Recognition of Protein Binding Events by Polarity-Sensitive Probes

Abstract

Polarity-dependent fluorescent probes are recently attracting interest for high-resolution cell imaging. The fluorescence enhancement of the solvatochromic dye, ideally located in a domain where polarity changes occur upon binding, allows for a fine detection of molecular recognition events even between non overexpressed proteins.1 We developed a toolbox of new solvatochromic coumarin derivatives, characterized by a donor-(coumarin core)-acceptor structure, tailored to in vivo imaging applications.After a preliminary screening by computational methods, we adopted a synthetic procedure tuneable on the substitution patterns to achieve. Our probes possess excellent fluorescence quantum yields (up to 0.95), high molar extinction coefficients (up to 46,000 M-1cm-1), and large Stokes shifts. Furthermore, they display strong solvatochromism, being almost non emissive in water and very fluorescent in less polar media (up to 780-fold enhancement in brightness).When tested on cultured cells, the developed coumarins resulted not harmful and their photophysical properties were unchanged compared to free solution. Due to both their strong solvatochromic properties, and their lipophilic character, the coumarin did fluoresce only in the most lipophilic environments of the cell. In particular, colocalization experiments with standard markers evidenced staining in ER, membranes and lysosomes, depending on the chemical structure of the solvatochromic probe.Finally, one compound (3-benzothiazenyl-4-ciano-6,7-dimethoxy coumarin) showed monoexponential decay of fluorescence with a lifetime which is linearly dependent on solvent polarity. This feature promotes its use as ratiometric indicator of cell polarity at nanoscale level. The prepared compounds are remarkable tools to investigate subtle biochemical processes in the cell environment after appropriate conjugation to biomolecules, and at the same time constitute the basis for further engineering of a new generation of biosensors.1) Nalbant, P.; Hodgson, L.; Kraynov, V.; Toutchkine, A.; Hahn, K. M. Science 2004, 305, 1615-1619.