Optical 2-benzyl-5-hydroxy-4-oxopentanoic acids against carboxypeptidase A: Synthesis, kinetic evaluation and X-ray crystallographic study

2-Benzyl-5-hydroxy-4-oxopentanoic acid 1 and its enantiomers were designed, synthesized and assayed for inhibitory activity against carboxypeptidase A (CPA, EC 3.4.17.1). To verify the role of the terminal hydroxyl group in 1 binding to CPA, 2-benzyl-5-benzyloxy-4-oxopentanoic acid 2 was also synthesized and evaluated. The inhibition constants show that both L-1 and D-1 were shown to have strong binding affinity with L-1 being more potent than its enantiomer by 165-fold. On the other hand, the inhibition constant of 2 increases 4-fold comparing with that of 1. In order to explore the exact binding mode of the hydroxyacteyl group of 1 to the active site zinc ion of CPA, we have solved the crystal structure of CPA complexed with L-1 up to 1.85Å resolution. In CPA·L-1 complex, the phenyl ring is fitted in the substrate recognition pocket at the S′1 subsite, and the carboxylate forms bifurcated hydrogen bonds with the guanidinium moiety of Arg-145 and Arg-127 and a hydrogen bond with the phenolic hydroxyl of the down-positioned Tyr-248. The carbonyl oxygen of L-1 does coordinate to the active site zinc ion of CPA as expectedly. Unexpectedly, the terminal hydroxyl group of L-1 is engaged in hydrogen bonding with carbonyl oxygen of Ser-197 instead of coordinating to the active site zinc ion.