Biocatalytic Transamination Research Articles

Step-Economic Synthesis of Tamsulosin Hydrochloride via Continuous Chlorosulfonation and Biocatalytic Transamination

Step-Economic Synthesis of Tamsulosin Hydrochloride via Continuous Chlorosulfonation and Biocatalytic Transamination

Front Cover Picture: Asymmetric Synthesis of Trisubstituted Piperidines via Biocatalytic Transamination and Diastereoselective Enamine or Imine Reduction (Adv. Synth. Catal. 13/2023)

The front cover picture shows the assembly of a trisubstituted piperidine derivative through the controlled formation of C−N and C−H bonds. Different stereoselective synthetic tools – namely transaminase and IRED enzymes, and platinum(0)-catalysed flow hydrogenation – are combined to obtain the target N-heterocycles from achiral, open-chain precursors with excellent enantio- and diastereocontrol. The cover design is by Verena Resch and used with her permission. Details can be found in the Research Article by Joerg Schrittwieser and co-workers (P. Petermeier, C. Kohlfuerst, A. Torvisco, R. C. Fischer, A. Mata, D. Dallinger, C. O. Kappe, J. H. Schrittwieser, W. Kroutil, Adv. Synth. Catal. 2023, 365, XXXX–XXXX; DOI: 10.1002/adsc.202300050)

Biocatalytic Transamination of Aldolase-Derived 3-Hydroxy Ketones.

Although optical pure amino alcohols are in high demand due to their widespread applicability, they still remain challenging to synthesize, since commonly elaborated protection strategies are required. Here, a multi-enzymatic methodology is presented that circumvents this obstacle furnishing enantioenriched 1,3-amino alcohols out of commodity chemicals. A Type I aldolase forged the carbon backbone with an enantioenriched aldol motif, which was subsequently subjected to enzymatic transamination. A panel of 194 TAs was tested on diverse nine aldol products prepared through different nucleophiles and electrophiles. Due to the availability of (R)- and (S)-selective TAs, both diastereomers of the 1,3-amino alcohol motif were accessible. A two-step process enabled the synthesis of the desired amino alcohols with up to three chiral centers with de up to >97 in the final products.

Asymmetric Synthesis of Trisubstituted Piperidines via Biocatalytic Transamination and Diastereoselective Enamine or Imine Reduction

AbstractSubstituted piperidine rings are a common motif in natural products and pharmaceutical drugs. The asymmetric synthesis of piperidines bearing multiple stereocentres remains a challenge, and current approaches often rely on lengthy reaction sequences and ‘chiral pool’ strategies. Herein, we report multi‐enzymatic and chemo‐enzymatic methods that allow the preparation of piperidines with three chirality centres in only two steps from achiral diketoester precursors. Stereocontrol is achieved by a highly enantioselective transamination leading to optically pure (ee >99%) enamine or imine intermediates, followed by diastereoselective reduction of these unsaturated N‐heterocycles using either platinum(0)‐catalysed flow hydrogenation or enzymatic imine reduction. In the latter case, coupling of the two biocatalytic reactions in a concurrent one‐pot process is possible, thus reducing the synthetic sequence to a single biotransformation. In total, nine trisubstituted piperidines were prepared in high stereoisomeric purities (dr ≥98:2) and isolated yields of up to 73%. Lead‐likeness analysis of five representative products using an open‐source webtool suggests that these compounds possess considerable application potential as building blocks in drug discovery.

Amine Transaminase Mediated Synthesis of Optically Pure Piperazinones and 1,4‐Diazepanones

AbstractA biocatalytic approach has been designed for the synthesis of optically active piperazinones and 1,4‐diazepanones in aqueous medium under mild conditions. The method described herein is based on a biocatalytic transamination of an easily accessible N‐(2‐oxopropyl) amino acid ester and the subsequent spontaneous cyclization of the initially formed amine. Both enantiomers of the synthesized piperazinones can be prepared by the selection of amine transaminases of opposite selectivity. The reaction conditions were optimised and eight selected processes were performed on a preparative scale. Thus, seven optically active piperazinones were synthesized in high isolated yields (70–90%) and a 1,4‐diazepanone in a moderate yield (51%), being the ee ≥99% in all the cases.magnified image

Simultaneous Preparation of (S)-2-Aminobutane and d-Alanine or d-Homoalanine via Biocatalytic Transamination at High Substrate Concentration

(S)-2-Aminobutane, d-alanine, and d-homoalanine are important intermediates for the production of various active pharmaceutical ingredients and food additives. The preparation of these small chiral amine or amino acids with high water solubility still demands searching for efficient methods. In this work, we identified an ω-transaminase (ω-TA) from Sinirhodobacter hungdaonensis (ShdTA) that catalyzed the kinetic resolution of racemic 2-aminobutane at a concentration of 800 mM using pyruvate as the amino acceptor, leading to the simultaneous isolation of enantiopure (S)-2-aminobutane and d-alanine in 46% and 90% yield, respectively. In addition, (S)-2-aminobutane (98% ee) and d-homoalanine (99% ee) were isolated in 45% and 93% yield, respectively, in the kinetic resolution of racemic 2-aminobutane at a concentration of 400 mM coupled with deamination of l-threonine by threonine deaminase. We thus developed a biocatalytic process for the practical synthesis of these valuable small chiral amine and d-amino acids.

Stereoselective Synthesis of β-Branched Aromatic α-Amino Acids by Biocatalytic Dynamic Kinetic Resolution*.

β-Branched noncanonical amino acids are valuable molecules in modern drug development efforts. However, they are still challenging to prepare due to the need to set multiple stereocenters in a stereoselective fashion, and contemporary methods for the synthesis of such compounds often rely on the use of rare-transition-metal catalysts with designer ligands. Herein, we report a highly diastereo- and enantioselective biocatalytic transamination method to prepare a broad range of aromatic β-branched α-amino acids. Mechanistic studies show that the transformation proceeds through dynamic kinetic resolution that is unique to the optimal enzyme. To highlight its utility and practicality, the biocatalytic reaction was applied to the synthesis of several sp3 -rich cyclic fragments and the first total synthesis of jomthonic acid A.

Stereoselective Synthesis of β‐Branched Aromatic α‐Amino Acids by Biocatalytic Dynamic Kinetic Resolution**

Abstractβ‐Branched noncanonical amino acids are valuable molecules in modern drug development efforts. However, they are still challenging to prepare due to the need to set multiple stereocenters in a stereoselective fashion, and contemporary methods for the synthesis of such compounds often rely on the use of rare‐transition‐metal catalysts with designer ligands. Herein, we report a highly diastereo‐ and enantioselective biocatalytic transamination method to prepare a broad range of aromatic β‐branched α‐amino acids. Mechanistic studies show that the transformation proceeds through dynamic kinetic resolution that is unique to the optimal enzyme. To highlight its utility and practicality, the biocatalytic reaction was applied to the synthesis of several sp3‐rich cyclic fragments and the first total synthesis of jomthonic acid A.

Multi-enzyme pyruvate removal system to enhance (R)-selective reductive amination of ketones.

Biocatalytic transamination is widely used in industrial production of chiral chemicals. Here, we constructed a novel multi-enzyme system to promote the conversion of the amination reaction. Firstly, we constructed the ArR-ωTA/TdcE/FDH/LDH multi-enzyme system, by combination of (R)-selective ω-transaminase derived from Arthrobacter sp. (ArR-ωTA), formate dehydrogenase (FDH) derived from Candida boidinii, formate acetyltransferase (TdcE) and lactate dehydrogenase (LDH) derived from E. coli MG1655. This multi-enzyme system was used to efficiently remove the by-product pyruvate by TdcE and LDH to facilitate the transamination reaction. The TdcE/FDH pathway was found to dominate the by-product pyruvate removal in the transamination reaction. Secondly, we optimized the reaction conditions, including d-alanine, DMSO, and pyridoxal phosphate (PLP) with different concentration of 2-pentanone (as a model substrate). Thirdly, by using the ArR-ωTA/TdcE/FDH/LDH system, the conversions of 2-pentanone, 4-phenyl-2-butanone and cyclohexanone were 84.5%, 98.2% and 79.3%, respectively.

Biocatalytic transamination in a monolithic flow reactor: improving enzyme grafting for enhanced performance.

Transaminases were immobilized onto macrocellular silica monoliths and used for carrying a continuous flow mode transamination reaction. Monoliths were prepared via an emulsion-templated sol–gel method and functionalised by amino-moieties (3-aminopropyl-triethoxysilane, APTES) in order to covalently immobilize the enzymes, using glutaraldehyde as a cross-linking agent. In order to obtain higher performance and improved reproducibility, we investigate the key parameters of APTES functionalisation and of enzyme grafting. Four functionalisation protocols were studied. We show that enhancing the homogeneity of the APTES grafting and controlling the moisture level during functionalisation led to a 3-fold increase in activity as compared to the previously reported data, and greatly improved the reproducibility. Additionally, we report a strong beneficial effect of running the enzyme immobilisation at room temperature instead of 4 °C, further enhancing the obtained activity. Finally, the popular method which consists of stabilizing the covalent attachment of the enzyme by reducing the imine bonds formed between the enzyme and the functionalised surface was investigated. We highlight a strong enzyme deactivation caused by cyanoborohydride, making this strategy irrelevant in this case. The improvements presented here led to more active macrocellular monoliths, of general interest for continuous flow mode biocatalysis.

N-Butylamine as an alternative amine donor for the stereoselective biocatalytic transamination of ketones

Formal reductive amination has been a main focus of biocatalysis research in recent times. Among the enzymes able to perform this transformation, pyridoxal-5′-phosphate-dependent transaminases have shown the greatest promise in terms of extensive substrate scope and industrial application. Despite concerted research efforts in this area, there exist relatively few options regarding efficient amino donor co-substrates capable of allowing high conversion and atom efficiency with stable enzyme systems. Herein we describe the implementation of the recently described spuC gene, coding for a putrescine transaminase, exploiting its unusual amine donor tolerance to allow use of inexpensive and readily-available n-butylamine as an alternative to traditional methods. Via the integration of SpuC homologues with tandem co-product removal and cofactor regeneration enzymes, high conversion could be achieved with just 1.5 equivalents of the amine with products displaying excellent enantiopurity.

Biocatalytic transamination with near-stoichiometric inexpensive amine donors mediated by bifunctional mono- and di-amine transaminases

Bifunctional transaminases enable a strategy for the production of chiral amines using small excesses of diamine donors.

Hydroxypyridyl Imines: Enhancing Chromatographic Separation and Stereochemical Analysis of Chiral Amines via Circular Dichroism.

Imine-bond formation between chiral amines and commercially available 3-hydroxypyridine-2-carboxaldehyde (HCA) was exploited for rapid determination of stereochemical composition. Chiral supercritical fluid chromatography (SFC) screening of the derivatized imine compounds led to the elucidation of multiple combinations of mobile and stationary phases that gave resolution of all members of a series of chiral amines. The first eluting enantiomer was generally the derivative of the (R)-amine enantiomer across the series that was studied, indicating that the imine formed from the (S)-amine has more favorable interaction with the chiral stationary phase of the column. These conditions were then applied to more challenging compounds, namely amino alcohols and diastereomers possessing more than one stereocenter. The approach was utilized to monitor stereoselective biocatalytic transamination and assign the absolute configuration of the enantiomeric products. Finally, hydrolysis of the imine bond of the derivative was shown to generate enantiopure amine starting materials without racemization. This further highlights the value of this approach for creating readily reversed derivatives that enhance chromatographic separation and aid in the determination of absolute configuration.

Biocatalytic Transamination for the Asymmetric Synthesis of Pyridylalkylamines. Structural and Activity Features in the Reactivity of Transaminases

A set of transaminases has been investigated for the biocatalytic amination of 1-(4-chloropyridin-2-yl)alkan-1-ones. The influence of the chain length of the n-1-alkanone at the C-2 position of the pyridine has been studied in the reaction with different (R)- and (S)-selective transaminases. Thus, enantiopure amines were isolated with high purity starting from a wide selection of prochiral ketones. On the one hand, excellent yields (from 97 to >99% conversion, up to 93% isolated yield) and stereoselectivity values (>99% ee for both amine enantiomers) were found for n-1-alkanone linear short chain substituents such as ethanone or propanone. On the other hand, more hindered substrates were accepted only when using evolved enzymes such as an evolved variant of (R)-Arthrobacter (ArRmut11-TA). An initial common structural feature was the presence of a chlorine atom on the C-4 position of the pyridine core, which was found to increase the reactivity of the starting ketone, giving extra versatility for the intro...

Synthesis of ((3R,6R)-6-Methylpiperidin-3-yl)methanol via Biocatalytic Transamination and Crystallization-Induced Dynamic Resolution

An asymmetric synthesis of orexin receptor antagonist MK-6096 piperidine core, ((3R,6R)-6-methylpiperidin-3-yl)methanol (3), is described. The target is synthesized in four steps and 40% overall yield from methyl vinyl ketone and diethyl malonate. The key operation is a practical crystallization-induced dynamic resolution for the conversion of a trans/cis mixture of lactam acid 17 into the desired trans-lactam acid salt in >95% de and 91% yield. The substrate lactam acid mixture was prepared via a solvent-free Michael reaction and a practical biocatalytic transamination process.

The Industrial Age of Biocatalytic Transamination.

During the last decade the use of ω-transaminases has been identified as a very powerful method for the preparation of optically pure amines from the corresponding ketones. Their immense potential for the preparation of chiral amines, together with their ease of use in combination with existing biocatalytic methods, have made these biocatalysts a competitor to any chemical methodology for (asymmetric) amination. An increasing number of examples, especially from industry, shows that this biocatalytic technology outmaneuvers existing chemical processes by its simple and flexible nature. In the last few years numerous publications and patents on synthetic routes, mainly to pharmaceuticals, involving ω-transaminases have been published. The review gives an overview of the application of ω-transaminases in organic synthesis with a focus on active pharmaceutical ingredients (APIs) and the developments during the last few years.

Economic Considerations for Selecting an Amine Donor in Biocatalytic Transamination

The industrial implementation of biocatalysis for production of pharma and fine chemicals has grown substantially over recent years. An upcoming application is that of chiral synthesis of optically pure amines, a technology known for many years but that is now seeing a renewed and wider interest in industry. The technology has been demonstrated in a few selected cases, but widespread implementation and for a broader range of target molecules requires a deeper understanding of the underlying thermodynamic as well as economic constraints for the different choices that can be made in designing the process, in particular the choice of amine donor. This paper discusses these constraints and demonstrates, through simple thermodynamic and economic models, the process targets that need to be set and achieved for a process dependent on allowed process costs and quality targets.

Amine donor and acceptor influence on the thermodynamics of ω-transaminase reactions

In recent years biocatalytic transamination using ω-transaminase has become established as one of the most interesting routes to synthesize chiral amines with a high enantiomeric purity, especially in the pharmaceutical sector where the demand for such compounds is high. Nevertheless, one limitation for successful implementation and scale-up is that the thermodynamics of such conversions are frequently found unfavourable. Herein we report experimental measurements of apparent equilibrium constants for several industrially relevant transamination reactions in a systematic manner to better understand the effect of amine acceptor and donor choice. For example, we have found that ortho-substitution of acetophenone like molecules, had a significant impact on the thermodynamic equilibrium. Likewise, the effect of cyclic amine acceptors was evaluated and compared to similar non-cyclic structures. It was found that an aliphatic six membered ring was favourable and a conjugated bicyclic five membered ring structure, unfavourable. Finally, we evaluated and compared the use of five different donor molecules, and calculated their ΔGapp values. This is particularly important in the further implementation of such reactions because it may be used to help select suitable donor/acceptor combinations. The results presented here give guidance, with respect to thermodynamics, in order to further extend the application of biocatalytic transamination.

Unusual pyrimidine participation: efficient stereoselective synthesis of potent dual orexin receptor antagonist MK-6096.

An asymmetric synthesis of dual orexin receptor antagonist MK-6096 (1) is described. Key steps for the trans-2,5-disubstituted piperidinyl ether fragment include a biocatalytic transamination, a trans-selective Mukaiyama aldol, and a regioselective pyridyl SNAr process. The pyrimidyl benzoic acid was synthesized via a Negishi coupling and a nitrile hydrolysis. Coupling of the two fragments via a catalytic T3P-mediated amidation completed the synthesis. Unusual behaviors in the hydrolysis of pyrimidyl benzonitrile and the amide coupling of the pyrimidyl benzoic acid are also described.

Biocatalytic potential of vanillin aminotransferase from Capsicum chinense

BackgroundThe conversion of vanillin to vanillylamine is a key step in the biosynthetic route towards capsaicinoids in pungent cultivars of Capsicum sp. The reaction has previously been annotated to be catalysed by PAMT (putative aminotransferase; [GenBank: AAC78480.1, Swiss-Prot: O82521]), however, the enzyme has previously not been biochemically characterised in vitro.ResultsThe biochemical activity of the transaminase was confirmed by direct measurement of the reaction with purified recombinant enzyme. The enzyme accepted pyruvate, and oxaloacetate but not 2-oxoglutarate as co-substrate, which is in accordance with other characterised transaminases from the plant kingdom. The enzyme was also able to convert (S)-1-phenylethylamine into acetophenone with high stereo-selectivity. Additionally, it was shown to be active at a broad pH range.ConclusionsWe suggest PAMT to be renamed to VAMT (vanillin aminotransferase, abbreviation used in this study) as formation of vanillin from vanillylamine could be demonstrated. Furthermore, due to high stereoselectivity and activity at physiological pH, VAMT is a suitable candidate for biocatalytic transamination in a recombinant whole-cell system.