C3 and C6 Modification‐Specific OYE Biotransformations of Synthetic Carvones and Sequential BVMO Chemoenzymatic Synthesis of Chiral Caprolactones

Abstract The scope for biocatalytic modification of non‐native carvone derivatives for speciality intermediates has hitherto been limited. Additionally, caprolactones are important feedstocks with diverse applications in the polymer industry and new non‐native terpenone‐derived biocatalytic caprolactone syntheses are thus of potential value for industrial biocatalytic materials applications. Biocatalytic reduction of synthetic analogues of R‐(−)‐carvone with additional substituents at C3 or C6, or both C3 and C6, using three types of OYEs (OYE2, PETNR and OYE3) shows significant impact of both regio‐substitution and the substrate diastereomer. Bioreduction of (−)‐carvone derivatives substituted with a Me and/or OH group at C6 is highly dependent on the diastereomer of the substrate. Derivatives bearing C6 substituents larger than methyl moieties are not substrates. Computer docking studies of PETNR with both (6S)‐Me and (6R)‐Me substituted (−)‐carvone provides a model consistent with the outcomes of bioconversion. The products of bioreduction were efficiently biotransformed by the Baeyer–Villiger monooxygenase (BVase) CHMO_Phi1 to afford novel trisubstituted lactones with complete regioselectivity to provide a new biocatalytic entry to these chiral caprolactones. This provides both new non‐native polymerization feedstock chemicals, but also with enhanced efficiency and selectivity over native (+)‐dihydrocarvone Baeyer–Villigerase expansion. Optimum enzymatic reactions were scaled up to 60–100 mg, demonstrating the utility for preparative biocatalytic synthesis of both new synthetic scaffold‐modified dihydrocarvones and efficient biocatalytic entry to new chiral caprolactones, which are potential single‐isomer chiral polymer feedstocks.


General enzymatic procedure for Baeyer−Villiger oxidation using CHMOs
The standard enzymatic reaction (1 mL
The organic layer was extracted with EtOAc (3 x 50 mL), dried over (MgSO4), and concentrated to afford a lactone 21 with yield (49 mg, 90%) as a colorless oil. respectively.

O OH
O OH   Resr. Sites = restriction sites through which the gene was cloned into the plasmid.

Expression and purification of recombinant OYE2
Expression and purification of OYE2 was performed using the method detailed below, based on the production and purification of OYE2 as described previously. 8
A filter-sterilised stock of glucose in distilled water was prepared (1M; 0.2 mm filtration), and added aseptically to the sterile medium to a final concentration of 20 mM. Lysogeny broth-Miller (LB) was prepared by combining tryptone (10 gL -1 ), yeast extract (5 gL -1 ) and NaCl (10 gL -1 ) followed by autoclaving. Lysogeny broth-Miller agar (LB agar) was prepared as for LB, except agarose (15 gL -1 ) was added to the medium prior to autoclaving. After sterilisation, the medium was cooled to ~ 50 °C, and a sterile ampicillin solution was added (100 mgmL -1 ) before pouring into sterile agar plates.

Transformation of competent cells
Purified OYE2 plasmid DNA (0.4 mL; Table S2) was mixed with pre-chilled competent E.
coli BL21 (DE3) cells (50 mL) and incubated on ice for 30 minutes. The suspension was heat shocked by incubating at 42 °C for 30 seconds, followed by cooling on ice for 2 minutes. Cell recovery and growth was initiated by the addition of SOC medium (0.45 mL) and the culture was incubated at 37 °C for 1 hour at 200 rpm agitation. The cell suspension was spread aseptically on to LB agar, containing ampicillin (100 mgmL -1 ), and incubated overnight at 37 °C.

Growth and expression of OYE2 clone
Starter cultures of OYE2 in E. coli BL21(DE3) were produced by inoculating LB media (5 mL) containing ampicillin (100 mgmL -1 ) with a colony from the transformation plate

BVMOs
The structures of both isomers were incompletely confirmed through proton NMR but this was complicated due to overlapping of the peaks. Batey and co-workers separated two diastereomers (23:22 = 7:5) of from radical opening ring of carvone-derived cyclopropyl ketone precursor, and proved the major one to be 23, and minor to be 22. 10 The proton NMR of 23 assigned therein is consistent with assignment of 23 (minor isomer of that mixture in the current study). This supports that the mixture of two isomers, separated in this work, consists of 22 and 23. Additionally, 22 and 23 have been reported (Siscovic and Roa 11 ) in a 1:1 ratio through conjugate addition to carvone.
Our comparative NMR data of a mixture of these two isomers, versus a total mixtue of all 4 below allows a cross-analysis of the other minor components herein.