1

“DESIGNING ‘TURN-ON’ LUMINESCENT PROBES

FOR IN VIVO PHOSPHOLIPID SENSING”

A PROJECT REPORT SUBMITTED TO SASTRA UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE

AWARD OF THE DEGREE OF

BACHELOR OF TECHNOLOGY

IN

INDUSTRIAL BIOTECHNOLOGY

SUBMITTED BY

SRISHTI RAMAKANT GUPTA (114016055)

Under the guidance of

Dr. Ankona Datta

Department of Chemical Sciences

TATA Institute of Fundamental Research, Mumbai

APRIL 2014

School of Chemical & Biotechnology

SASTRA UNIVERSITY

(A University established under section 3 of the UGC Act, 1956)

Thanjavur-613402

2

DECLARATION

I hereby declare that this submission “Designing ‘TURN-ON’ Luminescent Probes, for in-vivo

Phospholipid Sensing” is a record of my project work carried out in Tata Institute of Fundamental

Research, Mumbai, under the supervision of Dr. Ankona Datta, Department of Chemical Sciences,

and is my own work and that, to the best of my knowledge and belief, it contains no materials

previously published or written by another person, except where acknowledgement has been made

in text.

Date: 24/03/2014 SRISHTI RAMAKANT GUPTA

SASTRA University (114016055)

3

Certificate from TIFR to be attached

4

ACKNOWLEDGEMENT

I would first want to sincerely thank Dr. Ankona Datta for her guidance, motivation and support

throughout the course of my project. Her continuous efforts and valuable advice made it possible

for me to complete this work on time. She has been an inspiring and also a very approachable

guide throughout the course of my work in TIFR.

I want to express my heartfelt gratitude to Ms. Shafali Gupta and Mr. Samsuzzoha Mondal for

efficiently training me with patience and encouragement through the course of this project.

I would also like to sincerely thank all my lab mates Ms. Anindita Sarkar, Ms. Ananya Rakshit, Ms.

Subha B, Ms. Sayani Das, Dr. Manish Dhyani for their valuable inputs, help, whole-hearted

cooperation and constructive criticism. Several people are responsible for making my stay at TIFR

memorable, most important amongst them are Dr. Jyotishman Dasgupta and all his lab members.

My sincere thanks to Dr. Shayamalava Mazumdar, Kansara Ji, Mr. Rahul Gera for their help and

inputs. I cannot miss thanking the NMR facility, MALDI facility and staff, Biochemistry facility and all

the DCS lab members of TIFR.

I owe TIFR Mumbai for giving me this opportunity to be able to learn and work in a highly

motivated and progressive environment.

Date: 24/03/2014 SRISHTI RAMAKANT GUPTA

SASTRA University (114016055)

5

INDEX

A. Introduction

A.1. Background……………………………………………………….6

A.2. Lanthanide Based ‘TURN-ON’ approach………………………7

A.3. Aim and Strategy………………………………………………..9

B. RESULTS AND DISCUSSIONS

B.1. Protein expression and purification…………………………………………...11

B.2.Protein Labeling…………………………………………………………………………..13

B.3. Synthesis of chromophores capable of Lanthanide sensitization

a. Attachment through cysteine residues……………………………………..14

b. Attachment through tryptophan & tyrosine residues………………..24

C. EXPERIMENTAL SECTION

C.1.Instrumentation and General Procedures……………………………………..26

C.2. Protein Expression & Purification………………………………………………….27

C.3. Thrombin cleavage………………………………………………………………………..27

C.4. Protein labeling……………………………………………………………………………..28

C.5. Synthesis of chromophores capable of Lanthanide sensitization……28

REFERENCES……………………………………………………………………………..30

6

A. INTRODUCTION

The basic structure of biological membranes is the lipid bilayer, the main components of which are

phospholipids. Phospholipids maintain the integrity of the cell and organelles by creating a semi-

impermeable barrier from their outside environment. Phospholipids are composed of a glycerol

moiety linked to fatty acids and a phosphate group to which polar head groups like choline, serine or

inositol are attached. Based on the head group attached, phospholipids are named as

phosphatidylcholine (PC), phosphatidylserine (PS) and Phosphatidylinositol respectively (Figure

A.1). The chemical nature of the headgroups governs the self-assembly of phospholipids in the

membrane.

Figure A.1: Basic structure of phospholipids.

Phospholipids show asymmetric distribution on the membrane and studies indicate that this

asymmetry may play a critical role in many important biological and cellular processes.3 The

alteration of phospholipid distribution (for example, the externalization of PS on the cell membrane)

can also play important roles in activating cellular or biological processes such as blood coagulation,

recognition and removal of apoptotic cells, cytokinesis, and cell fusion. Phospholipids are thus not

mere spectators forming the matrix of the membrane. Several cell signaling pathways are mediated

through membrane interacting peripheral proteins that have specific interactions with PLs.11

Therefore visualizing and quantifying phospholipids on the cell membrane can provide mechanistic

insights into cell signaling processes.

A common strategy for PL detection involves the use of protein-dye conjugates incorporating

fluorophores. However, a strong background signal from unbound sensors is a major shortcoming of

this current approach. The potential of small molecules like Zn2+

based complexes (bis-ZnDPA) to

recognize phosphorylated biomolecules, has also been used in the recent past.10

But this approach is

based on electrostatic interactions and shows lack of specificity to target. The issues of high

7

background and low specificity can only be addressed by developing sensors that ‘turn-on’

specifically only in the presence of a particular phospholipid. Several membrane binding peripheral

proteins like, protein kinase C, annexin V, and synaptotagmin, interact with anionic phospholipids

via a Ca-bridging mechanism, where Ca2+

ions in the phospholipid binding sites interact with the

phosphate head-groups of the PLs. Selectivity towards a specific phospholipid arises from the

headgroup interacting with the binding pocket amino acid residues. Using this PL−metal ion

interaction, the ‘Datta Group’ of Chemical Biology & Molecular Imaging, TIFR, has developed a

unique “turn-on” detection strategy for anionic PLs via lanthanide (Ln) reconstituted proteins or

“lanthano” proteins.

Peripheral Ca2+

binding 36 kDa protein, human annexin V, has been exploited for sensing an

essential anionic signaling PL, phosphatidylserine by the ‘turn on’ PL detection strategy developed.1

PS comprises 5-15% of the total phospholipids in mammalian cells. Although PS is a quantitatively

minor component, PS is widely distributed in cellular organelles, indicating its fundamental

structural role in biological membranes. Specific binding of PS is known to be involved in full

activation of protein kinase C, a key mediator of diverse signal transduction pathways. The

proteolytic activity of blood coagulation factor Xa and prothrombin is facilitated in the presence PS.

Coagulation factor Va specifically undergoes conformational change upon the binding of PS, with

consequent activation of the protein. It is thus important to develop imaging agents for

phosphatidylserine (PS).

Figure A.2: Jablonski diagram depicting Lanthanide sensitization by chromophore.

8

A.2. Lanthanide Based ‘TURN-ON’ approach

Crystal structures of the protein AnxV have identified five Ca2+

binding sites. PS binds selectively to

Ca2+

bound AnxV. Strategically placed amino acids with positive and negative side chains, interact

with the carboxyl and amine moieties of the serine head group. These interactions provide the

specificity for PS. Crystallographic and modeling studies propose that phosphate groups of PS

replace inner sphere water molecules on Ca2+

ions. These Ca2+

ions in the protein can be replaced by

lanthanide ions since lanthanides have similar co-ordination preferences and also similar size as Ca2+

ions. Lanthanides can be sensitized to luminesce via energy transfer from aromatic chromophores.

AnxV has ample aromatic amino acids for Ln sensitization. Lanthanide emission is quenched by

inner sphere coordinated water molecules. Thus replacement of the inner sphere water molecules in

AnxV by PS should cause the sensor to ‘turn-on’ (Figure A.3). A great advantage of Ln-based

systems is that the emission wavelengths can be anywhere from the visible to the near-infrared

depending on the Ln used.1

Figure A.3: ‘Turn-on’ sensingstrategy for PS detection throughlanthanoAnxV. Communication

To test this strategy Anx V was expressed and purified, followed by desalting to remove metal ions.

The purified protein was reconstituted with Ln3+

to prepare the PL sensor. The natural sensitizers in

proteins being UV absorbers, Tb3+

(visible emitters which can be effectively sensitized by UV-

absorbing chromophores) was selected to prepare “lanthano” Anx V. A single tryptophan and 12

tyrosines provided the aromatic residues in Anx V for Ln sensitization. Thus a simple, but specific

“turn-on” detection strategy for PS, by using TbIII-AnxV was successfully demonstrated by the

Datta group.1

9

A.3. Aim and Strategy

The excitation wavelength of the developed sensor was at 280 nm. This excitation at 280 nm is not

compatible for biological imaging as it falls in the ultra violet region. Spectrum of ultraviolet

radiation damages many molecules in the biological system. Ultraviolet photon has the power to

alter chemical bonds in molecules.

In order to perform in vivo/cellular imaging, the excitation wavelength has to be shifted from UV to

the visible region (> 400 nm). This can be done by incorporation of a chromophore into the protein

that absorbs at visible wavelength and is capable of sensitizing lanthanides. (Figure A.4) The

chromophore absorbs the excitation energy and energy transfer to the metal center takes place, due

to this a metal centered, sensitized luminescence occurs. This process is also termed as antenna

effect. Energy transfer from chromophore to metal center takes place from triplet excited state T1

after intersystem crossing. The following criteria should be met for the above mentioned process to

occur. (Figure A.2)

Energy difference between T1 of the chromophore and excited state of the Lanthanide should

not be too large with T1 being the higher in energy.

Too small an energy gap could result in back energy transfer from Ln excited state to T1.9

Based on these conditions suitable chromophores for Lanthanide sensitization had been selected.9

Figure A.4: Chromophore attached to protein to sensitize Lanthanide.

The specific aim of the project was two fold

a. To express and purify a mutant of protein AnxV which has surface accessible cysteine

residues.

b. Synthesize chromophores which are capable of lanthanide sensitization and absorb in the

visible wavelength.

10

The acetamides of these chromophores can be attached to the reactive thiol sites in cysteine residues

present on the surface of the mutant protein. Attachment of chromophores could also be done

through tryptophan and tyrosine residues present in the protein by the formation of its diazonium

salts. (Figure A.5)

This would further be used to apply the developed ‘turn-on’ optical sensing strategy for anionic

phospholipids for in-cellulo measurments.

Figure A.5: Techniques to shift excitation wavelength from UV to Visible region

Biconjugation techniques

Conjugation of sensitizer through tyr and trp.

Conjugation of sensitizer

through cysteine.

11

B. RESULTS AND DISCUSSIONS

B.1. Protein expression and purification:

The first step was to express and purify mutants of human AnxV which have surface accessible

cysteine residues for attachment of the chromophores. Sites in human AnxV which do not interact

with PS or Ca, have hydrophobicity comparable to that of cysteine, and are also surface accessible

were identified.7 This was done by calculating the required distances from the crystal structures

using PyMOL, surface accessibilities were studied using ExPASy. These sites should also be in

appropriate distance from the Ca binding pockets so that efficient energy transfer can take place

from the sensitizer. These sites were identified to be Leu65 and Ala55. Plasmid pET28a-

hAnxVA55C, obtained from genscript had previously been used to transform BL21a E.coli cells.

Mutant proteins L65C-hAnxV and A55C-hAnx5 were expressed and then purified.

Figure B.1: SDS-PAGE gel depicting protein ladder in 1st lane from left and His-tag purified Human Anx V (36kDa)

in other lanes.

Figure B.2: SDS-PAGE gel depicting final purified protein obtained after gel filtration along with protein ladder in 1st lane.

12

Figure B.3: MALDI mass spectrum of His-tag purified protein indicating peaks at m/z 38082 (+1), 19033 (+2).

Disulfide bonds (R–S–S–R) in proteins are formed between the thiol groups of cysteine residues

by oxidation of sulfhydryl (–SH) groups. The other sulfur-containing amino acid, methionine, cannot

form disulfide bonds. Disulfide bonds play an important role in the folding and stability of some

proteins. Since most cellular compartments are reducing environments disulfide bonds are unstable

in the cytosol.

Transformed BL21aE.coli cells were used and the expression of protein was carried out by IPTG and

L-Arabinose induction. Protein was purified on a Ni-NTA column followed by Gel Filtration. The

expression and purification of A55C-hAnxV which have surface accessible cysteine residues was

successfully accomplished (experimental section C.2).

Figure B.4: SDS- PAGE gel depicting surface accessibility of cysteine residues in A55C hAnxV.

Lane 1: A55C-hAnxV with 10equivalence of βME as reducing agent, prominently depicting protein in its dimeric form. Lane 2: A55C-hAnxV with 50equivalence of βME as reducing agent, prominently depicting protein in its monomeric form.

13

B.2.Protein Labeling

Conjugation of acetamides of chromophores capable of lanthanide sensitization to surface accessible

cysteine residues in A55C-hAnxV and L65C-hAnxV was attempted. The protocol followed for

protein labeling is mentioned in the Experimental section (C.4 ).6

Figure B.5 Conjugation of chromophores to protein through Cysteine residue

Several attempts towards labeling the mutants A55C-hAnxV and L55C-hAnxV protein and

optimizing the labeling protocol were made.

100 equivalence of the dye was initially used with 10 equivalence of β mercaptoethanol (βME) as

the reducing agent. Later, the amount of reducing agent used was substantially increased (10

equivalence, 50 equivalence, 100 equivalence) while following labeling protocol.6

The labeled protein was not being detected in Matrix-assisted laser desorption/ionization (MALDI),

a soft ionization technique used in mass spectrometry, following which Bradford assay was

performed to detect the concentrations of protein present. This concentration was found to be very

low (0.4 µM). Loss of protein could have taken place during the process of gel filtration or dialysis

which was done to remove the unreacted dye. The labeled protein was then concentrated 10 times by

evaporation under vacuum after which its characterization by MALDI mass spectroscopy was

reattempted. In next attempt protein samples were concentrated by way of centrifugal ultrafiltration

using Amicon Ultracel 3000 Da molecular weight cutoff centrifugal filter units. The protein was

transferred to a new HEPES (20 mM HEPES, 100 mM NaCl) buffer from TRIS (TRIS 20 mM, 100

mM) buffer. The labeling protocol was followed again using (tris(2-carboxyethyl)phosphine)

(TCEP) as the reducing agent.

The reaction time for the protein reduction was also varied from 30 min to 12h. The ionization of

molecules and their desorption in MALDI MS could be affected by presence of contaminants like

salts present in the buffer or DMSO used with the dye. The DMSO present was evaporated under

high pressure, excess salts removed and characterization was reattempted. MALDI-MS was also

performed for 2 controls, one containing protein reacted with reducing agent (concentrations similar

to the labeling protocol) and other, protein with dye to be labeled (in DMSO, concentrations similar

to the labeling protocol). The labeled protein was not detected on characterization by MALDI mass

spectroscopy in all of the above cases.

14

B.3. Synthesis of chromophores capable of Lanthanide sensitization

a) Attachable through cysteine residue

Several chromophores capable of sensitizing lanthanides were synthesized in form of their

acetamides which would be used for incorporation into the protein by conjugation of the sensitizer

through surface accessible cysteine residues in A55C-hAnxV and L65C-hAnxV. The following

schemeS1 was used to synthesize 7-aminoacetamido-4-(trifluoromethyl) coumarin (compound 9).

Scheme 1 (S1)

7-amino-4-(trifluoromethyl) coumarin(compound 6) was acylated with bromoacetyl bromide. The

reaction was carried out in tetrahydrofuran using an external ice bath at 0ºC. This reaction was

quenched by addition of ice cold water which resulted in formation of bromoacetic acid form

bromoacetyl bromide. The resulting intermediate bromoacetamide was formed (compound 8) and

was characterized using Electrospray ionization (ESI) mass spectroscopy.8 The peak for the product

was observed at m/z =348,350 in the negative mode (Figure B.6).

Figure B.6: ESI-MS of Compound 8.

Finkelstein reaction was then carried out which involved the exchange of one halogen atom from

another by treatment with a solution of sodium iodide in acetone. Halide exchange is an equilibrium

reaction, but the reaction is driven to completion by exploiting of differential solubility of halide

salts. Sodium iodide is soluble in acetone while sodium bromide is not. The reaction is driven

towards products by mass action due to the precipitation of the insoluble salt. Bromoacetamide was

thus converted into corresponding iodide and this reaction was carried out in Argon atmosphere. The

resulting compound, coumrin iodoacetamide (compound 9) thus obtained was characterized using

Electrospray ionization (ESI) mass spectroscopy. The peak for the product was observed at m/z

=396 in the negative mode (Figure B.7). Characterization was also performed by 1H NMR of the

resulting compound 9 (Figure B.8)

15

Figure B.7: ESI-MS of Compound 9.

Figure B.8: 1H NMR of Compound 9.

Synthesis of N-((1,10-phenanthrolin-4-yl)methyl)-2-iodoacetamide (Compound 5) was attempted

following the scheme (S2) mentioned below.

Scheme 2 (S2)

16

Compound 2 was obtained through SeO2 mediated benzylic oxidation of 4-methyl-1,10

phenanthroline (Compound 1) and the reaction was performed under Argon atmosphere. The

oxidation using SeO2 follows a mechanism where the first step is a cycloaddition reaction. The

allylic seleninic acid produced undergoes a [2,3]-sigmatropic rearrangement to reinstate the double

bond position. Rapid decomposition of the selenium (II) intermediate leads to an allylic alcohol.

Oxidation continues to give the α,β-unsaturated carbonyl product (Compound 2). Presence of

Compound 2 was characterized using MALDI mass spectrometry. The expected peak was found at

m/z = 209 (M+H) (Figure B.9). Characterisation was also performed by 1H NMR (Figure B.10).

Compound 3 was then synthesised by performimg solid state reaction using Fmoc protected Rink

amide resin. Fmoc deprotection of resin was done using Piperidine after which coupling was

performed by addition of Compound 2 with 1:1 DMF:MeOH, AcOH and Na(CN)BH3 to the

deprotected resin. The reaction was performed for 18 h after which the resulting Compound 3 was

cleaved from the resin using trifluoroacetic acid (TFA), triisopropylsilane (TIS) and H2O. The

solvent was removed under a stream of N2, and the product obtained. The advantages of performing

solid state reaction were that only the compound of interest reacts with N-terminus in the resin.

Impurities can be easily removed by wash with DMF and MeOH and the resulting compound has

high purity.

Figure B.9: MALDI MS of Compound 2

17

Figure B.10: 1H NMR of Compound 2

Synthesis of 2-iodo-N-((5-oxo-5H-thiochromeni[2,3-b]pyridine-2-yl)methyl)acetamide (Compound

h) was attempted following the Scheme (S3) mentioned below.

Scheme 3 (S3)

18

Reaction of thiophenol (Compound a) with 2-chloro-6-methylnicotinic acid (Compound b) gave the

thioether (Compound c)2. This product was characterized with MALDI mass spectroscopy showing

peak at m/z = 246 (M+H) (Figure B.14). ESI Mass Spectroscopy was also used for characterization

giving a peak at m/z= 246 in the positive mode and 244 in the negative mode (Figure B.15).

Confirmation was done by performing 1H NMR (Figure B.16). Compound c then underwent

electrophilic cyclization in the presence of poly-(phosphoric acid) to yield the azathioxanthone

(Compound d)2.This compound was characterized by MALDI mass spectroscopy showing peak at

m/z = 228 (M+H), 250 (M+K) (Figure B.17). ESI Mass Spectroscopy was also used for

characterization giving peak at m/z = 228 in the positive mode and at 227 in the negative mode

(Figure B.18). Further confirmation was done by performing 1H NMR (Figure B.19).

Compound e was obtained through SeO2 mediated benzylic oxidation of Compound d and the

reaction was performed in Argon atmosphere. The oxidation using SeO2 follows a mechanism where

the first step is a cycloaddition reaction. The allylicseleninic acid produced undergoes a [2,3]-

sigmatropic rearrangement to reinstate the double bond position. Rapid decomposition of the

selenium (II) intermediate leads to an allylic alcohol. Oxidation gave the α,β-unsaturated carbonyl

product (Compound e). This step was repeated several times, monitored by performing thin layer

chromatography at regular intervals and then optimized. Conversion to Compound e occurred after

heating for 18h. Presence of Compound e was characterized using MALDI mass spectroscopy, the

expected peak was found at m/z = 242 (M+H) (Figure B.20). ESI Mass Spectroscopy was also used

for characterization and peak observed at m/z = 242 (M+H) (Figure B.21). Characterization was also

performed by 1H NMR where the signature peak of the aldehyde moiety was observed. (Figure

B.22). Compound e was found to be light sensitive and degraded over time.

Figure B.11: MALDI MS of Compound c

19

Figure B.12: ESI-MS of Compound c

Figure B.16: 1H NMR of compound c

20

Figure B.17: MALDI MS of compound d

Figure B.18: ESI-MS of compound d.

21

Figure B.19: 1H NMR of compound d.

Figure B.20: MALDI-MS of compound e.

22

Figure B.21: ESI-MS of compound e.

Figure B.22: 1H NMR of compound e

b) Attachable through tryptophan & tyrosine residues

As mentioned earlier chromophores capable of sensitizing lanthanides can be incorporated into the

protein by conjugation of the sensitizer through tryptophan and tyrosine residues present in A55C-

hAnxV and L65C-hAnxV. Control experiments have been performed to check the reactivity of three

dyes by this mechanism. (Compound 10, Compound 13, Compound 15) This was done by reacting

these dyes with free tryptophan by the formation of their diazonium salts through diazotization

reaction.

23

Scheme 4 (S4)

Protocol as that mentioned in the experimental section was followed (C.5)

The nitrosation of aromatic amines with nitrous acid lead to formation of diazonium salts5. In

aqueous solution diazonium salts are unstable at temperatures above +5 °C; the -N+≡N group tends

to be lost as N2 (nitrogen gas). Diazonium compound was not isolated once prepared, and used

immediately for further reaction. Azo coupling was performed where the diazonium compound was

attacked by electron-rich substrate (free tryptophan, Compound 11). 4,5

An enhancement in the fluorescence was observed in the reaction performed with 7-Amino-

4(trifluoromethyl) coumarin (Compound 13). 6-amino-fluorescein (Compound 15) was found to

fluoresce only after its conjugation with Tryptophan residues. To test that the fluorescence observed

in this case was due to formation of compound 16 and not due to free fluorescein which could be

formed on detachment of the amine group in compound 15, Liquid chromatography–mass

spectrometry (LCMS) was performed. No presence of free fluorescein was observed (Figure B.24).

To further confirm this result a thin layer chromatography was performed with compound 15,

compound 16 and free fluorescein. (Figure B.25)

24

Figure B.24: LCMS data showing presence of Compound 16 and absence of free fluorescein, thereby confirming

the observation.

Figure B.25: Thin Layer Chromatography. (5% MeOH in CH2Cl2).

Lane 1: Fluorescein

Lane 2: 6 amino fluorescein (Compound 15)

Lane 3: Reaction product (Compound 16)

25

C. EXPERIMENTAL

C.1.Instrumentation and General Procedures

Protein Purification. All cell cultures were incubated at 37°C in MaxQ* 8000 Incubated Stackable

Shakers (Thermo Scientific, Inc.). Cell lysis was performed by sonication in Sonics, Vibra Cell TM.

Protein purifications wereperformed on Ni SepharoseTM 6 Fast Flow BioProcess medium (GE

Healthcare Life Sciences). His-Tag removal viathrombin cleavage was achieved using Thrombin

Clean Cleave TM Kit from Sigma Aldrich®. General desalting andremoval of cleaved His-tag were

performed using Sephadex® G-75 gel filtration media (Sigma Aldrich®). Proteinsamples were

concentrated by way of centrifugal ultrafiltration using Amicon® Ultracel® 3000 Da molecular

weight cut off centrifugal filter units (Millipore, USA). Centrifugations were performed using the

following instruments: 1) AvantiTM J-20XP centrifuge (Beckman Coulter, Inc., USA); 2) Eppendorf

Centrifuge 5810R (Eppendorf AG, Germany); or 3) Sorvall Legend Micro 17R centrifuge (Thermo

Fisher Scientific, Inc.).

Gel Analyses. For protein analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis

(SDSPAGE) was run on a Mini-PROTEAN® gel running apparatus (Bio-Rad laboratories Inc.,

USA). Page Ruler Pluspre-stained protein ladder (Thermo Fisher Scientific, Inc.) was applied in at

least one lane of each gel to determinethe apparent molecular weight of the proteins. Protein bands

were visualized using Brilliant Blue R dye (Sigma Aldrich®).

UV-Visible spectrometric measurements for protein and phospholipid concentrations were

performed on SPECORD® 205 (Analytik Jena AG, Germany). Protein concentrations were also

determined on Nano Drop 1000spectrophotometer (Thermo Fisher Scientific, Inc.).

Mass Spectrometry Protein samples were analyzed on an Agilent 1200 SL RapidResolution LC

coupled to an ESI Agilent 6520 Q-TOF. Samples were desalted with ZipTip C18 Columns (Pierce,

Thermo Scientific Inc.) before injecting into the mass spectrometer. The samples were loaded onto

an Agilent nano CHIP C18 reverse phase column (150 mm, 300Å, C-18 chip w/40 nL trap column)

connected to a nano HPLC system.

Matrix Assisted Laser Desorption-Ionization Time of Flight (MALDI-TOF) mass spectra were

obtained ona BrukerUltrafleXtremeTM (BrukerDaltonics). Samples in buffer or water (0.5µL) were

mixed with a solution of3,5-Dimethoxy-4-hydroxycinnamic acid (1:2 MeCN: ddH2O with 0.1%

trifluoroacetic acid, 0.5µL) and applied onthe MALDI plate. Samples were allowed to dry

completely before analysis.

26

C.2. Protein Expression & Purification

Mutant A55C Human Annexin V (A55C-hAnx5) cDNA was obtained in vector that encoded a His-

Tag unit with a thrombin cleavage site which had been used to transform BL21a-E.coli cells.

LB medium (50mL) containing 30 µg/mL Kanamycin was incubated with the transformed colonies

at 37 °C overnight. The incubation was stopped once OD of 0.2 was reached. A secondary culture

was propagated in the LB Medium (1 L) containing 30 µg/mL of Kanamycin. This was done by

inoculating with the primary culture, followed by incubation at 37 °C until an OD of 0.6 was

reached. Protein expression was induced by addition of L-Arabinose (final concentration 0.2% w/v)

and isopropyl-1-thio-β-D-galactopyranoside (final concentration 1 mM) to the medium. This culture

was incubated at 37 °C for 4h. The bacterial culture obtained was pelleted down by centrifugation

(1.5 h at 7000 rpm, 4 °C). The supernatant was discarded and the pellet was resuspended into a

buffer containing 20 mM HEPES, 10 mM Imidazole, and 500 mM NaCl, at pH 7.4 (buffer a). This

was followed by sonication at 4 °C for 1.5 h at 40 % amplitude with 30 s of ‘ON’ and 20 s of ‘OFF’

cycles. The mixture was then centrifuged at 4000 rpm for 1 h at 4 °C and the supernatant obtained

was suspended in Ni Sepharose beads which were pre equilibrated with buffer a for 12 h at 4 °C.

The mixture was packed into a column and washed with a buffer containing 20 mM HEPES, 20 mM

Imidazole, and 500 mM NaCl at pH 7.4 to remove undesired protein impurities. The protein of

interest was then eluted using a buffer containing high concentration of Imidazole (500 mM). The

protein fractions were collected based on SDS-PAGE electrophoresis confirmation and taken

forward for His-Tag cleavage (Figure B.1). Thrombin cleavage was confirmed by SDS-PAGE. The

cleaved protein was purified and desalted by gel filtration using G-75 gel filtration media in a buffer

consisting of 20 mM TRIS, and 100 mM NaCl, at pH 6.8. Pure protein fractions were pooled based

on SDS-PAGE confirmation (Figure B.2). The protein was characterized by MALDI mass

spectrometry (Figure B.3). Protein concentrations were estimated from absorbance values at 280 nm

using NanoDrop 1000 spectrophotometer. A65C-hAnxV mutant with maximum concentration of

10µM was obtained.

C.3. Thrombin cleavage

Components:

Thrombin-Agarose: 50% suspension in 50% glycerol, 20mM Tris-HCl, pH 8.2

10x Cleavage Buffer: 500mM Tris-HCl, pH 8.0, 100mM CaCl2

The thrombin-agarose resin was thoroughly resuspended and made into homogeneous slurry. 100 µl

aliquot of a 50% (v/v) suspension of resin was removed and gently spun in a microcentrifuge at

2500rpm to pellet the resin. The supernatant was removed, 500 µl of 1x Cleavage Buffer was added

and gently resuspended. The system was centrifuged at 2500rpm and supernatant removed. The

above mentioned step was repeated again.

27

100 µl of 10x Cleavage Buffer was added to the centrifuged beads and gently resuspended. 1 mg of

the his-tag purified A55C-hAnxV was added and final volume brought to 1ml with water. The

cleavage reaction was incubated at 4ºC with gentle agitation to keep beads suspended. The aliquot

was removed after 16 h, gently centrifuged to remove resin and supernatant was analyzed for

cleavage.

C.4. Protein labeling

Reduction of disulfide bonds in the surface accessible cysteine residues of A55C-hAnx5 and L65C-

hAnx5 was performed by addition of 10 fold molar excess of freshly prepared solution of TCEP or β

Mercapto Ethanol to the mutant protein solution. The mixture was then allowed to stir for 30 min

following which a 10-fold molar excess of thiol reactive Compound 9 in DMSO was slowly added to

the protein solution while constantly stirring. Reactions were allowed to occur for 4h at RT in the

dark. Removal of the unreacted dye from the reaction mixtures was performed by passage through a

Sephadex G-25 column followed by dialysis.

C.5. Synthesis of chromophores capable of Lanthanide sensitization

SCHEME 1, (S1)

Synthesis of compound 8:

7 amino-4-(trifluoromethyl)coumarin (100 mg, 0.44 mmol) was added to ice cold solution of

bromoacetyl bromide (100 mg, 0.5 mmol) in THF. After 30 min at room temperature 3 ml ice cold

water was added to the reaction mixture.The product was then seperated, washed with water, dried

and recrystallised from Ethyl acetate to obtain crude 2-bromo-N-(2-oxo-(trifluoromethyl)-2H-

chromen-7-yl)acetamide (compound 8).

ESI-MS-: m/z 348, 350

Synthesis of compound 9:

2-bromo-N-(2-oxo-(trifluoromethyl)-2H-chromen-7-yl)acetamide (compound 8) was taken to which

NaI(90 mg, 0.60 mmol) and a few drops of acetone was added. The reaction mixture was refluxed

under Argon environment for 2 h. The mixture was cooled to room temperature, filtered and dried to

obtain bright yellow coloured 2-iodo-N-(2-oxo-4-(trifluoromethyl)-2H-chromen-7-yl) (compound 9).

ESI-MS-: m/z 396 (M-H)

1H NMR: (DMSO-D6, 500 MHz): δ 10.82 (1H, s, Hc); 7.86 (1H, s, Hd); 7.51 (1H, d, J=5.35 Hz, Ha);

6.92 (1H, s, He); 6.65 (1H, d, J= 5.6 Hz, Hb); 3.87(2H, s, Hf)

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SCHEME 2, S2

Synthesis of compound 2:

A mixture of 4-methyl-1,10-phenanthroline (compound1) (194.23 mg, 1 mmol) and Selenium

dioxide (443.84 mg, 4 mmol) in dioxane containing 4% water (20 ml) was heated under reflux for 60

min. The mixture was then filtered through celite while hot. A solid was found to be separated in

cold filterate and recrystallised from dioxane containing 4% water to give the crude product 1,10-

phenanthroline-4-carbaldehyde (compound 2).

MALDI MS: 209 (M+H)1H NMR: (DMSO-D6, 500 MHz): δ 10.71 (1H, s, Ha); 9.44 (1H,d,J=7.3 Hz,

Hb); 9.18(1H, d, J=5.1 Hz, Hf ); 9.00(1H, d, J=9.2 Hz, Hg); 8.69(1H, d, J=9.15 Hz, Hd); 8.58(1H, d,

J=7.7 Hz, Hc); 8.23-8.44(1H, m, He); 8.17(1H,d, J=8.7 Hz, Hh).

Synthesis of compound 3:

Compound 3 was synthesized using Fmoc protected Rink amide resin HL (100-200 mesh, 0.74

mmol/g resin). The resin was allowed to swell in DMF for 45 minutes. Fmoc deprotection of resin

was done using a solution of 20% Piperidine in DMF. After deprotection, coupling was performed

by taking 20 mg of the deprotected resin and addition of (15.29 mg, 5 equivalence) Compound 2, 1:1

DMF:MeOH (1 ml), AcOH (40 µL) and Na(CN)BH3 (40 mg, 0.637 mmol). These were placed in a

15 mL capped plastic tube and allowed to react for 18 h by placing this tube on a rotor with a speed

of 16 rpm. After 18 h, the solution was allowed to stand for some time and the supernatant solution

was decanted, leaving the resin in the plastic tube. The resin was washed twice, for 20 min each,

with 5 mL of DMF and once with 5 ml of MeOH for 20 min, by placing the tube on a rotor with a

speed of 16 rpm, and dried under reduced pressure. 20 mg of the resin was allowed to react

with trifluoroacetic acid (TFA): triisopropylsilane (TIS):H2O (700 µl: 20 µL: 20 µL) for 4 h, and the

resin was separated by filtration. The solvent was removed under a stream of N2, and the product

(Compound 3) was precipitated in cold Methyl tert-butyl ether, washed twice and dried under

reduced pressure to afford crude product

SCHEME 3, S3

Synthesis of compound c:

2-Chloro-6-methylnicotinic acid (compound a) (500 mg, 2.92 mmol) and thiophenol (compound b)

(380 mg, 3.45 mmol) were both taken and dissolved in DMF (3 ml) whilestirring continuously. This

was followed by the addition of copper(I) bromide (25 mg, 1.75 mmol) and K2CO3(600 mg, 4.35

mmol). The mixture was then heated for 30 min at 130 °C followed by heating for 18 h at 150 °C.

This resulted in the generation of a light yellow solution. The mixture was then cooled and treated

with water (170 ml) to give a yellow suspension, which was washed with ether (3 x 20 ml). The

aqueous solution was then acidified by the addition of acetic acid. This yielded a light yellow

precipitate on cooling. This precipitate was then filtered, washed with water, and then dried

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thoroughly to yield crude6-methyl-2-(phenylthio)nicotinic acid (compound c) as a pale yellow,

crystalline solid.

MALDI MS: m/z 246 (M+H).

ESMS+: m/z 246 (M+H), ESMS-: m/z 244(M-H).

1H NMR: (CDCl3, 500 MHz) δ 8.26(1H,d,J=7.95 Hz,Ha); 7.29-7.59(2H,m,Hd); 7.41-7.47(2H,m,He);

7.41-7.45(1H,m,Hf); 6.96(1H,d,J=7.95 Hz,Hb); 2.36(3H,s,Hc).

Synthesis of compound d:

Polyphosphoric acid (60 cm3) was added to 6-methyl-2-thiophenoxynicotinic acid. This mixture was

heated at 120 °C for 4 h under argon with stirring. The resulting brown liquid was cooled to room

temperature and then it was slowlypoured into cold concentrated aqueous sodium hydroxide solution

with stirringvigorously. The light yellow precipitate that was formed was collected through filtration.

The product was recrystallized from warm EtOH. The crystals that formed upon standing were

filtered and dried thoroughly to yield 2-methyl-5H-thiochromeno[2,3-b]pyridine-5-one (compound

d) as a pale yellow crystalline solid.

MALDI MS: m/z 228 (M+H), 250(M+K).ESMS+: m/z 228, ESMS-: m/z 226.1H NMR:

(CDCl3,500MHz) δ 8.74(1H,d,J=8Hz,Hc); 8.61(1H,d,J=8Hz,Hg); 7.67-7.72(1H,m,He); 7.63-

7.67(1H,m,Hd); 7.50-7.56(1H,m,Hf); 7.31(1H,d,J-8Hz,Hb); 2.72(3H,s,Ha).

Synthesis of compound e:

A mixture of 4-methyl-1,10-phenanthroline (compound d) and Selenium dioxide (443.84 mg, 10

mmol) in dioxane containing 4% water (20 ml) was heated under reflux for 18 h. The mixture was

then cooled to room temperature and filtered to obtain 5-oxo-5H-thiochromeno[2,3-b]pyridine-2-

carbaldehyde] (compound e) as a pale yellow solid.

MALDI MS: m/z 242(M+1).

ESMS-: m/z 241(M-H)

SCHEME 4, S4

Diazotisation reaction:

50 μL of an aqueous solution of p-TsOH (800 mM, 4 equivalence) was added to 100 μL of a 100

mM solution of compound 10/13/15in DMF in an Eppendorf tube. The resulting solution was

mixedby vortexing it and then cooled to 0 °C in an external ice bath. Thereafter5 x 10 μL aliquots of

an aqueous solution of NaNO2 (400 mM, 2 equivalence) was added to the cooled solutionat 0°C. The

solutionwas mixed thoroughly after every addition and incubated at 0 °C for 1 min. After the

addition of the last aliquot of NaNO2, the diazotizationreaction was allowed to proceed for 15 min

on addition of compound 11 to obtain compounds 12, 14, 16.

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