High Regio‐ and Stereoselective Multi‐enzymatic Synthesis of All Phenylpropanolamine Stereoisomers from β‐Methylstyrene

Abstract We present a one‐pot cascade for the synthesis of phenylpropanolamines (PPAs) in high optical purities (er and dr up to >99.5 %) and analytical yields (up to 95 %) by using 1‐phenylpropane‐1,2‐diols as key intermediates. This bioamination entails the combination of an alcohol dehydrogenase (ADH), an ω‐transaminase (ωTA) and an alanine dehydrogenase to create a redox‐neutral network, which harnesses the exquisite and complementary regio‐ and stereo‐selectivities of the selected ADHs and ωTAs. The requisite 1‐phenylpropane‐1,2‐diol intermediates were obtained from trans‐ or cis‐β‐methylstyrene by combining a styrene monooxygenase with epoxide hydrolases. Furthermore, in selected cases, the envisioned cascade enabled to obtain the structural isomer (1S,2R)‐1‐amino‐1‐phenylpropan‐2‐ol in high optical purity (er and dr >99.5 %). This is the first report on an enzymatic method that enables to obtain all of the four possible PPA stereoisomers in great enantio‐ and diastereo‐selectivity.


List of abbreviations
General Information. Enzymatic synthesis of the diol substrates, the selection of the secondary NAD +dependent ADHs and the chemical synthesis of amino alcohol references 5 and 5' are reported elsewhere. [1] All the enzymes used in this study are reported in Table S1 together with the source and expression conditions.  (2) Ref. [2] lyophilized whole cells [2] Sp(S)-EH Sphingomonas sp. HXN200 pET28b N-His6 1 mM IPTG, 25 °C 16 h lyophilized whole cells [3] St(R)-EH Solanum tuberosum pET28b N-His6 1 mM IPTG, 25 °C 16 h lyophilized whole cells [3] Aa-ADH Aromatoleum aromaticum pET28b N-His6

General procedures for expression and purification of enzymes
Note: in the case of transaminases, PLP (0.5 mM) was added to the cell lysate before loading on the column; protein concentration was determined according to Bradford assay using Ovalbumin as standard protein. [17] Expression of the enzymes: For recombinant expression, 800 mL of LB medium supplemented with the appropriate antibiotic (100 µg mL -1 ampicillin or 50 µg mL -1 kanamycin) were inoculated with 15 mL of an overnight culture harbouring the desired vector with genes for the expression of the enzymes. E. coli BL21 DE3 cells were used as host organism. Cells were grown at 37 °C until an OD600 of 0.6 to 1 was reached and the expression of the protein was induced by the addition of IPTG. Protein expression was carried out overnight and after harvesting of the cells by centrifugation (4 °C, 4500 rpm, 10 min), the remaining cell pellets were washed with buffer (for lyophilized cells: 50 mM Tris-HCl buffer, pH 8.0 for ADHs; 50 mM KPi, pH 8.0 for EHs and TAs; or lysis buffer for enzymes that were subsequently purified by affinity chromatography).
Purification by Ni 2+ affinity chromatography: His6-tagged proteins were resuspended in lysis buffer (50 mM KH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) prior to cell disruption, and protein purification was performed by Ni-NTA affinity chromatography using pre-packed Ni-NTA HisTrap HP columns (GE Healthcare), previously equilibrated with lysis buffer. After loading of the filtered lysate, the column was washed with sufficient amounts of washing buffer (50 mM KH2PO4, 300 mM NaCl, 25 mM imidazole, pH 8.0), and bound protein was recovered with elution buffer (50 mM KH2PO4, 300 mM NaCl, 200 mM imidazole, pH 8.0). Purity was analysed by SDS-PAGE and fractions showing >95% purity were combined and dialyzed overnight against Tris-HCl buffer (6 L, pH 8.0, 20 mM) or KPi buffer (6 L, pH 8.0, 50 mM). The enzyme solutions were concentrated and their concentration was determined spectrophotometrically based on their extinction coefficient at 280 nm.
Purification by Strep affinity chromatography: Strep-tagged proteins were resuspended in binding buffer (100 mM Tris-HCl, 150 mM NaCl and 1 mM EDTA, pH 8.0) prior to cell disruption, and protein purification was performed by Strep affinity chromatography using pre-packed columns with StrepTactin Sepharose (GE Healthcare), previously equilibrated with binding buffer. After washing of the column, the tagged protein was recovered with elution buffer (100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA and 2.5 mM desthiobiotin, pH 8.0). Purity was analysed by SDS-PAGE and fractions showing >95% purity were combined and dialyzed overnight against Tris-HCl buffer (6 L, pH 8.0, 20 mM). The enzyme solutions were concentrated and their concentration was determined spectrophotometrically based on their extinction coefficient at 280 nm. General procedure for the derivatization of the amino alcohol products to determine er and dr [18] . The aqueous reaction mixture (20 µL) was dissolved in acetonitrile (180 µL) to yield a final concentration of 0.5 mM. Then, GITC (1.5 mM) and Et3N (1.5 mM) were added as a solution in acetonitrile (200 µL). The mixture was incubated at room temperature at 1000 rpm for 35 min. Before injection into the RP-HPLC, the samples were centrifuged and filtered if required.

Screening of Aa-ADH with a panel of ω-transaminases with substrate (1S,2S)-3
The  Used as lyophilized E.coli whole cells (20 mg mL -1 ); [b] Determined by RP-HPLC (C18 column) after derivatization of the amino group with a chiral reagent (GITC); reactions were performed in duplicate and the reported results are the average of two samples.

Screening of Bs-BDHA with a panel of ω-transaminases with substrate (1S,2R)-3
The  [a] Used as lyophilized E.coli whole cells (20 mg mL -1 ); [b] determined by RP-HPLC (C18 column) after derivatization of the amino group with a chiral reagent (GITC); reactions were performed in duplicate and the reported results are the average of two samples.

One-pot biocatalytic synthesis of enantiomerically pure diols (1S,2R)-3 and (1R,2R)-3 from cisand trans-β-methylstyrene at hundreds of milligrams scale
General remark: The concentrations of coenzymes and co-substrate are always calculated on the volume of the aqueous phase, whereas the concentration of the substrate is referred to the organic phase.
Reaction conditions: Lyophilized E. coli cells co-expressing Fus-SMO/Cb-FDH (250 mg, 5 mg mL -1 ) were rehydrated in KPi buffer (50 mL, 50 mM, pH 8.0) in a baffled Erlenmeyer flask (500 mL). After that, NAD + (1 mM), HCOONa (5 eq.), FAD (50 µM) and catalase (0.1 mg mL -1 ) were added. Heptane (50 mL; 1:1 volumetric ratio with the buffer) was used as biphasic solvent. Finally, the biocatalytic reactions were initiated by the addition of substrate trans or cis-1 (50 mM, 2.5 mmol). The reactions were incubated at 30 °C and 200 rpm on an orbital shaker. After 5 hours, lyophilized E. coli cells expressing either Sp(S)-EH or St(R)-EH (1 g, 20 mg mL -1 ) were added and the reactions were further incubated at 30 °C and 170 rpm on an orbital shaker for 30 h. Heptane was removed, the aqueous phase was saturated with solid NaCl and the organic compounds extracted with MTBE (3 x 50 mL). After drying over MgSO4, the organic phase was removed under reduced pressure. The conversions and purity of the isolated products were determined by GC-FID, while er [%] and dr [%] were analysed by HPLC (Table S8).

One-pot biocatalytic amination of (1R,2R)-3 and (1S,2R)-3 at hundreds of milligrams scale
The one-pot biocatalytic amination on preparative scale was performed for two out of the four enantiomerically pure or enantiomerically enriched diols 3 as reported in Table S8 by applying the previously identified optimal conditions. Conversions and isolated products were analyzed by GC-FID (method A), whereas enantiomeric and as diastereomeric ratios were determined by RP-HPLC (method B) after derivatization with GITC.  Table S8.  Table S9.