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


(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.


Alcohol dehydrogenase Enantioselectivity Directed evolution 


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)


  1. Alanvert E, Doherty C, Moody TS, Nesbit N, Rowan AS, Taylor SJC, Vaughan F, Vaughan T, Wiffen J, Wilson I (2009) Highly stereoselective biocatalytic reduction of alpha-halo ketones. Tetrahedron Asymmetry 20(21):2462–2466CrossRefGoogle Scholar
  2. Cahn JKB, Werlang CA, Baumschlager A, Brinkmann-Chen S, Mayo SL, Arnold FH (2017) A general tool for engineering the NAD/NADP cofactor preference of oxidoreductases. ACS Synth Biol 6(2):326–333CrossRefGoogle Scholar
  3. Chen L-F, Fan H-Y, Zhang Y-P, Wu K, Wang H-L, Lin J-P, Wei D-Z (2017a) Development of a practical biocatalytic process for (S)-N-Boc-3-hydroxypiperidine synthesis. Tetrahedron Lett 58(16):1644–1650CrossRefGoogle Scholar
  4. Chen L-F, Zhang Y-P, Fan H-Y, Wu K, Lin J-P, Wang H-L, Wei D-Z (2017b) Efficient bioreductive production of (R)-N-Boc-3-hydroxypiperidine by a carbonyl reductase. Catal Commun 97:5–9CrossRefGoogle Scholar
  5. de Miranda AS, Simon RC, Grischek B, de Paula GC, Horta BAC, de Miranda LSM, Kroutil W, Kappe CO, de Souza ROMA (2015) Chiral Chlorohydrins from the biocatalyzed reduction of Chloroketones: chiral building blocks for antiretroviral drugs. ChemCatChem 7(6):984–992CrossRefGoogle Scholar
  6. Filling C, Berndt KD, Benach J, Knapp S, Prozorovski T, Nordling E, Ladenstein R, Jornvall H, Oppermann U (2002) Critical residues for structure and catalysis in short-chain dehydrogenases/reductases. J Biol Chem 277(28):25677–25684CrossRefGoogle Scholar
  7. Gong P-F, Xu J-H (2005) Bio-resolution of a chiral epoxide using whole cells of Bacillus megaterium ECU1001 in a biphasic system. Enzyme Eng 36(2):252–257Google Scholar
  8. Honda Y, Katayama S, Kojima M, Suzuki T, Kishibata N, Izawa K (2004) New approaches to the industrial synthesis of HIV protease inhibitors. Org Biomol Chem 2(14):2061–2070CrossRefGoogle Scholar
  9. Huisman GW, Liang J, Krebber A (2010) Practical chiral alcohol manufacture using ketoreductases. Curr Opin Chem Biol 14(2):122–129CrossRefGoogle Scholar
  10. Laane C, Boeren S, Vos K, Veeger C (2008) Rules for optimization of biocatalysis in organic solvents. Biotechnol Bioeng 102(1):1–8CrossRefGoogle Scholar
  11. Li H, Zhu D, Hua L, Biehl ER (2009) Enantioselective reduction of Diaryl ketones catalyzed by a carbonyl reductase from Sporobolomyces salmonicolorand its mutant enzymes. Adv Synth Catal 351(4):583–588CrossRefGoogle Scholar
  12. Liang J, Lalonde J, Borup B, Mitchell V, Mundorff E, Trinh N, Kochrekar DA, Nair Cherat R, Pai GG (2010) Development of a biocatalytic process as an alternative to the (−)-DIP-cl-mediated asymmetric reduction of a key intermediate of Montelukast. Org Process Res Dev 14(1):193–198CrossRefGoogle Scholar
  13. Liu Z-Q, Ye J-J, Shen Z-Y, Hong H-B, Yan J-B, Lin Y, Chen Z-X, Zheng Y-G, Shen Y-C (2014) Upscale production of ethyl (S)-4-chloro-3-hydroxybutanoate by using carbonyl reductase coupled with glucose dehydrogenase in aqueous-organic solvent system. Appl Microbiol Biotechnol 99(5):2119–2129CrossRefGoogle Scholar
  14. Liu ZQ, Wu L, Zheng L, Wang WZ, Zhang XJ, Jin LQ, Zheng YG (2018) Biosynthesis of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate by carbonyl reductase from Rhodosporidium toruloides in mono and biphasic media. Bioresour Technol 249:161–167CrossRefGoogle Scholar
  15. Man H, Loderer C, Ansorge-Schumacher MB, Grogan G (2014) Structure of NADH-dependent carbonyl reductase (CPCR2) from Candida parapsilosis provides insight into mutations that improve catalytic properties. ChemCatChem 6(4):1103–1111CrossRefGoogle Scholar
  16. Molina J-M, Andrade-Villanueva J, Echevarria J, Chetchotisakd P, Corral J, David N, Moyle G, Mancini M, Percival L, Yang R, Thiry A, McGrath D (2008) Once-daily atazanavir/ritonavir versus twice-daily lopinavir/ritonavir, each in combination with tenofovir and emtricitabine, for management of antiretroviral-naive HIV-1-infected patients: 48 week efficacy and safety results of the CASTLE study. Lancet 372(9639):646–655CrossRefGoogle Scholar
  17. Musa MM, Phillips RS (2011) Recent advances in alcohol dehydrogenase-catalyzed asymmetric production of hydrophobic alcohols. Catal Sci Technol 1(8):1311–1323CrossRefGoogle Scholar
  18. Patel RN (1999) Biocatalytic synthesis of chiral intermediates for antiviral and antihypertensive drugs. J Am Oil Chem Soc 76(11):1275–1281CrossRefGoogle Scholar
  19. Patel RN (2006) Biocatalysis: synthesis of chiral intermediates for pharmaceuticals. Curr Org Chem 10(11):1289–1321CrossRefGoogle Scholar
  20. Reetz MT, Carballeira JD (2007) Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes. Nat Protoc 2(4):891–903CrossRefGoogle Scholar
  21. Reetz MT, Bocola M, Carballeira JD, Zha D, Vogel A (2005) Expanding the range of substrate acceptance of enzymes: combinatorial active-site saturation test. Angew Chem Int Ed 44(27):4192–4196CrossRefGoogle Scholar
  22. Reetz MT, Prasad S, Carballeira JD, Gumulya Y, Bocola M (2010) Iterative saturation mutagenesis accelerates laboratory evolution of enzyme Stereoselectivity: rigorous comparison with traditional methods. J Am Chem Soc 132(26):9144–9152CrossRefGoogle Scholar
  23. Saag MS, Benson CA, Gandhi RT, Hoy JF, Landovitz RJ, Mugavero MJ, Sax PE, Smith DM, Thompson MA, Buchbinder SP, Del Rio C, Eron JJ Jr, Fatkenheuer G, Gunthard HF, Molina JM, Jacobsen DM, Volberding PA (2018) Antiretroviral drugs for treatment and prevention of HIV infection in adults: 2018 recommendations of the international antiviral society-USA panel. JAMA 320(4):379–396CrossRefGoogle Scholar
  24. Shang Y-P, Chen Q, Kong X-D, Zhang Y-J, Xu J-H, Yu H-L (2017) Efficient synthesis of (R)-2-Chloro-1-(2,4-dichlorophenyl)ethanol with a Ketoreductase from Scheffersomyces stipitis CBS 6045. Adv Synth Catal 359(3):426–431CrossRefGoogle Scholar
  25. Tang Y, Zhang G, Wang Z, Liu D, Zhang L, Zhou Y, Huang J, Yu F, Yang Z, Ding G (2018) Efficient synthesis of a (S)-fluoxetine intermediate using carbonyl reductase coupled with glucose dehydrogenase. Bioresour Technol 250:457–463CrossRefGoogle Scholar
  26. Wachtmeister J, Rother D (2016) Recent advances in whole cell biocatalysis techniques bridging from investigative to industrial scale. Curr Opin Biotechnol 42:169–177CrossRefGoogle Scholar
  27. Wang Y, Dai W, Liu Y, Zhang Z, Zhou J, Xu G, Ni Y (2018) Fine tuning the enantioselectivity and substrate specificity of alcohol dehydrogenase from Kluyveromyces polysporus by single residue at 237. Catal Commun 108:1–6CrossRefGoogle Scholar
  28. Wu K, Wang H, Chen L, Fan H, Zhao Z, Wei D (2016) Practical two-step synthesis of enantiopure styrene oxide through an optimized chemoenzymatic approach. Appl Microbiol Biotechnol 100(20):8757–8767CrossRefGoogle Scholar
  29. Xu Z, Singh J, Schwinden MD, Zheng B, Kissick TP, Patel B, Humora MJ, Quiroz F, Dong L, Hsieh D-M, Heikes JE, Pudipeddi M, Lindrud MD, Srivastava SK, Kronenthal DR, Mueller RH (2002) Process Research and Development for an efficient synthesis of the HIV protease inhibitor BMS-232632. Org Process Res Dev 6(3):323–328CrossRefGoogle Scholar
  30. Xu G-C, Yu H-L, Zhang X-Y, Xu J-H (2012) Access to optically active aryl Halohydrins using a substrate-tolerant carbonyl reductase discovered from Kluyveromyces thermotolerans. ACS Catal 2(12):2566–2571CrossRefGoogle Scholar
  31. Yang H, Huo N, Yang P, Pei H, Lv H, Zhang X (2015) Rhodium catalyzed asymmetric hydrogenation of 2-pyridine ketones. Org Lett 17(17):4144–4147CrossRefGoogle Scholar
  32. Zhou J, Wang Y, Xu G, Wu L, Han R, Schwaneberg U, Rao Y, Zhao Y-L, Zhou J, Ni Y (2018) Structural insight into enantioselective inversion of an alcohol dehydrogenase reveals a “polar gate” in Stereorecognition of Diaryl ketones. J Am Chem Soc 140(39):12645–12654CrossRefGoogle Scholar

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

Personalised recommendations