Enhanced stereoselectivity in pig liver esterase catalysed diester hydrolysis. The role of a competitive nucleophile.
The enantioselectivity of pig liver esterase catalysed hydrolysis of cis-N-benzyl-2,5-bis(methoxy-carbonyl)pyrrolidine (1) has previously been shown to be very dependent on the reaction conditions. Hydrolysis performed in media buffered with tris(hydroxymethyl)aminomethane (Tris) afforded a monoester with much higher optical purity than hydrolysis in media without Tris. Detailed product studies in a Tris-buffered medium have been performed using NMR-techniques and a 13C-labelled ester. The NMR-studies revealed the presence of (2S,5R)-N-benzyl-2-methoxycarbonyl-5-[[[2-hydroxy-1,1- bis(hydroxymethyl)ethyl]amino]carbonyl]pyrrolidine (4) as an intermediate, which together with the isolated product (2S,5R)-N-benzyl-2-carboxy-5-[[[2-hydroxy-1,1-bis(hydroxymethyl) ethyl]amino]carbonyl]pyrrolidine (3) suggested Tris as a competitive nucleophile to water. The increased enantioselectivity seen in the produced (2R,5S)-N-benzyl-2-methoxy-carbonyl-5-carboxypyrrolidine (2) was explained by the preference of Tris to react faster with one of the diastereomeric acyl enzymes over the other.