Development of sensitive esterase assays based on alpha-cyano-containing esters.
A novel approach is reported for the development of fluorogenic esterase reporters using alpha-cyano-containing esters as substrates. After ester hydrolysis, the released alcohol, a cyanohydrin, rapidly eliminates HCN to yield the corresponding aldehyde resulting in strong fluorescence. The pi conjugation of the resulting aldehyde also greatly enhances UV absorption and red shifts fluorescence emission relative to a corresponding alcohol or phenol. Two substrates, R/S-acetic acid cyano-(6-methoxynaphthalen-2-yl)-methyl ester (compound I) and trans/cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid R/S-cyano-(6-methoxynaphthalen-2-yl)-methyl ester (compound II), were synthesized and evaluated as substrates. Such alpha-cyano substrates possess very low background fluorescence and are more stable under enzyme assay conditions than phenolic substrates due to the aliphatic cyano group. The higher molar absorbtivity and quantum yield of the aldehyde, along 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, compound II showed high selectivity to pyrethroid-cleaving esterases and may provide a direct tool to monitor pyrethroid resistance in insects.
Shan
G
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Department of Entomology and Cancer Research Center, University of California, Davis, California 95616, USA.
Hammock
B D
BD
D000085:Acetates / Q000138:chemical synthesis / Q000737:chemistry*; D000818:Animals; D004950:Esterases / Q000032:analysis*; D004952:Esters / Q000138:chemical synthesis / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008862:Microsomes, Liver / Q000201:enzymology; D009036:Moths / Q000201:enzymology; D009570:Nitriles / Q000138:chemical synthesis / Q000737:chemistry*; D011722:Pyrethrins / Q000737:chemistry*; D051381:Rats; D013379:Substrate Specificity; D013552:Swine
C445061:3-(2,2-dichlorovinyl)-2,2--dimethylcyclopropanecarboxylic acid cyano-(6-methoxynaphthalen-2-yl)methyl ester; D000085:Acetates; D004952:Esters; D005456:Fluorescent Dyes; D009570:Nitriles; D011722:Pyrethrins; C445060:acetic acid cyano-(6-methoxynaphthalen-2-yl)methyl ester; C430593:cyanohydrin; C017160:cypermethrin; D004950:Esterases