Asymmetric Synthesis Of Chiral Amines Research Articles

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 optimizing biocatalysts. This concept paper explores the utilization of non-conventional media such as ether-type solvents, deep eutectic solvents, and micellar catalysis to enhance biocatalytic reactions for chiral amine synthesis. Each section focuses on the unique properties of these media, including their ability to stabilize enzymes, alter substrate solubility, and modulate enzyme selectivity. The paper aims to provide insights into how these innovative media can overcome traditional limitations, offering new avenues for sustainable and efficient chiral amine production through biocatalytic processes.

Engineering an (R)-selective transaminase for asymmetric synthesis of (R)-3-aminobutanol

(R)-selective transaminases show promise as catalysts for the asymmetric synthesis of chiral amines, which are building blocks of various small molecule drugs. However, their application is limited by poor substrate acceptance and low catalytic efficiency. Here, a potential (R)-selective transaminase from Fodinicurvata sediminis (FsTA) was identified through a substrate truncating strategy, and used as starting point for enzyme engineering toward catalysis of 4-hydroxy-2-butanone, a substrate that poses challenges in catalysis. Molecular docking and dynamics simulations revealed Y90 as the key residue responsible for poor substrate binding. Starting from the variant (Y90F, mut1) with initial activity, FsTA was systematically modified to improve substrate-binding through active site reshaping and consensus sequence strategy, yielding three variants (H30R, V152K, and Y156F) with improved activity. A quadruple mutation variant H30R/Y90F/V152K/Y156F (mut4) was also found to show a 7.95-fold greater catalytic efficiency (kcat/KM) than the initial variant mut1. Furthermore, mut4 also enhanced the thermostability of enzyme significantly, with the Tm value increasing by 10 °C. This variant also exhibited significantly improved activity toward a series of ketones that are either not accepted or poorly accepted by the wild-type. This study provides a basis for the rational design of an active to creating variants that can accommodate novel substrates.

Spiro-Josiphos Ligands for the Ir-Catalyzed Asymmetric Synthesis of Chiral Amines under Hydrogenation Conditions.

Transition metal-catalyzed asymmetric hydrogenation possesses unparalleled advantages to prepare chiral amines. Here we reported a novel ligand that combined Josiphos and a spirobiindane scaffold and simultaneously investigated its application in Ir-catalyzed asymmetric hydrogenation for the synthesis of chiral amines. Excellent catalytic activity (5000 TON), high enantioselectivity (up to 99% ee), and broad substrate scope (different C═N substrates) make it highly promising for both academic research and industrial applications.

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.

Considerations for the Scale‐up of in vitro Transaminase‐Catalyzed Asymmetric Synthesis of Chiral Amines

AbstractIn vitro transaminase catalyzed asymmetric synthesis is not a new phenomenon in the biocatalytic toolbox. Our group published a review on process consideration for these reactions in 2011, in which process metrics and the unfavorable reaction equilibrium was deduced to be the main bottlenecks at the time. Now, a decade later, this has been solved by different process methods presented in this review, with the development of enzymatic cascades one of the main solutions. Today, the new bottleneck is the downstream processing, due to the complexity of transaminase reactions, in which a ketone and an amine as substrates and a new ketone and a new amine as products. How to recover the produced amine from the resulting product mixture is considered and will be the key challenge for scale‐up of such systems in the near future.

Visible spectrophotometric assay for characterization of ω-transaminases

Omega-transaminases (ω-TAs) have attracted considerable interest for their use in asymmetric synthesis of chiral amines with a high degree of optical purity and yield. The rapid evaluation of the characteristics of newly identified or engineered ω-TAs is important for industrial applications. In this study, a visible spectrophotometric assay was developed for rapid quantitative determination of ω-TA activities based on the transamination of 2-(4-nitrophenyl)ethan-1-amine to generate a red product (E)-N-(4-nitrophenethyl)-2-(4-nitrophenyl)ethen-1-amine. After various co-solvents were evaluated, dimethyl sulfoxide (DMSO) was considered optimal because the red product exhibits good solubility and retains its original color. The red product dissolved in DMSO has its highest absorbance at 465 nm, and its concentration has a good linear relationship with the absorbance. A spectrophotometric assay was established and validated using conventional HPLC analysis (<10% divergence). This method was then used to characterize an ω-TA from thermophilic Geobacillus thermoleovorans and an ω-TA obtained from the metagenome of a soda lake. The results demonstrated that ω-transaminase enzymatic properties could be characterized simply, rapidly, and at low cost, using this newly established visible spectrophotometric assay method.

One pot cascade synthesis of L-2-aminobutyric acid employing ω-transaminase from Paracoccus pantotrophus

ω-transaminase can mediate the asymmetric synthesis of chiral amines from aldehydes and ketones, which has important value in the synthesis of pharmaceutical intermediates. A novel ω-transaminase derived from Paracoccus pantotrophus (PpTA) was obtained and cloned in E. coli BL21(DE3) for expression. The enzyme has high activity for 2-ketobutyric acid and benzylamine, as well as for aromatic compounds with side chains longer than ethyl aliphatic hydrocarbons. Molecular simulation showed that the S-pocket in the active center is larger than those of other ω-transaminases. Thereafter, a whole-cell catalytic system was designed to prepare l-2-aminobutyric acid by cascading PpTA and other enzymes. By using several strategies (regulation of RBS intensity, by-product decomposition and cofactor self-sufficiency), whole-cell cascade biocatalysis showed a high ee value (> 99%) and high yield (71%) in one pot reaction. This study therefore proposes an efficient biocatalyst for the synthesis of unnatural amino acids with the participation of ω-transaminase.

Chiral Synthesis of 3-Amino-1-phenylbutane by a Multi-Enzymatic Cascade System

Asymmetric synthesis of chiral amines from prochiral ketones using transaminases is an attractive biocatalytic strategy. Nevertheless, it is hampered by its unfavorable thermodynamic equilibrium. In the present work, an insitu by-product removal strategy was applied for the synthesis of 3-amino-1-phenylbutane (3-APB) by coupling a transaminase with a pyruvate decarboxylase (PDC), which does not require the use of any expensive additional cofactor. Using this strategy, the pyruvate obtained in the transamination reaction is transformed by PDC into acetaldehyde and CO2 which are of high volatility. Two different transaminases from Chromobacterium violaceum (CviTA) and Vibrio fluvialis (VflTA) were characterized to find out the appropriate pH conditions. In both cases, the addition of PDC dramatically enhanced 3-APB synthesis. Afterwards, different reaction conditions were tested to improve reaction conversion and yield. It was concluded that 30 °C and a 20-fold alanine excess lead to the best process metrics. Under the mentioned conditions, yields higher than 60% were reached with nearly 90% selectivity using both CviTA and VflTA. Moreover, high stereoselectivity for (S)-3-APB was obtained and ee of around 90% was achieved in both cases. For the first time, the asymmetric synthesis of 3-APB using PDC as by-product removal system using CviTA is reported.

In situ removal of inhibitory products with ion exchange resins for enhanced synthesis of chiral amines using ω-transaminase

ω-Transaminase (ω-TA) serves as an attractive biocatalyst for the asymmetric synthesis of chiral amines from prochiral ketones. However, the ω-TA reactions suffer from exceedingly unfavorable equilibrium and severe product inhibitions. Most reaction engineering strategies to overcome such limitations have focused on coproduct removal, which fails to mitigate enzyme inhibition by the desired amine product. Herein, we demonstrated in situ removal of both inhibitory products using ion exchange resin (IER). l-Alanine was chosen as a cosubstrate for S-selective ω-TA by considering a zwitterionic character, affording selective adsorption of the resulting products, i.e. (S)-amine and pyruvate on cation and anion exchange resin, respectively. The addition of both IERs to the reaction mixture containing 10 mM acetophenone and 200 mM l-alanine led to a 65 % yield of (S)-α-methylbenzylamine, whereas the same reaction without IER permitted only 3 % yield close to a thermodynamic limit. Computational prediction of the reaction yield was carried out using a Langmuir analysis, showing good agreements with the experimental results. The in situ product removal (ISPR) enabled amination of 2-octanone to reach even 84 % reaction yield. Preparative-scale synthesis of (S)-2-aminohexane was carried out using a batch recirculation reactor implemented with consecutive IER columns. The ISPR strategy could afford virtually inhibition-free reaction conditions and facile recovery of both valuable products.

Biovalorization of Lignin

Lignocellulosic biomass comprises three components – cellulose, hemicellulose and lignin. Biorefining is defined as the separation, isolation and conversion of cellulose, hemicellulose and lignin from lignocellulosic biomass into fuels, chemicals, materials and energy. While a number of processes have been developed over the years to produce valuable products from cellulose and hemicellulose, processes utilizing lignin have been few and far between, largely owing to the severe recalcitrance of lignin. Our inability to utilize lignin is a lost opportunity for the green economy. Lignin is abundant and can provide a myriad of chemicals that could be used as building blocks for life-saving pharmaceuticals or even as flavours and food ingredients. The development of greener conversion platforms that can efficiently convert lignin into valuable products is highly desired. This paper summarizes recent progress made towards the development of a family of biocatalytic processes that convert lignin to pharmaceutical building blocks, flavouring agents and drug delivery platforms. Biocatalytic processes are greener, emit lesser carbon dioxide and are more energy efficient than other alternatives. The first family of biocatalysts selectively degrades lignin to its monoaromatic constituents and the second family of biocatalysts then modifies the monoaromatic constituents to desired target molecules. In particular, this paper describes the novel use of functional metagenomics and whole-cell biosensors for the discovery of 147 new biocatalysts that convert lignin into vanillin and syringaldehyde, and the identification of a novel transaminase that catalyzes the asymmetric synthesis of chiral amines from vanillin, syringaldehyde and 12 other monoaromatic aldehyde and ketone degradation products of lignin. Recent progress on the assembly of a metabolic pathway for the biosynthesis of the spice molecule capsaicin from vanillylamine, and the integration of the entire pathway that valorizes lignin to capsaicin into the metabolic network of E. coli has also been discussed. This work will eventually lead to the development of consolidated bioprocesses that convert lignin into value-added chemicals.

Co-immobilization of metal and enzyme into hydrophobic nanopores for highly improved chemoenzymatic asymmetric synthesis.

Chemoenzymatic catalysts with hydrophobic nanopores were fabricated by co-immobilizing metal nanoparticles and enzymes into the dendritic organosilica nanoparticles. They demonstrated highly improved catalytic performance in chemoenzymatic asymmetric synthesis of chiral amines and alcohols. The hydrophobic microenvironment proved to be critical to enhanced stability, activity and cascade efficiency.

Rapid and Quantitative Profiling of Substrate Specificity of ω‐Transaminases for Ketones

Abstractω‐Transaminases (ω‐TAs) have gained growing attention owing to their capability for asymmetric synthesis of chiral amines from ketones. Reliable high‐throughput activity assay of ω‐TAs is essential in carrying out extensive substrate profiling and establishing a robust screening platform. Here we report spectrophotometric and colorimetric methods enabling rapid quantitation of ω‐TA activities toward ketones in a 96‐well microplate format. The assay methods employ benzylamine, a reactive amino donor for ω‐TAs, as a cosubstrate and exploit aldehyde dehydrogenase (ALDH) as a reporter enzyme, leading to formation of benzaldehyde detectable by ALDH owing to concomitant NADH generation. Spectrophotometric substrate profiling of two wild‐type ω‐TAs of opposite stereoselectivity was carried out at 340 nm with 22 ketones, revealing subtle differences in substrate specificities that were consistent with docking simulation results obtained with cognate amines. Colorimetric readout for naked eye detection of the ω‐TA activity was also demonstrated by supplementing the assay mixture with color‐developing reagents whose color reaction could be quantified at 580 nm. The colorimetric assay was applied to substrate profiling of an engineered ω‐TA for 24 ketones, leading to rapid identification of reactive ketones. The ALDH‐based assay is expected to be promising for high‐throughput screening of enzyme collections and mutant libraries to fish out the best ω‐TA candidate as well as to tailor enzyme properties for efficient amination of a target ketone.

A Single Mutation Increases the Thermostability and Activity of Aspergillus terreus Amine Transaminase.

Enhancing the thermostability of (R)-selective amine transaminases (AT-ATA) will expand its application in the asymmetric synthesis of chiral amines. In this study, mutual information and coevolution networks of ATAs were analyzed by the Mutual Information Server to Infer Coevolution (MISTIC). Subsequently, the amino acids most likely to influence the stability and function of the protein were investigated by alanine scanning and saturation mutagenesis. Four stabilized mutants (L118T, L118A, L118I, and L118V) were successfully obtained. The best mutant, L118T, exhibited an improved thermal stability with a 3.7-fold enhancement in its half-life (t1/2) at 40 °C and a 5.3 °C increase in T5010 compared to the values for the wild-type protein. By the differential scanning fluorimetry (DSF) analysis, the best mutant, L118T, showed a melting temperature (Tm) of 46.4 °C, which corresponded to a 5.0 °C increase relative to the wild-type AT-ATA (41.4 °C). Furthermore, the most stable mutant L118T displayed the highest catalytic efficiency among the four stabilized mutants.

Activity Improvements of an Engineered ω-transaminase for Ketones Are Positively Correlated with Those for Cognate Amines

Chiral amines are broadly used as essential building blocks of diverse pharmaceutical drugs. ω-Transaminase (ω-TA) is one of the promising enzymes for biocatalytic preparation of chiral amines owing to its capability for reductive amination of ketones. However, industrial application of ω-TA to asymmetric synthesis of chiral amines is often limited by marginal activities for ketones. Here, we explore whether activity improvements of ω-TA for ketones caused by active site engineering are correlated with those for cognate amines. We examined amino donor reactivities of 20 amines with ω-TA from Ochrobactrum anthropi (OATA) and its engineered variant carrying a W58L mutation (OATAW58L). Consistent with the previously observed activity increases for ketones, OATAW58L showed 3 to 79-fold activity improvements for amines. Docking simulations suggested that the activity improvements resulted from increased proximity of a bound amine to an internal aldimine owing to relocation of a small binding pocket. Activity comparison indicated that the W58L mutation induced activity increases for ketones better than it did those for amines. This result motivated us to carry out Pearson correlation analysis among the activity improvements for amines and ketones, revealing a strong positive correlation (r = 0.84). This suggests that active site engineering beneficial for activity of ω-TA toward a target amine is likely to evoke an activity improvement for its cognate ketone. Our finding is expected to provide a clue to design a robust high-throughput screening method required for creation of an engineered mutant displaying a better activity toward a target ketone.

Navigating around Patented Routes by Preserving Specific Motifs along Computer-Planned Retrosynthetic Pathways

Summary By keeping track of lists of specific bonds one wishes to preserve, a computer program is able to identify the key disconnections used in the patented syntheses and design synthetic routes that circumvent these approaches. Here, we provide examples of computer-designed syntheses relevant to medicinal chemistry, in which the machine avoids “strategic” disconnections common to industrial patents and is forced to use different starting materials. The ability of modern retrosynthetic planners to navigate around patented solutions may have significant implications for the ways in which intellectual property related to multistep syntheses is protected and/or challenged.

Active Site Engineering of ω-Transaminase Guided by Docking Orientation Analysis and Virtual Activity Screening

Creation of enzyme variants displaying desirable catalytic performance usually necessitates tedious and time-consuming procedures for library generation and selection, which may be circumvented by a computational method based on a precise understanding of the reaction mechanism in the context of active site environment. Despite the great potential of ω-transaminases (ω-TAs) for asymmetric synthesis of chiral amines from ketones, it remains elusive why ω-TAs exhibit marginal activities for most ketones in contrast to their high activities for α-keto acids and aldehydes. To address this puzzling question, crystal structure determination and molecular modeling of ω-TAs were carried out to analyze docking orientations of the amino acceptors in the Michaelis complex. We found that ketones, unlike the reactive substrates, led to nonproductive binding complexes where the bound substrate was hardly accessible to a nucleophilic attack by the pyridoxamine cofactor to initiate reductive amination of the amino accept...

Encapsulation of amine dehydrogenase in hybrid titania nanoparticles by polyethylenimine coating and templated biomineralization

Asymmetric synthesis of chiral amines by amine dehydrogenases (AmDH), which catalyzed reductive amination of ketones with high enantioselectivity, is an ideal route for production of chiral amine. In this study, a facile approach is proposed to immobilize unstable amine dehydrogenase by two steps. Firstly, polyethylenimine (PEI), a cationic polymer, was applied for coating enzyme to inhibit the dissociation of multimeric enzymes. Strong interaction of PEI with AmDH were analyzed and indicated PEI provided the hydrophilic microenvironment for enzyme. The half-life was improved about 18 fold in 50°C. Secondly, to further stabilize AmDH-PEI, PEI coated on AmDH surface was used as a biological template inducing the hydrolysis and condensation of titanium precursor to form titania. AmDH-PEI-Ti possessed more than 80% of the catalytic activity of free enzyme and the entrapment efficiency can be high up to 90%. A mechanistic illustration of the formation of AmDH-PEI-Ti nanoparticles were proposed. Titania provided a rigid cage pocket for the protection from structure unfolding. This generally applicable strategy offers a potential technique for multimeric enzyme immobilization with the advantages of low cost, easy operation, high reservation of activity and high stability.

A facile method to determine intrinsic kinetic parameters of ω-transaminase displaying substrate inhibition

It is usually time-consuming to determine intrinsic kinetic parameters of bisubstrate enzymes, especially when experimental kinetic data deviate from a linear Lineweaver-Burk plot due to complex inhibition patterns. A typical example is ω-transaminase (ω-TA) which is an industrially important enzyme for asymmetric synthesis of chiral amines. ω-TA catalyzes transfer of an amino group between a donor (D) and an acceptor (A) via a ping-pong bi-bi mechanism and often displays substrate inhibitions by reactive amino acceptors, which leads one to prefer to determine apparent kinetic parameters rather than intrinsic ones despite limited applicability for precise understanding of enzyme properties. Here, we developed a new method to determine intrinsic kinetic parameters of ω-TA by double-reciprocal analysis using only two sets of kinetic data. First, linear regression of 1/initial rate (vi) against 1/[A] was carried out with one set of kinetic data measured at a fixed [D] while [A] lay far below the concentration range under the influence of substrate inhibition. Second, another linear regression of 1/[D] vs 1/vi was conducted with one set of kinetic data obtained at a fixed [A] within a substantial substrate inhibition range. The resulting four equations obtained from the y-intercepts and slopes of the two regression lines were used for determination of four intrinsic kinetic parameters, i.e. turnover number (kcat), substrate inhibition constant (KSI) for A and Michaelis constants (KM) for D and A. To evaluate reliability of the intrinsic parameters, a validity test was taken by comparing experimental and computational results for the maximum point on a concave-down substrate inhibition curve. Once the intrinsic parameters were determined for a substrate pair, intrinsic parameters for other substrates were simply assessed by constituting a new substrate pair with the kinetically characterized substrate and carrying out linear regression with one set of kinetic data. Our method is expected to be applicable to a wide range of bisubstrate enzymes for facile determination of intrinsic kinetic parameters including KSI.

An improved process for biocatalytic asymmetric amine synthesis by in situ product removal using a supported liquid membrane

Chiral amines are important building blocks in the pharmaceutical industry, and the biocatalytic synthesis of these compounds using ω-transaminases has been increasingly studied in recent years. In principal, asymmetric synthesis of chiral amines from a prochiral ketone is the preferable route, but it is often hampered by an unfavourable equilibrium position and product inhibition. An effective method for product removal is therefore necessary to drive the reaction towards product formation. In a recent study (Rehn et al., 2014) [29] we reported on the successful use of a supported liquid membrane (SLM) for the in situ product removal (ISPR) of (S)-α-methylbenzylamine (MBA) produced by Arthrobacter citreus ω-transaminase present in immobilized Escherichia coli cells.In the present work, we thoroughly discuss the factors influencing the performance of the SLM system and considerations for its successful use. Moreover, the system is further improved by implementing continuous control of the reactor pH using the amine donor substrate, and regeneration of the SLM unit at regular intervals to maintain the extraction performance, allowing the accumulation of 1.0M (121g/l) product in the stripping phase during operation for 91h.

Asymmetric Amination of Secondary Alcohols by using a Redox‐Neutral Two‐Enzyme Cascade

AbstractMultienzyme cascade approaches for the synthesis of optically pure molecules from simple achiral compounds are desired. Herein, a cofactor self‐sufficient cascade protocol for the asymmetric amination of racemic secondary alcohols to the corresponding chiral amines was successfully constructed by employing an alcohol dehydrogenase and a newly developed amine dehydrogenase. The compatibility and the identical cofactor dependence of the two enzymes led to an ingenious in situ cofactor recycling system in the one‐pot synthesis. The artificial redox‐neutral cascade process allowed the transformation of racemic secondary alcohols into enantiopure amines with considerable conversions (up to 94 %) and &gt;99 % enantiomeric excess at the expense of only ammonia; this method thus represents a concise and efficient route for the asymmetric synthesis of chiral amines.