Synthesis and spectroscopic characterisation of new ESIPT fluorescent protein probes

Abstract

Three new benzazole isothiocyanate fluorescent dyes, 2-(4′-isothiocyanate-2′-hydroxyphenyl)benzoxazole, 2-(4′-isothiocyanate-2′-hydroxyphenyl)benzothiazole and 2-(4′-isothiocyanate-2′-hydroxyphenyl)benzimidazole were synthesised, purified until optical purity grade and characterised by spectroscopic techniques. UV/VIS and steady-state fluorescence were also applied to characterise the photophysical behaviour of the dyes. These dyes exhibit an intense fluorescence emission with a large Stokes shift, inherent to the class of benzazoles which relax by the excited state intramolecular proton transfer (ESIPT) mechanism. The dyes were studied for labeling bovine serum albumin (BSA), resulting conjugates BSA-dye with a remarkable photostability under UV/VIS radiation in relation to classical protein labels. The resulting conjugates presented fluorescence in the blue–green region. Direct fluorescence detection of protein-labeled with those dyes after polyacrylamide gel electrophoresis indicates their potential use as fluorescent probes for proteins. © 2005 The Royal Society of Chemistry and Owner Societies.