Enantioselective Fluorogenic Assay of Acetate Hydrolysis for Detecting Lipase Catalytic Antibodies

Abstract

An enantioselective fluorogenic assay for the kinetic resolution of chiral alkyl acetates is demonstrated with 7-(3-acetoxybutoxy)-2H-1-benzopyran-2-ones (R)- and (S)-4 or 7-(3-acetoxy-2-methylpropoxy)-2H-1benzopyran-2-ones (R)-4 and (S)-6. The alcohols released by hydrolysis of these acetates are oxidized by horseliver alcohol dehydrogenase to unstable b-(aryloxy)carbonyl compounds, which undergo b-elimination of the strongly fluorescent product umbelliferone (a 7-hydroxy-2H-1-benzopyran-2-one; 3 )( l ema 460 20 nm, lexa 360 20 nm). Enantioselectivities are calculated from the reaction rates for each enantiomeric acetate. For a series of representative lipases, the reactivities and enantioselectivities under preparative conditions are predicted accurately. This highly sensitive enantioselective assay detects as little as 10 mg/ml of hydrolytic enzyme, can be carried out in 96-well microtiter plates, and is compatible with cell-culture media. It is, therefore, suited for screening libraries of antibodies for enantioselective lipase catalytic antibodies.