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

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 bondforming 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 7membered 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.

Iron catalysts with N-ligands for carbene transfer of diazo reagents

“…Please do not adjust margins Please do not adjust margins based procedures for the activation of less reactive hydrocarbons and for the formation of C-heteroatom bonds. In this context, the catalytic application of artificial metalloenzymes (ArMs) is assuming a particular importance in view of the efficiency of ArMs in mediating carbene insertion reactions into both C-H [159][160][161] and X-H [162][163][164][165][166][167][168] bonds (X = N, S, Si, B). However, all the ArMs applied in carbene insertion reactions have been synthesized by replacing aminoacidic residues in heme proteins (pathway a of Scheme 30) rather than embedding a synthetic molecule into the biological framework.…”

Exploring and Adapting the Molecular Selectivity of Artificial Metalloenzymes

“…Summarized in Figure 2, a plethora of research in literature has focused on expanding heme-containing proteins for carbene transfer reactions. With the number of literature examples growing constantly, these new-to-nature reactions have included aldehyde olefination, [14][15] alkyne cyclopropenation, [16][17] alkene cyclopropanation, [18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36] the Doyle-Kirmse reaction, 37 N−H bond insertion, [38][39][40] S−H bond insertion, 27,41 Si−H bond insertion, [42][43] B−H bond insertion, [44][45][46] and C-H insertion. [47][48][49] Other works have also shown high enantioselectivity with C(sp 3 )−H bond aminations that can proceed either intramolecularly, 36,[50][51][52][53][54][55][56]…”

“…As shown in Figure 2C, evolved variants were found to be effective catalysts for asymmetric Si-H bond, 42 and B−H bond insertion reactions. [44][45][46] For example, a triple mutant (V75T/M100D/M103E) Rma cyt c variant was shown to effectively catalyze the formation of organosilicon compounds under physiological conditions. 42 Compared to the wild type, this was done with an over 40-fold increase in reactivity and higher enantioselectivity (>99% ee).…”

The Power of Iron Catalysis in Diazo Chemistry

“…Cyclic lactone diazo compounds were the next choice of compounds to experience the power of directed evolution (Scheme 2c). 23 The rigidity of the resulting iron carbene intermediate was expected to exhibit distinctive conformational and electronic properties when compared to acyclic analogues. Indeed, when employing the variant BOR R1 which was used for B-H insertions with ethyl 2-diazopropanoate, high turnover and yield of borylation was obtained but with poor enantioselectivity (36% ee).…”