ABSTRACT
An efficient synthesis of original bio-reductive probes suitable for the detection of azoreductases from the fluorescent rhodamine 110 dye is presented. A "turn-on" green fluorescence response upon reduction of the two diazo bonds of these latent fluorophores was observed both in vitro and in the context of bacterial cultures.
Subject(s)
Bacteria/enzymology , Fluorescent Dyes/metabolism , NADH, NADPH Oxidoreductases/metabolism , Rhodamines/metabolism , Azo Compounds/analysis , Azo Compounds/metabolism , Enzyme Assays , Fluorescent Dyes/analysis , Models, Molecular , NADH, NADPH Oxidoreductases/analysis , Nitroreductases , Oxidation-Reduction , Rhodamines/analysisABSTRACT
Organophosphorus nerve agents irreversibly inhibit cholinesterases. Phosphylation of the catalytic serine can be reversed by the mean of powerful nucleophiles like oximes. But the phosphyl adduct can undergo a rapid spontaneous reaction leading to an aged enzyme, i.e., a conjugated enzyme that is no longer reactivable by oximes. One strategy to regain reactivability is to alkylate the phosphylic adduct. Specific alkylating molecules were synthesized and the crystal structures of the complexes they form with soman-aged human butyrylcholinesterase were solved. Although the compounds bind in the active site gorge of the aged enzyme, the orientation of the alkylating function appears to be unsuitable for efficient alkylation of the phosphylic adduct. However, these crystal structures provide key information to design efficient alkylators of aged-butyrylcholinesterase and specific reactivators of butyrylcholinesterase.