Asymmetric Enantio-complementary Synthesis of Thioethers via Ene-Reductase-Catalyzed C-C Bond Formation

Abstract

Enzymes are attractive catalysts due to their high chemo-, regio-, and enantioselectivity. In recent years, the application of enzymes in organic synthesis has expanded dramatically, especially for the synthesis of chiral alcohols and amines, two very important functional groups found in many active pharmaceutical ingredients (APIs). Indeed, many elegant routes employing such compounds have been described by industry. Yet, for the synthesis of chiral thiols and thioethers, likewise found in APIs albeit less ubiquitous, only very few biocatalytic syntheses have been reported, and stereocontrol has proved challenging. Here, we apply ene-reductases (EREDs), whose ability to initiate and control chemically challenging radical chemistries has recently emerged, to the synthesis of chiral thioethers from α-bromoacetophenones and pro-chiral vinyl sulfides, without requiring light. Depending on the choice of ERED either enantiomer of the product could be accessed. The highest conversion and selectivity were achieved with GluER T36A using fluorinated substrates, reaching up to 82% conversion and >99.5% ee. With α-bromoacetophenone and α-(methylthio)styrene, the reaction could be performed on a 100 mg scale, affording the product in a 46% isolated yield with a 93% ee. Finally, mechanistic studies were carried out using stopped-flow spectroscopy and protein mass spectrometry, providing insight into the preference of the enzyme for the intermolecular reaction. This work paves the way for new routes for the synthesis of thioether-containing compounds.

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(A) Chiral alcohols and chiral amines and the main enzyme classes that have been applied in biocatalytic approaches for their production. ADH: alcohol dehydrogenase, PDC: pyruvate decarboxylase, UPO: unspecific peroxygenase, HHDH: halohydrin dehalogenase, HNL: hydroxynitrile lyase, IRED: imine reductase, AmDH: amine dehydrogenase, ATA: amine transaminase, RedAm: reductive aminase, ODH: opine dehydrogenase, MAO: monoamine oxidase. (B) Examples of APIs containing chiral thioethers. (C) Examples of chiral thiols and thioethers generated biocatalytically. HMFO: hydroxymethylfurfural oxidase, ERED: ene reductase. (D) Selected examples of recently developed enzymatically controlled radical reactions. GDH: glucose dehydrogenase, TAld: threonine aldolase, PC: photocatalyst, Ox.: oxidant. (E) Proposed synthesis of chiral thioethers from pro-chiral vinyl sulfides.

1. Proposed Side Product Formation

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Time course of the GluER T36A-catalyzed reaction between 1a and 2a. Conditions: d-glucose (55 mM), NADP⁺ (0.5 mM), JM GDH-101 (0.5 mg mL^–1), 1a–d (10 mM), 2a (10 mM), ERED (1.4 mol %), Tris-HBr (50 mM), pH 7.5, 25 °C, 750 rpm, anoxic. Independent reactions for each time point. Time points are averages; error bars are standard deviations (n = 2).

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Substrate scope. Conversions based on relative HPLC areas. Conditions: d-glucose (55 mM), NADP⁺ (0.5 mM), JM GDH-101 (0.5 mg mL^–1), 1a–d (10 mM), 2a–h (10 mM), ERED (1.4 mol %), Tris-HBr (50 mM), pH 7.5, 25 °C, 750 rpm, anoxic, 24 h. ^a isolated yield (49.5 mg), d-glucose (100 mM), NADP⁺ (0.5 mM), JM GDH-101 (0.5 mg mL^–1), 1a (20 mM), 2a (20 mM), ERED (1.0 mol %), Tris-HBr (100 mM), pH 7.5, 23–24 °C, anoxic, 24 h. (R)-3aa: CCDC 2410188.

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Mechanistic investigations. (A) Proposed catalytic cycle. SET: single electron transfer. HAT: hydrogen atom transfer. (B) Presteady state kinetics measured by stopped-flow. No signal for the semiquinone was observed (see Figure S5), implying that the SET is the rate-determining step. The reaction was thus modeled as a single step (rate constant k _ox). An acceleration of the reaction was observed in the presence of the vinyl cosubstrate (2a). The affinity for 2a was 1 order of magnitude higher compared to 1a, explaining the exquisite selectivity of the enzyme for the formation of 3aa. (C) Deuterium incorporation experiments, showing that the hydrogen at the benzylic position originates from the FMN cofactor.

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(A) Outcome of biotransformations with GluER T36A with 2a only, 1a only, or both 1a and 2a. Concentrations determined by HPLC using a calibration curve. It is apparent that in the absence of 2a, the fate of the α-acyl intermediate is not predominantly 4a. Also shown is the appearance of biotransformations with GluER T36A after 24 h with no substrate, 2a only, 1a only, or both 1a and 2a. The yellow color in the reaction with 1a is indicative of oxidized flavin, implying enzyme inactivation. (B) UV–vis spectra of reactions containing 1a, as well as controls without the substrate, GluER T36A, or GDH. The spectrum of FMN after a reaction with 1a only matches that of enzyme-bound oxidized flavin (no substrate, no GDH control). The control containing 1a but no GDH showed extensive precipitation of GluER T36A resulting in a lower flavin peak. In the absence of 1a, FMN remains fully reduced, with some slight turbidity. (C) Coomassie-stained SDS-PAGE gel of biotransformations with GluER T36A after 24 h with no substrate, 2a only, 1a only, or both 1a and 2a. The orange box shows GluER T36A. (D) Difference in peptide abundance after tryptic digest of the band of GluER T36A in the reaction with 1a only and without the substrate. For the difference in peptide abundance of 2a only and 1a and 2a, see Figures S5 and S6. (E) Active site of GluER T36A (PDB: 6MYW) with acetate bound, showing peptides with decreased abundance by color, putative amino acids in the active site that may become modified by radical reactions, and C97 which is distal from the active site but may have formed an adduct with the α-acyl radical.