Production of a Doubly Chiral Compound, (4 R ,6 R )-4-Hydroxy-2,2,6-Trimethylcyclohexanone, by Two-Step Enzymatic Asymmetric Reduction

Abstract

A practical enzymatic synthesis of a doubly chiral key compound, (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone, starting from the readily available 2,6,6-trimethyl-2-cyclohexen-1,4-dione is described. Chirality is first introduced at the C-6 position by a stereoselective enzymatic hydrogenation of the double bond using old yellow enzyme 2 of Saccharomyces cerevisiae, expressed in Escherichia coli, as a biocatalyst. Thereafter, the carbonyl group at the C-4 position is reduced selectively and stereospecifically by levodione reductase of Corynebacterium aquaticum M-13, expressed in E. coli, to the corresponding alcohol. Commercially available glucose dehydrogenase was also used for cofactor regeneration in both steps. Using this two-step enzymatic asymmetric reduction system, 9.5 mg of (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone/ml was produced almost stoichiometrically, with 94% enantiomeric excess in the presence of glucose, NAD ؉ , and glucose dehydrogenase. To our knowledge, this is the first report of the application of S. cerevisiae old yellow enzyme for the production of a useful compound.(4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone (actinol) is an ideal precursor for the synthesis of naturally occurring, optically active hydroxylated carotenoids, such as zeaxanthin (8), cryptoxanthin, and structurally related compounds (1, 2). Two-step conversion of 2,6,6-trimethyl-2-cyclohexen-1,4-dione (ketoisophorone) to 4-hydroxy-2,2,6-trimethylcyclohexanone ( Fig. 1) by bacterial cells has been reported previously (3,11,12,19). However, in those cases, mixtures of isomers, (4R,6S), (4S,6R), (4R,6R), and (4S,6S), were produced and the productivity was low (ϳ2.5 mg/ml). The enzymes involved in these reactions have not yet been identified.The discovery, purification, and characterization of levodione reductase (LVR), which catalyzes the regio-and stereospecific reduction of (6R)-2,2,6-trimethylcyclohexane-1,4-dione [(6R)-levodione] to actinol, has been reported (17). This LVR has also been cloned and expressed in Escherichia coli (21). Recently, it was also reported that old yellow enzyme (OYE; EC 1.6.99.1) of Candida macedoniensis catalyzes the stereospecific hydrogenation of ketoisophorone to (6R)-levodione (4). However, this enzyme has not yet been cloned, and enzyme purification from C. macedoniensis is needed for (6R)-levodione production. Thus, we tried to use OYE of Saccharomyces cerevisiae, for which the whole-genome sequence is now available. S. cerevisiae has two OYEs: OYE2, encoded by the OYE2 gene (15), and OYE3, encoded by the OYE3 gene (10). Both of them were cloned and expressed in E. coli, and the conversion efficiencies of ketoisophorone to (6R)-levodione were then compared.Here, we report actinol production by the stereoselective hydrogenation of the double bond of ketoisophorone and by the stereoselective reduction of the carbonyl group at the C-4 position of (6R)-levodione. For the first step, hydrogenation, the OYE2 of S. cerevisiae expressed in E. coli was used, and for the second step, carbon...

Photoenzymatic Reduction of CC Double Bonds

“…[21][22][23][24][25][26] To avoid additional efforts and expenditures of external NAD(P)H regeneration, asymmetric bioreductions using OYEs are preferentially performed in vivo, thus utilizing the metabolic NAD(P)H regeneration capacity of the living cell. [9][10][11]14,27] However, ubiquitous NAD(P)H-dependent ketoreductases can cause undesired carbonyl reduction of substrates and products leading to lower yields and complex product mixtures. Even using isolated OYEs together with an enzymatic NAD(P)H regeneration system the procedure can suffer from this side reaction due to contaminating ketoreductases.…”

Comparison of Three Enoate Reductases and their Potential Use for Biotransformations

“…Enoate reductases (ERs) perform such reactions and have been recognized for decades, but the stereoselective application was only recently published. [5][6][7] Enoate reductase from Saccharomyces carlsbergensis, also known as Old Yellow Enzyme (OYE), was the first isolated flavin-containing enzyme in 1933. [8] Despite the fact that OYE has been characterized thoroughly, [9] its physiological role has only recently begun to emerge.…”

Guidelines for the Application of NAD(P)H Regenerating Glucose Dehydrogenase in Synthetic Processes

“…Accumulation of glucono-1,5-lactone in GDH regenerated processes over a time period of 2 h. Solid lines, NADH regeneration; dashed lines, NADPH regeneration. Data for modeling (glucose conversion rates, coenzyme concentration, pH) are taken from the indicated references: a, synthesis of l-6-hydroxynorleucine; [13] b, synthesis of actinol; [9] c, synthesis of l-leucovorin; [16] d, synthesis of methyl (R)-o-chloromandelate; [4] e, synthesis of l-carnitine; [12] f, synthesis of l-glyceraldehyde; [11] g, synthesis of (R)-ethyl 2-methyl 4-oxo-2-pentanoate; [10] h, synthesis of (R)-1-phenyl-A C H T U N G T R E N N U N G ethanol. [14] The pH-dependent glucono-1,5-lactone hydrolysis rates were taken from.…”

“…[4,[9][10][11][12][13][14] The hydrolysis rate of the oxidation intermediate glucono-1,5-lactone to gluconic acid depends on the applied pH and buffer system. Also, the concentration of glucono-1,5-lactone affects the rate of hydrolysis.…”