Enzymatic Routes for Chiral Amine Synthesis: Protein Engineering and Process Optimization.

Chiral amines are essential motifs in pharmaceuticals, agrochemicals, and specialty chemicals. While traditional chemical routes to chiral amines often lack stereoselectivity and require harsh conditions, biocatalytic methods using engineered enzymes can offer high efficiency and selectivity under sustainable conditions. This review discusses recent advances in protein engineering of transaminases, oxidases, and other enzymes to improve catalytic performance. Strategies such as directed evolution, immobilization, and computational redesign have expanded substrate scope and enhanced efficiency. Furthermore, process optimization guided by techno-economic assessments has been crucial for establishing viable biomanufacturing routes. Combining state-of-the-art enzyme engineering with multifaceted process development will enable scalable, economical enzymatic synthesis of diverse chiral amine targets.

Biocatalysis in Non-Conventional Media: Unlocking the Potential for Sustainable Chiral Amine Synthesis.

The application of biocatalysis has become essential in both academic and industrial domains for the asymmetric synthesis of chiral amines, and it serves as an alternative tool to transition-metal catalysis and complements traditional chemical methods. It relies on the swift expansion of available processes, primarily as a result of advanced tools for enzyme discovery, combined with high-throughput laboratory evolution techniques for optimising biocatalysts. This manuscript highlights recent chemical and technological developments contributing to the sustainable applications of biocatalysis with industrial interest. Specifically, the use of non-conventional reaction media and the combination with photocatalysis can enhance production of chiral amines by allowing higher working concentrations and cascade transformations, leading to high yields and enantiomeric excesses. Furthermore, a selection of both known and modern strategies for enzyme immobilisation, along with the use of fed-batch and flow synthesis, demonstrates the potential to translate laboratory synthesis to effective scaled-up applications and improve the processing of large reaction volumes.

Construction of Multi-Enzyme Integrated Catalysts for Deracemization of Cyclic Chiral Amines.

Multi-enzyme cascade catalysis has become an important technique for chemical reactions used in manufacturing and scientific study. In this research, we designed a four-enzyme integrated catalyst and used it to catalyse the deracemization reaction of cyclic chiral amines, where monoamine oxidase (MAO) catalyses the enantioselective oxidation of 1-methyl-1,2,3,4-tetrahydroisoquinoline (MTQ), imine reductase (IRED) catalyses the stereo selective reduction of 1-methyl-3,4-dihydroisoquinoline (MDQ), formate dehydrogenase (FDH) is used for the cyclic regeneration of cofactors, and catalase (CAT) is used for decomposition of oxidative reactions. The four enzymes were immobilized via polydopamine (PDA)-encapsulated dendritic organosilica nanoparticles (DONs) as carriers, resulting in the amphiphilic core-shell catalysts. The hydrophilic PDA shell ensures the dispersion of the catalyst in water, and the hydrophobic DON core creates a microenvironment with the spatial confinement effect of the organic substrate and the preconcentration effect to enhance the stability of the enzymes and the catalytic efficiency. The core-shell structure improves the stability and reusability of the catalyst and rationally arranges the position of different enzymes according to the reaction sequence to improve the cascade catalytic performance and cofactor recovery efficiency.

Cyclometallated Imides as Templates for the H-Bond Directed Iridium-Catalyzed Asymmetric Hydrogenation of N-Methyl, N-Alkyl and N-Aryl Imines.

A combined computational and experimental approach allowed us to develop overall the most selective catalyst for the direct hydrogenation of N-methyl, N-alkyl and N-aryl imines described to date. Iridium catalysts with a cyclometallated cyclic imide group provide selectivity of up to 99 % enantiomeric excess. Computational studies show that the selectivity results from the combined effect of H-bonding of the imide C=O with the substrate iminium ion and a stabilizing π-π interaction with the cyclometallated ligand. The cyclometallated ligand thus exhibits a unique mode of action, serving as a template for the H-bond directed approach of the substrate which results in enhanced selectivity. The catalyst (2) has been synthesized and isolated as a crystalline air-stable solid. X-ray analysis of 2 confirmed the structure of the catalyst and the correct position of the imide C=O groups to engage in an H-bond with the substrate. 19F NMR real-time monitoring showed the hydrogenation of N-methyl imines catalyzed by 2 is very fast, with a TOF of approx. 3500 h-1.

Identification, characterization and application of M16AT, a new organic solvent-tolerant (R)-enantio-selective type IV amine transaminase from Mycobacterium sp. ACS1612.

Biocatalysis has emerged as a powerful alternative to traditional chemical methods, especially for asymmetric synthesis. As biocatalysts usually exhibit excellent chemical, regio- and enantioselectivity, they facilitate and simplify many chemical processes for the production of a broad range of products. Here, a new biocatalyst called, R-selective amine transaminases (R-ATAs), was obtained from Mycobacterium sp. ACS1612 (M16AT) using in-silico prediction combined with a genome and protein database. A two-step simple purification process could yield a high concentration of pure enzyme, suggesting that industrial application would be inexpensive. Additionally, the newly identified and characterized R-ATAs displayed a broad substrate spectrum and strong tolerance to organic solvents. Moreover, the synthetic applicability of M16AT has been demonstrated by the asymmetric synthesis of (R)-fendiline from of (R)-1-phenylethan-1-amine.

Heterologous Expression and Partial Characterization of a Putative Opine Dehydrogenase from a Metagenomic Sequence of Desulfohalobium retbaense.

The aim of this research was to prove the function of the putative opine dehydrogenase from Desulfohalobium retbaense and to characterize the enzyme in terms of functional and kinetic parameters. A putative opine dehydrogenase was identified from a metagenomic library by a sequence-based technique search of the metagenomic library, and afterward was successfully heterologously produced in Escherichia coli. In order to examine its potential for applications in the synthesis of secondary amines, first the substrate specificity of the enzyme towards different amino donors and amino acceptors was determined. The highest affinity was observed towards small amino acids, preferentially L-alanine, and when it comes to α-keto acids, pyruvate proved to be a preferential amino acceptor. The highest activity was observed at pH 6.5 in the absence of salts. The enzyme showed remarkable stability in a wide range of experimental conditions, such as broad pH stability (from 6.0-11.0 after 30 min incubation in buffers at a certain pH), stability in the presence of NaCl up to 3.0 M for 24 h, it retained 80 % of the initial activity after 1 h incubation at 45 °C, and 65 % of the initial activity after 24 h incubation in 30 % dimethyl sulfoxide.

Immobilization of Lipase B from Candida antarctica on Magnetic Nanoparticles Enhances Its Selectivity in Kinetic Resolutions of Chiral Amines with Several Acylating Agents.

In lipase-catalyzed kinetic resolutions (KRs), the choice of immobilization support and acylating agents (AAs) is crucial. Lipase B from Candida antarctica immobilized onto magnetic nanoparticles (CaLB-MNPs) has been successfully used for diverse KRs of racemic compounds, but there is a lack of studies of the utilization of this potent biocatalyst in the KR of chiral amines, important pharmaceutical building blocks. Therefore, in this work, several racemic amines (heptane-2-amine, 1-methoxypropan-2-amine, 1-phenylethan-1-amine, and 4-phenylbutan-2-amine, (±)-1a-d, respectively) were studied in batch and continuous-flow mode utilizing different AAs, such as diisopropyl malonate 2A, isopropyl 2-cyanoacetate 2B, and isopropyl 2-ethoxyacetate 2C. The reactions performed with CaLB-MNPs were compared with Novozym 435 (N435) and the results in the literature. CaLB-MNPs were less active than N435, leading to lower conversion, but demonstrated a higher enantiomer selectivity, proving to be a good alternative to the commercial form. Compound 2C resulted in the best balance between conversion and enantiomer selectivity among the acylating agents. CaLB-MNPs proved to be efficient in the KR of chiral amines, having comparable or superior properties to other CaLB forms utilizing porous matrices for immobilization. An additional advantage of using CaLB-MNPs is that the purification and reuse processes are facilitated via magnetic retention/separation. In the continuous-flow mode, the usability and operational stability of CaLB-MNPs were reaffirmed, corroborating with previous studies, and the results overall improve our understanding of this potent biocatalyst and the convenient U-shape reactor used.

[Semi-rational evolution of ω-transaminase from Aspergillus terreus for enhancing the thermostability].

ω-transaminase (ω-TA) is a natural biocatalyst that has good application potential in the synthesis of chiral amines. However, the poor stability and low activity of ω-TA in the process of catalyzing unnatural substrates greatly hampers its application. To overcome these shortcomings, the thermostability of (R)-ω-TA (AtTA) from Aspergillus terreus was engineered by combining molecular dynamics simulation assisted computer-aided design with random and combinatorial mutation. An optimal mutant AtTA-E104D/A246V/R266Q (M3) with synchronously enhanced thermostability and activity was obtained. Compared with the wild- type (WT) enzyme, the half-life t1/2 (35 ℃) of M3 was prolonged by 4.8-time (from 17.8 min to 102.7 min), and the half deactivation temperature (T1050) was increased from 38.1 ℃ to 40.3 ℃. The catalytic efficiencies toward pyruvate and 1-(R)-phenylethylamine of M3 were 1.59- and 1.56-fold that of WT. Molecular dynamics simulation and molecular docking showed that the reinforced stability of α-helix caused by the increase of hydrogen bond and hydrophobic interaction in molecules was the main reason for the improvement of enzyme thermostability. The enhanced hydrogen bond of substrate with surrounding amino acid residues and the enlarged substrate binding pocket contributed to the increased catalytic efficiency of M3. Substrate spectrum analysis revealed that the catalytic performance of M3 on 11 aromatic ketones were higher than that of WT, which further showed the application potential of M3 in the synthesis of chiral amines.

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 H2 O2 -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.

Chiral Carbon Nanorings: Synthesis, Properties and Hierarchical Self-assembly of Chiral Ternary Complexes Featuring a Narcissistic Chiral Self-Recognition for Chiral Amines.

We report the synthesis and chiroptical properties of novel chiral carbon nanorings Sp-/Rp-[12]PCPP containing a planar chiral [2.2]PCP unit, and demonstrate that Sp-/Rp-[12]PCPP can not only host crown ether 18-Crown-6 to form ring-in-ring complexes with a binding constant 3.35×103  M-1 , but also accommodate the complexes of 18-Crown-6 and S/R-protonated amines to form homochiral S@Sp-/R@Rp- and heterochiral S@Rp-/R@Sp- ternary complexes, displaying significantly larger binding constants of up to 3.31×105  M-1 depending on the chiral guests. Importantly, homochiral S@Sp-/R@Rp- ternary complexes exhibit an enhanced CD signal, while the heterochiral S@Rp-/R@Sp- ones have a constant CD signal compared with the chiral carbon nanorings, respectively, which suggests that homochiral S@Sp-/R@Rp- ternary complexes display a highly narcissistic chiral self-recognition for S/R-protonated chiral amines, respectively. Finally, the chiral ternary complexes can be further applied to determine the ee values of chiral guests. The findings highlight a new application of carbon nanorings in supramolecular sensors, beyond the common recognition of π-conjugated molecules.

Cobalt-Catalyzed Efficient Convergent Asymmetric Hydrogenation of E/Z-Enamides.

Using the diphosphine-cobalt-zinc catalytic system, an efficient asymmetric hydrogenation of internal simple enamides has been realized. In particular, the Ph-BPE ligand can achieve convergent asymmetric hydrogenation of E/Z-substrates. High yields and excellent enantioselectivities were obtained for both acyclic and cyclic enamides bearing α-alkyl-ß-aryl, α-aryl-ß-aryl, and α-aryl-ß-alkyl substituents. Hydrogenated products can be applied for the synthesis of useful chiral drugs such as Arfromoterol, Rotigotine, and Norsertraline. In addition, reasonable catalytic mechanism and stereocontrol mode are proposed based on DFT calculations.

Highly Enantioselective Brønsted Acid Catalyzed Heyns Rearrangement.

Herein we report the first method for highly enantioselective Brønsted acid catalyzed Heyns rearrangements. These reactions, catalyzed by a chiral spiro phosphoric acid, afforded synthetically valuable chiral α-aryl-α-aminoketones which cannot be obtained by means of previously reported Heyns rearrangement methods. This method features low catalyst loadings, high yields and high enantioselectivities, making these reactions highly practical. We used the method to efficiently synthesize various chiral amines, including some biologically active molecules. We experimentally proved that these acid-catalyzed Heyns rearrangements proceeded via a proton-transfer process involving an enol intermediate and the stereocontrol was realized during the proton-transfer step.

Semi-rational evolution of ω-transaminase from Aspergillus terreus for enhancing the thermostability / 生物工程学报

ω-transaminase (ω-TA) is a natural biocatalyst that has good application potential in the synthesis of chiral amines. However, the poor stability and low activity of ω-TA in the process of catalyzing unnatural substrates greatly hampers its application. To overcome these shortcomings, the thermostability of (R)-ω-TA (AtTA) from Aspergillus terreus was engineered by combining molecular dynamics simulation assisted computer-aided design with random and combinatorial mutation. An optimal mutant AtTA-E104D/A246V/R266Q (M3) with synchronously enhanced thermostability and activity was obtained. Compared with the wild- type (WT) enzyme, the half-life t1/2 (35 ℃) of M3 was prolonged by 4.8-time (from 17.8 min to 102.7 min), and the half deactivation temperature (T1050) was increased from 38.1 ℃ to 40.3 ℃. The catalytic efficiencies toward pyruvate and 1-(R)-phenylethylamine of M3 were 1.59- and 1.56-fold that of WT. Molecular dynamics simulation and molecular docking showed that the reinforced stability of α-helix caused by the increase of hydrogen bond and hydrophobic interaction in molecules was the main reason for the improvement of enzyme thermostability. The enhanced hydrogen bond of substrate with surrounding amino acid residues and the enlarged substrate binding pocket contributed to the increased catalytic efficiency of M3. Substrate spectrum analysis revealed that the catalytic performance of M3 on 11 aromatic ketones were higher than that of WT, which further showed the application potential of M3 in the synthesis of chiral amines.

Efficient Synthesis of Key Chiral Intermediate in Painkillers (R)-1-[3,5-Bis(trifluoromethyl)phenyl]ethanamine by Bienzyme Cascade System with R-ω-Transaminase and Alcohol Dehydrogenase Functions.

(R)-1-[3,5-bis(trifluoromethyl)phenyl]ethanamine, a key chiral intermediate of selective tetrodotoxin-sensitive blockers, was efficiently synthesized by a bienzyme cascade system formed by with R-ω-transaminase (ATA117) and an alcohol dehydrogenase (ADH) co-expression system. Herein, we report that the use of ATA117 as the biocatalyst for the amination of 3,5-bistrifluoromethylacetophenone led to the highest efficiency in product performance (enantiomeric excess > 99.9%). Moreover, to further improve the product yield, ADH was introduced into the reaction system to promote an equilibrium shift. Additionally, bienzyme cascade system was constructed by five different expression systems, including two tandem expression recombinant plasmids (pETDuet-ATA117-ADH and pACYCDuet-ATA117-ADH) and three co-expressed dual-plasmids (pETDuet-ATA117/pET28a-ADH, pACYCDuet-ATA117/pET28a-ADH, and pACYCDuet-ATA117/pETDuet-ADH), utilizing recombinant engineered bacteria. Subsequent studies revealed that as compared with ATA117 single enzyme, the substrate handling capacity of BL21(DE3)/pETDuet-ATA117-ADH (0.25 g wet weight) developed for bienzyme cascade system was increased by 1.50 folds under the condition of 40 °C, 180 rpm, 0.1 M pH9 Tris-HCl for 24 h. To the best of our knowledge, ours is the first report demonstrating the production of (R)-1-[3,5-bis(trifluoromethyl)phenyl]ethanamine using a bienzyme cascade system, thus providing valuable insights into the biosynthesis of chiral amines.

Mechanism-Guided Computational Design of ω-Transaminase by Reprograming of High-Energy-Barrier Steps.

ω-Transaminases (ω-TAs) show considerable potential for the synthesis of chiral amines. However, their low catalytic efficiency towards bulky substrates limits their application, and complicated catalytic mechanisms prevent precise enzyme design. Herein, we address this challenge using a mechanism-guided computational enzyme design strategy by reprograming the transition and ground states in key reaction steps. The common features among the three high-energy-barrier steps responsible for the low catalytic efficiency were revealed using quantum mechanics (QM). Five key residues were simultaneously tailored to stabilize the rate-limiting transition state with the aid of the Rosetta design. The 14 top-ranked variants showed 16.9-143-fold improved catalytic activity. The catalytic efficiency of the best variant, M9 (Q25F/M60W/W64F/I266A), was significantly increased, with a 1660-fold increase in kcat /Km and a 1.5-26.8-fold increase in turnover number (TON) towards various indanone derivatives.

Tuning an Imine Reductase for the Asymmetric Synthesis of Azacycloalkylamines by Concise Structure-Guided Engineering.

Although imine reductases (IREDs) are emerging as attractive reductive aminases (RedAms), their substrate scope is still narrow, and rational engineering is rare. Focusing on hydrogen bond reorganization and cavity expansion, a concise strategy combining rational cavity design, combinatorial active-site saturation test (CAST), and thermostability engineering was designed, that transformed the weakly active IR-G36 into a variant M5 with superior performance for the synthesis of (R)-3-benzylamino-1-Boc-piperidine, with a 4193-fold improvement in catalytic efficiency, a 16.2 °C improvement in Tm , and a significant increase in the e.e. value from 78 % (R) to >99 % (R). M5 exhibits broad substrate scope for the synthesis of diverse azacycloalkylamines, and the reaction was demonstrated on a hectogram-scale under industrially relevant conditions. Our study provides a compelling example of the preparation of versatile and efficient IREDs, with exciting opportunities in medicinal and process chemistry as well as synthetic biology.

Direct Asymmetric Reductive Amination of Alkyl (Hetero)Aryl Ketones by an Engineered Amine Dehydrogenase.

The direct asymmetric reductive amination of heteroaryl ketones has been a long-standing synthetic challenge. Here we report the engineering of an amine dehydrogenase (AmDH) from Jeotgalicoccus aerolatus for the asymmetric synthesis of chiral α-(hetero)aryl primary amines in excellent conversions (up to 99 %) and enantioselectivities (up to 99 % ee). The best AmDH variant (Ja-AmDH-M33 ) exhibited high activity and specificity toward alkyl (hetero)aryl ketones, even for those bearing a bulky alkyl chain. An efficient directed evolution approach based on molecular docking was implemented to enlarge the active pocket with a more hydrophobic entrance, which is responsible for the high activity. The Ja-AmDH-M33 was also used for preparative-scale synthesis of pharmaceutically relevant amines and a key intermediate of chiral pincer ligands, which highlighted its practical application in synthetic chemistry.

Crystal structure and Hirshfeld surface analysis of (S)-N-methyl-1-phenyl-ethan-1-aminium chloride.

The title compound C9H14N+·Cl-, (1), can be synthesized starting from (S)-N-methyl-1-phenyl-ethan-1-amine (2). Compound 2 upon addition of HCl·Et2O leads to crystallization of compound 1 as colorless blocks. The configuration of compound 1 is stable as well as preserved in space group P212121. Ammonium chlorides, like the title compound, are often observed as undesirable by-products in amino-silylation of chloro-silanes. Additionally, these by-products are usually soluble in selected organic solvents, which require difficult separation steps. Therefore, detailed studies on structural features and inter-molecular inter-actions performed by Hirshfeld atom refinement (HAR) using NoSpherA2 [Kleemiss et al. (2021 ▸). Chem. Sci. 12, 1675-1692] and Hirshfeld surface analysis were used to address structural issues on that separation problem.

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.

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,ß-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,ß-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.