Vitamin B₆ cofactor based fluorescent probe for sensing an anion (F⁻) and cation (Co²⁺) independently in a pure aqueous medium

Abstract

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.

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Introduction

Fluorescent chemosensors can serve as effective tools in molecular sensing as is evident from their prominent role in medicinal diagnostics, biological labeling and optoelectronic materials.¹ In particular, single molecular sensors with varied responses towards different analytes are cost effective and convenient for real applications. Among metal ions, sensing of cobalt has received increasing attention, since Co²⁺ is a component of vitamin B₁₂, a vitamin essential for DNA synthesis, the formation of red blood cells, maintenance of the nervous system, and the growth and development of children.² Fluoride is also an essential nutrient for the normal development and growth of a human being.³ Excess accumulation of cobalt in the body can result in cardiomyopathy, hypothyroidism, and neurological damage, while a high level of fluoride can cause dental and skeletal fluorosis.⁴

A number of fluorescent sensors have already been established for sensing individually either cobalt⁵ or fluoride.⁶ But reports about a single probe that senses fluoride and cobalt(II) ions independently are rare. Recently, chromogenic recognition of Co²⁺ or fluoride ions has been achieved in a DMSO–CH₃CN system using a calixarene based ditopic receptor by H. M. Chawla et al.⁷ However, it suffers from an interference with Ag⁺ and Cu²⁺ while fluoride ions bind to the receptor. Furthermore, most of the reported sensors often feature a tedious synthetic protocol, non-aqueous media requirement, lack of dual response and cross sensitivities towards other ions. All these limitations restrict their potential use in environmental and biological applications.

Amongst different organic scaffolds, the scaffold that facilitates Excited State Intramolecular Proton Transfer (ESIPT) as a sensing mechanism is a perfect candidate as a fluorescent probe because of its significant photostability, large stoke shift and intense luminescence.⁸ Pyridoxal phosphate (PLP), the active form of vitamin B₆, functions as a coenzyme in numerous enzyme-catalyzed reactions, such as transamination, α- and β-decarboxylations, β- and γ-eliminations, racemizations, and aldol reactions.⁹ Although PLP is well known for its coordination and optical properties, very few reports have shown the design of a fluorescent chemosensor utilizing a PLP platform.¹⁰ The fluorescence detection of ions using small-molecule sensors in an aqueous environment is still a difficult task. Only a small number of fluorescent probes that have been used successfully for this purpose have appeared in the literature.¹¹ In continuation of our ongoing research to develop fluorescent chemosensors,¹² herein, we have designed a pyridoxal linked aminoethylaminoethanol (PYET) receptor for the selective recognition of Co²⁺ and F⁻ in an aqueous solution via enhanced fluorescence emission. Interestingly, compared to various other fluorescent chemosensors, probe PYET exhibits good water solubility, optical sensitivity in a buffer medium, dual emission, a low detection limit and visible strong fluorescence under UV light when adding guest species. To the best of our knowledge, probe PYET is the first bifunctional chromogenic and fluorogenic chemosensor that enables independent sensing of fluoride and cobalt(II) ions in an aqueous medium.

Results and discussion

Chemosensor PYET was synthesized from Vitamin B₆ cofactor pyridoxal phosphate with 2-(2-aminoethylamino)-ethanol in a methanol solution (Fig. S1–S6†). Aminoethylethanolamine has been selected as a side chain because of its hydrophilic character, while pyridoxal phosphate (PLP) acts as a signalling moiety, due to its low fluorescence quantum yield and good binding sites (Scheme 1). Furthermore, this type of structural arrangement can undergo keto–enol tautomerism through an excited state intramolecular proton transfer (ESIPT) mechanism. The probe PYET was characterized by NMR and other spectroscopic methods.

Scheme 1 Synthetic route of probe PYET.

The photonic properties of PYET were investigated by UV-vis and fluorescence 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 the presence of HEPES buffer at pH 7.4. The probe showed λ_max at 328 nm and 387 nm in its absorption spectrum. Systematic titration of sensor PYET with increasing concentrations of Co²⁺ revealed a new absorption band at 369 nm with the disappearance of the bands at 328 and 387 nm (Fig. 1).

Fig. 1 Absorption spectra of PYET (5 μM) upon gradual addition of Co²⁺ in pH 7.4 HEPES buffered water.

Upon addition of F⁻ (Fig. 2), the intensity of the absorption band centered at 328 nm decreased while that of the band at 387 nm increased. The addition of other biologically important metal ions and anions led to no obvious change in the ground state behaviour of probe PYET (Fig. S7 and S8†). When excited at 350 nm in the same solvent system, PYET exhibits fluorescence emission bands at 445 nm and 510 nm with a quantum yield of 0.010 at room temperature. The presence of dual emission indicates the possibility of the ESIPT mechanism. It also enables a new way for the ratiometric analysis in which the ratio between the two emission intensities can be used to evaluate the analyte concentration and provide a built-in correction for the receptor concentration, photobleaching and environmental effects.¹³ The emission band at 445 nm was attributed to the enol form and the emission at 510 nm was assigned to the keto tautomer, produced by the ESIPT process (Scheme 2).¹⁴

Fig. 2 Absorption spectra of PYET (10 μM) upon gradual addition of F⁻ in pH 7.4 HEPES buffered water.

Scheme 2 Schematic illustration of the photophysical cycle of probe PYET.

The pH sensitivity of the probe was tested by recording fluorescence spectra at different pH values (Fig. S9†). At low pH, the dual emission underwent a significant red shift with an increase in fluorescence intensity due to the protonation of the probe. However, at high pH, the emission bands at 445 nm and 510 nm disappeared and a striking new peak emerged at 457 nm due to the deprotonation of the hydroxyl group. The fluorescence intensity remained weak at intermediate pH (HEPES buffer 20 mM, pH 7.4). The pH titration indicates that the probe exhibits a remarkable pH-dependent behaviour.

Interestingly, the addition of Co²⁺ to the solution of PYET caused a marked fluorescence enhancement (Φ_f = 0.28), with an increase in intensity of the emission band at 445 nm along with a concomitant decrease of the band at 510 nm, thus allowing a rapid quantification of Co²⁺ based on the intensity ratio of the enol and keto tautomers (Fig. 3 and S10a†). These changes led to the blocking of intramolecular hydrogen bonds by coordination of phenolic O–H with Co²⁺. This in turn led to the inhibition of the ESIPT feature. Therefore, the two nitrogen atoms (N–H group, CH=N), phenolic O–H and methylene O–H play a crucial role in the efficient binding of PYET with Co²⁺. From the Job plot analysis and mass spectrum, these spectral changes 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 M and 6.89 × 10⁴ M⁻¹ in a pure aqueous medium. The probe as expected was insensitive to the addition of other metal ions (Fig. S12†).

Fig. 3 Fluorescence spectra of PYET (5 μM) upon gradual addition of Co²⁺ in pH 7.4 HEPES buffered water. Excitation at 350 nm. The slit width is 5 nm.

In contrast, the addition of F⁻ resulted in a slight change of the keto emission band at 510 nm accompanied by a strong increase of the enol emission band at 445 nm with a quantum yield of Φ_f = 0.48 (Fig. 4 and S10b†). Hence, it is inferred that ESIPT is inhibited by the interaction of the F⁻ anion with the O–H group of PYET. The binding constant of the PYET–F⁻ system was estimated to be 1.46 × 10⁵ M⁻¹ by linear fitting of the fluorescence titration curve. The Job plot (Fig. S11b†) and mass spectrum (Fig. S6†) of [PYET–F⁻] supports the 1 : 1 binding stoichiometry. The detection limit was measured to be 7.77 × 10⁻⁸ M. Furthermore, the optical response of PYET is insignificant for anions other than fluoride under identical conditions (Fig. S13†).

Fig. 4 Fluorescence spectra of PYET (10 μM) upon gradual addition of F⁻ in pH 7.4 HEPES buffered water. Excitation at 350 nm. The slit width is 5 nm.

To test the practical applicability of PYET, a competitive binding experiment was carried out in the presence of varying concentrations of Co²⁺/F⁻ (0–20 μM), treated with 100 μM of competing analytes. No significant variation was observed in the presence of other competitive ions in comparison to a solution containing only Co²⁺/F⁻ (Fig. S14†). These results suggest that the Co²⁺/F⁻ recognition by the probe is barely affected by other coexisting metal ions/anions.

Furthermore, the binding mode of the probe with F⁻ was investigated by running proton NMR titrations in the presence and absence of F⁻ in DMSO-d₆ (Fig. S15†). Upon addition of F⁻, the signals of the aromatic rings changed slightly and the resonance signals corresponding to N–H and phenolic O–H at 6.5 ppm and 10.4 ppm shifted downfield and eventually disappeared when the concentration of F⁻ ions increased. This supported the deprotonation interaction between F⁻, phenolic O–H and the N–H group.

To provide an insight into the photonic properties of the probe, DFT studies were performed. The optimized structures of 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,† the HOMO is positioned on the pyridoxal scaffold while the LUMO spreads over the pyridoxal phosphate with an imine group. After the appendage of the Co²⁺ ion to the probe, the HOMO spreads over both the pyridoxal scaffold and the metal center whereas the pyridoxal phosphate with the imine group still retains its LUMO character. Upon addition of F⁻ to the probe, aminoethylethanolamine with fluoride behaves as the HOMO, while the pyridoxal scaffold with aminoethylethanolamine behaves as the LUMO. The HOMO–LUMO energy difference of probe PYET is 5.17 eV and the binding of Co²⁺/F⁻ results in lowering the energy gap to 1.86 eV and 3.33 eV, respectively.

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

Conclusion

In summary, a fluorescent chemosensor based on a vitamin B₆ cofactor has been designed and synthesized. Probe PYET reveals a fluorescence turn-on response to F⁻, and a ratiometric response towards Co²⁺ in an aqueous medium. Notably, the absorption change and turn-on fluorescence response render the sensor suitable for the detection of Co²⁺ and F⁻ by simple visual inspection. Hence, the promising characters, such as facile synthetic methodology, good water solubility, ratiometric response and high selectivity, constitute desirable features for the 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 and PURSE for instrumentation facilities. The authors gratefully acknowledge DBT-IPLS, School of Biological Sciences, MKU for providing instrumentation facilities.

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Footnote

† Electronic supplementary information (ESI) available: NMR and MS spectral data, UV-vis data and computational details. See DOI: 10.1039/c4ra02778g

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