A Biocatalytic Platform for Synthesis of Chiral α- Trifluoromethylated Organoborons

Abstract

There are few biocatalytic transformations that produce fluorine-containing molecules prevalent in modern pharmaceuticals. To expand the scope of biocatalysis for organofluorine synthesis, we have developed an enzymatic platform for highly enantioselective carbene B-H bond insertion to yield versatile α-trifluoromethylated (α-CF₃) organoborons, an important class of organofluorine molecules that contain stereogenic centers bearing both CF₃ and boron groups. In contrast to current "carbene transferase" enzymes that use a limited set of simple diazo compounds as carbene precursors, this system based on Rhodothermus marinus cytochrome c (Rma cyt c) can accept a broad range of trifluorodiazo alkanes and deliver versatile chiral α-CF₃ organoborons with total turnovers up to 2870 and enantiomeric ratios up to 98.5:1.5. Computational modeling reveals that this broad diazo scope is enabled by an active-site environment that directs the alkyl substituent on the heme CF₃-carbene intermediate toward the solvent-exposed face, thereby allowing the protein to accommodate diazo compounds with diverse structural features.

Figure 1

(a) Design of a general enzymatic platform for synthesis of chiral α-CF₃ organoborons. (b) On the left, overlay of Rma cyt c (PDB 3CP5, front loop region in blue), Rma TDE (PDB 6CUK, front loop region in orange), and Rma TDE with iron porphyrin carbene structure obtained from computational modeling (ref (52), front loop region in green). The large change in front loop structure highlights the impact loop mutations have on access to the active site of Rma cyt c. On the right, we propose that the active-site environment can be tuned to orient the heme-carbene intermediate such that the alkyl substituent R is solvent-exposed.

Figure 2

(a) X-ray crystal structure of wild-type Rma cyt c (PDB 3CP5). Residues targeted for site-saturation mutagenesis (V75, M99, M100, M103, and Y44) are shown in sticks. (b) Directed evolution of Rma cyt c for enantioselective synthesis of α-CF₃ organoborons with NHC borane 1 and diazo compound 2 as the model substrates. Reactions were performed in M9-N (pH 7.4) suspensions of Escherichia coli cells expressing Rma cyt c variants (OD₆₀₀ = 20). Standard reaction conditions were 10 mM borane substrate 1, 7.5 mM diazo substrate 2, room temperature under anaerobic conditions. Total turnovers (TTN) were defined as the amount of α-CF₃ organoboron product divided by the total amount of expressed Rma cyt c protein as determined by the hemochrome assay. The absolute configuration of product 2a was determined to be R based on the optical rotation of the derivatized alcohol. See the Supporting Information, section VII, for detailed experimental procedures. wt refers to wild-type Rma cyt c. Single-letter abbreviations for the amino acid residues: V, Val; S, Ser; M, Met; L, Leu; D, Asp; A, Ala; Y, Tyr; I, Ile.

Figure 3

(a) Scope of trifluorodiazo alkanes for carbene B–H insertion catalyzed by BOR-CF₃. Reactions were performed in M9-N (pH 7.4) suspensions of E. coli cells expressing BOR-CF₃ (OD₆₀₀ = 20). Standard reaction conditions were 10 mM borane substrate 1, 7.5 mM diazo substrate n, room temperature under anaerobic conditions. The absolute configuration of 2a was determined to be R based on the optical rotation of the derivatized alcohol (see the Supporting Information, section VII). (b) Preparative scale synthesis (0.2 mmol scale) and the derivatization of 2a to boronic acid 2c.

Figure 4

(a) The hydrogen bonding interactions between 75S and 71Y amide backbone and the empty surface area in a representative snapshot obtained from MD trajectories of BOR-CF₃ in the absence of diazo compound (see also Figures S1 and S2 in the Supporting Information). (b) Binding pose of diazo substrate 2 bound into the active site of BOR-CF₃, obtained from docking and constrained MD simulations (see Figure S3 and computational details in the SI). Direct comparison between the shape of the BOR-CF₃ active site and the docked diazo 2 shows the high complementarity achieved by evolution and introduction of V75S mutation. (c) Comparison of diazo 2 bound in the BOR-CF₃ structure and the DFT-optimized model transition state (TS) geometry for formation of the iron porphyrin carbene (IPC) intermediate (see the SI, Figure S8). The diazo 2 in this binding pose represents a near attack conformation that leads to the transition state for the generation of the iron porphyrin carbene intermediate.

Figure 5

(a) Overlay of snapshots extracted from a 500 ns MD trajectory of BOR-CF₃-bound trifluoroalkyl-carbene intermediate 2b (see also Figures S4 and S5 in the Supporting Information). (b) Representative snapshots of the two main conformations explored by trifluoroalkyl-carbene intermediate 2b in BOR-CF₃ during MD trajectories. Blue surface represents the inner void cavity generated in the protein active site next to the iron porphyrin carbene. (c) DFT-optimized transition state (TS) for B–H carbene insertion in a model system (see also Figure S7). Key distances and angles are given in Å and degrees.