Elsevier

Tetrahedron

Volume 65, Issue 11, 14 March 2009, Pages 2307-2312
Tetrahedron

An NBD-based colorimetric and fluorescent chemosensor for Zn2+ and its use for detection of intracellular zinc ions

https://doi.org/10.1016/j.tet.2009.01.035Get rights and content

Abstract

A new 7-nitrobenz-2-oxa-1,3-diazole (NBD) based colorimetric and fluorescence chemosensor for Zn2+, an ion involved in many biological processes, was designed and synthesized. The NBD-probe 1 displays a red-to-yellow color change and an enhancement of fluorescent intensity in the presence of an aqueous solution of Zn2+ ions (pH 7.2). Internal charge transfer (ICT) and photoinduced electron transfer (PET) mechanisms are responsible for these changes. The practical use of this probe was demonstrated by its application to the biologically relevant detection of Zn2+ ions in pancreatic β-cells.

Introduction

The ability to detect metal ions with high specificity under physiological conditions is an important criterion that must be met in the design of fluorescent chemosensors for biological and environmental applications.1, 1(a), 1(b), 1(c), 1(d), 1(e) In particular, the development of a fluorescent probe for zinc ion in the presence of a variety of other metal ions has received great attention. These efforts are stimulated by the fact that Zn2+ is involved in a variety of physiological and pathological processes. For example, Zn2+ is an essential component of many enzymes and it is involved in maintaining key structural features of gene transcription proteins.2, 2(a), 2(b) Interestingly, in comparison with other tissues, high concentrations of Zn2+ are present in pancreatic islets, which play a critical role in insulin biosynthesis, storage, and secretion.3 A decrease in the concentration of Zn2+ causes a reduction of the ability of the islet cells to produce and secrete insulin.4 It is also reported that zinc ion is a potent killer of neurons via oxidative stress, the cause of neurodegenerative disorders.5 Owing to the biological significance of zinc, a considerable effort has been devoted to the development of efficient and selective methods to detect Zn2+.

Fluorescence based chemosensors have a key advantage over those that rely on other detection techniques, such as atomic absorption, X-ray fluorescence, and radioisotopes, in that they can be used to readily detect intracellular ion levels without the need for sophisticated instrumentation or time-consuming sample preparation.1, 1(a), 1(b), 1(c), 1(d), 1(e), 6, 6(a), 6(b), 6(c), 6(d), 6(e), 6(f), 6(g), 6(h), 6(i) Moreover, a dual colorimetric-fluorescent probe combines the sensitivity of fluorescence with the convenience and esthetic appeal of a colorimetric assay.7, 7(a), 7(b), 7(c), 7(d), 7(e)

Several fluorescent probes, such as Zinquin and 6-methoxy-8-quinolyl-p-toluenesulfonamide (TSQ), have been designed for the detection of intracellular zinc ions.6(a), 6(b) However, these probes have relatively low selectivities for zinc ion in that they also respond to calcium ions. Therefore, the search continues for a Zn2+ fluorescent probe that has improved selectivity. Additional criteria applied in the design of these probes include ready synthesis, easy detection, good water solubility, high cell permeability, and long wavelength absorbance to avoid cell damage, and a high fluorescence background.

7-Nitrobenz-2-oxa-1,3-diazole (NBD) has been widely utilized as a fluorophore in various fluorescent chemosensors owing to its emission at long wavelengths and good cell permeability.8, 8(a), 8(b) However, only one example of an NBD-based Zn2+ fluorescent probe has been described to date.9 Although, not applied to monitor intracellular zinc ions, this PET mechanism based chemosensor shows a high selectivity and sensitivity for Zn2+ in aqueous solution with a significant ‘off–on’ fluorescence response.

Below, we describe the results of a study that has led to the development of the NBD-based probe 1, which displays a red-to-yellow color change and a selective fluorescent enhancement in aqueous solutions (pH 7.2) containing Zn2+. A combination of PET (photoinduced electron transfer) and ICT (internal charge transfer) processes are involved in promoting the color and fluorescence changes. By using NMR titration as well as cyclic voltammetry (CV) experiments, we have demonstrated that a unique stepwise binding mechanism is involved in this system. Furthermore, we have shown that 1 has excellent cell permeability. This conclusion comes from the observation that zinc ions present in pancreatic β-cells can be efficiently detected.

Section snippets

Results and discussion

The route used to synthesize 1 is initiated by coupling of N,N′-bis(2-pyridylmethyl)-ethane-1,2-diamine (3)10 and NBD–Cl (4) to produce 2 in 86% yield (Scheme 1). Reaction of 2 with 2-(bromomethyl)pyridine under basic conditions gives 1 in 92% yield. The detailed procedures and spectroscopic characterization of products are given in the Experimental section and Supplementary data. By using this route, the complicated synthesis of the Zn2+ chelator N-bis-pyridin-2-ylmethylethane-1,2-diamine is

Conclusion

In conclusion, this effort has led to the design and synthesis of the new NBD-based fluorescent sensor 1 that can be used for the detection of Zn2+. This displays a selective fluorescent enhancement and colorimetric change with Zn2+ ions in aqueous solution. The binding mode of probe 1 with Zn2+ was probed by using NMR and CV experiments. Furthermore, the practical use of this probe is demonstrated by its application to the detection of Zn2+ ions in pancreatic β-cells. The findings summarized

General methods

Unless otherwise noted, materials were obtained from commercial suppliers and were used without further purification. Flash chromatography was carried out on silica gel 60 (230–400 mesh ASTM; Merck). Thin layer chromatography (TLC) was carried out using Merck 60 F254 plates with a thickness of 0.25 mm. Preparative TLC was performed using Merck 60 F254 plates with a thickness of 1 mm. Melting points were measured using a Büchi 530 melting point apparatus, and are uncorrected. 1H NMR and 13C NMR

Acknowledgements

This work was supported by grants from the NRL program (R04-2007-000-2007-0 and R0A-2005-000-10027-0), the SRC program of KOSEF/MEST (R11-2005-008 and R11-2000-070), and WCU (R31-2008-000-10010-0) program. G.-H.K and M.J.C. also thank the BK21 program (KRF). Mass spectral data were obtained from the Korea Basic Science Institute (Daegu) on a Jeol JMS 700 high resolution mass spectrometer.

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