Abstract
Ketoreductases capable of performing chiral selective reduction in tert-butyl[5-(4-cyanobenzoyl)-2-fluorophenyl]carbamate to tert-butyl{5-[(4-cyanophenyl)(hydroxy)methyl]-2-fluorophenyl}carbamate were screened, and ES-KRED-213 and KRED-P1-H01 were the best ones for targeted biotransformation. Dimethylsulfoxide (10% V/V) was the best co-solvent, and the optimum parameters were 40 °C, pH 7.0, 10% enzyme loading, and 100 g/L substrate loading for maximum conversion and > 99% chiral selectivity. Gram scale batches were performed at different temperature (30 °C, 40 °C, and 50 °C), enzyme loading (5%, 10%, 15%, and 20%), and substrate loading (50 g/L, 100 g/L, and 150 g/L) and assessed for timely progression of reaction, and then, product formation and yield were calculated. ES-KRED-213 showed 99.4% conversion, 4.9 g/50 mL product formation, with an actual product recovery of 4.2 g corresponding to a product yield 85% with respect to the product formed after reaction. The purity of tert-butyl{5-[(4-cyanophenyl)(hydroxy)methyl]-2-fluorophenyl}carbamate formed was ≥ 99% (RP-HPLC) and chiral purity ≥ 99% (NP-HPLC). The recovered product was confirmed and characterized with instrumental analysis using HPLC analysis, specific optical rotation, melting point and boiling point, LC–MS, ATR-FTIR, 1H NMR, and 13C NMR.
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Acknowledgements
The authors are grateful to the Dean, School of Basic and Applied Sciences, and Vice-Chancellor, Galgotias University, Greater Noida for providing required facilities to carry out this research work.
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Design of experiments was contributed by AGK and ABM; performance of experiment was contributed by AGK; analysis and interpretation of data were contributed by AGK, AKJ, and ABM; manuscript draft and compilation were contributed by AGK and ABM; manuscript correction and revision were contributed by AGK, AKJ, and ABM.
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Khatik, A.G., Muley, A.B. & Jain, A.K. Ketoreductase-assisted synthesis of chiral selective tert-butyl{5-[(4-cyanophenyl)(hydroxy)methyl]-2-fluorophenyl}carbamate: process minutiae, optimization and characterization. Chem. Pap. 77, 3213–3227 (2023). https://doi.org/10.1007/s11696-023-02698-3
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DOI: https://doi.org/10.1007/s11696-023-02698-3