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School of Chemistry, Madurai Kamaraj U

India. E-mail: dcmku123@gmail.com

† Electronic supplementary informationdata, UV-vis data and computational deta

Cite this: RSC Adv., 2014, 4, 25393

Received 29th March 2014Accepted 28th April 2014

DOI: 10.1039/c4ra02778g

www.rsc.org/advances

This journal is © The Royal Society of C

Vitamin B6 cofactor based fluorescent probe forsensing an anion (F�) and cation (Co2+)independently in a pure aqueous medium†

Murugan Iniya, Dharmaraj Jeyanthi, Karuppiah Krishnaveni and Duraisamy Chellappa*

A new highly selective and sensitive bifunctional fluorescent probe for Co2+ and F� ions has been derived

from a vitamin B6 cofactor and the response mechanism has been analyzed using DFT calculations. The

probe features a facile synthetic protocol, good water solubility, high selectivity and sensitivity, a

fluorescence turn-on response to F�, and a ratiometric response towards Co2+ in an aqueous medium.

Introduction

Fluorescent chemosensors can serve as effective tools inmolecular sensing as is evident from their prominent role inmedicinal diagnostics, biological labeling and optoelectronicmaterials.1 In particular, single molecular sensors with variedresponses towards different analytes are cost effective andconvenient for real applications. Among metal ions, sensing ofcobalt has received increasing attention, since Co2+ is acomponent of vitamin B12, a vitamin essential for DNAsynthesis, the formation of red blood cells, maintenance of thenervous system, and the growth and development of children.2

Fluoride is also an essential nutrient for the normal develop-ment and growth of a human being.3 Excess accumulation ofcobalt in the body can result in cardiomyopathy, hypothy-roidism, and neurological damage, while a high level of uoridecan cause dental and skeletal uorosis.4

A number of uorescent sensors have already been estab-lished for sensing individually either cobalt5 or uoride.6 Butreports about a single probe that senses uoride and cobalt(II)ions independently are rare. Recently, chromogenic recognitionof Co2+ or uoride ions has been achieved in a DMSO–CH3CNsystem using a calixarene based ditopic receptor by H. M.Chawla et al.7 However, it suffers from an interference with Ag+

and Cu2+ while uoride ions bind to the receptor. Furthermore,most of the reported sensors oen feature a tedious syntheticprotocol, non-aqueous media requirement, lack of dualresponse and cross sensitivities towards other ions. All theselimitations restrict their potential use in environmental andbiological applications.

Amongst different organic scaffolds, the scaffold that facili-tates Excited State Intramolecular Proton Transfer (ESIPT) as a

niversity, Madurai-625021, Tamilnadu,

(ESI) available: NMR and MS spectralils. See DOI: 10.1039/c4ra02778g

hemistry 2014

sensing mechanism is a perfect candidate as a uorescentprobe because of its signicant photostability, large stoke shiand intense luminescence.8 Pyridoxal phosphate (PLP), theactive form of vitamin B6, functions as a coenzyme in numerousenzyme-catalyzed reactions, such as transamination, a- and b-decarboxylations, b- and g-eliminations, racemizations, andaldol reactions.9 Although PLP is well known for its coordina-tion and optical properties, very few reports have shown thedesign of a uorescent chemosensor utilizing a PLP platform.10

The uorescence detection of ions using small-moleculesensors in an aqueous environment is still a difficult task. Onlya small number of uorescent probes that have been usedsuccessfully for this purpose have appeared in the literature.11

In continuation of our ongoing research to develop uorescentchemosensors,12 herein, we have designed a pyridoxal linkedaminoethylaminoethanol (PYET) receptor for the selectiverecognition of Co2+ and F� in an aqueous solution via enhanceduorescence emission. Interestingly, compared to various otheruorescent chemosensors, probe PYET exhibits good watersolubility, optical sensitivity in a buffer medium, dual emission,a low detection limit and visible strong uorescence under UVlight when adding guest species. To the best of our knowledge,probe PYET is the rst bifunctional chromogenic and uoro-genic chemosensor that enables independent sensing of uo-ride and cobalt(II) ions in an aqueous medium.

Results and discussion

Chemosensor PYET was synthesized from Vitamin B6 cofactorpyridoxal phosphate with 2-(2-aminoethylamino)-ethanol in amethanol solution (Fig. S1–S6†). Aminoethylethanolamine hasbeen selected as a side chain because of its hydrophilic char-acter, while pyridoxal phosphate (PLP) acts as a signallingmoiety, due to its low uorescence quantum yield and goodbinding sites (Scheme 1). Furthermore, this type of structuralarrangement can undergo keto–enol tautomerism through anexcited state intramolecular proton transfer (ESIPT)

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Fig. 1 Absorption spectra of PYET (5 mM) upon gradual addition ofCo2+ in pH 7.4 HEPES buffered water.

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mechanism. The probe PYET was characterized by NMR andother spectroscopic methods.

The photonic properties of PYET were investigated by UV-visand uorescence measurements with different metal ions, i.e.Na+, Mg2+, K+, Ca2+, Mn2+, Ni2+, Fe3+, Cu2+, Ag+, Cd2+, Cr3+, Zn2+,Fe2+, Hg2+, Pb2+, Al3+, and anions, such as Cl�, Br�, I�, OAc�,NO3

�, HSO4�, H3PO4

� and CN�, in an aqueous solution in thepresence of HEPES buffer at pH 7.4. The probe showed lmax at328 nm and 387 nm in its absorption spectrum. Systematictitration of sensor PYET with increasing concentrations of Co2+

revealed a new absorption band at 369 nm with the disap-pearance of the bands at 328 and 387 nm (Fig. 1).

Upon addition of F� (Fig. 2), the intensity of the absorptionband centered at 328 nm decreased while that of the band at387 nm increased. The addition of other biologically importantmetal ions and anions led to no obvious change in the groundstate behaviour of probe PYET (Fig. S7 and S8†). When excited at350 nm in the same solvent system, PYET exhibits uorescenceemission bands at 445 nm and 510 nm with a quantum yield of0.010 at room temperature. The presence of dual emissionindicates the possibility of the ESIPT mechanism. It alsoenables a new way for the ratiometric analysis in which the ratiobetween the two emission intensities can be used to evaluatethe analyte concentration and provide a built-in correction forthe receptor concentration, photobleaching and environmentaleffects.13 The emission band at 445 nm was attributed to theenol form and the emission at 510 nm was assigned to the ketotautomer, produced by the ESIPT process (Scheme 2).14

The pH sensitivity of the probe was tested by recordinguorescence spectra at different pH values (Fig. S9†). At low pH,the dual emission underwent a signicant red shi with anincrease in uorescence intensity due to the protonation of theprobe. However, at high pH, the emission bands at 445 nm and510 nm disappeared and a striking new peak emerged at457 nm due to the deprotonation of the hydroxyl group. Theuorescence intensity remained weak at intermediate pH(HEPES buffer 20 mM, pH 7.4). The pH titration indicates thatthe probe exhibits a remarkable pH-dependent behaviour.

Interestingly, the addition of Co2+ to the solution of PYETcaused amarked uorescence enhancement (Ff¼ 0.28), with anincrease in intensity of the emission band at 445 nm along witha concomitant decrease of the band at 510 nm, thus allowing arapid quantication of Co2+ based on the intensity ratio of theenol and keto tautomers (Fig. 3 and S10a†). These changes ledto the blocking of intramolecular hydrogen bonds by coordi-nation of phenolic O–H with Co2+. This in turn led to theinhibition of the ESIPT feature. Therefore, the two nitrogen

Scheme 1 Synthetic route of probe PYET.

25394 | RSC Adv., 2014, 4, 25393–25397

atoms (N–H group, CH]N), phenolic O–H and methylene O–Hplay a crucial role in the efficient binding of PYET with Co2+.From the Job plot analysis and mass spectrum, these spectralchanges were also attributed to the formation of a 1 : 1 complex(Fig. S5 and S11a†). It is noteworthy that the detection limit15

and binding constant16 of this probe for Co2+ is 6.42 � 10�7 Mand 6.89 � 104 M�1 in a pure aqueous medium. The probe asexpected was insensitive to the addition of other metal ions(Fig. S12†).

In contrast, the addition of F� resulted in a slight change ofthe keto emission band at 510 nm accompanied by a strongincrease of the enol emission band at 445 nm with a quantumyield of Ff ¼ 0.48 (Fig. 4 and S10b†). Hence, it is inferred thatESIPT is inhibited by the interaction of the F� anion with the

Fig. 2 Absorption spectra of PYET (10 mM) upon gradual addition ofF� in pH 7.4 HEPES buffered water.

This journal is © The Royal Society of Chemistry 2014

Scheme 2 Schematic illustration of the photophysical cycle of probePYET.

Fig. 3 Fluorescence spectra of PYET (5 mM) upon gradual addition ofCo2+ in pH 7.4 HEPES buffered water. Excitation at 350 nm. The slitwidth is 5 nm.

Fig. 4 Fluorescence spectra of PYET (10 mM) upon gradual addition ofF� in pH 7.4 HEPES buffered water. Excitation at 350 nm. The slit widthis 5 nm.

Fig. 5 Optimized structures of the (a) PYET-enol form, (b) PYET-ketoform, (c) PYET–Co2+, and (d) PYET–F�.

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O–H group of PYET. The binding constant of the PYET–F�

system was estimated to be 1.46 � 105 M�1 by linear tting ofthe uorescence titration curve. The Job plot (Fig. S11b†) andmass spectrum (Fig. S6†) of [PYET–F�] supports the 1 : 1binding stoichiometry. The detection limit was measured to be7.77 � 10�8 M. Furthermore, the optical response of PYET isinsignicant for anions other than uoride under identicalconditions (Fig. S13†).

To test the practical applicability of PYET, a competitivebinding experiment was carried out in the presence of varyingconcentrations of Co2+/F� (0–20 mM), treated with 100 mM ofcompeting analytes. No signicant variation was observed inthe presence of other competitive ions in comparison to asolution containing only Co2+/F� (Fig. S14†). These resultssuggest that the Co2+/F� recognition by the probe is barelyaffected by other coexisting metal ions/anions.

Furthermore, the binding mode of the probe with F� wasinvestigated by running proton NMR titrations in the presenceand absence of F� in DMSO-d6 (Fig. S15†). Upon addition of F�,the signals of the aromatic rings changed slightly and theresonance signals corresponding to N–H and phenolic O–H at

This journal is © The Royal Society of Chemistry 2014

6.5 ppm and 10.4 ppm shied downeld and eventually dis-appeared when the concentration of F� ions increased. Thissupported the deprotonation interaction between F�, phenolicO–H and the N–H group.

To provide an insight into the photonic properties of theprobe, DFT studies were performed. The optimized structuresof the tautomers of probe PYET, PYET–Co2+ and PYET–F�

(Fig. 5) were obtained using DFT/B3LYP-6-31G and B3LYP/LanL2DZ basis sets,17 respectively. As shown in Fig. S16,† theHOMO is positioned on the pyridoxal scaffold while the LUMOspreads over the pyridoxal phosphate with an imine group. Aerthe appendage of the Co2+ ion to the probe, the HOMO spreadsover both the pyridoxal scaffold and the metal center whereasthe pyridoxal phosphate with the imine group still retains itsLUMO character. Upon addition of F� to the probe,

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aminoethylethanolamine with uoride behaves as the HOMO,while the pyridoxal scaffold with aminoethylethanolaminebehaves as the LUMO. The HOMO–LUMO energy difference ofprobe PYET is 5.17 eV and the binding of Co2+/F� results inlowering the energy gap to 1.86 eV and 3.33 eV, respectively.

Conclusion

In summary, a uorescent chemosensor based on a vitamin B6

cofactor has been designed and synthesized. Probe PYETreveals a uorescence turn-on response to F�, and a ratiometricresponse towards Co2+ in an aqueous medium. Notably, theabsorption change and turn-on uorescence response renderthe sensor suitable for the detection of Co2+ and F� by simplevisual inspection. Hence, the promising characters, such asfacile synthetic methodology, good water solubility, ratiometricresponse and high selectivity, constitute desirable features forthe chemosensor reported in this manuscript.

Acknowledgements

M.I., D.J. and K.K. thank UGC-BSR for research fellowships.M.I., D.J., K.K. and D.C. also acknowledge DST-IRHPA, FIST andPURSE for instrumentation facilities. The authors gratefullyacknowledge DBT-IPLS, School of Biological Sciences, MKU forproviding instrumentation facilities.

References

1 (a) Chemosensors of ion and molecule recognition, ed. J. P.Desvergne and A. W. Czarnik, 1997, Kluwer Academic,Dordrecht, The Netherlands, p. 492; (b) A. P. De Silva,H. Q. Gunaratne, N. T. A. Gunnlaugsson, T. M. Huxley,C. P. McCoy, J. T. Rademacher and T. E. Rice, Chem. Rev.,1997, 97, 1515; (c) C. Caltagirone and P. A. Gale, Chem. Soc.Rev., 2009, 38, 520; (d) P. A. Gale, Coord. Chem. Rev., 2003,240, 1; (e) P. D. Beer and P. A. Gale, Angew. Chem., Int. Ed.,2001, 40, 486; (f) F. P. Schmidtchen and M. Berger, Chem.Rev., 1997, 97, 1609; (g) X. Chen, Y. Zhou, X. Peng andJ. Yoon, Chem. Soc. Rev., 2010, 39, 2120; (h) Z. Xu, X. Chen,H. N. Kim and J. Yoon, Chem. Soc. Rev., 2010, 39, 127; (i)Y. Zhou and J. Yoon, Chem. Soc. Rev., 2012, 41, 52.

2 (a) E. J. Underwood, Trace Elements in Human and AnimalNutrition, Academic Press, New York, 4th edn, 1977; (b)D. Purves, Trace Element Contamination of the Environments,Elsevier Science Publishers BV, Netherlands, 1985; (c)C. D. Klaassen, Casarett, Doull's Toxicology the Basic Scienceof Poisoning, McGraw-Hill, Medical Publishing Division,New York, 6th edn, 1986, p. 839.

3 (a) K. L. Kirk, Biochemistry of the Halogens and InorganicHalides, Plenum Press, New York, 1991; (b) M. Kleerekoper,Endocrinol. Metab. Clin. North Am., 1998, 27, 441; (c)Environmental Health Criteria, IPCS InternationalProgramme on Chemical Safety, World Health Organization,New York, 2002.

4 (a) B. Venugopal and T. D. Luckey, Metal Toxicity inMammals, Plenum Press, New York, vol. 2, 1979; (b)

25396 | RSC Adv., 2014, 4, 25393–25397

K. L. Kirk, Biochemistry of the Elemental Halogens andInorganic Halides, Plenum Press, New York, 1991, p. 19; (c)H. S. Horowitz, J. Publ. Health Dent., 2003, 63, 3; (d)J. R. Farley, J. E. Wergedal and D. J. Baylink, Science, 1983,222, 330; (e) C. E. Housecro and A. G. Sharpe, InorganicChemistry, Pearson Education Ltd, Harlow, 3rd edn, 2008,p. 872; (f) Y. C. Yang, J. A. Baker and J. R. Ward, Chem.Rev., 1992, 92, 1729.

5 (a) C. Y. Li, X. B. Zhang, Z. J. Rui Han, G. L. Shen and R. Q. Yu,Anal. Chim. Acta, 2006, 580, 143; (b) A. Basoglu, S. Parlayan,M. Ocak, H. Alp, H. Kantekin, M. Ozdemir and U. Ocak, J.Fluoresc., 2009, 19, 655; (c) H. Y. Luo, X. B. Zhang, C. L. He,G. L. Shen and R. Q. Yu, Spectrochim. Acta, Part A, 2008, 70,337.

6 (a) P. Chinna Ayya Swamy, S. Mukherjee and P. Thilagar,Chem. Commun., 2013, 49, 993; (b) J. Wang, L. Yang,C. Hou and H. Cao, Org. Biomol. Chem., 2012, 10, 6271; (c)L. Gai, H. Chen, B. Zou, H. Lu, G. Lai, Z. Li and Z. Shen,Chem. Commun., 2012, 48, 10721; (d) P. Sokkalingam andC. H. Lee, J. Org. Chem., 2011, 76, 3820; (e) Z. Xu, N. JitenSingh, S. K. Kim, D. R. Spring, K. S. Kim and J. Yoon,Chem. –Eur. J., 2011, 17, 1163; (f) M. Cametti andK. Rissanen, Chem. Commun., 2009, 2809; (g)H. Lenormand, J. P. Goddard and L. Fensterbank, Org.Lett., 2013, 15, 748; (h) L. Zang, D. Wei, S. Wang andS. Jiang, Tetrahedron, 2012, 68, 636.

7 H. M. Chawla, S. N. Sahu, R. Shrivastava and S. Kumar,Tetrahedron Lett., 2012, 53, 2244.

8 (a) D. L. Williams and A. Heller, J. Phys. Chem., 1970, 74,4473; (b) J. Catalan, F. Fabero, M. S. Guijarro,R. M. Claramunt, M. D. S. Maria, M. C. Foces-Foces,F. H. Cano, J. Elguero and R. Sastre, J. Am. Chem. Soc.,1990, 112, 747; (c) L. M. Tolbert and K. M. Solntsev, Acc.Chem. Res., 2002, 35, 19.

9 (a) Biochemistry of Vitamin B6 and PQQ, ed. G. Marino, G.Sannia and F. Bossa, Birkauser Verlag, Basel, Switzerland,1994; (b) A. E. Evangelopoulos, Chemical and BiologicalAspects of Vitamin B6 Catalysis, A. R. Liss, New York, 1984.

10 (a) M. Strianese, S. Milione, V. Bertolasi and C. Pellecchia,Inorg. Chem., 2013, 52, 11778; (b) E. Oliveira, C. Santos,P. Poeta, J. L. Capelo and C. Lodeiro, Analyst, 2013, 138,3642; (c) D. Sharma, S. K. Sahoo, S. Chaudhary, R. K. Beraand J. F. Callan, Analyst, 2013, 138, 3646; (d) L. Qu, C. Yin,F. Huo, J. Chao, Y. Zhang and F. Cheng, Sens. Actuators, B,2014, 191, 158.

11 (a) J. Li, H. Lin, Z. Cai and H. Lin, Spectrochim. Acta, Part A,2009, 72, 1062; (b) S. Y. Kim, J. Park, M. Koh, S. B. Park andJ. I. Hong, Chem. Commun., 2009, 4735.

12 (a) G. Sivaraman, T. Anand and D. Chellappa, Analyst, 2012,137, 5881; (b) G. Sivaraman, V. Sathiyaraja and D. Chellappa,J. Lumin., 2014, 145, 480; (c) G. Sivaraman, T. Anand andD. Chellappa, RSC Adv., 2013, 3, 17029; (d) G. Sivaramanand D. Chellappa, J. Mater. Chem. B, 2013, 1, 5768; (e)D. Jeyanthi, M. Iniya, K. Krishnaveni and D. Chellappa,RSC Adv., 2013, 3, 20984; (f) M. Iniya, D. Jeyanthi,K. Krishnaveni, A. Mahesh and D. Chellappa, Spectrochim.Acta, Part A, 2014, 120, 40; (g) T. Anand, G. Sivaraman and

This journal is © The Royal Society of Chemistry 2014

Paper RSC Advances

Publ

ishe

d on

29

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D. Chellappa, Spectrochim. Acta, Part A, 2014, 123, 18; (h)T. Anand, G. Sivaraman, P. Anandh, D. Chellappa andS. Govindarajan, Tetrahedron Lett., 2014, 55, 671; (i)G. Sivaraman, T. Anand and D. Chellappa, RSC Adv., 2012,2, 10605; (j) G. Sivaraman, T. Anand and D. Chellappa,Anal. Methods, 2014, 6, 2343.

13 (a) G. Grynkiewicz, M. Poenie and R. Y. Tsien, J. Biol. Chem.,1985, 260, 3440; (b) S. Banthia and A. Samanta, J. Phys. Chem.B, 2006, 110, 6437.

14 W. R. Cordoba, C. Fregoso, E. Zugozagoitia and J. Peon, J.Phys. Chem. A, 2007, 111, 6241.

15 M. Shortreed, R. Kopelman, M. Kuhn and B. Hoyland, Anal.Chem., 1996, 68, 1414.

16 K. A. Conners, Binding Constants, Wiley, New York, 1987.17 M. J. Frisch, G. Trucks, H. B. Schlegel, G. E. Scuseria,

M. A. Robb, J. R. Cheeseman, J. A. Montgomery, T. Vreven,K. N. Kudin, J. C. Burant, J. M. Millam, S. S. Iyengar,J. Tomasi, V. Barone, B. Mennucci, M. Cossi, G. Scalmani,

This journal is © The Royal Society of Chemistry 2014

N. Rega, G. A. Petersson, H. Nakatsuji, M. Hada, M. Ehara,K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima,Y. Honda, O. Kitao, H. Nakai, M. Klene, X. Li, J. E. Knox,H. P. Hratchian, J. B. Cross, V. Bakken, C. Adamo,J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev,A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski,P. Y. Ayala, K. Morokuma, G. A. Voth, P. Salvador,J. J. Dannenberg, V. G. Zakrzewski, S. Dapprich,A. D. Daniels, M. C. Strain, O. Farkas, D. K. Malick,A. D. Rabuck, K. Raghavachari, J. B. Foresman, J. V. Ortiz,Q. Cui, A. G. Baboul, S. Clifford, J. Cioslowski,B. B. Stefanov, G. Liu, A. Liashenko, P. Piskorz,I. Komaromi, R. L. Martin, D. J. Fox, T. Keith,M. A. AlLaham, C. Y. Peng, A. Nanayakkara,M. Challacombe, P. M. W. Gill, B. Johnson, W. Chen,M. W. Wong, C. Gonzalez, J. A. Pople, Gaussian 03, RevisionC.02, Gaussian, Inc., Wallingford CT, 2004.

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