Synthesis of Enantiopure Sulfoxides by Concurrent Photocatalytic Oxidation and Biocatalytic Reduction

Abstract The concurrent operation of chemical and biocatalytic reactions in one pot is still a challenging task, and, in particular for chemical photocatalysts, examples besides simple cofactor recycling systems are rare. However, especially due to the complementary chemistry that the two fields of catalysis promote, their combination in one pot has the potential to unlock intriguing, unprecedented overall reactivities. Herein we demonstrate a concurrent biocatalytic reduction and photocatalytic oxidation process. Specifically, the enantioselective biocatalytic sulfoxide reduction using (S)‐selective methionine sulfoxide reductases was coupled to an unselective light‐dependent sulfoxidation. Protochlorophyllide was established as a new green photocatalyst for the sulfoxidation. Overall, a cyclic deracemization process to produce nonracemic sulfoxides was achieved and the target compounds were obtained with excellent conversions (up to 91 %) and superb optical purity (>99 % ee).

Note for photon fluxes that the actinometry measurements were done on a reaction volume of 1 mL, whereas all experiments described herein were run on a 0.5 mL scale, except stated otherwise. [1] General procedures

Kinetic resolution of rac-1a
Scheme S1.Kinetic resolution of rac-1a using lyophilized cell free extracts of Msrs or purified paMsr.
For work-up a spatula tip of NaCl was added to the reaction mixture prior to extraction with EtOAc (500 µL) containing decane (10 mM) as internal standard.After mixing and centrifugation (14680 rpm, 3 min), the organic phase (300 µL) was transferred to a fresh vial containing Na2SO4 for drying.The extraction step was repeated (500 µL EtOAc without internal standard) and the combined and dried organic phases were centrifuged (14680 rpm, 5 min) and transferred to a glass crimp vial (1.5 mL) for analysis on GC-FID.

Deracemization and stereoinversion of sulfoxides
Scheme S3.Cyclic deracemization process of rac-sulfoxides based on an enantioselective biocatalytic reduction followed by a photocatalytic sulfide oxidation.
Work-up for GC analysis was done as described in section 2.1.
The conditions for substrate 10a were slightly varied due to its poor solubility and light instability.For the deracemisation the substrate 10a was dissolved in KPi buffer (50 mM substrate in 0.5 M KPi buffer pH 8.0, final pH 7.5).Water (up to 500 µL), substrate (50 mM in KPi buffer, 10 mM final substrate concentration, 100 mM final buffer concentration), DTT (1M stock in H2O, 25 µL, 50 mM) and P6 (2.57mM stock in MeOH, 10 µL, 51 µM final concentration) were pipetted into a screw neck glass vial before the reaction was started by the addition of paMsr CFE (5 mg dissolved in 50 µL H2O).The vials were then placed in the custom photoreactor developed by C. K. Winkler et al. [1] at 30 °C, 400 rpm and illuminated at 455 nm (0.018 µmol photons s -1 ) for 5 min.
For work-up, the samples were extracted with EtOAc (500 µL) without internal standard.After vortexing the samples were centrifuged (14680 rpm, 2 min) and the organic phase (300 µL) was transferred to a fresh microcentrifuge tube containing Na2SO4 for drying.The extraction step was repeated, the dried combined organic phases were centrifuged (14680 rpm, 5 min) and transferred to a glass vial for HPLC analysis.

Cloning of methionine sulfoxide reductases
The enzymes were selected based on a literature search.The corresponding genes were ordered from General Biosystems as optimized sequence for overexpression in E. coli and cloned into a pET28a vector either by restriction enzyme cloning (MsrA, restriction sites EcoRI and XhoI) or via TEDA [2] cloning (paMsr, pmMsr).Successful cloning was confirmed by sequencing.

Heterologous expression and cell lysis of methionine sulfoxide reductases
The recombinant plasmid was transformed into E. coli BL21(DE3) for heterologous expression of the corresponding enzyme.For the overnight culture, lysogeny broth (LB, 10 mL) containing kanamycin (50 mg/mL, 10 µL, 50 µg/mL) was inoculated from an agar plate or from glycerol stock and incubated overnight (37 °C, 120 rpm).For the main culture, sterile LB medium in a non-baffled flask (700 mL in a 2 L flask), containing kanamycin (50 mg/mL, 700 µL, 50 µg/mL) was inoculated with the E. coli overnight culture (1% v/v).The culture was incubated at 37 °C and 120 rpm until an OD600 of 0.4-0.6 was reached, followed by induction adding IPTG (0.2 mM for paMsr and pmMsr, 0.5 mM for MsrA).
Further incubation took place at 20 °C and 120 rpm overnight.The cell culture was harvested by centrifugation (5000 rpm, 20 min, 4 °C), the obtained supernatant discarded, and the remaining pellet washed with KPi buffer (50 mM, pH 7.5, 30 mL).The suspension was centrifuged (8 °C, 4500 rpm, 20 min), the supernatant discarded and the remaining pellet either lysed or shock frozen in liquid nitrogen and stored at -20 °C until further usage.
Lysis of the resuspended whole cells in either KPi buffer (50 mM, pH 7.5, 10 mL/g cell pellet; when the CFE was required) or in lysis buffer (see Table S2; for further purification), was done on ice by ultrasonication (three times 2 min 30 sec, 30% amplitude, 2.0 sec pulse on, 4.0 sec pulse off; 1 min pause on ice between the sonications; Digital sonifier, BRANSON).The cell suspension was centrifuged (20 min, 18 000 rpm, 4 °C) and the clear slightly yellow cell free extract (CFE) was filtered (0.45 µm syringe filter) and lyophilized or stored on ice for further protein purification using metal ion affinity chromatography.
Successful expression of soluble enzyme was verified by SDS page analysis (Figure S1).

Protein purification via nickel-affinity chromatography
The His6-tag containing paMsr was purified by nickel affinity chromatography using a HisTrap TM FF 5 mL column (GE HEALTHCARE) in combination with Äkta TM pure (GE Healthcare).The purification was performed at 4 °C using a flow rate of 5 mL/min.The column was equilibrated with 5 column volumes (CV) of lysis buffer (see Table S2) prior to loading the CFE onto the column at a flow rate of 2 mL/min.The column was washed with 5 CV lysis buffer (5 mL/min) containing 1% elution buffer (see Table S2) before the elution buffer was applied in a gradient (1-100% elution buffer) in order to elute the protein.
As the eluted protein was colorless, absorption at 280 nm was used to identify the fractions (each 5 mL) containing protein.The protein containing fractions were combined and concentrated to 2.5 mL using Vivaspin® 20 mL (SARTORIUS, 10 kDa cut-off).To exchange the buffer, a Sephadex G-25 PD10 desalting column (GE Healthcare) was equilibrated with KPi buffer (50 mM, pH 7.5).The concentrated protein solution (2.5 mL) was loaded onto the column and eluted with 3.5 mL KPi buffer (50 mM, pH 7.5).The final enzyme solution was aliquoted (100 µL portions in pcr tubes), shock frozen in liquid nitrogen and stored at -20 °C.

Synthesis and purification of protochlorophyllide
Protochlorophyllide P6 (pchlide) was isolated and purified from Rhodobacter capsulatus ZY5 strain as described in literature (cf. Figure S3, B-G). [3]Cells from a Rhodobacter capsulatus ZY5 glycerol stock were plated on a VN-agar plate (10 g/L yeast extract, 1 g/L K2HPO4, 0.5 g MgSO4, pH 7.0) containing rifampicin [25 µg/mL, rifampicin stock in DMSO (50 mg/mL)].The agar plate was incubated at 32-36 °C for 40 h, before VN-medium (100 mL, containing 25 µg/mL) was inoculated with several colonies.The preculture was incubated at 34 °C for 30 h.For the main culture, the preculture was added to 1 L of VN-medium (containing 25 µg/mL rifampicin) and additionally three white polyurethane foam bungs (height/diameter 50/35 mm) were added.Further incubation was done at 34 °C and 120 rpm for another 48 h.The dark green foam bungs were exchanged after 24 h and dried at room temperature in the dark.The absorbed dark green pchlide was washed from the 6 foam bungs with MeOH (~800 mL), which was then removed under reduced pressure.The crude pchlide was then resuspended in acetone (800 mL) supplemented with 1.5% (v/v) MeOH.
A CM Sepharose Fast Flow column (Sigma Aldrich) was prepared by washing the resin (50-75 mL) with deionized water (dH2O, 500 mL).The resin was then resuspended in acetone, stirred, and dried under suction in a Buchner funnel, which was repeated three times.The final slurry was resuspended in acetone (100 mL) and poured into a glass column (5 cm width).
The pchlide suspension was loaded onto the column.The column was then washed with acetone (800 mL) until the flowthrough became clear.In order to remove phytol or pheophorbide the column was subsequently washed with acetone containing MeOH (5% v/v, 500 mL).The purified pchlide was eluted from the column with MeOH in acetone (25% v/v, 500 mL) and concentrated to 50 mL under reduced pressure.The pchlide solution was aliquoted (1 mL fractions in 1.5 mL microcentrifuge tubes) and the remaining solvent was removed under pressure.The aliquotes were stored at -21 °C until further usage.
A yield of 37.9 mg pchlide was determined by recording a UV-Vis spectrum (Figure S3, A) on the photometer (Cary 60 UV-Vis, Agilent Technologies) and calculation following Lambert Beer`s Law with ε630= 23.95 mM -1 cm -1 . [4]B C

D E F G
. A: UV-Vis absorption spectrum of the purified pchlide (ε630= 23.95 mM -1 cm -1 ) [4] ; B: Rhodobacter capsulatus ZY5 culture with white polyurethane foam bungs; C: Rhodobacter capsulatus ZY5 culture incubated for 24 h with the pchlide absorbed onto the foam bungs; D: Pchlide washed from the foam bungs with MeOH; E: CM Sepharose Fast Flow column with the pchlide suspension loaded; F: Flowthrough containing carotenoids; G: Final purified concentrated pchlide solution.

Reference synthesis of enantiopure (R)-9a
In order to assign the absolute configuration of the deracemized 9a, a reference compound was synthesized by biocatalytic kinetic resolution.The absolute configuration was assigned via comparison of the specific optical rotation of the isolated and purified reaction product.

Deracemization without photocatalyst at 385 nm
In order to evaluate the stability of the enzyme at 385 nm, purified paMsr and rac-1a were incubated for 8h under illumination at 385 nm, prior to the addition of the reducing equivalents (DTT) which is required for the reduction step.Successful reduction to the corresponding sulfide 1b confirmed that the enzyme was still active (Figure S5).Reaction conditions: rac-1a (10 mM), DTT after 8 h (10 mM), paMsr (0.5 mg purified), KPi buffer (50 mM, pH 7.5 for a total volume of 0.5 mL), 385 nm, 0.75 µmol photons s -1 , 400 rpm, 30 °C.

Blank and control reactions
Several control reactions were performed (see Table S4) to confirm that the oxidation of the corresponding sulfide intermediate 1b only occurred in presence of light and photocatalyst (P6).The qualitative analysis of the blank and control experiments is based on the quantification of 1a and 1b as well as the ee of 1a.Starting from rac-1a, no oxidation and consequently no deracemization occurred in the absence of either light (entry 4) or photocatalyst (P6, pchlide, entry 1) or photocatalyst and methanol (entry 3), while in absence of MeOH and presence of photocatalyst the oxidation was still occurring.MeOH is therefore not responsible for the oxidation.Standard deracemization conditions (section 2.3) were applied for the positive control (entry 5).+: reaction taking place -: reaction not taking place Reaction conditions: 1a (10 mM), P6 (14 µM), MeOH (1% v/v), paMsr (30 mg CFE), DTT (20 mM), KPi buffer (50 mM, pH 7.5 for a total volume of 0.5 mL), 455 nm, 0.036 µmol photons s -1 , 30 °C, 400 rpm, overnight.

Light source screening for the deracemization process with P6
Protochlorophyllide (P6) shows absorption in the region of 400-480 nm as well as low absorption around 580 nm and 630 nm, respectively.Therefore, several light sources including white light as well as various single wavelengths were evaluated (see Figure S6) in the deracemization process.Similar S12 results were obtained independent from the applied light sources.For photon and luminous flux see Table S5.

Protochlorophyllide concentration screening
Examination of the protochlorophyllide (P6) concentration in the deracemization process showed a slight improvement at higher concentrations (7 µM vs 43 µM, Figure S7).

Redox equivalents in the cell free extract
A kinetic resolution of rac-1a was performed with and without the addition of external redox equivalents (Figure S8).It is shown that 30 mg CFE of paMsr provides enough redox equivalents for the reduction of the corresponding sulfoxide, since the addition of DTT as external reducing agent did not result in better resulst.However, decreasing the amount of CFE (only 10 mg instead of 30 mg) led to an incomplete kinetic resolution.

Time study
The deracemization process of rac-1a was followed over time monitoring the concentration of the sulfoxide 1a, sulfide intermetiate 1b and the ee of 1a (Figure S9).

Figure S1 .
Figure S1.SDS-Page to verify expression of methionine sulfoxide reductases.L = cell free extract, P = insoluble fraction.

Table S1 .
Illumination conditions for the photocatalysts screening in the deracemization process.

Table S2 .
Buffer compositions used for protein purification.

Table S4 .
Blank and control reactions to confirm the light and photocatalyst dependency of the sulfide oxidation and deracemization, respectively.

Table S7 .
GC-FID method and retention times for the analysis of 1a-1c and 9a.

Table S8 .
HPLC methods for the analysis of 2a-8a and 10a.