Experimental and computational analyses were performed on the corepressor (L-tryptophan) binding site of the trp-repressor of Escherichia coli to investigate the ligand-protein interactions. Gly85, one of the residues forming the hydrophobic pocket of the binding site, was systematically replaced with Ala, Val, Leu and Trp by cassette mutagenesis. Biochemical characterization showed that all these mutations caused significant decreases in tryptophan binding activity. Free energy perturbation calculations were performed for the mutants and were consistent with the experimental results. The lack of a side chain at position 85 was concluded to be essential for binding the corepressor; the structure of the binding pocket was suggested to be tight in the vicinity of Gly85.
The reduction of α-keto amides derived from (2R,5R)-trans-2,5-bis(methoxymethoxymethyl)pyrrolidine [(R)-BMOMP, 1] with LiBEt3H or KBEt3H proceeded with high diastereoselectivity (up to 99% ds) to afford the corresponding α-hydroxy amides in good yields. The additive effect of crown ethers or metallic salts on the stereoselectivity was also examined.
The nucleophilic addition of organometallics to α-keto amides having (2R,5R)-trans-2,5-bis(methoxymethoxymethyl)pyrrolidine as a chiral auxiliary was studied and it was found that the sense and degree of diastereoselection strongly depended on organometallics and the reaction conditions used and that the diastereomeric hydroxy amides was obtained with high stereoselectivity by the choice of appropriate reaction conditions.
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