A strategy to identify a ketoreductase that preferentially synthesizes pharmaceutically relevant (S)-alcohols using whole-cell biotransformation

Abstract Introduction Chemical industries are constantly in search of an expeditious and environmentally benign method for producing chiral synthons. Ketoreductases have been used as catalysts for enantioselective conversion of desired prochiral ketones to their corresponding alcohol. We chose reported promiscuous ketoreductases belonging to different protein families and expressed them in E. coli to evaluate their ability as whole-cell catalysts for obtaining chiral alcohol intermediates of pharmaceutical importance. Apart from establishing a method to produce high value (S)-specific alcohols that have not been evaluated before, we propose an in silico analysis procedure to predict product chirality. Results Six enzymes originating from Sulfolobus sulfotaricus, Zygosaccharomyces rouxii, Hansenula polymorpha, Corynebacterium sp. ST-10, Synechococcus sp. PCC 7942 and Bacillus sp. ECU0013 with reported efficient activity for dissimilar substrates are compared here to arrive at an optimal enzyme for the method. Whole–cell catalysis of ketone intermediates for drugs like Aprepitant, Sitagliptin and Dolastatin using E. coli over-expressing these enzymes yielded (S)-specific chiral alcohols. We explain this chiral specificity for the best-performing enzyme, i.e., Z. rouxii ketoreductase using in silico modelling and MD simulations. This rationale was applied to five additional ketones that are used in the synthesis of Crizotinib, MA-20565 (an antifungal agent), Sulopenem, Rivastigmine, Talampanel and Barnidipine and predicted the yield of (S) enantiomers. Experimental evaluation matched the in silico analysis wherein ~ 95% (S)-specific alcohol with a chemical yield of 23–79% was obtained through biotransformation. Further, the cofactor re-cycling was optimized by switching the carbon source from glucose to sorbitol that improved the chemical yield to 85–99%. Conclusions Here, we present a strategy to synthesize pharmaceutically relevant chiral alcohols by ketoreductases using a cofactor balanced whole-cell catalysis scheme that is useful for the industry. Based on the results obtained in these trials, Zygosaccharomyces rouxii ketoreductase was identified as a proficient enzyme to obtain (S)-specific alcohols from their respective ketones. The whole–cell catalyst when combined with nutrient modulation of using sorbitol as a carbon source helped obtain high enantiomeric and chemical yield

Structure-Activity Relationship Studies, SPR Affinity Characterization, and Conformational Analysis of Peptides That Mimic the HNK-1 Carbohydrate Epitope

The design of molecules that mimic biologically relevant glycans is a significant goal for understanding important biological processes and may lead to new therapeutic and diagnostic agents. In this study we focused our attention on the trisaccharide human natural killer cell-1 (HNK-1), considered the antigenic determinant of myelin-associated glycoprotein and the target of clinically relevant auto-antibodies in autoimmune neurological disorders such as IgM monoclonal gammopathy and demyelinating polyneuropathy. We describe a structure-activity relationship study based on surface plasmon resonance binding affinities aimed at the optimization of a peptide that mimics the HNK-1 minimal epitope. We developed a cyclic heptapeptide that shows an affinity of 1.09×10(-7)  m for a commercial anti-HNK1 mouse monoclonal antibody. Detailed conformational analysis gave possible explanations for the good affinity displayed by this novel analogue, which was subsequently used as an immunological probe. However, preliminary screening indicates that patients' sera do not specifically recognize this peptide, showing that murine monoclonal antibodies cannot be used as a guide to select immunological probes for the detection of clinically relevant human auto-antibodies