New 4-styrylcoumarin derivatives as potentials fluorescent labels for biomolecules: application in RNA-FISH probes.

Abstract

Currently, fluorescence microscopy is one of the highly sensitive imaging techniques that allow visualize, detect, and track biomolecules in analytical studies in many important scientific areas as cellular biology, environmental sciences, medicine, pharmacy, among others. One of the great advantages of fluorescence microscopy is the possibility to use several fluorescent labels to detect different biomolecules and produce multicolour images allowing the identification, in vitro and in vivo, of specific components of complex biomolecular assemblies, as well analysis of their interactions. Fluorescent labels can produce chemically stable and small bioconjugates, with insignificant interference on the structure and biological functions of the unlabelled biomolecules. The amine-reactive fluorescent labels, due to the abundance of amino groups or its easy insertion into biomolecules, are the most frequently used to prepare bioconjugates for a multiplicity of biological applications as fluorescence in situ hybridization (FISH), histochemistry, cell tracing, receptor binding, or direct and indirect immunochemistry. Presently, the most widely used available fluorescent labels are very expensive and that's why coumarin derivatives can be a solution to develop low-cost new brightness fluorophores. In this work, we developed a low cost and effective synthetic strategy to synthesize six new 4-styrylcoumarin derivatives (Figure 1), as potentials fluorescent labels for biomolecules, using the inexpensive 7-diethylamino-4-methylcoumarin as a starting material. New fluorescent oligonucleotide probes have been obtained, six directed to the rRNA region of eukaryotic cells (EUK516) and six to the rRNA region of prokaryotic cells (EUB338). The developed fluorescent probes were tested on microorganisms belonging to the culture collection of the Laboratory of Biodegradation and Biotechnology of the HERCULES Laboratory (University of Évora), namely Saccharomyces cerevisiae and Bacillus showing effective performance as RNA-FISH probes. These findings evidenced the applicability of the new 4-styrylcoumarin derivatives in labeling of biomolecules and bioimaging.