Development of sensitive esterase assays based on alpha-cyano-containing esters

A novel approach is reported for the development of fluorogenic esterase re porters using alpha -cyano-containing esters as substrates. After ester hyd rolysis, the released alcohol, a cyanohydrin, rapidly eliminates HCN to yie ld the corresponding aldehyde resulting in strong fluorescence. The Tr conj ugation of the resulting aldehyde also greatly enhances UV absorption and r ed shifts fluorescence emission relative to a corresponding alcohol or phen ol. Two substrates, R/S-acetic acid cyano-(6-methoxynaphthalen-2-yl)-methyl ester (compound II) and trans/cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclop ropanecarboxylic acid R/S-cyano-(6-methoxynaphthalen-2-yl)-methyl ester (co mpound II), were synthesized and evaluated as substrates. Such a-cyano subs trates possess very low background fluorescence and are more stable under e nzyme assay conditions than phenolic substrates due to the aliphatic cyano group. The higher molar absorbtivity and quantum yield of the aldehyde, alo ng with its larger Stokes' shift combined with the increased stability and lower background signal of the cyanohydrin substrate, increases the utility and sensitivity of the resulting assays over current methods. Moreover, co mpound II showed high selectivity to pyrethroid-cleaving esterases and may provide a direct tool to monitor pyrethroid resistance in insects. (C) 2001 Elsevier Science.