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.