CC BY-ND-NC 4.0 · Synlett 2019; 30(04): 378-382
DOI: 10.1055/s-0037-1611662
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Engineered Cytochrome c-Catalyzed Lactone-Carbene B–H Insertion

Kai Chen 
a   Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena, CA 91125, USA   Email: frances@cheme.caltech.edu
,
Xiongyi Huang 
a   Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena, CA 91125, USA   Email: frances@cheme.caltech.edu
,
Shuo-Qing Zhang 
b   Department of Chemistry, Zhejiang University, Hangzhou, Zhejiang 31007, P. R. of China   Email: hxchem@zju.edu.cn
,
Andrew Z. Zhou
a   Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena, CA 91125, USA   Email: frances@cheme.caltech.edu
,
S. B. Jennifer Kan 
a   Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena, CA 91125, USA   Email: frances@cheme.caltech.edu
,
Xin Hong  *
b   Department of Chemistry, Zhejiang University, Hangzhou, Zhejiang 31007, P. R. of China   Email: hxchem@zju.edu.cn
,
Frances H. Arnold  *
a   Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena, CA 91125, USA   Email: frances@cheme.caltech.edu
› Author Affiliations

Financial support from the NSF Division of Molecular and Cellular Biosciences grant MCB-1513007, National Natural Science Foundation of China (21702182), Zhejiang University, the Chinese “Thousand Youth Talents Plan”, and the “Fundamental Research Funds for the Central Universities” is gratefully acknowledged. K.C. thanks the Resnick Sustainability Institute at Caltech for fellowship support. X.H. is supported by an NIH pathway to independence award (grant K99GM129419).
Further Information

Publication History

Received: 26 November 2018

Accepted after revision: 05 January 2019

Publication Date:
14 January 2019 (online)

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Published as part of the 30 Years SYNLETT – Pearl Anniversary Issue

Abstract

Previous work has demonstrated that variants of a heme protein, Rhodothermus marinus cytochrome c (Rma cyt c), catalyze abiological carbene boron–hydrogen (B–H) bond insertion with high efficiency and selectivity. Here we investigated this carbon–boron bond-forming chemistry with cyclic, lactone-based carbenes. Using directed evolution, we obtained a Rma cyt c variant BORLAC that shows high selectivity and efficiency for B–H insertion of 5- and 6-membered lactone carbenes (up to 24,500 total turnovers and 97.1:2.9 enantiomeric ratio). The enzyme shows low activity with a 7-membered lactone carbene. Computational studies revealed a highly twisted geometry of the 7-membered lactone carbene intermediate relative to 5- and 6-membered ones. Directed evolution of cytochrome c together with computational characterization of key iron-carbene intermediates has allowed us to expand the scope of enzymatic carbene B–H insertion to produce new lactone-based organoborons.

Key words

cytochrome c - carbene - organoboron - lactones - biocatalyst - directed evolution

Supporting Information

    Supporting information for this article is available online at https://doi.org/10.1055/s-0037-1611662.
  • Supporting Information
 

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  • 37 General ProcedureTo a 20 mL vial were added a suspension of E. coli expressing Rma cytochrome c variant BORLAC (5 mL, OD600 = 15), borane (150 μL of a 400 mM stock solution in MeCN, 0.06 mmol), lactone diazo compound (150 μL of a 400 mM stock solution in MeCN, 0.06 mmol), and d-glucose (50 mM) in M9-N buffer (pH 7.4) under anaerobic conditions. The vial was capped and shaken (480 rpm) at room temperature for 18 h. Reactions were set up in quadruplicate. Upon completion, reactions in replicate were combined and transferred to 50 mL centrifuge tubes. The reaction vials were washed with water (2 × 2 mL) followed by a mixed organic solvent (hexane/ethyl acetate = 1:1, 3 × 2 mL). The washing solution was combined with the reaction mixture in the centrifuge tubes. An additional 15 mL of hexane/ethyl acetate solvent was added to every tube. The tube was then vortexed (3 × 1 min) and shaken vigorously, and centrifuged (5,000 × g, 5 min). The organic layer was separated and the aqueous layer was subjected to three more rounds of extraction. The organic layers were combined, dried over Na2SO4 and concentrated under reduced pressure. Purification by silica gel column chromatography with hexane/(ethyl acetate/acetone 3:7) as eluent afforded the desired lactone-based organo­boranes. Enantiomeric ratio (e.r.) was measured by chiral HPLC. TTN was calculated based on measured protein concentration and isolated product yield. Product 3a was obtained as a white solid (41.4 mg, 89%, 1290 TTN). 1H NMR (400 MHz, CDCl3): δ = 6.83 (s, 2 H), 4.45 (dddd, J = 10.7, 8.1, 6.8, 1.0 Hz, 1 H), 4.27 (tq, J = 9.0, 1.1 Hz, 1 H), 3.83–3.69 (m, 6 H), 2.53–2.25 (m, 1 H), 2.01–1.93 (m, 1 H), 1.88–1.80 (m, 1 H), 1.79–1.16 (m, 2 H); 13C NMR (101 MHz, CDCl3): δ = 187.91, 120.62, 67.86, 36.15, 30.86; 11B NMR (128 MHz, CDCl3): δ = –27.08 (t, J = 89.6 Hz); HRMS (FAB+): m/z [(M + H+) – H2] calcd for C9H14O2N2B: 193.1148; found: 193.1144; Chiral HPLC (Chiralpak IC, 40% i-PrOH in hexane, flow rate: 1.5 mL/min, temperature: 32 °C, λ = 235 nm): t R (major) = 12.857 min, t R (minor) = 10.991 min; 96.2:3.8 e.r.