Presentation Information
[4ASBA-09]Database Mining-driven Enzyme Discovery for Stereodivergent
Cyclopropanation
○Shunsuke Kato1 (1. Kobe University (Japan))
Keywords:
Biocatalysis,Enzyme,Heme,Asymmetric Synthesis,Cyclopropanation
With the rapid advancement of biotechnology, biocatalysis has emerged as a promising and sustainable approach for the synthesis of valuable chemicals. A major challenge associated with biocatalysis is to expand the catalytic repertoire of enzymes to meet the requirements of synthetic chemistry. In this context, our research group is working toward the discovery of novel enzymes capable of catalyzing abiotic chemical transformation by leveraging principles of organic chemistry. In this presentation, we will introduce our recent progress in identifying new bacterial enzymes capable of catalyzing the stereodivergent synthesis of 1,2-disubstituted cyclopropanes.
We first developed a new in vitro high-throughput screening platform, designated as CSAP system, to accelerate throughput of the enzyme discovery.1 Since this CSAP system enables rapid preparation of purified enzyme libraries, catalytic performance of the target enzymes can be easily assessed in a chemically defined in vitro condition. Taking this advantage of the CSAP system, we next performed in vitro screening of purified hemoprotein libraries to discover a novel enzyme which can catalyze asymmetric cyclopropanation between styrene (1) and ethyl diazoacetate (2). After screening various heme-dependent enzymes, which include cytochrome P450s, DyP-type peroxidases, catalases, and tryptophan 2,3-dioxygenases, two distinct hemoproteins capable of catalyzing diastereodivergent synthesis of 1,2-disubstituted cyclopropanes were successfully identified. Bacterial hemoglobin from Starkeya. novella (SnVHb) was found to produce trans-isomer of cyclopropane (S,S)-3 with high yield and excellent stereoselectivity (kcat = 4.9 × 104 min−1, KM = 3.4 mM, >96% de, >98%ee). In contrast, truncated hemoglobin from Streptosporangium roseum (SrTrHb) gave a thermodynamically unfavored cis-isomer (S,R)-4 with notable stereoselectivity (kcat = 68 min−1, KM = 4.2 mM, >96% de, >98%ee). Furthermore, additional screening using principal component analysis (PCA)-based clustering has afforded two hemoproteins which can selectively produce two another stereoisomer (S,S)-3 and (R,S)-4, ultimately leading to the stereodivergent synthesis of all the four possible stereoisomers of ethyl 2-phenylcyclopropanecarboxylates.2 These results highlight that natural sequence diversity can serve as a source of highly active and stereoselective catalysts for abiotic transformations.
1 S. Kato et al. Angew. Chem. Int. Ed. 2023, 62, e202303764.
2 S. Kato et al. Angew. Chem. Int. Ed. 2026, 65, e202526025.
We first developed a new in vitro high-throughput screening platform, designated as CSAP system, to accelerate throughput of the enzyme discovery.1 Since this CSAP system enables rapid preparation of purified enzyme libraries, catalytic performance of the target enzymes can be easily assessed in a chemically defined in vitro condition. Taking this advantage of the CSAP system, we next performed in vitro screening of purified hemoprotein libraries to discover a novel enzyme which can catalyze asymmetric cyclopropanation between styrene (1) and ethyl diazoacetate (2). After screening various heme-dependent enzymes, which include cytochrome P450s, DyP-type peroxidases, catalases, and tryptophan 2,3-dioxygenases, two distinct hemoproteins capable of catalyzing diastereodivergent synthesis of 1,2-disubstituted cyclopropanes were successfully identified. Bacterial hemoglobin from Starkeya. novella (SnVHb) was found to produce trans-isomer of cyclopropane (S,S)-3 with high yield and excellent stereoselectivity (kcat = 4.9 × 104 min−1, KM = 3.4 mM, >96% de, >98%ee). In contrast, truncated hemoglobin from Streptosporangium roseum (SrTrHb) gave a thermodynamically unfavored cis-isomer (S,R)-4 with notable stereoselectivity (kcat = 68 min−1, KM = 4.2 mM, >96% de, >98%ee). Furthermore, additional screening using principal component analysis (PCA)-based clustering has afforded two hemoproteins which can selectively produce two another stereoisomer (S,S)-3 and (R,S)-4, ultimately leading to the stereodivergent synthesis of all the four possible stereoisomers of ethyl 2-phenylcyclopropanecarboxylates.2 These results highlight that natural sequence diversity can serve as a source of highly active and stereoselective catalysts for abiotic transformations.
1 S. Kato et al. Angew. Chem. Int. Ed. 2023, 62, e202303764.
2 S. Kato et al. Angew. Chem. Int. Ed. 2026, 65, e202526025.
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