to transition metals, Chem. Rev.
1979, 79, 53.
5. Johnson, B. F. G.; Bhaduri, S. The nitrosyl ligand as an oxidant, J. Chem. Soc., Chem.
Commun. 1973, 650.
Prajnan O Sadhona ……., Vol. 2, 2015
176
6. Goodrich, L. E.; Paulat, F.; Praneeth, V. K. K.; Lehnert, N. Electronic Structure of
Heme-Nitrosyls and Its Significance for Nitric Oxide Reactivity, Sensing, Transport,
and Toxicity in Biological Systems, Inorg. Chem. 2010, 49, 6293.
7. Roncaroli, F.; Videla, M.; Slep, L. D.; Olabe, J. A. New features in the redox
coordination chemistry of metal nitrosyls {M-NO+; M-NO•; M-NO– (HNO)}, Coord.
Chem. Rev. 2007, 251, 1903.
8. Legzdins, P.; Richter-Addo, G. B. Metal Nitrosyls; Oxford University Press: New York,
1992.
9. Evans, W. J.; Fang, M.; Bates, J. E.; Furche, F.; Ziller, J.W.; Kiesz, M. D.; Zink, J. I.
Isolation of a Radical Dianion of Nitrogen Oxide (NO)2-, Nat. Chem. 2010, 2, 644.
10. Enemark, J. H.; Feltham, R. D. Principles of structure, bonding, and reactivity for metal
nitrosyl complexes, Coord. Chem. Rev. 1974, 13, 339.
11. Conradie, J.; Quarless, D. A.; Hsu, H.-F.; Harrop, T. C.; Lippard, S. J.; Koch, S. A.;
Ghosh, A. Electronic Structure and FeNO Conformation of Nonheme
Iron−Thiolate−NO Complexes: An Experimental and DFT Study, J. Am. Chem. Soc.
2007, 129, 10446.
12. Franz, K. J., Lippard, S. J. NO Disproportionation Reactivity of Fe Tropocoronand
Complexes, J. Am. Chem. Soc. 1999, 121, 10504.
13. Siladke, N. A.; Meihaus, K. R.; Ziller, J. W.; Fang, M.; Furche, F.; Long, J. R.; Evans,
W. J. Synthesis, Structure, and Magnetism of an f Element Nitrosyl Complex, J. Am.
Chem. Soc. 2011, 134, 1243.
14. Griffith, W. P.; Lewis, J.; Wilkinson, G. Über die Einwirkung von Dipyridyl-(2.2′) auf
Chrom(II)-acetat und die Darstellung des Tris-dipyridyl-(2.2′)-chrom(0) [CrDipy3], J.
Chem. Soc. 1959, 872.
15. Vannerberg, N. G. The Crystal Structure of K3[Cr(CN)5NO], Acta. Chem. Scand. 1966,
20, 1571.
16. Bhattacharyya, R. G., Bhattacharjee, G. P.; Roy, P. S.; Ghosh, N. Complexes of the
Types: K3[Cr(NO)(CN)5] and [Cr(NO)(NCS)2(LL)] (LL = 2,2′-Bipyridine; 1,10-
Phenanthroline, Inorg. Synth. 1985, 23, 182.
17. Griffith, Y. P. Studies on transition-metal–nitric oxide complexes. Part VII. Nitric oxide
complexes of chromium and molybdenum, J. Chem. Soc. 1963, 3286.
18. Bhattacharyya, R. G.; Bhattacharjee, G. P.; Ghosh, N. Reductive nitrosylation of
tetraoxometallates. part V1. single pot and A virtually single step synthesis of
Ghosh, S.: Some synthetic and structural aspects …….
177
chromium(l) cyanonitrosyl derivatives directly from chromate(VI) in aqueous-aerobic
media, Polyhedron. 1983, 2, 543.
19. Muller, A.; Sarkar, S. A Contribution on the Synthesis and Reactivity of Nitrosyl
Complexes. Direct Preparation of Thiocyanatonitrosvl Complexes and of
[Mo(NO)(CN)5]3 , Z. Maturfcrsch. 1978, 33b, 1053.
20. Bhattacharyya, R. G.; Bhattacharjee, G. P.; Roy, P, S. Reductive nitrosylation of
tetraoxometallates. Part I. Generation of [Cr(NO)2+] moiety: Single step synthesis of
complexes of the type [Cr(NO)(NCS)2LL] (LL = 2,2-́bipyridine or 1,10-
phenanthroline) and [Cr(NO)(dtc)2] (dtc = N,N-́diethydithiocarbamate) directly from
chromat(VI) in aqueous and aerobic media, Inorg. Chimca. Acta. 1981, 54, L 263.
21. Bhattacharyya, R. G.; Bhattacharjee, G, P. Reductive nitrosylation of tetraoxometalates.
single-pot and a virtually single-step synthesis of [Os(NO)(NCS)5]2- and its 1,10-
phenanthroline and 2,2'-bipyridine derivatives directly from OsO4 in aqueous aerobic
media, Polyhedron. 1983, 2, 1221.
22. Ardon, M.; Herman, J. I. The complex ion Cr(H2O)5NO2+ J. Chem. Soc. 1962, 507.
23. Mori, M.; Ueshlba, S.; Kawaguchi, S. Brown Nitrosylpentamminechromium(III) Salts
and Green Nitrosylpentamminechromium(III) Perchlorate, Bull. Chem, Soc, Japan.
1963, 36, 796.
24. Takanaka, A.; Sasada, Y.; Omura, T.; Ogoshi, H.; Yoshida, Z. The Kinetics and
Mechanisms of the Reactions of Nitric and Nitrous Acids with Hexaaquochromium(II)
Ions, Bull. Chem, Soc. Jap. 1974, 47, 308.
25. Satija, S. K.; Swanson, B. I. Tetranitrosylchromium and
Carbonyltrinitrosylmanganese, Inorg. Synth. 1976, 16, 1.
26. Feltham, R. D.; Silverthom, W.; MePherson, G. Inorg. Chem. 1969, 8, 344.
27. Bradley, D. C.; Newing, C. W. Low spin four-co-ordinated chromium nitrosyl
dialkylamides and alkoxides, J. Chem. Soc., Chem. Commun. 1970, 219.
28. Beck, W.; Lottes, K. Chem. Ber. 1963, 96, 10.
29. Connelly, N. G.; Dahl, L. F. Acetonitrilecarbonyl and acetonitrilenitrosyl metal cations
from the reactions of the nitrosonium ion, NO+, with transition-metal organometallics
in acetonitrile, J. Chem. Soc. Chem. Commun. 1970, 880.
30. Carlin, R. L.; Canziani, F.; Bralton, W. K. Properties of some metal nitrosyl
dithiocarbamates, J. Inorg. Nucl. Chem. 1964, 26, 898.
31. Pandey, D. S.; Khan, M. I.; Agarwala, U. C. Indian J. Chem. Sect. A. 1987, 26A, 570.
Prajnan O Sadhona ……., Vol. 2, 2015
178
32. Maurya, R. C.; Shukla, R.; Shukla, R. K.; Anandam, N.; Srivastava, S. K.; Maurya, M.
R. Trans. Met. Chem. 1987, 12(3), 203.
33. Akashi, H.; Yamauchi, T.; Shibahara, T. Hydrogen bonding: further evidence for the
cause of the color change of nitrosylpentaamminechromium (III) compounds. Crystal
structures of [Cr(NO)(NH3)5](PF6)2 and [Cr(NO)(NH3)5]Cl(PF6), Inorg. Chimica Acta.
2004, 357, 325.
34. Akashi, H.; Mori, M.; Shibahara, T. Pentaamminenitrosylchromium(III) dichloride,
Acta Crystallogr. Sect. E. 2001, 57, i75.
35. Shibahara, T.; Akashi, H.; Asano, M.; Wakamatsu, K.; Nishimoto, K.; Mori, M. DFT
calculation and X-ray structure of nitrosyl pentaammine chromium complex, Inrog.
Chem. Commun. 2001, 4, 413.
36. Akashi, H.; Nishiura, M.; Mori, M.; Shibahara, T. Effect of outer sphere anions on the
structure and color of nitrosylpentaamminechromium complex, Inorg. Chim. Acta.
2002, 331, 290.
37. Lin, R.; Farmer, P. J. The HNO Adduct of Myoglobin: Synthesis and Characterization,
J. Am. Chem. Soc. 2000, 122, 2393.
Mandal, T. K.: Biological importance of 4H-1-benzopyran …….
179
Review Article
Biological importance of 4H-1-benzopyran and derivatives
Tapas Kumar Mandal
Assistant Professor, Fakir Chand College, Diamond Harbour - 743331, South 24 Parganas,
West Bengal, India
Correspondence should be addressed to Tapas Kumar Mandal; [email protected]
Abstract
Compounds containing pyran, benzopyran and benzothiopyran ring systems display
interesting biological activities such as antiallergic, antitumor, antiviral, antioxidant and
antiinflammatory etc. The aim of the present paper was to review the available information on
this field.
Key Words: 4H-1-Benzopyran and derivatives, Biological importance
Introduction
Six-membered oxygen and sulfur containing heterocycles are widespread through both
the domains of natural and synthetic compounds. Compounds containing pyran, benzopyran
and benzothiopyran ring systems display interesting biological activities, which have
motivated the chemical community towards the studies on their isolation, structure, reactivity
and synthesis. The important group of natural benzopyran compounds is the flavonoids which
abundantly occur in the plant kingdom. Their intake takes place through foods and they perform
many important biological functions. The main objectives of synthesis of chromones and
related compounds are not only for the development of more diverse and complex compounds
having wide range of biological activities and their structure-activity relationship (SAR) studies
but also for other applications in medicinal chemistry and material science, such as
development of various fluorescence probes due to interesting photophysical and
photochemical properties of these compounds. Benzothiopyrans are mainly synthetic
compounds and in the recent past the studies on their syntheses have experienced a surge due
identification of a number of biological activities of compounds containing this ring system.
Prajnan O Sadhona ……., Vol. 2, 2015
180
Biological Importance of Chroman [ ≡ 2,3-Dihydro-4H-1-benzopyran] Derivatives
Chromans have considerable biological importance, especially as potentially useful
pesticides and drugs1a. Chromans have gained recent interest because of their broad biological
and pharmacological activities. Disodium cromoglycate marked as Intal or Cromolyn sodium
bears some structural resemblance to khelin, the spasmolytic component of seeds of
Ammivisnaga. Intal is one of the successful drugs for the prevention of asthmatic attacks,
though it is not effective in the treatment of an acute attack of asthma. It appears to prevent the
release of histamine and other substances which mediate hypersensitive reactions but is
ineffective once these substances have been released1b-c.
A number of 3-(1H-tetrazol-5-yl)chromones were synthesized and found to have
antiallergic activity2. Yang et al. synthesized series of chromanones and chromones analogues
of diacylhydrazine derivatives which have broad insecticidal activities3. The insecticidal
activity of such compounds against Aphis medicagini, Nilaparvata legen, Mythima separata,
Tetranychus cinnabarinus etc. were investigated. Heilmann et al. isolated chromanone acids
from Calophyllum brasiliense Cambers. All compounds showed moderate to strong
antibacterial4 activity against Bacillus cereus and Staphylococus epidermidis. Bis-chromans 1
show potential biological activity against human pathogenic bacteria5. Bis-chromanones have
been found to exhibit significant activity towards S. aureus and S. faecalis.
Park et al. synthesized6 a new series of compounds with chromone and chromanone
which have ability to inhibit HIV-1. A series of chroman and chromanone derivatives have
antioxidant activities. Lee et al. synthesized and evaluated7 6-hydroxy-7-methoxy-4-
chromanone and chroman-2-carboxamides as antioxidant. Gamal-Eldeen et al. isolated8 a
O
O
O
O
R
R
1
O
O
O
NaO2C
CH2 CH
OH
CH2 O
OO
CO2Na
Intal
Mandal, T. K.: Biological importance of 4H-1-benzopyran …….
181
chromone derivative, 2-(hydroxymethyl)-8-methoxy-3-methyl-4H-chroman-4-one, which has
modulatory effect on carcinogen metabolizing enzyme CYP1A (Cytochrome P-450 1A). The
results indicate that the chroman is a promising inhibitor of CYP1A activity up to 60% of the
stimulated-CYP1A in murine hepatoma cells and significantly reduced GST (Glutathione S-
transferases). The isolated chroman possesses a potent specific radical scavenging activity
against hydroxyl radicals and induced DNA damage. Prakash et al.9 synthesized 3-hydroxy-
2-(1-phenyl-3-aryl-4-pyrazolyl) chromone 2 which has antifungal activity against three
phylopathogenic fungi, namely Helminthosporium species, Fusarium oxysporum and
Alternaria alternata. Ishar et al. synthesized novel 6-chloro/flurochromone derivatives 310 as
potential topoisomerase inhibitor anticancer agents.
Tkaishi et al. isolated11 chromone glucosides, takanechromones 4 and chromanone
glucosides, named takanechromanones 5 from the methanolic extracts of Hypericium
sikokumontanum together with twenty seven compounds. The isolated compounds were
assayed for antimicrobial activity against Helicobacter pylori and cytotoxicity against human
cancer cell line.
The significant antipyretic activity of 2-methylchromones has been recognized fifteen
years ago. They have the same antipyretic effect as paracetamol and analgesic effect as
novalgin. Also, 2-methylchromones 6 and 7 play vital role in the replication cycle of AIDS
virus and thus act as HIV-1 protease inhibitors12
.
4 5
O
O
R
OH
GClO O
O
R
OH
GClO OClG
O
OMe
O
OMe
8
O
CH3
O
O
N
NAr
Ph
OH
2 3
O NX
n
O O
HX1
Prajnan O Sadhona ……., Vol. 2, 2015
182
Khellin 8 is the principal constituent of Ammivisnaga L. It is 2-methyl- chromone with
a linearly fused furan ring system and has been found to be a potent coronary vasodilator in
bronchial action on bronchial muscle, gall bladder and bile duct. Additionally, it has been
reported to be used as antispasmodic13. 5,6,7-Trihydroxy-2-methylchromone 9 showed a high
inhibition activity towards α-glycosidase (the α-glycosidase enzyme catalyses the final step in
the digestive process of carbohydrate, hence, α-glycosidase inhibitors can retard the
decomposition and absorption of dietary carbohydrates to suppress postprandial
hyperglycemia14. Chromone derivatives have capacity to act as antioxidant. Many chromone
derivatives
such as luteolin 10, quercetin 11, catechins 12 are better antioxidants than the nutrients
antioxidants such as vitamin C, vitamin E and β–carotene15. The function of antioxidant is to
intercept and react with free radicals at a rate faster than the substrate. Since free radicals are
able to attack at variety of targets including lipids, fats, and proteins, it is believed that they
may damage organisms leading to disease poising including aging. Quercetin 11, kaempferol,
morin, myricetin and rutin, by acting as antioxidants, exhibited anti-inflammatory, antiallergic,
antiviral as well as anticancer activity. They have also protective role in lever and
cardiovascular disease. Quercetin and silybin act as radical scavengers and protect liver from
reperfusion ischemic tissue damage16,17. Quercertin has been reported to completely inhibit the
growth of Staphylococcusaureus18. Chromone derivatives have been investigated for their
antibacterial, antifungal and antiviral activities. Y. B. Vibhute et al. showed that chromones of
the type 13 available from a new class of chalcones have antibacterial activity against
Xanthomanas citri, Ervinia carotovara, Escherichia coli, Bacillus subtilis using ampicillin as
a standard drug19. Murty et al. synthesized 7,4´-dihydroxy-3´-methoxyflavone 14, a chromone
9
O
HO
HO
O
CH3
OH
O
OH
OH
OOH
HO
Luteolin
10
12
O
OH
OH
OH
OH
HO
catechinsQuercetin
O
OH
OH
OH
OOH
HO
11
Mandal, T. K.: Biological importance of 4H-1-benzopyran …….
183
derivative, which has antibacterial activity against gram positive bacteria like Staphylococcus
aureus, Bacillus subtilis and Staphylococcus griseus and gram negative bacteria like
Escherichia coli and Psuedomonas aureagionsa20.
Subramanyam et al. isolated the trimethoxyflavone 15 from Andrographis viscosula
and used it in the treatment of dyspepsia, influenza, malaria, respiratory functions and as an
astringent and antidote for poisonous stings of some insects21. B. S. Dawane et al. synthesized
chromone derivatives22 of the type 16 containing substituted naphthalene moiety having
antibacterial activities against Staphylococcus aureus and Escherichia coli, by disk diffusion
method, using tetracycline antibiotic for comparison of activity.
A number of chromones were isolated from the peelings of tangerine orange, having
fungistatic activity against Deuterophoma tracheiphila. Nobiletin 17, a citrus flavonoid,
isolated from tangerine orange exhibited strong activities. Chlorflavonin was the first chlorine-
containing flavonoid-type antifungal antibiotic produced by strains of Aspergillus candidus23.
Naturally occurring chromone derivatives with antiviral activity have been recognized
since the 1940s, but only recently attempts have been made to make synthetic modification of
natural compounds to improve antiviral activity. Quercetin, morin, rutin, dihydroquercetin,
apigenin, catechin, hesperidin have been reported to possess antiviral activity against some of
the 11 types of viruses24. It has been found that flavonols are more active than flavones against
Herpes simplex virus type 1. Calsalpinia pulcherrima showing antiviral activity has been
found to possess quercetin. The mode of action of quercetin against HSV-1(herpes virus) and
ADV-3 (adeno-virus) was found to be at the early state of multiplication, and this compound
can be used for the treatment of infection caused by these two virus30. Because of world-wide
O N
R1
R2
R2
O
OCH3
Cl
13
O
O
R2
R1
16
O
OCH3
OOCH3
H3COOCH3
OCH3
H3CO
17
Prajnan O Sadhona ……., Vol. 2, 2015
184
spread of HIV, the investigation of antiviral activity of chromanone and chromone derivatives
was focused mainly on HIV25. There have been several recent reports on anti-AIDS activity
of such derivatives26.
Chromanone derivatives have effect on gastrointestinal system. Hesperidine 18, citrus
flavonoids, possesses significant anti-inflammatory and analgesic effects27. Recently, apigenin
19, luteolin 10 and quercetin 11 have been reported to exhibit anti-inflammatory activity28.
Some recent studies have indicated that flavonoids possess antiulcerogenic activity. Flavonoid
lycosides of Ocimum basilicum decreased ulcer index and inhibited gastric acid and pepsin
secretions in aspirin-induced ulcers in rats29.
The liver is subjected to acute and potentially lethal injury by several substances
including phalloidin (the toxic constituent of the musroom, Amanita phalloides), CCl4,
galactosamine, ethanol and other compounds. Chromone derivatives have also been found to
possess hepatoprotective activity. The derivatives apigenin 19, quercetin 11 and naringenin 20
can act as putative therapeutic agents against microcrystin LR-induced hepatotoxicity,
silymarin was found to be most effective one30. The flavonoid, rutin 21 and venoruton 22
showed hepatoprotective effects in experimental cirrhosis31.
O
OOH
HOOCH3
OO
OH
O
HOHO
OHHO
H3CHO
Hesperidin
18
O
OOH
HO
OH
Apigenin
19
O
OOH
HO
OH
Naringenin
20
Mandal, T. K.: Biological importance of 4H-1-benzopyran …….
185
Chromone derivatives, especially quercetin, have been reported to possess antidiabetic
activity. Vessal et al. reported that quercetin brings about the regeneration of pancreatic islets
and probably increases insulin release in strptozotocin-induced diabetic rats32. Hif and Howell
reported that quercetin stimulate insulin release and enhances Ca2+ uptake from isolated islets
cell which suggest a place for chromones in non-insulin dependent diabetes33,34.
The consumption of flavonoids prevents endothelial dysfunction by enhancing the
vasorelaxant process leading to a reduction of arterial pressure35. Some derivatives of
chromones can prevent a number cardiovascular disease including hypertension and
atherosclerosis36.
It was found in the 1960s that tea pigment can reduce blood coagualability, increase
fibrinolysis and prevent platlet adhesion and aggregation37. Selected chromanone derivatives
such as quercetin 11, kaempferol 23 and myricetin 24 were shown to be effective inhibitors of
platelet aggregation in dogs and monkeys38.
Recent interest in different chromanone derivatives has been stimulated by the
potential health benefits arising from the antioxidant activity of these polyphenolic compounds.
Some derivatives have been considered as potential protectors against chronic cardiotoxicity
caused by the cytostatic drug doxorubicin. Doxorubicin is a very effective antitumor agent but
its clinical use is limited by the occurrence of a cumulative dose-related cadiotoxicity, resulting
in congestive heart failure. In a recent report, the cadiotoxicity of doxorubicin on the mouse
left atrium has been inhibited by flavonoids 39, 40, 41.
Some chromone derivatives have antineoplastic activity. Quercetin exerted a dose-
dependent inhibition of growth and colony formation. The flavonoids, kaempferol, catechin,
toxifolin 25 and fisetin 26, also suppressed cell growth42.
O
OOH
HO
OH
OH
OO
HOOH
O
OH
O
OHHO
HO
H3C
Rutin
21
O
OH
O
O
O
O
O
OH
OH
OO
OH OH
CH3
OHO
OH
OH
OH
Venoruton
22
Prajnan O Sadhona ……., Vol. 2, 2015
186
Conclusion
4H-1-Benzothiopyran derivatives show vast array of biologically activity and have been
used in traditional eastern medicine for thousands of years. They also constitute unavoidable
components of the diet. In the present review, I have reviewed biological properties of 4H-1-
Benzothiopyran derivatives. Their widespread occurrence, broad spectrum diversity and
natural origin make them appropriate chemical scaffolds for novel therapeutic agents.
Acknowledgment
The author acknowledges Prof. Asok Kumar Mallik, Research guide, Department of
Chemistry, Jadavpur University, for giving valuable suggestions during writing this review.
Author also thanks departmental colleague for their support.
References
1. McClure, J. W.; Harborne, J. B.; Mabry, T. J.; Mabry (Eds.), H. The Flavonoids,
Chapman and Hall: London, 1975, p. 970. (b) Middleton Jr, E.; Kandaswami, C.;
Harborne (Eds.), J. B. The Flavonoids Advances in Research since 1986, Chapman and
Hall: London, 1994, 619, (c) Bruneton, J. Pharmacognosy, Phytochemistry and
Medicinal Plants, English Translation by C. K. Hatton, Lavoisier Publishing, Paris,
1995, 265.
2. Groot, H. D.; Raven, U. Tissue injury by reactive oxygen species and protective effects
of flavonoids. Fundam Clin Pharma Col. 1998, 12, 249.
3. Zhao, P. L.; Li, J.; Yang, G. –F. Synthesis and Insecticidal Activity of Chromanone and
Chromone Analogues of Diacylhydrazines. Bioorg. and Med. Chem. 2007, 15, 1888.
4. Cottiglia, F.; Dhanapal, B.; Stcher, O.; Heilmann, J. Thermochemistry of chromone-
and coumarin-3-carboxylic acid. J. Nat. Prod. 2004, 67, 537.
5. Ranjan, Y. C.; Kanakam, C. C.; Selvam, S. P.; Murugesan, K. A study on the synthesis
and biological and optical properties of methylene-dinaphthyl bis-chromanones: the
utility of Baylis–Hillman adducts. Tetrahedron Lett. 2007, 48, 8562.
OHO
OH
OH
O
OH
OH
25
Toxifolin
Mandal, T. K.: Biological importance of 4H-1-benzopyran …….
187
6. Park, J. H.; Lee, S. U.; Kim, S. H.; Shin, S. Y.; Lee, J. Y.; Shin, C.-G.; Yoo, K. H.;
Lee, Y. S. Chromone and Chromanone Derivatives as Strand Transfer Inhibitors of
HIV-1 Integrase. Arch. Pharm. Res. 2008, 31, 1.
7. Lee, H.; Lee, K.; Jung, J.-K.; Cho, J.; Theodorakis, E. A. Synthesis and evaluation of
6-hydroxy-7-methoxy-4-chromanone and chroman-2-carboxamides as antioxidants.
Bioorg. Med. Chem. Lett. 2005, 15, 2745.
8. Gamal-Eldeen, A. M.; Lateff, A. A.; Okino, T. Modulation of carcinogen metabolizing
enzymes by chromanone A; a new chromone derivative from algicolous marine fungus
Penicillium sp. Environmental Toxicology and Pharmacology. 2009, 28, 317.
9. Prakash, O.; Kumar, R.; Prakash, V. Synthesis and Antimicrobial Activity of Some
New 2-(3-(4-Aryl)-1-phenyl-1H-pyrazol-4-yl) Chroman-4-ones. Eur. J. Med. Chem.
2008, 43, 435.
10. Ishar. M. P. S.; Singh, S.; Singh, S.; Sreenivasan, K. K.; Singh,G. Design, synthesis,
and evaluation of novel 6-chloro-/fluorochromone derivatives as potential
topoisomerase inhibitor anticancer agents. Bioorg. Med. Chem. Lett. 2006, 16, 1366
11. Tanaka, N.; Kashiwada, Y.; Nakano, T.; Shibata, H.; Higuch, T.; Sekiya, M.; Tikeshiro,
Y.; Takaishi. Y. Chromone and chromanone glucosides from Hypericum
sikokumontanum and their anti-Helicobacter pyloriactivitie. Phytochemistry. 2009, 70,
141.
12. Ibrahim, M. A.; Ali, T. E.; Alnamer Y. A.; Gabr, Y.A. Synthesis and chemical reactivity
of 2-methylchromones. ARKIVOC, 2010 (i), 98.
13. Rajanna, K. C.; Solomon, F.; Ali, M. M.; Saiprakash, P. K. Kinetics and Mechanism of
Vilsmeir-Haack Synthesis of 3-formylchromone………. Tetrahedron. 1996, 52, 3669.
14. Gao, H.; Kawabata, J. Importance of the B ring and its substitution on the alpha-
glucosidase inhibitory activity of baicalein, 5,6 ............. . Biosci. Biotechnol. Biochem.
2004, 68, 1858.
15. Wegener, T.; Fintelmann, V. Gastroprotective agents for the prevention ...……..
Flavonoids and bioactivity, Wein Med. Wochem. Schr. 1999, 149, 241.
16. Amic, D.; Davidovic-Amic, D.; Beslo, D.; Trinajstic, N. Structure radical scavenging
activity relationships of flavonoids. Croat Chem Act. 2003, 76, 55.
17. Farkas, O.; Jakus, J.; Heberger, K. Quantitative structure-antioxidant activity
relationship of flavonoid compounds. Molecules. 2004, 9, 1079.
18. Havsteen, B. Flavonoids, a class of natural products of high pharmacological potency.
Biochem. Pharmacol. 1983, 32, 1141.
Prajnan O Sadhona ……., Vol. 2, 2015
188
19. Vibhute Y. B.; Mokle, S. S.; Khansole, S. V.; Patil, R. B. Solvent Extraction of
Chromium (VI) From Mineral Aci.... International Journal of Pharma and Bioscience.
2010, VI (i).
20. Murty, Y. L. N.; Kasi Viswanath, I. V.; Pandit, E. N. P. Synthesis, Characterization &
Antibacterial Activityof 7, 4– Dihydroxy, 3-Methoxy Flavones. Inter. J. Chem. Tech.
Res. 2010, 2, 1097.
21. Gokara, M.; Sudhamala, B.; Amooru, D. G.; Subramanyam, R. Luminescence
quenching effect for the...…. PLoS ONE. 2010, 5, 137.
22. Zangade, S. B.; Jadav, J. D.; Vibute, Y. B. Vibute.; Dawane, B. S. Synthesis and
antimicrobial activity of some new chalcones and flavones containing ..... J. Chem.
Pharm. Res. 2010, 2, 310.
23. Tencate, J. W.; van Hoeringen, N.; Gerritsen, J.; Glasius, E. Biological activity of
semisynthetic flavonoid O-(-hydroxyethyl) rutosine: Light scattering and metabolic
studies of human red cells and platelets. Clin. Chem. 1973, 19, 31.
24. Selway, J. W. T.; Cody, V.; Middleton, E.; Harborne, J. B. (eds). Plant flavonoids in
biology and medicine: Biochemical, pharmacological and structure activity
relationships. New York: Alan R Liss, Inc; 1986. 521.
25. Huang, B.; Fong, W. P.; Yeung, H. W. Anti-human immunodeficiency virus (anti-HIV)
natural products with special emphasis on HIV transcriptase inhibitors. Life Sci. 1997,
61, 933.
26. Gerdin, B.; Srensso, E. Inhibitory effect of flavonoids on increased microvascular
permeabilityinduced by various agents in rat skin. Int. J. Micro Cir. Clin. Exp. 1983, 2,
39.
27. Shahid, F.; Yang, Z. Saleemi, Z. O. Natural flavonoids as stabilizers. J. Food Lipids
1998, 1, 69.
28. Farmica, J. V.; Regelson, W. Review of the biology of quercetin and related
bioflavonoids. Fd. Chem. Toxic. 1995, 33, 1061.
29. Alarcon, D. L.; Martin, M. J.; Locasa, C.; Motilva, V. Antiulcerogenic activity of
flavonoids and gastric protection. Ethnopharmacol. 1994, 42,161.
30. Izzo, A. A.; Carlo, G. D.; Mascolo, N.; Capasso, F.; Autore, G. Effect of quercetin on
gastrointestinal tract. Phyto. Ther. Res. 1994, 8, 179.
31. Carlo, G. D.; Autore, G. Izzo A. A. Inhibition of intestinal motility and secretion by
flavonoids in mice and rats; structure activity relationships. J. Pharm. Pharmcol. 1993,
45, 1045.
Mandal, T. K.: Biological importance of 4H-1-benzopyran …….
189
32. Vessal, M.; Hemmati, M.; Vasei, M. Antidiabetic effects of quercetin in streptozocin
induced diabetic rats. Comp. Biochem. Physiol. 2003, 135, 357.
33. Hif, C. S.; Howell, S. L. Effects of epicatechin on rat islets of langerhans. Diabetes
1984, 33, 291.
34. Hif, C. S.; Howell, S. L. Effects of flavonoids on insulin secretion and 45 Ca+2 handling
in rat islets of langerhans. J. Endocrinol. 1985, 107, 1.
35. Iijima, K.; Aviram, M. Flavonoids protect LDL from oxidation and attenuate
atherosclerosis. Curr. Opin. Lipidol. 2001, 12, 41.
36. Bernatova, I.; Pechanova, O.; Balal, P. Wine polyphenols improve cardiovascular
remodeling and vascular function in NO-deficient hypertension. Am. J. Physiol. Heart
Cir. Physiol. 2002, 282, 942.
37. Lou, F. Q.; Zhang, M. F.; Zhang, X. G.; Liu, J. M.; Yuan, W. L. A study on tea pigment
in prevention of antherosclerosis. Chin. Med. J. (Engl.) 1989, 102, 579.
38. Osman, H. E.; Maalej, N.; Shanmuganayagam, D.; Folts J. D. Flavonoids as
Nutraceuticals: A Review. Trop. J. Pharm. Res. 2008, 1099, 7.
39. Lackeman, G. M.; Claeys, M.; Rwanagabo, P.C.; A. V. Herman, A.V. Chronotropic
effect of quercetin on guinea pig right atrium. J. Planta. Med. 1986, 52, 433.
40. Huesken, B. C. P.; Dejong, J.; Beekman, B. Flavonoids as cardio protective agents.
Cancer Chemotherapy Pharmacol. 1995, 37, 55.
41. Bast, A.; Kaiserov, H.; den Hartog, G. J. M.; Haenen, G. R. M. M.; W. J. F. van der
Vijgh, w. J. F. The minor structural difference between the antioxidants quercetin and
... The flavonoid 7-mono-O-(β-hydroxyethyl)-rutoside……………… Flavonoids. Cell
Biol. Toxicol. 2007, 23, 39.
42. Kim, H. K.; Namgoong, S. Y.; Kim, H. P. Biological Actions of Flavonoids-I. Arch.
Pharmacol. Res. 1993, 16, 18.
Prajnan O Sadhona ……., Vol. 2, 2015
190
INSTRUCTION FOR SUBMISSION OF MANUSCRIPTS
‘Prajnan O Sadhona – A Science Annual’ is a multidisciplinary science journal and therefore
research and review papers of general significance that are written clearly and well organized will be
given preference. Submission of an article implies that it has not been previously published and is not
under consideration for publication elsewhere; and further, that if accepted, it will not be published
elsewhere. All papers, solicited and unsolicited, will be first assessed by our Editorial Board and the
decision of the Board members are final for publishing the submitted paper. Authors of these
papers will be notified of acceptance, need for revision or rejection of the paper. Papers found
unsuitable in terms of the overall requirements of the journal will be redirected to the authors for proper
revision as suggested by the reviewers/editors. If the paper is not properly revised following the
comments of the reviewer/editors, the Editorial Board has every right to reject it for publication.
It may be noted that the same papers once rejected cannot be resubmitted. Illustrations and other
materials to be reproduced from other publications must be properly credited; it is the authors’
responsibility to obtain permission for reproduction of figures, tables, etc. from published sources.
Declarations are to be made regarding ethical issues.
All manuscripts should be addressed to ‘The Editors, Prajnan O Sadhona’ and be submitted
electronically to the e-mail i.d. [email protected] .
Manuscripts of all categories are to be submitted with a letter of transmittal, giving (i) title of
the contribution and (ii) names and complete addresses for communication of all the authors with
telephone/mobile numbers, e-mail, fax etc. with the following format. No other format is acceptable
for review and publication in the journal.
MANUSCRIPT PREPARATION
1. Manuscripts should be typed in A4 paper size (21 cm × 29.7 cm, in portrait orientation) with
NORMAL margins (2.54 cm) in all sides only in Microsoft word file format.
2. Manuscripts should be written in ‘Times New Roman’ font with font size 12 and should
maintain 1.5 line spacing in the paragraph. No extra line spacing should be there. Spacing
before and after paragraph should be adjusted to ‘0’ point.
3. The pages should be numbered consecutively, starting with the title page and through the text,
reference list, tables and figure legends.
4. Each new paragraph should be started by inserting a default single ‘Tab’ (1.27 cm) spacing.
5. The manuscript should be ordered as follows: Title, Authors with affiliation, address and e.
mail i.d. for correspondence, abstract, key words, text with properly labeled figure, table,
diagram etc., acknowledgement and references.
The title should be brief, specific and amenable to indexing. Not more than five keywords
should be indicated separately; these should be chosen carefully and must not be phrases of
several words. Abstract and summary should be limited to 100 words and convey the main
points of the paper, outline the results and conclusions, and explain the significance of the
results.
Text: All papers should have a brief introduction. The text should be intelligible to readers in
different disciplines and technical terms should be defined. Tables and figures should be
referred to in numerical order. All symbols and abbreviations must be defined, and used only
when necessary. Superscripts, subscripts and ambiguous characters should be clearly indicated.
Units of measure should be metric or, preferably, SI.
Instruction for submission of manuscripts
191
Tables: All the tables must be incorporated in the main MS-WORD manuscript at proper places
in Portrait style. Table should be inserted from the ‘Insert Table’ menus and properly aligned
both horizontally and vertically. If the Table is extended in more than one page, proper ‘Page
Break’ and ‘Column Heading’ in the next page is desirable.
Figures: All Figures/Photographs should be pasted in the main MS-WORD manuscript file at
suitable places (in Portrait-style). Those documents should preferably be in black & white/gray
scale. Coloured documents will only be accepted if it is absolutely necessary for
proper/healthier documentation. All the figures/photographs should be sharp and clear with
proper heading/label. Chemical/biological structures drawn in “Chemdraw software” with
standard line size/thickness and text in ‘Times New Roman font’ with font size 12 is preferable.
Photomicrographs and other photographs that require must have a scale bar, which should be
defined clearly in the legend. Primary data should be submitted as far as possible (e.g. actual
photographs of electrophoretic gels rather than idealized diagrams).
References: References should be numbered in superscript without bracket, serially in the
order in which they appear, first through the text and then through table and figure legends.
References should not include unpublished source materials. The list of References at the end
of the text should be in the following format.
[Surname 1, first alphabet of Name 1.; Surname 2, first alphabet of Name 2. Title of the paper.
Journal in abbreviated form. Year, Volume, Page number.], e.g.,
1. Whittle, S.; Allen, N. B.; Lubman, D. I.; Yücel, M. The neurobiological basis of
temperament: towards a better under understanding of psychopathology. Neurosci.
Biobehav. Rev. 2005, 30, 511.
2. Rao, K. N.; Vaidyanadhan, R. Geomorphic features in Krishna Delta and its evolution. In
Proceedings of the National Symposium on Morphology and Evolution of Landforms,
Department of Geology, Delhi University, New Delhi, 1978.
6. Footnotes are not normally allowed except to identify the author for correspondence.
7. Clinical Study: When publishing clinical studies, the publisher aims to comply with the
recommendations of the International Committee of Medical Journal Editors (ICMJE) on trials
registration. Therefore, authors are requested to register the clinical trial presented in the
manuscript in a public trials registry and include the trial registration number at the end of the
abstract. Trials initiated after July 1, 2005, must be registered prospectively before patient
recruitment has begun. For trials initiated before July 1, 2005, the trial must be registered
before submission.
8. Ethical Guidelines: In any studies that involve experiments on human or animal subjects, the
following ethical guidelines must be observed. For any human experiments, all work must be
conducted in accordance with the Declaration of Helsinki (1964). Papers describing
experimental work on human subjects who carry a risk of harm must include a statement that
the experiment was conducted with the understanding and the consent of the human subject, as
well as a statement that the responsible Ethical Committee has approved the experiments. In
the case of any animal experiments, the authors should provide a full description of any
anesthetic and surgical procedure used, as well as evidence that all possible steps were taken to
avoid animal suffering at each stage of the experiment.