Abstract
The biocatalytic toolbox has recently been expanded to include enzyme-catalyzed carbene transfer reactions not occurring in Nature. Herein, we report the development of a biocatalytic strategy for the synthesis of enantioenriched α-trifluoromethyl amines through an asymmetric N–H carbene insertion reaction catalyzed by engineered variants of cytochrome c552 from Hydrogenobacter thermophilus. Using a combination of protein and substrate engineering, this metalloprotein scaffold was redesigned to enable the synthesis of chiral α-trifluoromethyl amino esters with up to >99% yield and 95:5 er using benzyl 2-diazotrifluoropropanoate as the carbene donor. When the diazo reagent was varied, the enantioselectivity of the enzyme could be inverted to produce the opposite enantiomers of these products with up to 99.5:0.5 er. This methodology is applicable to a broad range of aryl amine substrates, and it can be leveraged to obtain chemoenzymatic access to enantioenriched β-trifluoromethyl-β-amino alcohols and halides. Computational analyses provide insights into the interplay of protein- and reagent-mediated control on the enantioselectivity of this reaction. This work introduces the first example of a biocatalytic N–H carbenoid insertion with an acceptor–acceptor carbene donor, and it offers a biocatalytic solution for the enantioselective synthesis of α-trifluoromethylated amines as valuable synthons for medicinal chemistry and the synthesis of bioactive molecules.
Highlights
The incorporation of fluorine can favorably alter the physicochemical and biological properties of bioactive molecules.[1,2] Fluorine-containing building blocks are increasingly used in medicinal chemistry, as the introduction of fluorine substituents can improve the pharmacokinetic and pharmacological properties of small-molecule drugs, including their potency, cell permeability, and metabolic stability.[3,4]One group of organofluorines of great interest in drug discovery and development are chiral α-trifluoromethyl amine derivatives, such as substituted trifluoroethylamines[5,6] and αtrifluoromethyl amino esters.[7]
We tested the activity of wild-type sperm whale Mb and variants thereof toward catalyzing the conversion of p-anisidine 1a into αtrifluoromethyl amino ester 1b in the presence of ethyl 2diazo-3,3,3-trifluoropropanoate (EtDTP, 2a), under anaerobic and reducing conditions using sodium dithionite as a reductant (Table 1, entries 2 and 4)
We developed a biocatalytic platform for the asymmetric synthesis of α-trifluoromethyl amines via an abiological N−H carbene insertion
Summary
The incorporation of fluorine can favorably alter the physicochemical and biological properties of bioactive molecules.[1,2] Fluorine-containing building blocks are increasingly used in medicinal chemistry, as the introduction of fluorine substituents can improve the pharmacokinetic and pharmacological properties of small-molecule drugs, including their potency, cell permeability, and metabolic stability.[3,4]. The lower energy of the dominant state of the ylides derived from the less bulky diazo esters (2a,c,h vs 2e−g; Table 2) tend to correlate with the higher yields of the corresponding reactions (Scheme 2), other factors can contribute to these differences These analyses show that a combination of structural and energetic factors, mediated by all four beneficial mutations as well as other residues surrounding the heme c cofactor, contribute to destabilize the proR configurations of the 2gderived ylide over the proS state, resulting in the dramatic switch in enantioselectivity observed experimentally in the HtCc552(G50T,M59G,P60E,Q62R)-catalyzed N−H insertion reactions with 2g. LAH reduction of 3c followed by exposure to XtalFluor-E and tetraethylammonium bromide afforded the trifluoromethylated β-amino alkyl bromide 10 in enantioenriched form (86:14 er; Scheme 5)
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