Enantioselective fluorogenic assay of acetate hydrolysis for detecting lipase catalytic antibodies

An enantioselective fluorogenic assay for the kinetic resolution of chiral alkyl acetates is demonstrated with 7-(3-aceroxybutoxy)-2H-1-henzopyran-2-o nes (R)- and (S)-4 or 7-(3-acetoxy-2-methylpropoxy)-2H-1-benzopyran-2-ones (R)-4 and (S)-6. The alcohols released by hydrolysis of these acetates are oxidized by horse-liver alcohol dehydrogenase to unstable beta-(aryloxy)car bonyl compounds: which undergo beta-elimination of the strongly fluorescent product umbelliferone (= 7-hydroxy-2H-1-benzopyran-2-one; 3) (lambda(em) = 460 +/- 20 nm, lambda(ex) = 360 +/- 20 nm). Enantioselectivities are calcu lated from the reaction rates for each enantiomeric acetate. For a series o f representative lipases, the reactivities and enantioselectivities under p reparative conditions are predicted accurately. This highly sensitive enant ioselective assay detects as little as 10 mu g/ml of hydrolytic enzyme, can be carried out in 96-well microtiter plates. and is compatible with cell-c ult Ire media. It is, therefore, suited for screening libraries of antibodi es for enantioselective lipase catalytic antibodies.