Asymmetric Synthesis of Chiral 2-Cyclohexenones with Quaternary Stereocenters via Ene-Reductase Catalyzed Desymmetrization of 2,5-Cyclohexadienones

Abstract

Stereoselective synthesis of quaternary stereocenters represents a significant challenge in organic chemistry. Herein, we describe the use of ene-reductases OPR3 and YqjM for the efficient asymmetric synthesis of chiral 4,4-disubstituted 2-cyclohexenones via desymmetrizing hydrogenation of prochiral 4,4-disubstituted 2,5-cyclohexadienones. This transformation breaks the symmetry of the cyclohexadienone substrates, generating valuable quaternary stereocenters with high enantioselectivities (ee, up to >99%). The mechanistic causes for the observed high enantioselectivities were investigated both experimentally (stopped-flow kinetics) as well as theoretically (quantum mechanics/molecular mechanics calculations). The synthetic potential of the resulting chiral enones was demonstrated in several diversification reactions in which the stereochemical integrity of the quaternary stereocenter could be preserved.

Scheme 1. Strategies for the Desymmetrizing Hydrogenation of Cyclohexadienones; (A) Principle of Desymmetrizing Hydrogenation; (B) Previous Work with Transition Metal-Catalyzed Reactions; (C) This Work: Employing Ene-Reductases for the Desymmetrizing Hydrogenation of 2,5-Cyclohexadienones under Environmentally Benign Conditions

Scheme 2. Substrate Scope of the Ene-Reductase Catalyzed Cyclohexadienone Desymmetrization

Reactions were performed in triplicates. Cyclohexenone yields were determined by HPLC at a wavelength of 254 nm. For quantification, external calibrations aided by 1,3,5-tribromobenzene (TBB) as an external standard were used. Substrate-recoveries as determined by HPLC-MS are summarized in the Supporting Information (Table S3). Enantioselectivities were determined by chiral HPLC (CHIRACEL OJ-H): (a) For the biotransformation of 1t, only 1.1 eq NADH were used, and the reaction time was reduced to 3 h. n.d. = not determined.

Scheme 3. Preparative Scale Ene-Reductase Catalyzed Desymmetrizations

Scheme 4. Crude Cell Lysate-Based Biocatalytic Desymmetrization of 1a with and without a FDH-Cofactor Recycling System

Scheme 5. Diversification Reactions with Cyclohexenones 2a

Figure 1

Stopped-flow measurements of dienone 1a and enone 2a with YqjM. Measurements were performed against prereduced YqjM under anoxic conditions.

Figure 2

Conformational changes in residue R336 of the second protomer observed during the MD simulations. (a) Docked ligand in the crystal structure of the enzyme shows an “in” conformation of R336 restricting the space in the binding site. (b) MD simulations reveal an “out” conformation of R336, enabling more space in the binding site. The two protomers of the YqjM dimer are shown in gray and yellow nontransparent surface representation. Residue R336 is shown as sticks with carbon atoms colored gray and also has a more transparent surface representation, whereas the substrate and flavin are only shown as sticks with their carbon atoms colored yellow.

Figure 3

(a–e) (Top) QM/MM geometry optimized molecular structures corresponding to stationary points along the lowest energy reaction coordinate for the reduction of 1a. TS1 depicts the transition state for the hydride transfer step, and TS2 displays the transition state for the proton transfer step. The substrate and flavin are shown in yellow sticks, and side chains of active site residues are shown in gray sticks. In TS1 and TS2, the transient hydride and proton are also depicted as a gray ball, respectively. (Bottom) Schematic of each configuration showing key distances (red).