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

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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• 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.