Asymmetric Synthesis of Optically Pure Pharmacologically Relevant Amines Employing ω‐Transaminases

Various ω‐transaminases were tested for the synthesis of enantiomerically pure amines from the corresponding ketones employing D ‐ or L ‐alanine as amino donor and lactate dehydrogenase to remove the side‐product pyruvate to shift the unfavourable reaction equilibrium to the product side. Both enantiomers, (R)‐ and (S)‐amines, could be prepared with up to 99% ee and >99% conversions within 24 h at 50 mM substrate concentration. The activity and stereoselectivity of the amination reaction depended on the ω‐transaminase and substrate employed; furthermore the co‐solvent significantly influenced both the stereoselectivity and activity of the transaminases. Best results were obtained by employing ATA‐117 to obtain the (R)‐enantiomer and ATA‐113 or ATA‐103 to access the (S)‐enantiomer with 15% v v⁻¹ DMSO.     

Regio‐ and Stereoselective Biocatalytic Monoamination of a Triketone Enables Asymmetric Synthesis of Both Enantiomers of the Pyrrolizidine Alkaloid Xenovenine Employing Transaminases

The (+)‐ as well as the (−)‐enantiomer of the pyrrolizidine alkaloid xenovenine were prepared within five steps with 17 and 30% overall yields, respectively, in optically pure form, >99% ee as well as >99% de. In the asymmetric key step a transaminase performed a regio‐ and stereoselective monoamination of a triketone. By employing two enantiocomplementary transaminases from Arthrobacter sp. both enantiomers were accessible. The triketone was readily prepared via two steps starting from commercially available, achiral 2‐(n‐heptyl)furan. In the final catalytic hydrogenation step, the newly introduced chiral centre directed hydrogen addition to form preferentially the desired (5Z,8E)‐diastereomer. The regio‐ and stereoselective amination of a single ketone moiety out of three allowed the performance of the shortest and highest yielding total synthesis of the bicyclic showcase pyrrolizidine alkaloid without the need for protecting strategies.

     

Cutting Short the Asymmetric Synthesis of the Ramatroban Precursor by Employing ω‐Transaminases

Starting from an adequate ketone precursor previous reports required three steps for the preparation of (R)‐2,3,4,9‐tetrahydro‐1H‐carbazol‐3‐amine, a key intermediate for the synthesis of the antiallergic drug ramatroban. A single biocatalytic step was sufficient to prepare the target amine with >97% ee (HPLC) via reductive amination of the corresponding ketone using an ω‐transaminase as biocatalyst. Since the ketone was barely soluble under the reaction conditions employed, it was provided as a solid and still the reaction went to completion within 4 h at 50 mM substrate concentration. Although 2‐propylamine is regarded as an ideal amine donor, it turned out to be detrimental for the specific ketone precursor leading to the formation of various side products. These could be avoided by using (R)‐1‐phenylethylamine as the best suited amine donor. An alternative work‐up was developed via freeze‐drying of the reaction mixture, enabling the isolation of the desired (R)‐amine in excellent yield (96%) and enantiopure form on a preparative scale (500 mg). No purification steps (e.g., column chromatography, crystallisation) were required.

     

Computer Modeling Explains the Structural Reasons for the Difference in Reactivity of Amine Transaminases Regarding Prochiral Methylketones

Amine transaminases (ATAs) are pyridoxal-5′-phosphate (PLP)-dependent enzymes that catalyze the transfer of an amino group from an amino donor to an aldehyde and/or ketone. In the past decade, the enzymatic reductive amination of prochiral ketones catalyzed by ATAs has attracted the attention of researchers, and more traditional chemical routes were replaced by enzymatic ones in industrial manufacturing. In the present work, the influence of the presence of an α,β-unsaturated system in a methylketone model substrate was investigated, using a set of five wild-type ATAs, the (R)-selective from Aspergillus terreus (Atr-TA) and Mycobacterium vanbaalenii (Mva-TA), the (S)-selective from Chromobacterium violaceum (Cvi-TA), Ruegeria pomeroyi (Rpo-TA), V. fluvialis (Vfl-TA) and an engineered variant of V. fluvialis (ATA-256 from Codexis). The high conversion rate (80 to 99%) and optical purity (78 to 99% ee) of both (R)- and (S)-ATAs for the substrate 1-phenyl-3-butanone, using isopropylamine (IPA) as an amino donor, were observed. However, the double bond in the α,β-position of 4-phenylbut-3-en-2-one dramatically reduced wild-type ATA reactivity, leading to conversions of <10% (without affecting the enantioselectivity). In contrast, the commercially engineered V. fluvialis variant, ATA-256, still enabled an 87% conversion, yielding a corresponding amine with >99% ee. Computational docking simulations showed the differences in orientation and intermolecular interactions in the active sites, providing insights to rationalize the observed experimental results.     

Cooperative Catalysis with Iron and a Chiral Brønsted Acid for Asymmetric Reductive Amination of Ketones

The direct asymmetric reductive amination (DARA) of ketones with anilines is described by combining a chiral Brønsted acid (TRIP) and the non‐chiral Knölker iron complex as the catalyst system. In situ‐formed imines are reduced with molecular hydrogen to give chiral amines in high yields (90%) and enantioselectivities of up to 99% ee.

     

Sequential Two-Step Stereoselective Amination of Allylic Alcohols through the Combination of Laccases and Amine Transaminases

A sequential two-step chemoenzymatic methodology for the stereoselective synthesis of (3E)-4-(het)arylbut-3-en-2-amines in a highly selective manner and under mild reaction conditions is described. The approach consists of oxidation of the corresponding racemic alcohol precursors by the use of a catalytic system made up of the laccase from Trametes versicolor and the oxy-radical TEMPO, followed by the asymmetric reductive bio-transamination of the corresponding ketone intermediates. Optimisation of the oxidation reaction, exhaustive amine transaminase screening for the bio-transaminations and the compatibility of the two enzymatic reactions were studied in depth in search of a design of a compatible sequential cascade. This synthetic strategy was successful and the combinations of enzymes displayed a broad substrate scope, with 16 chiral amines being obtained in moderate to good isolated yields (29–75 %) and with excellent enantiomeric excess values (94 to >99 %). Interestingly, both amine enantiomers can be achieved, depending on the selectivity of the amine transaminase employed in the system.     

Direct Synthesis of Primary Amines via Ruthenium‐Catalysed Amination of Ketones with Ammonia and Hydrogen

A highly selective reductive amination of ketones to primary amines with ammonia and hydrogen using a simple ruthenium catalyst has been developed. The protocol described constitutes an efficient and direct atom‐economical approach en route to α‐methylbenzylamine derivatives in good to high yields. The presence of catalytic amounts of aluminum triflate turned out to be crucial for achieving high conversion towards primary amines.

     

Preparation of (R)‐Amines from Racemic Amines with an (S)‐Amine Transaminase from Bacillus megaterium

Screening was carried out to identify strains useful for the preparation of (R)‐1‐cyclopropylethylamine and (R)‐sec‐butylamine by resolution of the racemic amines with an (S)‐specific transaminase. Several Bacillus megaterium strains from our culture collection as well as several soil isolates were found to have the desired activity for the resolution of the racemic amines to give the (R)‐enantiomers. Using an extract of the best strain, Bacillus megaterium SC6394, the reaction was shown to be a transamination requiring pyruvate as amino acceptor and pyridoxal phosphate as a cofactor. Initial batches of both amines were produced using whole cells of Bacillus megaterium SC6394. The transaminase was purified to homogeneity to obtain N‐terminal as well as internal amino acid sequences. The sequences were used to design polymerase chain reaction (PCR) primers to enable cloning and expression of the transaminase in E. coli SC16578. In contrast to the original B. megaterium process, pH control and aeration were not required for the resolution of sec‐butylamine and an excess of pyruvate was not consumed by the recombinant cells. The resolution of sec‐butylamine (0.68 M) using whole cells of E. coli SC16578 was scaled up to give (R)‐sec‐butylamine⋅1/2 H₂SO₄ in 46.6% isolated yield with 99.2% ee. An alternative isolation procedure was also used to isolate (R)‐sec‐butylamine as the free base.     

Asymmetric Reduction of (R)-Carvone through a Thermostable and Organic-Solvent-Tolerant Ene-Reductase

Ene-reductases allow regio- and stereoselective reduction of activated C=C double bonds at the expense of nicotinamide adenine dinucleotide cofactors [NAD(P)H]. Biological NAD(P)H can be replaced by synthetic mimics to facilitate enzyme screening and process optimization. The ene-reductase FOYE-1, originating from an acidophilic iron oxidizer, has been described as a promising candidate and is now being explored for applied biocatalysis. Biological and synthetic nicotinamide cofactors were evaluated to fuel FOYE-1 to produce valuable compounds. A maximum activity of (319.7±3.2) U mg⁻¹ with NADPH or of (206.7±3.4) U mg⁻¹ with 1-benzyl-1,4-dihydronicotinamide (BNAH) for the reduction of N-methylmaleimide was observed at 30 °C. Notably, BNAH was found to be a promising reductant but exhibits poor solubility in water. Different organic solvents were therefore assayed: FOYE-1 showed excellent performance in most systems with up to 20 vol% solvent and at temperatures up to 40 °C. Purification and application strategies were evaluated on a small scale to optimize the process. Finally, a 200 mL biotransformation of 750 mg (R)-carvone afforded 495 mg of (2R,5R)-dihydrocarvone (>95 % ee), demonstrating the simplicity of handling and application of FOYE-1.     

Highly Efficient Chemoenzymatic Synthesis of Methyl (R)‐o‐Chloromandelate,a Key Intermediate for Clopidogrel,via Asymmetric Reduction with Recombinant Escherichia coli

Methyl (R)‐o‐chloromandelate [(R)‐ 1 ], which is an intermediate for a platelet aggregation inhibitor named clopidogrel, was obtained in >99% ee by the asymmetric reduction of methyl o‐chlorobenzoylformate ( 2 ) with recombinant Escherichia coli overproducing a versatile carbonyl reductase. A remarkable temperature effect on productivity was observed in the whole‐cell reduction of 2 , and the optimum productivity as high as 178 g/L was attained at 20 °C on a 2‐g scale (1.0 M). The optimized reaction could be scaled up easily to transform 20 g of 2 in 100 mL of buffer. Three synthetic methods for 2 are compared.     

Efficient Biocatalytic Synthesis of (R)‐Pantolactone

Screening for stereoselective cyanohydrin synthesis in 96‐well plates was employed in the development of an efficient, pH‐stable hydroxynitrile lyase for the conversion of sterically hindered aliphatic aldehydes. Site‐saturation mutagenesis (SSM) resulted in a powerful catalyst for the stereoselective conversion of hydroxypivalaldehyde and pivalaldehyde to their corresponding (R)‐cyanohydrins (ee >97%) which are used as chiral building blocks (e.g., for pantothenic acid production). Furthermore, redesigning the PaHNL5 gene and improving its expression by Pichia pastoris with the help of a new P_AOX1 promoter variant and the helper protein PDI (protein disulfide isomerase) led to elevated amounts of today’s most efficient biocatalyst for vitamin B₅ synthesis.     

A Novel (2,2‐Diarylvinyl)phosphine/Palladium Catalyst for Effective Aromatic Amination

A new series of diarylvinylphosphine ligands was designed and synthesized. A catalyst system, consisting of the ligands and palladium species, effectively catalyzed the coupling reaction of aryl bromides and chlorides with amines to afford the corresponding products in good to excellent yields. The efficiency is likely derived from an interaction between the palladium center and the cis‐aryl moiety on the diarylvinylphosphine ligand stabilizing a catalytic intermediate during the coupling reaction.     

Asymmetric Bio‐amination of Ketones in Organic Solvents

ω‐Transaminases, employed as a lyophilised crude cell‐free extract, were successfully employed in organic solvent for the asymmetric amination of ketones without the need for immobilisation. Best activity was found for methyl tert‐butyl ether (MTBE) at a water activity of 0.6. The ω‐transaminases (9 different enzymes) accepted efficiently 2‐propylamine as amine donor when used in the solvent, which is not the case when they are used in aqueous solution. The bio‐amination in organic solvent showed several advantages such as higher reaction rates (up to 17‐fold), general acceptance of 2‐propylamine as amine donor, simple work‐up procedure (i.e., no basification and extraction required), easy recycling of the catalyst and lack of substrate inhibition. The biocatalysts maintained their excellent stereoselectivity in MTBE allowing the preparation of optically pure amines (ee >99%) with up to >99% conversion.     

Efficient and Practical Syntheses of (R)‐(5‐Amino‐2,3‐dihydro‐1H‐inden‐2‐yl)‐carbamic Acid Methyl Ester

Efficient and practical syntheses of enantiomerically pure (R)‐(5‐amino‐2,3‐dihydro‐1H‐inden‐2‐yl)‐carbamic acid methyl ester ( 1 ) by three different routes via the resolution of different aminoindan intermediates are described.     

Structure,Activity and Stereoselectivity of NADPH‐Dependent Oxidoreductases Catalysing the S‐Selective Reduction of the Imine Substrate 2‐Methylpyrroline

Oxidoreductases from Streptomyces sp. GF3546 [3546‐IRED], Bacillus cereus BAG3X2 (BcIRED) and Nocardiopsis halophila (NhIRED) each reduce prochiral 2‐methylpyrroline (2MPN) to (S)‐2‐methylpyrrolidine with >95 % ee and also a number of other imine substrates with good selectivity. Structures of BcIRED and NhIRED have helped to identify conserved active site residues within this subgroup of imine reductases that have S selectivity towards 2MPN, including a tyrosine residue that has a possible role in catalysis and superimposes with an aspartate in related enzymes that display R selectivity towards the same substrate. Mutation of this tyrosine residue—Tyr169—in 3546‐IRED to Phe resulted in a mutant of negligible activity. The data together provide structural evidence for the location and significance of the Tyr residue in this group of imine reductases, and permit a comparison of the active sites of enzymes that reduce 2MPN with either R or S selectivity.     

Dynamic Kinetic Resolution of 2‐Phenylpropanal Derivatives to Yield β‐Chiral Primary Amines via Bioamination

The amination of racemic α‐chiral aldehydes, 2‐phenylpropanal derivatives, was investigated employing ω‐transaminases. By medium and substrate engineering the optical purity of the resulting β‐chiral chiral amine could be enhanced to reach optical purities up to 99% ee. Using enantiocomplementary ω‐transaminases allowed us to access the (R)‐ as well as the (S)‐enantiomer in most cases. It is important to note that the stereopreference of the ω‐transaminases found for α‐chiral aldehydes did not correlate with the stereopreference previously observed for the amination of methyl ketones. In one case the stereopreference switched even upon exchanging a methyl substituent to a methoxy group.

     

Co-Immobilization of a Multi-Enzyme Cascade: (S)-Selective Amine Transaminases,l-Amino Acid Oxidase and Catalase

An enzyme cascade was established previously consisting of a recycling system with an l -amino acid oxidase (hcLAAO4) and a catalase (hCAT) for different α-keto acid co-substrates of (S)-selective amine transaminases (ATAs) in kinetic resolutions of racemic amines. Only 1 mol % of the co-substrate was required and l -amino acids instead of α-keto acids could be applied. However, soluble enzymes cannot be reused easily. Immobilization of hcLAAO4, hCAT and the (S)-selective ATA from Vibrio fluvialis (ATA-Vfl) was addressed here. Immobilization of the enzymes together rather than on separate beads showed higher reaction rates most likely due to fast co-substrate channeling between ATA-Vfl and hcLAAO4 due to their close proximity. Co-immobilization allowed further reduction of the co-substrate amount to 0.1 mol % most likely due to a more efficient H₂O₂-removal caused by the stabilized hCAT and its proximity to hcLAAO4. Finally, the co-immobilized enzyme cascade was reused in 3 cycles of preparative kinetic resolutions to produce (R)-1-PEA with high enantiomeric purity (97.3 %ee). Further recycling was inefficient due to the instability of ATA-Vfl, while hcLAAO4 and hCAT revealed high stability. An engineered ATA-Vfl-8M was used in the co-immobilized enzyme cascade to produce (R)-1-(3-ethoxy-4-methoxyphenyl)-2-(methylsulfonyl)ethanamine, an apremilast-intermediate, with a 1,000 fold lower input of the co-substrate.     

Efficient Biosynthesis of (R)‐ or (S)‐2‐Hydroxybutyrate from l‐Threonine through a Synthetic Biology Approach

An efficient multi‐enzyme cascade reaction for the synthesis of (R)‐ or (S)‐2‐hydroxybutyric acid [(R)‐ or (S)‐2‐HB] from l ‐threonine was developed by using recombinant Escherichia coli cells expressing separately or co‐expressing l ‐threonine deaminase from Escherichia coli K‐12 (ilvA), formate dehydrogenase (FDH) from Candida boidinii and l ‐lactate dehydrogenase (l ‐LDH) from Oryctolagus cuniculus or d ‐lactate dehydrogenase (d ‐LDH) from Staphylococcus epidermidis ATCC 12228. Up to 750 mM of l ‐threonine were completely transformed to (R)‐ or (S)‐2‐HB in optically pure form (>99% ee) with high isolated yields. This one‐pot multi‐enzyme transformation provides a new practical method for the synthesis of these important optically pure compounds.

     

Stereo-Divergent Enzyme Cascades to Convert Racemic 4-Phenyl-2-Butanol into either (S)- or (R)-Corresponding Chiral Amine

The synthesis of enantiopure chiral amines from racemic alcohols is a key transformation in the chemical industry, e. g., in the production of active pharmaceutical ingredients (APIs). However, this reaction remains challenging. In this work, we propose a one-pot enzymatic cascade for the direct conversion of a racemic alcohol into either (S)- or (R)-enantiomers of the corresponding amine, with in-situ cofactor recycling. This enzymatic cascade consists of two enantio-complementary alcohol dehydrogenases, both NADH and NADPH oxidase for in-situ recycling of NAD(P)⁺ cofactors, and either (S)- or (R)-enantioselective transaminase. This cell-free biocatalytic system has been successfully applied to the conversion of racemic 4-phenyl-2-butanol into the high value (S)- or (R)-enantiomers of the amine reaching good (73 % (S)) and excellent (>99 % (R)) enantioselectivities.     

Extended Catalytic Scope of a Well‐Known Enzyme: Asymmetric Reduction of Iminium Substrates by Glucose Dehydrogenase

NADP(H)‐dependent imine reductases (IREDs) are of interest in biocatalytic research due to their ability to generate chiral amines from imine/iminium substrates. In reaction protocols involving IREDs, glucose dehydrogenase (GDH) is generally used to regenerate the expensive cofactor NADPH by oxidation of d ‐glucose to gluconolactone. We have characterized different IREDs with regard to reduction of a set of bicyclic iminium compounds and have utilized ¹H NMR and GC analyses to determine degree of substrate conversion and product enantiomeric excess (ee). All IREDs reduced the tested iminium compounds to the corresponding chiral amines. Blank experiments without IREDs also showed substrate conversion, however, thus suggesting an iminium reductase activity of GDH. This unexpected observation was confirmed by additional experiments with GDHs of different origin. The reduction of C=N bonds with good levels of conversion (>50 %) and excellent enantioselectivity (up to >99 % ee) by GDH represents a promiscuous catalytic activity of this enzyme.