C=C-Ene-Reductases Reduce the C=N Bond of Oximes

Although enzymes have been found for many reactions, there are still transformations for which no enzyme is known. For instance, not a single defined enzyme has been described for the reduction of the C=N bond of an oxime, only whole organisms. Such an enzymatic reduction of an oxime may give access to (chiral) amines. By serendipity, we found that the oxime moiety adjacent to a ketone as well as an ester group can be reduced by ene-reductases (ERs) to an intermediate amino group. ERs are well-known enzymes for the reduction of activated alkenes, as of α,β-unsaturated ketones. For the specific substrate used here, the amine intermediate spontaneously reacts further to tetrasubstituted pyrazines. This reduction reaction represents an unexpected promiscuous activity of ERs expanding the toolkit of transformations using enzymes.

General 1 H-and 13 C-NMR spectra were recorded using a 300 MHz instrument. Chemical shifts are given in parts per million (ppm) and coupling constants (J) are reported in Hertz (Hz). Melting points were determined in open capillary tubes and are uncorrected. Thin layer chromatography was carried out on silica gel 60 F 254 plates and compounds were visualised either by dipping into basic permanganate reagent (10 g/L KMnO 4 , 50 g/L Na 2 CO 3 , 0.85 g/L NaOH, in H 2 O), or by UV. High Resolution Electrospray Ionization Mass Spectrometry (HR-ESMS) measurements were performed on a Q-Exactive Hybrid Quadrupole-Orbitrap MS after flow injection with a Dionex Ultimate 3000 series HPLC-system (Thermo Fisher Sci., Erlangen, Germany). The mobile phase was methanol containing 0.1 % (v/v) formic acid delivered with a flow rate of 0.2 mL/min, and the injection volume was set to 10 µL. The HR-MS was furnished with a HESI-II atmospheric pressure electrospray ionization source (ES), using nitrogen as nebulizer and drying gas. Measurements were performed in positive or negative ionization mode, the spray voltage was +3.5 kV or -2.5 kV, capillary temperature 250 °C, sheath gas flow rate 45 AU, auxiliary gas temperature 400 °C, auxiliary gas flow rate 10 AU, and the resolution was 70,000 (FWHM). The observed mass range was set to m/z 100-350 with data dependent fragmentation at various normalized collision energies (NCE: 20, 30, 40) and subsequent recording of MS/MS fragment ions. Recorded mass differences were always <3 ppm compared to calculated masses. Unless otherwise noted, reagents and organic solvents were obtained from commercial suppliers in reagent grade quality and used without further purification. For GC-FID analysis: conversions were measured on an Agilent 7890A GC with FID detector and Agilent J&W HP-5 column (30 m, 320 μm, 0.25 μm) with helium as carrier gas. Conversions were calculated from the ratios between internal standard (tetramethylpyrazine, 10 mM) and product areas. Injection volume: 1 μL, split ration 90:1. GC parameters: injector 300 °C, flow 31.202 cm/sec, 1.1913 mL/min. Temperature programme (standard): 100 °C, hold 0.5 min, ramp 10 °C/min to 300 °C. Temperature programme (highboilers): 200 °C, hold 0.5 min, ramp 5 °C to 300 °C. For quantification of remaining oxime, the method was adapted in the following manner: injection volume: 3 μL, split ration 50:1, temperature programme (standard) was left unchanged. Chiral HPLC analysis was performed on a Shimadzu HPLC system using a Daicel Chiralpak IC column as chiral stationary phase, n-heptane/2-PrOH 90:10 as eluent, flow rate 1 mL/min, oven temperature 30 °C, 30 min.

General procedure for analytical scale biotransformations
In a 2 mL microcentrifuge tube 100 µg of purified ene reductase, NADPH (stock solution: 10 mM, final concentration: 0.5 mM), glucose (stock solution: 1.0 M, final concentration: 50 mM) and lyophilized GDH preparation (2 mg) were mixed in 50 mM phosphate buffer, pH 7.5. The reactions were started by adding the oxime substrate [final concentration: 10 mM, 5% (v/v) DMSO, total reaction volume: 500 µL]. Samples were shaken at 30 °C, 120 rpm for 24 h in a shaking incubator. The biotransformations were extracted twice with 0.5 mL ethyl acetate containing 10 mM tetramethyl pyrazine as internal standard and the organic phase was dried over MgSO 4 , centrifuged (13000 rpm, 1 min) and the supernatant transferred to a glass vial for GC-FID measurements.

General procedure for cascade biotransformations
In a 2 mL microcentrifuge tube 100 µg of purified ene reductase (added from a stock solution at varied concentration depending on the specific enzyme in buffer), NADPH (stock solution: 10 mM, final concentration: 0.5 mM), NADH (stock solution: 10 mM, final concentrations: 0.5 mM), ADH-A (heatpurified CFE, 1 mg), glucose (stock solution: 1.0 M, final concentration: 50 mM) and lyophilized GDH preparation (2 mg) were mixed in 50 mM phosphate buffer, pH 7.5. The reactions were started by adding the oxime substrate [final concentration: 10 mM, 5% (v/v) DMSO, total reaction volume: 500 µL]. Samples were shaken at 30 °C, 120 rpm for 24 h in a shaking incubator. A solution of benzoyl chloride (30 mM) in EtOAc containing 10 mM acetanilide as internal standard was added and the mixtures were shaken for 45 minutes (900 rpm, 30 °C). The layers were separated and the aqueous phase extracted once more with EtOAc containing 10 mM acetanilide. The organic phases were combined, dried over Na 2 SO 4 , centrifuged (13000 rpm, 1 min) and the supernatant transferred to a glass vial for HPLC-UV measurements.
General procedure for imine reductase screening In a 2 mL microcentrifuge tube 2 mg of lyophilized imine reductase cell free extract, NAD(P)H (stock solution: 20 mM, final concentration: 0.5 mM), glucose (stock solution: 500 mM, final concentration: 50 mM) and lyophilized GDH preparation (1 mg) were mixed in 100 mM Tris-HCl buffer, pH 7.5. The reactions were started by adding the oxime substrate (final concentration: 10 mM, 5 % (v/v) DMSO, total reaction volume: 500 µL). Samples were shaken at 30 °C, 120 rpm for 24 h in a shaking incubator. The biotransformations were extracted with 1 mL ethyl acetate containing 10 mM tetramethyl pyrazine as internal standard and the organic phase was dried over MgSO 4 , centrifuged (13000 rpm, 1 min) and the supernatant transferred to a glass vial for GC-FID measurements.

Procedure for control experiments
To prove the role of the ERs in oxime reduction and pyrazine formation, a series of control experiments was performed. The procedure corresponds to the one described under "General procedure for analytical scale biotransformations", with the adaptations and results as listed in Table S2. NB: The ER used was OYE3.

Synthesis of oxime substrates
General procedure A: To a stirred solution of the -keto-ester or diketone (1.0 eq) in glacial acetic acid (5-10 mL) a solution of NaNO 2 in water (1.5 eq in 5 mL H 2 O) was added dropwise. The reaction temperature was kept at 0 °C in an ice-water bath. The reaction was stirred until full conversion was detected via TLC. Afterwards the mixture was diluted with water (20 mL) and extracted with EtOAc (3 x 20 mL). The combined organic phases were washed with a saturated NaHCO 3 solution (2 x 20 mL) and dried over Na 2 SO 4 . The remaining solvent was removed under reduced pressure to yield the crude product.
General procedure B: To a stirred solution of the -keto-ester or diketone (1.0 eq) in 15 % H 2 SO 4 (5-10 mL) a solution of NaNO 2 in water (1.5-2.0 eq) was added dropwise. The reaction temperature was kept at 0 °C in an ice-water bath. The reaction was stirred until full conversion was detected via TLC. Afterwards the mixture was diluted with water (10 mL) and neutralized with solid Na 2 CO 3 . The solution was extracted with EtOAc (3 x 20 mL) and the combined organic phases were dried over Na 2 SO 4 . The remaining solvent was removed under reduced pressure to yield the crude product.
General procedure C: To a stirred solution of the diketone (1.0 eq.) in 25 mL 15 % KOH, NaNO 2 (1.2 eq.) was added and the mixture cooled to 0 °C in an ice-water bath. 15 % H 2 SO 4 (40 mL) was added dropwise over a period of 30 min and the reaction was allowed to stir at the low temperature for 30 min. After that time the formed precipitate was filtered off and dried under reduced pressure.

Synthesis of liguzinediol
Pyrazine ester 2d (204 mg, 0.66 mmol, 1.0 eq.) was added to a 100 mL round bottom flask and dissolved in MeOH. Sodium methoxide (1.8 mg, 5 mol%) was added and the reaction was stirred at room temperature for 5 min. Sodium borohydride (150 mg, 4.0 mmol, 6.0 eq.) was added at once to the reaction and the mixture was stirred at room temperature for 5 h. Excess metal hydride was quenched with water and the mixture was extracted with ethyl acetate (4x 15 mL). The combined organic phases were dried over Na 2 SO 4 and the solvent was removed under reduced pressure. The crude product was purified via flash chromatography (CHCl 3 /MeOH = 10:1) to obtain the title compound as orange oil (80 mg, 72%). [

N-Benzoyl-L-threonine
A solution of L-threonine (1.19 g; 10 mmol, 1 eq) in aqueous NaOH (1 M, 21 mL; 21 mmol; 2.1 eq) was cooled to 0 °C and a solution of benzoyl chloride (1.40 mL; 12 mmol; 1.2 eq) in 1,4-dioxane (10 mL) was carefully added. After thirty minutes, the cooling was removed and the mixture was stirred at room temperature overnight. Then, the 1,4-dioxane was removed under reduced pressure (40 °C) and, when needed, the pH of the solution was adjusted to approx. pH 1 using concentrated hydrochloric acid. The suspension was extracted with EtOAc (3x 25 mL), the combined organic layers dried over Na 2 SO 4 , filtered and solvent was removed under reduced pressure (40 °C). The obtained white solids were stirred in cyclohexane (40 mL) for thirty minutes to dissolve any residual benzoic acid and then filtered. The residue was triturated with pentane to obtain white solids (1.67 g; 7.5 mmol; 75%). 1

N-Benzoyl-L-allothreonine
A solution of L-allo-threonine (250 mg; 2.1 mmol, 1 eq) in 1 M aqueous NaOH (4.4 mL; 4.4 mmol; 2.1 eq) was cooled to 0 °C and a solution of benzoyl chloride (0.3 mL; 2.52 mmol; 1.2 eq) in 1,4-dioxane (2 mL) was carefully added. After thirty minutes, the cooling was removed and the mixture was stirred at room temperature overnight. Then, the 1,4-dioxane was removed under reduced pressure (40 °C) and, when needed, the pH of the solution was adjusted to approx. pH 1 using concentrated hydrochloric acid. The suspension was extracted with EtOAc (3x 10 mL), the combined organic layers dried over Na 2 SO 4 , filtered and solvent was removed under reduced pressure (40 °C). The obtained oil was treated with cyclohexane, but contained too much water to precipitate the pure product. After removing the solvent under reduced pressure (40 °C), the oil was redissolved in EtOAc (50 mL) and dried over a substantial amount of Na 2 SO 4 , giving 760 mg of solid crude product after removing the solvent. This crude product was used without further purification or analysis.

N-Benzoyl-D-allothreonine
A solution of D-allo-threonine (100 mg; 0.84 mmol, 1 eq) in 1 M aqueous NaOH (1.76 mL; 1.76 mmol; 2.1 eq) was cooled to 0 °C and a solution of benzoyl chloride (117 μL; 1.0 mmol; 1.2 eq) in 1,4-dioxane (1 mL) was carefully added. After thirty minutes, the cooling was removed and the mixture was stirred at room temperature overnight. Then, the 1,4-dioxane was removed under reduced pressure (40 °C) and, when needed, the pH of the solution was adjusted to approx. pH 1 using concentrated hydrochloric acid. The suspension was extracted with EtOAc (3x 5 mL), the combined organic layers dried over Na 2 SO 4 , filtered and solvent was removed under reduced pressure (40 °C). The obtained oil was treated with cyclohexane, stirred overnight and then filtered. The white sticky residue was recovered from the filter by dissolving it in MeOH to yield 158 mg of crude product as a yellow/brown oil, which was used without further purification or analysis.
The synthesis of the ester was performed according to a protocol from literature. [9] N-Benzoyl-L-threonine ethyl ester To a solution of the carboxylic acid (893 mg; 4.0 mmol; 1.0 eq) in anhydrous DMF (22 mL) was added K 2 CO 3 (1.55 g; 11.2 mmol; 2.8 eq) and ethyl iodide (900 μL; 11.2 mmol; 2.8 eq). The suspension was flushed with argon for at least fifteen minutes, and the reaction was stirred at room temperature overnight. The mixture was filtered, the filtrate poured into water (80 mL) and extracted with EtOAc (3x 100 mL). The combined organic layers were washed with brine (2x 100 mL), dried over Na 2 SO 4 , and solvent was removed under reduced pressure (40 °C) to yield 1.57 grams of crude product.

N-Benzoyl-D-threonine ethyl ester
To a solution of the carboxylic acid (893 mg; 4.0 mmol; 1.0 eq) in anhydrous DMF (22 mL) was added K 2 CO 3 (1.55 g; 11.2 mmol; 2.8 eq) and ethyl iodide (900 μL; 11.2 mmol; 2.8 eq). The suspension was flushed with argon for at least fifteen minutes, and the reaction was stirred at room temperature overnight. The mixture was filtered, the filtrate poured into water (80 mL) and extracted with EtOAc (3x 100 mL). The combined organic layers were washed with brine (2x 100 mL), dried over Na2SO4, and solvent was removed under reduced pressure (40 °C) to yield 3.38 grams of crude product. The mixture was purified by column chromatography (SiO 2 ; EtOAc/cyclohexane 1:2 to 1:1, R f 0.1) to yield the desired product as a yellow oil that crystallised upon standing in 72% yield (720 mg; 2.78 mmol).

N-Benzoyl-L-allo-threonine ethyl ester
To a solution of the crude carboxylic acid (760 mg) in anhydrous DMF (11 mL) was added K 2 CO 3 (813 mg; 5.88 mmol; 2.8 eq) and ethyl iodide (473 μL; 5.88 mmol; 2.8 eq). The suspension was flushed with argon for at least fifteen minutes, and the reaction was stirred at room temperature overnight. The mixture was filtered, the filtrate poured into water/brine 1:1 (60 mL) and extracted with EtOAc (3x 60 mL). The combined organic layers were washed with brine (2x 100 mL), dried over Na 2 SO 4 , and solvent was removed under reduced pressure (40 °C) to yield 590 mg of crude product. The mixture was purified by column chromatography (SiO2; EtOAc/cyclohexane 1:2, R f 0.18) to yield the desired product as a yellow oil that crystallised upon standing in 18% yield over two steps (94 mg; 0.374 mmol

N-Benzoyl-D-allo-threonine ethyl ester
To a solution of the crude carboxylic acid (158 mg) in anhydrous DMF (5 mL) was added K 2 CO 3 (325 mg; 2.35 mmol; 2.8 eq) and ethyl iodide (189 μL; 2.35 mmol; 2.8 eq). The suspension was flushed with argon for at least fifteen minutes, and the reaction was stirred at room temperature overnight. The mixture was filtered, the filtrate poured into water/brine 1:1 (30 mL) and extracted with EtOAc (3x 30 mL). The combined organic layers were washed with brine (2x 50 mL), dried over Na 2 SO 4 , and solvent was removed under reduced pressure (40 °C) to yield 124 mg of crude product. The mixture was purified by column chromatography (SiO 2 ; EtOAc/cyclohexane 1:2, R f 0.18) to yield the desired product as a yellow oil that crystallised upon standing in 10% yield over two steps (23 mg; 0.091 mmol). 1

Synthesis of ethyl 3-hydroxy-2-(hydroxyimino)butanoate
A solution of oxime 1a (318 mg; 2.0 mmol; 1.0 eq) in absolute ethanol (10 mL) was cooled to 0 °C and sodium borohydride (76 mg; 4.0 mmol; 2.0 eq) was added in one portion. The bubbling solution quickly turned white and then lightly yellow. TLC analysis (SiO 2 , EtOAc/Cy 1:1, R f alcohol 0.33) showed full consumption of the oxime within ten minutes, and only one product formed. The reaction mixture was quenched by addition of water (5 mL) and then 1 M HCl (aqueous, 5 mL) while still cooled to 0 °C. The mixture was extracted with EtOAc (3x 25 mL), the combined organic layers dried over Na2SO4, filtered and solvent was removed under reduced pressure to yield the pure oxime alcohol in 50 % yield (161 mg; 1.0 mmol) as a slightly yellow oil. 1

Gene synthesis, subcloning and protein expression
The gene sequence encoding the investigated FOYE was obtained from the Genbank database entry associated with the corresponding protein sequence entry in the UniProt database. Synthetic genes were obtained from Invitrogen as linear double-stranded strings optimized for expression in E. coli containing an NdeI and XhoI restriction site on the 5' and the 3' end, respectively for subcloning into a pET28a(+) vector. Successful insertion of the target gene into the vector was confirmed by DNA sequencing. The protein of interest was expressed in E. coli BL21(DE3).

Protein expression and Protein Purification of FOYE
The expression plasmid of FOYE was transformed into chemically competent E. coli BL21(DE3) cells following the supplier's manual. A single colony of the transformation plate was used for an over-night culture (10 mL LB medium, 50 µg/mL kanamycin) which was incubated at 30 °C, 120 rpm in a shaking incubator. 2 mL of the over-night culture were used on the next day to inoculate the main culture (300 mL LB medium, 50 µg/mL kanamycin). The resulting culture was incubated at 37 °C, 120 rpm until an OD 600 of around 0.6-0.8 was reached (3-4 hours). Protein expression was then induced with isopropylβ-D-thiogalactopyranosid (IPTG) at a final concentration of 0.05 mM for 18 h at 25°C and 120 rpm. Cells were harvested via centrifugation (5000 rpm, 4 °C, 20 min) and the supernatant discarded. The pellet was resuspended in 100 mM Tris-HCl buffer, pH 7.5 and transferred to a 50 mL plastic tube. The suspension was centrifuged again (5000 rpm, 4 °C, 20 min) and the pellet stored at -20 °C until further use.
The cell pellet was thawed and resuspended in HisTrap buffer A (10 mL/g cell weight, Tris-HCl buffer, 100 mM, pH 7.5, 50 mM imidazole) supplemented with 1 mg/mL lysozyme and 1 mg FMN for cell disruption. This suspension was incubated at 30 °C, 120 rpm for 45 min and then cooled to 0 °C in an ice-water bath. Cell disruption was performed by ultrasonication using a Branson Digital Sonifier 250 at 30% amplitude, 2 s pulse, 4 s pause, for a total pulse time of 3 min (90 cycles). Cell debris was removed by centrifugation (16000 rpm, 4 °C, 20 min) and the supernatant used for protein purification via immobilized metal affinity chromatography (IMAC) as described below.
A 5 mL HisTrap FF column was equilibrated with 10 column volumes (CV) of HisTrap buffer A (Tris-HCl buffer, 100 mM, pH 7.5, 50 mM imidazole). The supernatant obtained from the cell disruption was filtered through a 0.45 µm syringe filter prior to the application onto the column. Unbound proteins were removed from the HisTrap column by washing with 10 CV of HisTrap buffer A. The protein of interest was then eluted with 5-10 mL of HisTrap buffer B (Tris-HCl buffer, 100 mM, pH 7.5, 500 mM imidazole) and concentrated using a Sartorius VivaSpin 20 centrifugal filter with a molecular weight cut-off of 10 kDa.
The concentrated protein solution was desalted using a GE Healthcare PD 10 desalting column with Tris HCl buffer, 100 mM, pH 7.5 as the eluent. Protein concentration of the desalted sample was determined using the Biorad Bradford assay following the supplier's instructions. For exchange of the protein into phosphate buffer (50 mM, pH 7.5), the purified protein was loaded onto a PD10 column and eluted following the gravity protocol described by the supplier.
All other enzymes were expressed as reported previously. [10]

Analytics
The retention times of various products analysed on GC-FID can be found in Table S4. Retention times of the compounds analysed after cascade reactions can be found in Table S5. For the HPLC parameters, see the General section.  A chromatogram showing the pyrazine, internal standard and all four enantiomers of the threonine product is shown in Figure S3. NB: an peak due to ethyl acetate is present at 4.25 min.