Improving the Enantioselectivity of CHMOBrevi1 for Asymmetric Synthesis of Podophyllotoxin Precursor |
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Authors: | Shou-Cheng Huang Yi-Ke Zhang Qiang Geng Qi-Kang Huang Prof Jian-He Xu Prof Yi-Feng Chen Prof Hui-Lei Yu |
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Affiliation: | 1. State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing, School of Biotechnology, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237 China;2. Key Laboratory for Advanced Materials, Joint International Research Laboratory of Precision Chemistry and Molecular Engineering, School of Chemistry and Molecular Engineering, East China University of Science & Technology, 130 Meilong Road, Shanghai, 200237 China |
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Abstract: | (R)-β-piperonyl-γ-butyrolactones are key building blocks for the synthesis of podophyllotoxin, which have demonstrated remarkable potential in cancer treatment. Baeyer-Villiger monooxygenases (BVMOs)-mediated asymmetric oxidation is a green approach to produce chiral lactones. While several BVMOs were able to oxidize the corresponding cyclobutanone, most BVMOs gave the (S) enantiomer while Cyclohexanone monooxygenase (CHMO) from Brevibacterium sp. HCU1 gave (R) enantiomer, but with a low enantioselectivity (75 % ee). In this study, we use a strategy called “focused rational iterative site-specific mutagenesis” (FRISM) at residues ranging from 6 Å from substrate. The mutations by using a restricted set of rationally chosen amino acids allow the formation of a small mutant library. By generating and screening less than 60 variants, we achieved a high ee of 96.8 %. Coupled with the cofactor regeneration system, 9.3 mM substrate was converted completely in a 100-mL scale reaction. Therefore, our work reveals a promising synthetic method for (R)-β-piperonyl-γ-butyrolactone with the highest enantioselectivity, and provides a new opportunity for the chem-enzymatic synthesis of podophyllotoxin. |
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Keywords: | asymmetric synthesis Baeyer-Villiger monooxygenases enantioselective modification podophyllotoxin (R)-β-piperonyl-γ-butyrolactone |
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