Organic Letters
Letter
the para-halogen atom type, the activity was decreased in order
of F, Cl, and Br (substrates 1e, 1h, and 1i). The ortho-
substitution, such as CH3 (1b) and Cl (1f), showed positive
effects on enzyme activity and stereoselectivity for SSCR wild-
type. For mutant M4, ortho-substitution showed less effect on
the enzyme activity but a positive effect on the stereoselectivity.
When the 2-substituent was bulky, such as β-naphthyl (1j), the
stereoselectivity was reduced. The lower stereoselectivity was
also observed when the 2-substituent was a less steric group,
such as a vinyl (1k) or ethynyl (1l) group. Similar stereo-
selectivity for the reduction of 1k and 1l was observed using
baker’s yeast.25 The preparative-scale reduction showed that by
monitoring the reaction the diol products could be controlled
below 2% when the conversion reached >98% (1a−1h). Mutant
M4 serves as an excellent catalyst for the synthesis of (2S,3S)-
2,2-substituted 3-hydroxycyclopentanones via reductive desym-
metrization of 1a and its derivatives.
In summary, the carbonyl reductase SSCR was engineered
through structure-guided site-subsaturation mutagenesis, and
the high-throughput colorimetric screening resulted in mutant
M4 (T134I/V135I/P243F/Q245G) with enhanced activity and
improved stereoselectivity for the desymmetric reduction of
various 2-benzyl-substituted 1,3-cyclopentanediones into the
corresponding (2S,3S)-ketols. In some cases, the (2S,3S)-ketols
were obtained as the sole product. Interestingly, the reduction of
the second carbonyl group in these diketones was observed only
after the reduction of the first one proceeded to completion. The
enzyme activity and stereoselectivity of SSCR were affected by
residues of the substrate binding site and the 2-substituent of
1,3-cyclopentanediones. While the chemical reduction of 2,2-
disubstituted-1,3-cyclopentanediones leads to the preparation
of (2R,3R)-ketols and the enzymatic reduction by mutant
RasADH affords (2R,3S)-ketols, the current study offers an
efficient method to access the (2S,3S)-ketols, a class of versatile
chiral building blocks for the synthesis of many bioactive natural
products and pharmaceuticals. Until now, three of the four
stereoisomers for the reduction of 2,2-disubstituted-1,3-cyclo-
pentanediones have been prepared by chemical or enzymatic
approaches, and the preparation of the only inaccessible
(2S,3R)-stereoisomer is currently being pursued in our
laboratory.
Academy of Sciences, Tianjin 300308, China; Email: wu_qq@
Dunming Zhu − National Engineering Laboratory for Industrial
Enzymes and Tianjin Engineering Research Center of Biocatalytic
Technology, Tianjin Institute of Industrial Biotechnology, Chinese
Academy of Sciences, Tianjin 300308, China; University of
Chinese Academy of Sciences, Beijing 100049, China;
Authors
Juan Li − National Engineering Laboratory for Industrial Enzymes
and Tianjin Engineering Research Center of Biocatalytic
Technology, Tianjin Institute of Industrial Biotechnology, Chinese
Academy of Sciences, Tianjin 300308, China; University of
Chinese Academy of Sciences, Beijing 100049, China
Jinhui Feng − National Engineering Laboratory for Industrial
Enzymes and Tianjin Engineering Research Center of Biocatalytic
Technology, Tianjin Institute of Industrial Biotechnology, Chinese
Academy of Sciences, Tianjin 300308, China
Jingyao Gong − National Engineering Laboratory for Industrial
Enzymes and Tianjin Engineering Research Center of Biocatalytic
Technology, Tianjin Institute of Industrial Biotechnology, Chinese
Academy of Sciences, Tianjin 300308, China; Department of
Medicinal Chemistry, School of Pharmacy Southwest Medical
University, Luzhou, Sichuan 646000, China
Yunfeng Cui − National Engineering Laboratory for Industrial
Enzymes and Tianjin Engineering Research Center of Biocatalytic
Technology, Tianjin Institute of Industrial Biotechnology, Chinese
Academy of Sciences, Tianjin 300308, China
Hongliu Zhang − National Engineering Laboratory for Industrial
Enzymes and Tianjin Engineering Research Center of Biocatalytic
Technology, Tianjin Institute of Industrial Biotechnology, Chinese
Academy of Sciences, Tianjin 300308, China
Dandan Bu − National Engineering Laboratory for Industrial
Enzymes and Tianjin Engineering Research Center of Biocatalytic
Technology, Tianjin Institute of Industrial Biotechnology, Chinese
Academy of Sciences, Tianjin 300308, China
Complete contact information is available at:
Notes
ASSOCIATED CONTENT
* Supporting Information
■
The authors declare no competing financial interest.
sı
The Supporting Information is available free of charge at
ACKNOWLEDGMENTS
■
This work was financially supported by the National Key R&D
Program of China (no. 2018YFA0901602), the National
Natural Science Foundation of China (Nos. 1602246 and
21778072), and Tianjin Municipal Science and Technology
Commission (No. 15PTCYSY00020).
Detailed experimental procedures; screening results of
carbonyl reductases toward substrate 1a, primers used for
mutation, HPLC separation conditions, NMR data and
spectra, HRMS data, and HPLC spectra, respectively
REFERENCES
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AUTHOR INFORMATION
Corresponding Authors
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