Colton_2011_Adv.Synth.Catal_353_2529

Lipase from Candida rugosa shows high enantioselectivity toward alpha-substituted chiral acids such as 2-arylpropionic acids. To understand how Candida rugosa lipase (CRL) distinguishes between enantiomers of chiral acids, we determined the X-ray crystal structure of a transition-state analog covalently linked to CRL. CRL shows moderate enantioselectivity (E=23) toward methyl 2-methoxy-2-phenylacetate, 1-methyl ester, favoring the (S)-enantiomer. We synthesized phosphonate (RC,RPSP)-3, which, upon reaction with CRL, mimics the transition state for hydrolysis of (S)-1-methyl ester, the fast-reacting enantiomer. An X-ray crystal structure of this complex shows a catalytically productive orientation with the phenyl ring in the hydrophobic tunnel of the lipase. Phe345 crowds the region near the substrate stereocenter. Computer modeling of the slow-reacting enantiomer examined four possible conformations for the corresponding slow-reacting enantiomer: three conformations where two substituents at the stereocenter have been exchanged relative to the fast-reacting enantiomer and one conformation with an umbrella-like inversion orientation. Each of these orientations disrupts the orientation of the catalytic histidine, but the molecular basis for disruption differs in each case showing that multiple mechanisms are required for high enantioselectivity.