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

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.


Materials and Methods
Chemicals were purchased from Sigma Aldrich.NAD(P) + , ADHs, ERs, and GDH-101 were kindly provided by Johnson Matthey in the form of lyophilized cell-free extracts (CFEs).The panel of UPOs (produced by recombinant expression in Pichia pastoris) 1 was purchased from Aminoverse.LbADH, LkADH, TsOYE, TsOYE C25D/I67T, OYE2, OYE3 and AaeUPO PaDa-I (rAaeUPO) 2 were used from frozen stock solutions or lyophilized powders available from previous work. 3,4lylic oxidation using PhI(OAc)2 and t BuOOH Based on a previous reference: 5 To (diacetoxyiodo)benzene (484 mg, 1.50 mmol) and potassium carbonate (35 mg, 0.25 mmol) was added n-butyl n-butanoate (1 mL) and the mixture was cooled to -20 or -15 °C, after which cyclohex-1-enecarbonitrile 1 (56.6 µL, 0.5 mmol) was added.With vigorous stirring, t-butyl hydroperoxide (400 µL of 5-6 M solution in decane) was added slowly over 30 min.The reaction was stirred at -20 or -15 °C for 24 h and filtered through a cotton plug prior to use in the ER screening or dilution in EtOAc for GC analysis (see analytical methods) or CDCl3 for NMR analysis.

Screening of UPOs
One replicate of the enzymes provided in the UPO enzyme panel purchased from Aminoverse, as well as lyophilized rAaeUPO (2-5 mg) were resuspended in MilliQ H2O (100 µL).Reactions were assembled in a 96-deep-weel-plate (DWP) by sequentially adding potassium phosphate buffer (KPi-buffer 100 mM, pH 7.0; 97.5 µL), resuspended UPO (50 µL), and substrate 1 (200 mM in acetonitrile; 12.5 µL).Hydrogen peroxide (66.7 mM; 10 µL) was added once per hour over the course of 9 h.The DWP was sealed with an aluminium seal and incubated at ambient temperature (20 °C) with shaking (600 rpm) in between hydrogen peroxide additions.After 9 h, EtOAc (1 mL) was added to each well and each well was transferred to a microfuge tube for extraction.The EtOAc supernatant was dried with Na2SO4, and analysed by chiral GC-FID as described under analytical methods (split ratio 10:1).

Analytical methods
The identity of product 5 was confirmed by chemical reduction of 3 with sodium borohydride NaBH4: racemic 4 (20 µL), and NaBH4 (20 mg) were added to methanol (1 mL), on ice.The mixture was brought to ambient temperature (20 °C) over 15 min.After addition of aqueous sodium hydroxide (0.1 M; 1 mL), the mixture was concentrated in vacuo, neutralized with hydrochloric acid and extracted with EtOAc (1 mL).

Supplementary Tables
Table S2.Screening of a panel of UPOs for the allylic oxidation of cyclohexene-1-nitrile 1, sorted by relative amounts of desired product 3.Initial screening at 10 mM scale.Negative controls are based on different expression strains, without overexpressed UPO.Concentrations are 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.
Table S4.The full cascade from 1 to 5, using MorUPO, ENE-101, ADH-20.A total of 12 reactions were carried out, 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.

Figure S3 .
Figure S3.Time course of the allylic oxidation catalysed by rAaeUPO of cyclohexene-1-nitrile 1. Analysis by GC-FID following extraction with EtOAc on an Agilent CP-Sil 8 CB column for conversion and Hydrodex β-6TBDM column for ee.

Figure S4. A :
Figure S4.A: Combined cascade starting from the chemical allylic oxidation of cyclohexene-1-nitrile 1 (using diacetoxyiodo)benzene and t-butyl hydroperoxide).B: Fully enzymatic cascade with rAaeUPO and ENE-101, comparing ADH-19 and ADH-20 in the last step.C: Comparison of the ER step, starting from rAaeUPO or MorUPO with varying conditions.Analysis by GC-FID following extraction with EtOAc on a Hydrodex β-6TBDM column for conversion and ee.

Figure S6 .
Figure S6.Stacked GC chromatograms of the time course of the allylic oxidation of 1 using (diacetoxyiodo)benzene and t-butyl hydroperoxide (CP-Sil 8 CB).

Figure S14 .
Figure S14.GC chromatograms of cascade stopped after ER step (black) and authentic standard of racemic 4 (pink).

Figure S15 .
Figure S15.GC chromatograms of cascade stopped after ADH step (black) and 5 produced with NaBH4.