Engineered Cytochrome c-Catalyzed Lactone-Carbene B–H Insertion

Kai Chen; Xiongyi Huang; Shuo-Qing Zhang; Andrew Z. Zhou; S. B. Jennifer Kan; Xin Hong; Frances H. Arnold

doi:10.1055/s-0037-1611662

Synlett, Table of Contents

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

^aDivision of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena, CA 91125, USA Email: frances@cheme.caltech.edu

,

Xiongyi Huang

^aDivision of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena, CA 91125, USA Email: frances@cheme.caltech.edu

,

Shuo-Qing Zhang

^bDepartment of Chemistry, Zhejiang University, Hangzhou, Zhejiang 31007, P. R. of China Email: hxchem@zju.edu.cn

,

Andrew Z. Zhou

^aDivision of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena, CA 91125, USA Email: frances@cheme.caltech.edu

,

S. B. Jennifer Kan

^aDivision of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena, CA 91125, USA Email: frances@cheme.caltech.edu

,

Xin Hong *

^bDepartment of Chemistry, Zhejiang University, Hangzhou, Zhejiang 31007, P. R. of China Email: hxchem@zju.edu.cn

,

Frances H. Arnold *

^aDivision of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena, CA 91125, USA Email: frances@cheme.caltech.edu

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Abstract

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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 BOR^LAC 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

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References

References
1 Fyfe JW. B, Watson AJ. B. Chem 2017; 3: 31
2 Leonori D, Aggarwal VK. Acc. Chem. Res. 2014; 47: 3174
3 Lennox AJ. J, Lloyd-Jones GC. Chem. Soc. Rev. 2014; 43: 412
4 Brown HC, Ramachandran PV. Pure Appl. Chem. 1991; 63: 307
5 Brown HC, Jadhav PK, Mandal AK. Tetrahedron 1981; 37: 3547
6 Miyaura N, Suzuki A. Chem. Rev. 1995; 95: 2457
7 Martin R, Buchwald SL. Acc. Chem. Res. 2008; 41: 1461
8 Boronic Acids: Preparation and Applications in Organic Synthesis and Medicine. Hall DG. Wiley-VCH; Weinheim: 2005
9 Das BC, Thapa P, Karki R, Schinke C, Das S, Kambhampati S, Banerjee SK, Veldhuizen PV, Verma A, Weiss LM, Evans T. Future Med. Chem. 2013; 5: 653
10 Dembitsky VM, Al Quntar AA, Srebnik M. Chem. Rev. 2011; 111: 209
11 Li X, Curran DP. J. Am. Chem. Soc. 2013; 135: 12076
12 Cheng Q.-Q, Zhu S.-F, Zhang Y.-Z, Xie X.-L, Zhou Q.-L. J. Am. Chem. Soc. 2013; 135: 14094
13 Chen D, Zhang X, Qi W.-Y, Xu B, Xu M.-H. J. Am. Chem. Soc. 2015; 137: 5268
14 Hyde S, Veliks J, Liégault B, Grassi D, Taillefer M, Gouverneur V. Angew. Chem. Int. Ed. 2016; 55: 3785
15 Yang J.-M, Li Z.-Q, Li M.-L, He Q, Zhu S.-F, Zhou Q.-L. J. Am. Chem. Soc. 2017; 139: 3784
16 Pang Y, He Q, Li Z.-Q, Yang J.-M, Yu J.-H, Zhu S.-F, Zhou Q.-L. J. Am. Chem. Soc. 2018; 140: 10663
17 Allen TH, Kawamoto T, Gardner S, Geib SJ, Curran DP. Org. Lett. 2017; 19: 3680
18 Yang J.-M, Zhao Y.-T, Li Z.-Q, Gu X.-S, Zhu S.-F, Zhou Q.-L. ACS Catal. 2018; 8: 7351
19 Kan SB. J, Huang X, Gumulya Y, Chen K, Arnold FH. Nature 2017; 552: 132
20 Huang X, Garcia-Borràs M, Miao K, Kan SB. J, Zutshi A, Houk KN, Arnold FH. ChemRxiv 2018;
21 Chen K, Zhang S.-Q, Brandenberg OF, Hong X, Arnold FH. J. Am. Chem. Soc. 2018; 140: 16402
22 Coelho PS, Brustad EM, Kannan A, Arnold FH. Science 2013; 339: 307
23 Coelho PS, Wang ZJ, Ener ME, Baril SA, Kannan A, Arnold FH, Brustad EM. Nat. Chem. Biol. 2013; 9: 485
24 Brandenberg OF, Fasan R, Arnold FH. Curr. Opin. Biotechnol. 2017; 47: 102
25 Chen K, Huang X, Kan SB. J, Zhang RK, Arnold FH. Science 2018; 360: 71
26 Kan SB. J, Lewis RD, Chen K, Arnold FH. Science 2016; 354: 1048
27 Lewis RD, Garcia-Borras M, Chalkley MJ, Buller AR, Houk KN, Kan SB. J, Arnold FH. Proc. Natl. Acad. Sci. U.S.A. 2018; 115: 7308
28 Khade RL, Fan W, Ling Y, Yang L, Oldfield E, Zhang Y. Angew. Chem. Int. Ed. 2014; 53: 7574
29 Sharon DA, Mallick D, Wang B, Shaik S. J. Am. Chem. Soc. 2016; 138: 9597
30 Wei Y, Tinoco A, Steck V, Fasan R, Zhang Y. J. Am. Chem. Soc. 2018; 140: 1649
31 Stelter M, Melo AM. P, Pereira MM, Gomes CM, Hreggvidsson GO, Hjorleifsdottir S, Saraiva LM, Teixeira M, Archer M. Biochemistry 2008; 47: 11953
32 Kille S, Acevedo-Rocha CG, Parra LP, Zhang Z.-G, Opperman DJ, Reetz MT, Acevedo JP. ACS Synth. Biol. 2013; 2: 83
33 Yang J.-M, Li Z.-Q, Zhu S.-F. Chin. J. Org. Chem. 2017; 37: 2481
34 Tehfe M.-A, Monot J, Malacria M, Fensterbank L, Fouassier J. P, Curran DP, Lacôte E, Lalevée J. ACS Macro Lett. 2012; 1: 92
35 Renata H, Lewis RD, Sweredoski MJ, Moradian A, Hess S, Wang ZJ, Arnold FH. J. Am. Chem. Soc. 2016; 138: 12527
36 DeAngelis A, Dmitrenko O, Fox JM. J. Am. Chem. Soc. 2012; 134: 11035
37 General ProcedureTo a 20 mL vial were added a suspension of E. coli expressing Rma cytochrome c variant BOR^LAC (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.

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