Peroxygenase-Catalyzed Allylic Oxidation Unlocks Telescoped Synthesis of (1 S,3 R)-3-Hydroxycyclohexanecarbonitrile

Abstract

The unmatched chemo-, regio-, and stereoselectivity of enzymes renders them powerful catalysts in the synthesis of chiral active pharmaceutical ingredients (APIs). Inspired by the discovery route toward the LPA₁-antagonist BMS-986278, access to the API building block (1S,3R)-3-hydroxycyclohexanecarbonitrile was envisaged using an ene reductase (ER) and alcohol dehydrogenase (ADH) to set both stereocenters. Starting from the commercially available cyclohexene-1-nitrile, a C-H oxyfunctionalization step was required to introduce the ketone functional group, yet several chemical allylic oxidation strategies proved unsuccessful. Enzymatic strategies for allylic oxidation are underdeveloped, with few examples on selected substrates with cytochrome P450s and unspecific peroxygenases (UPOs). In this case, UPOs were found to catalyze the desired allylic oxidation with high chemo- and regioselectivity, at substrate loadings of up to 200 mM, without the addition of organic cosolvents, thus enabling the subsequent ER and ADH steps in a three-step one-pot cascade. UPOs even displayed unreported enantioselective oxyfunctionalization and overoxidation of the substituted cyclohexene. After screening of enzyme panels, the final product was obtained at titers of 85% with 97% ee and 99% de, with a substrate loading of 50 mM, the ER being the limiting step. This synthetic approach provides the first example of a three-step, one-pot UPO-ER-ADH cascade and highlights the potential for UPOs to catalyze diverse enantioselective allylic hydroxylations and oxidations that are otherwise difficult to achieve.

Scheme 1. (A) Abbreviated Discovery Route to BMS-986278 and Retrosynthetic Analysis

(A) Abbreviated discovery route to BMS-986278. A chiral substituted cyclohexanol is prepared from (S)-cyclohex-3-enecarboxylic acid, which is then coupled to the pyridine moiety and deprotected to give BMS-986278. (B) Retrosynthetic Analysis of a Simplified BMS-986278 Analogue. The acid may be obtained by hydrolysis of a nitrile group, which would be compatible with the Mitsunobu reaction. The chiral 3-hydroxycyclohexanecarbonitrile 5 may be prepared from the pro-chiral cyclohex-1-enecarbonitrile 1. (C) The Proposed Enzymatic Synthesis of (1S,3R)-5, Where UPO = Unspecific Peroxygenase, ER = Ene Reductase, ADH = Alcohol Dehydrogenase, and GDH = Glucose Dehydrogenase.

Figure 1

Chemical allylic oxidation of cyclohexene-1-nitrile 1, followed by screening of a panel of ERs for the reduction of 3-oxocyclohex-1-ene-1-carbonitrile 3 to 3-oxocyclohexane-1-carbonitrile 4. Analysis by GC-FID followed extraction with EtOAc on an Agilent CP-Sil 8 CB column for conversion and a Hydrodex β-6TBDM column for ee. Conditions: allylic oxidation: ^tBuOOH (4–5 equiv), PhI(OAc)₂ (1.5 equiv), K₂CO₃ (0.5 equiv), butyl butyrate (1 mL), decane (0.4 mL), −20 °C, 19 h. ER step: Filtered reaction mixture from previous step (140 μL), ER (2 mg/mL), GDH-101 (1 mg/mL), NAD⁺ (1 mol %), D-Glc (1.1 equiv), MOPS-NaOH (200 mM), pH 7, 0.5 mL, 30 °C, 900 rpm, 24 h. ^a purified enzyme, 0.2 mg/mL protein content; ^b −15 °C, 24 h allylic oxidation; ^c unidentified peaks; ^d overlapping with an unidentified impurity in ^tBuOOH. Data are also shown in Table S1.

Figure 2

Screening of a panel of UPOs for the allylic oxidation of cyclohexene-1-nitrile 1, sorted by relative amounts of the desired product 3. A: initial screening at the 10 mM scale. Conditions: UPO (4–10 mg/mL), H₂O₂ (10 × 0.24 equiv), acetonitrile (5% v/v), KP_i-buffer (100 mM), pH 7, 0.25 mL, RT, 600 rpm, 9 h in 96-deepwell plate. Negative controls are based on different expression strains without overexpressed UPO. B: Rescreening of top variants at targeted reaction scale. Conditions: UPO (10 mg/mL), H₂O₂ (0.1 eq./h), KP_i-buffer (300 mM), pH 7, 1 mL, 30 °C, 600 rpm, 24 h. Concentrations are given with respect to the final reaction volume. Analysis by GC-FID following extraction with EtOAc on a Hydrodex β-6TBDM column for conversion and ee. Data are also shown in Table S2.

Figure 3

Screening of a panel of ADHs for the reduction of 3-oxocyclohexane-1-carbonitrile 4 to chiral 3-hydroxycyclohexanecarbonitrile 5. Analyses by GC-FID following extraction with EtOAc on a Hydrodex β-6TBDM column for conversion and ee. Conditions: ADH (4 mg/mL), GDH-101 (2 mg/mL), NAD(P)⁺ (1 mol %), D-Glc (1.1 equiv), DMSO (10% v/v), MOPS (200 mM), pH 7, 0.5 mL, 30 °C, 900 rpm, 24 h. ^a cell-free extract, 4 mg/mL protein content; ^b purified enzyme, 0.4 mg/mL protein content; ^c ADH (2 mg/mL), and GDH-101 (1 mg/mL). Data are also shown in Table S3.

Figure 4

Full cascade from 1 to 5, using MorUPO, ENE-101, ADH-20. A total of 12 reactions were set up and sets of three were quenched and analyzed at the start of the cascade, and after each of the three enzymatic steps. Analyses by GC-FID following extraction with EtOAc on a Hydrodex β-6TBDM column for conversion and ee. Conditions: UPO-step: MorUPO (3.75 mg/mL), H₂O₂ (0.1 equiv/h for 22 h), KP_i-buffer (300 mM), pH 7, 30 °C, 600 rpm, 24 h, final volume 990 μL. ER step: addition of ER (3 mg), GDH-101 (1.5 mg), NAD⁺ (2 mol %), D-Glc (1.1 equiv), 30 °C, 900 rpm, 20 h, final volume 1245 μL. ADH step: addition of ADH (3 mg), GDH-101 (1.5 mg), NAD⁺ (2 mol %), D-Glc (1.1 equiv), 30 °C, 900 rpm, 24 h, final volume 1500 μL. Data are also shown in Table S4.