Reductive amination is one of the most important methods for the synthesis of chiral amines Here we report the discovery of an NADP(H)-dependent reductive aminase from Aspergillus oryzae (AspRedAm, Uniprot code Q2TW47) that can catalyse the reductive coupling of a broad set of carbonyl compounds with a variety of primary and secondary amines with up to >98% conversion and with up to >98% enantiomeric excess In cases where both carbonyl and amine show high reactivity, it is possible to employ a 1:1 ratio of the substrates, forming amine products with up to 94% conversion Steady-state kinetic studies establish that the enzyme is capable of catalysing imine formation as well as reduction Crystal structures of AspRedAm in complex with NADP(H) and also with both NADP(H) and the pharmaceutical ingredient (R)-rasagiline are reported We also demonstrate preparative scale reductive aminations with wild-type and Q240A variant biocatalysts displaying total turnover numbers of up to 32,000 and space time yields up to 373 g l−1 d−1 An enzyme (AspRedAm) capable of coupling carbonyls with a variety of amines in a reductive amination has now been discovered Kinetic studies revealed that the enzyme catalysed both the imine formation step, as well as the reduction step Structure and mutagenesis studies have highlighted essential catalytic residues and preparative scale examples have demonstrated total turnover numbers of up to 32,000

A reductive aminase from Aspergillus oryzae

Imine reductases catalyse the reductive amination of aldehydes or ketones with amines to produce chiral amines—a key transformation in the preparation of fine chemicals and active pharmaceutical ingredients. Although significant progress has been recently made in the field, their industrial application has not been demonstrated. Herein, we describe a wild-type imine reductase that was engineered to perform reductive amination with concomitant substrate amine resolution to give a commercially relevant manufacturing process to lysine-specific demethylase-1 inhibitor GSK2879552. Three rounds of evolution resulted in an enzyme variant showing a >38,000-fold improvement over wild type. The engineering of a more stable and active enzyme variant enabled process optimization to an economic, high quality and sustainable operating space. Using the evolved enzyme, kilogram quantities of a key intermediate to GSK2879552 were produced in 84% yield, at 99.9% purity and >99.7% enantiomeric excess, with improved process mass intensity. Imine reductases have been regarded as one of the most promising enzymes by the pharmaceutical industry—but their industrial application is still lacking. This work reports the successful industrial application of an imine reductase, enabled by directed evolution.

Chiral synthesis of LSD1 inhibitor GSK2879552 enabled by directed evolution of an imine reductase

Biocatalysis, using enzymes for organic synthesis, has emerged as powerful tool for the synthesis of active pharmaceutical ingredients (APIs). The first industrial biocatalytic processes launched in the first half of the last century exploited whole-cell microorganisms where the specific enzyme at work was not known. In the meantime, novel molecular biology methods, such as efficient gene sequencing and synthesis, triggered breakthroughs in directed evolution for the rapid development of process-stable enzymes with broad substrate scope and good selectivities tailored for specific substrates. To date, enzymes are employed to enable shorter, more efficient, and more sustainable alternative routes toward (established) small molecule APIs, and are additionally used to perform standard reactions in API synthesis more efficiently. Herein, large-scale synthetic routes containing biocatalytic key steps toward >130 APIs of approved drugs and drug candidates are compared with the corresponding chemical protocols (if available) regarding the steps, reaction conditions, and scale. The review is structured according to the functional group formed in the reaction.

https://pubs.acs.org/doi/pdf/10.1021/acs.chemrev.1c00574

Shortening Synthetic Routes to Small Molecule Active Pharmaceutical Ingredients Employing Biocatalytic Methods

The broad interdisciplinary nature of biocatalysis fosters innovation, as different technical fields are interconnected and synergized. A way to depict that innovation is by conducting a survey on patent activities. This paper analyses the intellectual property activities of the last five years (2014–2019) with a specific focus on biocatalysis applied to asymmetric synthesis. Furthermore, to reflect the inventive and innovative steps, only patents that were granted during that period are considered. Patent searches using several keywords (e.g., enzyme names) have been conducted by using several patent engine servers (e.g., Espacenet, SciFinder, Google Patents), with focus on granted patents during the period 2014–2019. Around 200 granted patents have been identified, covering all enzyme types. The inventive pattern focuses on the protection of novel protein sequences, as well as on new substrates. In some other cases, combined processes, multi-step enzymatic reactions, as well as process conditions are the innovative basis. Both industries and academic groups are active in patenting. As a conclusion of this survey, we can assert that biocatalysis is increasingly recognized as a useful tool for asymmetric synthesis and being considered as an innovative option to build IP and protect synthetic routes.

/pdf/biocatalysis-as-useful-tool-in-asymmetric-synthesis-an-3r60txktuu.pdf

Biocatalysis as Useful Tool in Asymmetric Synthesis: An Assessment of Recently Granted Patents (2014–2019)

Enzymatic reductive amination, being a direct, selective and green methodology, has attracted significant interest in a short period of time and is emerging as a powerful tool for the synthesis of chiral alkylated amines. The discovery of an increasing number of imine reductases with reductive aminase (RedAm) activity has enabled mechanistic and substrate profiling studies. However, their potential for commercial applications has not been realized. Here, we report the discovery of RedAm activity in an imine reductase enzyme for the direct reductive amination of a cyclic ketone with methylamine. We also investigate engineering the enzyme to access a cis-cyclobutyl-N-methylamine for the manufacturing of a late-stage drug candidate, Janus kinase 1 (JAK1) inhibitor abrocitinib. The engineered enzyme, SpRedAm-R3-V6, showed >200-fold improvement in performance over the wild-type enzyme and was successfully used to develop a commercial manufacturing process with 73% isolated yield at 99.5% purity and high selectivity (>99:1 cis:trans). This process has been successfully used to manufacture multi-metric tons of the amine, demonstrating the potential of RedAm technology for commercial manufacturing. Reductive aminases show strong potential for the sustainable synthesis of chiral amines, but their application in industrial scale processes is lacking. Now, such an enzyme is screened and engineered allowing its use in commercial manufacturing of abrocitinib JAK1 inhibitor in multi-metric tons.

/pdf/biocatalytic-reductive-amination-from-discovery-to-1xfwz1s1r3.pdf

Biocatalytic reductive amination from discovery to commercial manufacturing applied to abrocitinib JAK1 inhibitor

The present disclosure provides engineered polypeptides having imine reductase activity, polynucleotides encoding the engineered imine reductases, host cells capable of expressing the engineered imine reductases, and methods of using these engineered polypeptides with a range of ketone and amine substrate compounds to prepare secondary and tertiary amine product compounds.

Engineered imine reductases and methods for the reductive amination of ketone and amine compounds

The disclosure provides transaminase polypeptides capable of converting the substrate, 2-(3,4-dimethoxyphenethoxy)cyclohexanone to the trans diastereomer product (1R,2R)-2-(3,4-dimethoxyphenethoxy)cyclohexanamine in at least a 2:1 diastereomeric ratio relative to the cis diastereomer (1R,2S)-2-(3,4-dimethoxyphenethoxy)cyclohexanamine. The disclosure also provides polynucleotides, vectors, host cells, and methods of making and using the transaminase polypeptides in processes for preparing (1R,2R)-2-(3,4-dimethoxyphenethoxy)cyclohexanamine and its analogs, which can product compounds can be further used to prepare the aminocyclohexylether compound, (3R)-1-[(1R,2R)-2-[2-(3,4-dimethoxyphenyl)ethoxy]cyclohexyl]pyrrolidin-3-ol, which is an ion channel blocker.

Biocatalysts and methods for the synthesis of (1r,2r)-2-(3,4-dimethoxyphenethoxy)cyclohexanamine

The present disclosure relates to engineered ketoreductase polypeptides for the preparation of hydroxyl substituted carbamate compounds, and polynucleotides, vectors, host cells, and methods of making and using the ketoreductase polypeptides.

Biocatalysts for the preparation of hydroxy substituted carbamates

The present disclosure relates to transaminase polypeptides capable of aminating a dicarbonyl substrate, and polynucleotides, vectors, host cells, and methods of making and using the transaminase polypeptides.

Biocatalysts and methods for the synthesis of substituted lactams

La presente invention concerne des polypeptides transaminases capables d'effectuer l'amination d'un substrat dicarbonyle, ainsi que des polynucleotides, des vecteurs, des cellules hotes et des procedes de fabrication et d'utilisation des polypeptides transaminases.

Biocatalyseurs et procédés pour synthèse de lactames substitués

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