Enantioselective Fluorogenic Assay of Acetate Hydrolysis for Detecting Lipase Catalytic Antibodies |
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Authors: | Gé rard Klein,Jean-Louis Reymond |
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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-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 β-(aryloxy)carbonyl compounds, which undergo β-elimination of the strongly fluorescent product umbelliferone (=7-hydroxy-2H-1-benzopyran-2-one; 3 ) (λem=460±20 nm, λex=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 μg/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. |
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