Enantioselective reduction of sulfur-containing cyclic imines through biocatalysis

The 3-thiazolidine ring represents an important structural motif in life sciences molecules. However, up to now reduction of 3-thiazolines as an attractive approach failed by means of nearly all chemical reduction technologies for imines. Thus, the development of an efficient general and enantioselective synthetic technology giving access to a range of such heterocycles remained a challenge. Here we present a method enabling the reduction of 3-thiazolines with high conversion and high to excellent enantioselectivity (at least 96% and up to 99% enantiomeric excess). This technology is based on the use of imine reductases as catalysts, has a broad substrate range, and is also applied successfully to other sulfur-containing heterocyclic imines such as 2H-1,4-benzothiazines. Moreover the effiency of this biocatalytic technology platform is demonstrated in an initial process development leading to 99% conversion and 99% enantiomeric excess at a substrate loading of 18 g/L in the presence of designer cells.


General experimental information
Reactions that were sensitive to moisture were performed in dried glassware and under argon atmosphere. All commercially available reagents were used as received. Solvents were either used in high-grade purity or purified by distillation.
Column Chromatography was performed by manual column chromatography with silica 60 (0.04-0.063 μm particle size) or by Biotage "Isolera One" flash chromatography system with cyclohexane/ethyl acetate mixtures.
NMR spectra were recorded on Bruker Avance III 500 or Bruker Advance III 500HD at a frequence of 500 MHz ( 1 H) or 125 MHz ( 13 C). The chemical shift δ is given in ppm and referenced to the corresponding solvent signal (CDCl3). Coupling constants (J) are given in Hz.
Nano-ESI mass spectra were recorded using an Esquire 3000 ion trap mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) equipped with a standard nano-ESI source. Samples were introduced by static nano-ESI using in-house pulled glass emitters. Nitrogen served both as the nebulizer gas and the dry gas. Nitrogen was generated by a Bruker nitrogen generator NGM 11. Helium served as cooling gas for the ion trap and collision gas for MS n experiments.
HRMS-ESI mass spectra are recorded using an Agilent 6220 time-of-flight mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) in extended dynamic range mode equipped with a Dual-ESI source, operating with a spray voltage of 2.5 kV. Nitrogen served both as the nebulizer gas and the dry gas. Nitrogen was generated by a nitrogen generator NGM 11. Samples are introduced with a 1200 HPLC system consisting of an autosampler, degasser, binary pump, column oven and diode array detector (Agilent Technologies, Santa Clara, CA, USA) using a C18 Hypersil Gold column (length: 50 mm, diameter: 2.1 mm, particle size: 1,9 μm) with a short gradient (in 4 min from 0% B to 98% B, back to 0% B in 0.2 min, total run time 7.5 min) at a flow rate of 250 μL/min and column oven temperature of 40°C. HPLC solvent A consists of 94.9% water, 5% acetonitrile and 0.1% formic acid, solvent B of 5% water, 94.9% acetonitrile and 0.1% formic acid. The mass axis was externally calibrated with ESI-L Tuning Mix (Agilent Technologies, Santa Clara, CA, USA) as calibration standard.
EI mass spectra were recorded using an Autospec X magnetic sector mass spectrometer with EBE geometry (Vacuum Generators, Manchester, UK) equipped with a standard EI source. Samples were introduced by push rod in aluminium crucibles if not otherwise noted. Ions were accelerated by 8 kV in EI mode.

Chemical attempts towards reduction of 3-thiazolines
Reduction with hydrogen and palladium on activated carbon 2,2,4-trimethyl-3-thiazoline (1c) (500 mg, 3.87 mmol) is dissolved in methanol (5 mL) and palladium on activated carbon (10%) (41.0 mg, 0.39 mmol, 10 mol%) is added. The reaction mixture is stirred for 18 h under hydrogen atmosphere at room temperature. Palladium on carbon is filtered off, washed with cold methanol und the solvent evaporated in vacuo. 1c was not converted.

Reduction using LiAlH4
Finely crushed LiAlH4 (58.8 mg, 1.55 mmol) is dissolved in diethylether (4 mL) and cooled to 4 °C. 2,2,4trimethyl-3-thiazoline (1c) (200 mg, 1.55 mmol) is dissolved in diethylether (2 mL) and added to the suspension at 4 °C. The reaction mixture is stirred at room temperature for 2 h. Ice water and diethylether are added and the organic phase is decanted carefully. The salts are washed three times with diethylether. The combined organic phases are dried over magnesium sulfate and the solvent is evaporated in vacuo. 1c was not reduced to the desired 3-thiazolidine. In contrast to this ring opening of the N,S-acetal and other cleavage products were observed.

DNA and Protein Sequences of imine reductases
Codon optimized DNA sequences and protein sequenes of imine reductases (IREDs) containing N-or C-terminal His6-Tag on pET-22b(+) vector.

General Procedure 1 (GP 1): α-chlorination of aldehydes and ketones
The synthesis is conducted to an adapted procedure from Rodig et. al. 4 Sulfuryl chloride (0.50 mol, 1.00 equ.) is added under cooling at vacuum of 900 mbar to the aldehyde or ketone, keeping the temperature stable at 40 °C. The reaction mixture is stirred afterwards for 2 h at 45 °C and 900 mbar. The crude product is purified by fractional distillation.

GP 2: Synthesis of 3-thiazolines
The synthesis is conducted according to Martens et al. 6

GP 3: Synthesis of 2H-1,4-benzothiazines
The synthesis is conducted according to an adapted procedure from Stalling et al. 9 Sodium (   The analytical data corresponds with literature data. 9      (2 x CH3).

Synthesis of reference compounds GP4: Synthesis of 3-thiazolidines
The synthesis is conducted according to an adapted procedure from Reiners et al.

GP 5: Derivatization of 3-thiazolidines with phenylisocyanate
The synthesis is conducted according to Reiners et al.

GC analytics
Conversions for biotransformations of 3-thiazolines 1b-f to the corresponding 3-thiazolidines 2b-f were determined by analyzing the organic phase directly after extraction. Analysis was carried out using
Enantiomeric excess was determined based on area% of the enantiomers. Retention Times are given in Supplementary Table 6, HPLC chromatograms are shown in Supplementary Figures 79-82.

Chiral HPLC analytics for 2H-1,4-benzothiazines/3,4-dihydro-2H-1,4-benzothiazines
For the analysis of the biotransformations of 2H-1,4-benzothiazines a combined approach for the determination of the conversions and the enantiomeric excess was used. HPLC measurements were performed by analyzing the organic phase directly after extraction. Analysis was carried out by LC2000

Colorimetric pH shift assay
The colorimetric pH shift assay is conducted according to Pick et al. 12 This assay is an indirect screening method, based on a color change of bromthymolblue depending on the pH. A decrease of pH under 5 leads to a color change from blue/green to yellow. The formation of gluconic acid due to the consumption of the substrate and the regeneration of NADPH decreases the pH, resulting in the color change (Supplementary Figure 3).

Spectrophotometric activity assay
For the determination of the specific activity, a spectrophotometric activity study was performed, The results for the spectrophotometric activity assay are shown in Supplementary Table 3   In addition, calculations utilizing the SMD intrinsic solvation model 16 were also performed, and parameters for water and chloroform were used. All geometries were reoptimized with the intrinsic solvation model using the functional and basis set mentioned above. Coordinates for the initial complexes of the starting materials and the transition states are given below. Methods; synthesis of racemic 3-3,4-dihydro-2H-1,4-benzothiazine reference compounds is described in Supplementary Methods and related NMR data are shown in Supplementary Figures 96-101). The results of these experiments are shown in Table 2 (main manuscript).

Benzothiazin: complex of starting materials in water
Negative controls were performed on 0.5 mL scale at 30 °C and 850 rpm in 100 mM KPi buffer pH 7, with 4% methanol (in case of 3a and 3b) or dimethylsulfoxide (in case of 3c) as cosolvent containing 40 mM D-glucose, 20 mM 2H-1,4-benzothiazine 3a-c, 12 U of GDH and 0.1 mM NADP + . After 4, 6 or 8 h, the reaction was stopped by adding 10 µL of 32% NaOH solution and 300 µL of dichloromethane.
Phase separation was promoted by centrifugation and the conversion was determined by analyzing the organic phase by means of LC2000 SFC-HPLC system from Jasco (Easton, USA) (Supplementary Table 7 and Supplementary Methods).

Determination of the absolute configuration of (S)-2f
The absolute configuration of (S)-2,2,3-Trimethyl-1-thia-4-azaspiro [4.4]nonane ((S)-2f) was determined by vibrational circular dichroism (VCD) spectroscopy. The IR and VCD spectra were recorded for a 0.3 M solution of (S)-2f in CDCl3 at a pathlength of 100 µm over the course of 8 hrs accumation time (~35000 scans). The VCD baseline was corrected by subtraction of the spectrum of the racemic mixture 2f recorded under identical condition. The experimentally obtained spectra are shown in Figure 4 (main manuscript).
In order to determine the absolute configuration, a conformational analysis was carried out for (S)-2f at the MMFF level of theory using Spartan 14 (Spartan 14, Wavefunction Inc., Irvine, CA, USA (2014)).
Subsequently, all eight obtained conformers were subjected to further geometry optimizations followed by spectra calculations at the B3LYP/6-311g++(2d,p)/IEFPCM(CHCl3) level of theory (Gaussian 09 Rev. Boltzmann-averaging of the spectra. Direct comparison of the resulting simulated IR and VCD spectra with the experimental data, as indicated by the assignments given in Figure 4 (main manuscript), reveals a very good agreement. Therefore, the absolute configuration can with very high confidence be assigned as (S)-2f.

Construction and preparation of whole cell-catalyst
Escherichia coli strain BL21(DE3), which was used for expression, and pACYCDuet-1 vector were purchased from Novagen (Madison, USA). The whole-cell catalyst was constructed as a two-plasmid-  Table   4 and Supplementary Methods). For isolation of 2,2,3-trimethyl-1-thia-4-azaspiro [4.4]nonane (2f) the crude product was dissolved in dichloromethane and was washed with dH2O (2 x 30 mL) and brine (30 mL). The organic phase was dried over magnesium sulfate and the solvent was evaporated in vacuo.