Enantiocomplementary Michael Additions of Acetaldehyde to Aliphatic Nitroalkenes Catalyzed by Proline‐Based Carboligases

Abstract The blockbuster drug Pregabalin is widely prescribed for the treatment of painful diabetic neuropathy. Given the continuous epidemic growth of diabetes, the development of sustainable synthesis routes for Pregabalin and structurally related pharmaceutically active γ‐aminobutyric acid (GABA) derivatives is of high interest. Enantioenriched γ‐nitroaldehydes are versatile synthons for the production of GABA derivatives, which can be prepared through a Michael‐type addition of acetaldehyde to α,β‐unsaturated nitroalkenes. Here we report that tailored variants of the promiscuous enzyme 4‐oxalocrotonate tautomerase (4‐OT) can accept diverse aliphatic α,β‐unsaturated nitroalkenes as substrates for acetaldehyde addition. Highly enantioenriched aliphatic (R)‐ and (S)‐γ‐nitroaldehydes were obtained in good yields using two enantiocomplementary 4‐OT variants. Our results underscore the synthetic potential of 4‐OT for the preparation of structurally diverse synthons for bioactive analogues of Pregabalin.


Synthesis of nitroalkenes
General procedure for the synthesis of compounds 2b,c,f,h To an oven-dried round-bottom flask charged with a magnetic stir-bar was added TEMPO (0.4 equiv.), olefin (4) (1.0 equiv.), t BuONO (2.0 equiv.) and 1,4-dioxane (20 mL). [1] After the addition of all the starting material, the mixture was stirred at 90 °C for 12 h. After the completion of starting material (monitored by TLC), the reaction mixture was cooled to room temperature followed by work-up using EtOAc/H2O (3 x 20 mL). The organic extracts were combined, dried over Na2SO4, and evaporated using a rotary evaporator. The crude products were further purified by silica gel column chromatography (using petroleum ether/ethyl acetate 95:5) as an eluent to give the corresponding nitroalkenes 2.
General procedure for the synthesis of compounds 2d,e To a mixture of aldehyde (5) (1.0 equiv.) and nitromethane (6) (1.0 equiv.) in methanol (10 mL) at 0 °C, a solution of NaOH in H2O (1.2 equiv.) was added dropwise. Further methanol (2 mL) was added and the resulting yellow slurry stirred at 0 °C for 1 h. Water (30 mL) was added and the clear yellow solution was poured into hydrochloric acid (20 mL conc. hydrochloric acid in 30 mL H2O) and stirred for 15 min. The aqueous mixture was extracted with DCM (10 mL x 3), the combined organic layers dried over sodium sulfate, and the solvent removed using a rotary evaporator. The residue was purified by column chromatography (using petroleum ether/ethyl acetate 95:5) to give the nitro-olefins 2.

Synthesis of racemic reference compounds 3b-f
Aldehyde (S7) (1.0 equiv.) was dissolved in 5 mL anhydrous DCM and treated with carbethoxymethylene triphenylphosphorane (S8) (1.1 equiv.) at room temperature. After the complete addition of all the starting material, the mixture was stirred at rt for 12 h. After the completion of starting material (monitored by TLC, KMnO4 staining), the reaction mixture was quenched with saturated aqueous ammonium chloride, and extracted 3 times with DCM. The organic extracts were combined, dried over Na2SO4, and evaporated using a rotary evaporator. The crude products were further purified by silica gel column chromatography (using petroleum ether/ethyl acetate 95:5) as an eluent to give the corresponding α,β-unsaturated ester S9.
The 1 H NMR data of compound S9b match with earlier reported NMR data. [  dried over Na2SO4, and evaporated using a rotary evaporator. The crude products were further purified by silica gel column chromatography (using petroleum ether/ethyl acetate from 95:5 to 90:10) as an eluent to give the corresponding Michael adducts S10.
After the complete addition of DIBAL-H, the mixture was stirred at the same temperature for 3 h and then at room temperature for 1 h. After the completion of starting material (monitored by TLC, KMnO4 staining), the reaction mixture was quenched with saturated aqueous ammonium chloride, and extracted 3 times with DCM. The organic extracts were combined, dried over Na2SO4, and evaporated using a rotary evaporator. The crude products were further purified by silica gel column chromatography (using petroleum ether/ethyl acetate from 90:10 to 80:20) as an eluent to give the corresponding alcohols S11.

Expression and purification of 4-OT
The expression and purification of 4-OT mutants were performed using slightly modified previously reported procedures. [16] Briefly, 1 l of LB medium substituted with 0.5 % glycerol and 100 µg/ml ampicillin was inoculated from a glycerol stock with E. coli BL21(DE3) harboring a pJexpress 414 vector with the respective 4-OT gene. After 1 h at 37 °C, 200 rpm the expression was induced with 100 µM IPTG and continued at 37 °C, 200 rpm overnight.
After the expression of 4-OT the cells were collected and the enzymes were purified according to previously reported methods. [17,18] The enzyme concentration was determined using the Waddell method. [19] The purified enzymes were flash frozen in liquid nitrogen and stored at -