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Efficient synthesis of an antiviral drug intermediate using an enhanced short-chain dehydrogenase in an aqueous-organic solvent system

  • Kai Wu
  • Kun Zheng
  • Liangbin Xiong
  • Zhijun Yang
  • Zhiteng Jiang
  • Xiangguo Meng
  • Lei ShaoEmail author
Biotechnologically relevant enzymes and proteins
  • 58 Downloads

Abstract

(2R,3S)-N-tert-Butoxycarbonyl-3-amino-1-chloro-2-hydroxy-4-phenylbutane (1b) is key for the synthesis of the antiviral drug atazanavir. It can be obtained via the stereoselective bioreduction of (3S)-3-(N-Boc-amino)-1-chloro-4-phenyl-butanone (1a) with short-chain dehydrogenase/reductase (SDR). However, the stereoselective bioreduction of this hydrophobic and bulky substrate still remained a challenge because of the steric hindrance effect and low mass transfer rate. In this study, SDR isolated from Novosphingobium aromaticivorans (NaSDR) having low activity to 1a, which was engineered to enhance catalytic efficiency through active pocket iterative saturation mutagenesis (ISM). The obtained mutant (muSDR) (G141V/I195L) had 3.57 times higher kcat than the wild type (WT) towards 1a. Molecular docking analysis revealed considerable differences in the distance between the substrate and catalytic residues in WT and mutant SDR. Moreover, muSDR reduced 15 ketones with excellent enantioselectivity, indicating broad substrate acceptance. After optimization of expression and reaction conditions, the conversion was completed in a scale-up reaction (500 mL) using 50% toluene with 500 mM substrate without additional NADH. These results show that muSDR may be a valuable biocatalyst for future industrial applications.

Keywords

Alcohol dehydrogenase Enantioselectivity Directed evolution 

Notes

Funding information

This work was supported in part by grants from the National Natural Science Foundation of China (No. 81773616), Natural Science Foundation of Shanghai (No. 16ZR1435400), Program of Shanghai Technology Research Leader (No. 17XD1423200), and the Seed Fund Program of Shanghai University of Medicine and health Sciences (SFP-18-22-07-002).

Compliance with ethical standards

Conflict of interests

The authors declare that they have no conflicts of interests.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2019_9781_MOESM1_ESM.pdf (521 kb)
ESM 1 (PDF 520 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Kai Wu
    • 1
    • 2
  • Kun Zheng
    • 3
  • Liangbin Xiong
    • 2
  • Zhijun Yang
    • 1
    • 2
  • Zhiteng Jiang
    • 1
    • 2
  • Xiangguo Meng
    • 1
    • 2
  • Lei Shao
    • 2
    • 3
    Email author
  1. 1.School of PharmacyShanghai University of Medicine and Health SciencesShanghaiChina
  2. 2.Microbial Pharmacology LaboratoryShanghai University of Medicine and Health SciencesShanghaiChina
  3. 3.State Key Laboratory of New Drug and Pharmaceutical ProcessShanghai Institute of Pharmaceutical IndustryShanghaiChina

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