ABSTRACT
Process intensification is crucial for industrial implementation of biocatalysis and can be achieved by continuous process operation in miniaturized reactors with efficiently immobilized biocatalysts, enabling their long-term use. Due to their extremely large surface-to-volume ratio, nanomaterials are promising supports for enzyme immobilization. In this work, different functionalized nanofibrous nonwoven membranes were embedded in a two-plate microreactor to enable immobilization of hexahistidine (His6)-tagged amine transaminases (ATAs) in flow. A membrane coated with Cu2+ ions gave the best results regarding His6-tagged ATAs immobilization among the membranes tested yielding an immobilization yield of up to 95.3 % for the purified N-His6-ATA-wt enzyme. Moreover, an efficient one-step enzyme immobilization process from overproduced enzyme in Escherichia coli cell lysate was developed and yielded enzyme loads up to 1088 U mL-1. High enzyme loads resulted in up to 80 % yields of acetophenone produced from 40 mM (S)-α-methylbenzylamine in less than 4 min using a continuously operated microreactor. Up to 81 % of the initial activity was maintained in a 5-day continuous microreactor operation with immobilized His6-tagged ATA constructs. The highest turnover number within the indicated time was 7.23·106, which indicates that this immobilization approach using advanced material and reactor system is highly relevant for industrial implementation.
ABSTRACT
Biocatalysis is becoming an indispensable tool in organic synthesis due to high enzymatic catalytic efficiency as well as exquisite chemo- and stereoselectivity. Some biocatalysts display great promiscuity including a broad substrate scope as well as the ability to catalyze more than one type of transformation. These promiscuous activities have been applied individually to efficiently access numerous valuable target molecules. However, systems in which enzymes possessing multiple different catalytic activities are applied in the synthesis are less well developed. Such multifunctional biocatalysts (MFBs) would simplify chemical synthesis by reducing the number of operational steps and enzyme count, as well as simplifying the sequence space that needs to be engineered to develop an efficient biocatalyst. In this Perspective, we highlight recently reported MFBs focusing on their synthetic utility and mechanism. We also offer insight into their origin as well as comment on potential strategies for their discovery and engineering.
Subject(s)
Biocatalysis , Catalysis , Chemistry Techniques, SyntheticABSTRACT
In the ever-growing demand for sustainable ways to produce high-value small molecules, biocatalysis has come to the forefront of greener routes to these chemicals. As such, the need to constantly find and optimise suitable biocatalysts for specific transformations has never been greater. Metagenome mining has been shown to rapidly expand the toolkit of promiscuous enzymes needed for new transformations, without requiring protein engineering steps. If protein engineering is needed, the metagenomic candidate can often provide a better starting point for engineering than a previously discovered enzyme on the open database or from literature, for instance. In this review, we highlight where metagenomics has made substantial impact on the area of biocatalysis in recent years. We review the discovery of enzymes in previously unexplored or 'hidden' sequence space, leading to the characterisation of enzymes with enhanced properties that originate from natural selection pressures in native environments.
Subject(s)
Biocatalysis , Metagenomics , Enzymes/metabolism , Enzymes/chemistry , Enzymes/genetics , Protein EngineeringABSTRACT
Chiral amines are pivotal building blocks for the pharmaceutical industry. Asymmetric reductive amination is one of the most efficient and atom economic methodologies for the synthesis of optically active amines. Among the various strategies available, NAD(P)H-dependent amine dehydrogenases (AmDHs) and imine reductases (IREDs) are robust enzymes that are available from various sources and capable of utilizing a broad range of substrates with high activities and stereoselectivities. AmDHs and IREDs operate via similar mechanisms, both involving a carbinolamine intermediate followed by hydride transfer from the co-factor. In addition, both groups catalyze the formation of primary and secondary amines utilizing both organic and inorganic amine donors. In this review, we discuss advances in developing AmDHs and IREDs as biocatalysts and focus on evolutionary history, substrate scope and applications of the enzymes to provide an outlook on emerging industrial biotechnologies of chiral amine production.
Subject(s)
NAD , Oxidoreductases , Amination , Oxidoreductases/metabolism , Amines , Biocatalysis , Imines , StereoisomerismABSTRACT
Novel building blocks are in constant demand during the search for innovative bioactive small molecule therapeutics by enabling the construction of structure-activity-property-toxicology relationships. Complex chiral molecules containing multiple stereocenters are an important component in compound library expansion but can be difficult to access by traditional organic synthesis. Herein, we report a biocatalytic process to access a specific diastereomer of a chiral amine building block used in drug discovery. A reductive aminase (RedAm) was engineered following a structure-guided mutagenesis strategy to produce the desired isomer. The engineered RedAm (IR-09 W204R) was able to generate the (S,S,S)-isomer 3 in 45% conversion and 95% ee from the racemic ketone 2. Subsequent palladium-catalyzed deallylation of 3 yielded the target primary amine 4 in a 73% yield. This engineered biocatalyst was used at preparative scale and represents a potential starting point for further engineering and process development.
Subject(s)
Amines , Drug Design , Biocatalysis , StereoisomerismABSTRACT
Despite the increasing use of biocatalysis for organic synthesis, there are currently no databases that adequately capture synthetic biotransformations. The lack of a biocatalysis database prevents accelerating biocatalyst characterization efforts from being leveraged to quickly identify candidate enzymes for reactions or cascades, slowing their development. The RetroBioCat Database (available at retrobiocat.com) addresses this gap by capturing information on synthetic biotransformations and providing an analysis platform that allows biocatalysis data to be searched and explored through a range of highly interactive data visualization tools. This database makes it simple to explore available enzymes, their substrate scopes, and how characterized enzymes are related to each other and the wider sequence space. Data entry is facilitated through an openly accessible curation platform, featuring automated tools to accelerate the process. The RetroBioCat Database democratizes biocatalysis knowledge and has the potential to accelerate biocatalytic reaction development, making it a valuable resource for the community.
ABSTRACT
Biocatalysis is important in the discovery, development, and manufacture of pharmaceuticals. However, the identification of enzymes for target transformations of interest requires major screening efforts. Here, we report a structure-based computational workflow to prioritize protein sequences by a score based on predicted activities on substrates, thereby reducing a resource-intensive laboratory-based biocatalyst screening. We selected imine reductases (IREDs) as a class of biocatalysts to illustrate the application of the computational workflow termed IREDFisher. Validation by using published data showed that IREDFisher can retrieve the best enzymes and increase the hit rate by identifying the top 20 ranked sequences. The power of IREDFisher is confirmed by computationally screening 1400 sequences for chosen reductive amination reactions with different levels of complexity. Highly active IREDs were identified by only testing 20 samples in vitro. Our speed test shows that it only takes 90 min to rank 85 sequences from user input and 30 min for the established IREDFisher database containing 591 IRED sequences. IREDFisher is available as a user-friendly web interface (https://enzymeevolver.com/IREDFisher). IREDFisher enables the rapid discovery of IREDs for applications in synthesis and directed evolution studies, with minimal time and resource expenditure. Future use of the workflow with other enzyme families could be implemented following the modification of the workflow scoring function.
ABSTRACT
In the wake of the Covid-19 pandemic, it has become clear that global access to efficacious antiviral drugs will be critical to combat future outbreaks of SARS-CoV-2 or related viruses. The orally available SARS-CoV-2 main protease inhibitor nirmatrelvir has proven an effective treatment option for Covid-19, especially in compromised patients. We report a new synthesis of nirmatrelvir featuring a highly enantioselective biocatalytic desymmetrization (>99% ee) and a highly diastereoselective multicomponent reaction (>25:1 dr) as the key steps. Our route avoids the use of transition metals and peptide coupling reagents, resulting in an overall highly efficient and atom-economic process.
Subject(s)
COVID-19 , Humans , Pandemics , SARS-CoV-2 , Lactams , Leucine , NitrilesABSTRACT
The oxidative aromatization of aliphatic N-heterocycles is a fundamental organic transformation for the preparation of a diverse array of heteroaromatic compounds. Despite many attempts to improve the efficiency and practicality of this transformation, most synthetic methodologies still require toxic and expensive reagents as well as harsh conditions. Herein, we describe two enzymatic strategies for the oxidation of 1,2,3,4-tetrahydroquinolines (THQs) and N-cyclopropyl-N-alkylanilines into quinolines and 2-quinolones, respectively. Whole cells and purified monoamine oxidase (MAO-N) enzymes were used to effectively catalyze the biotransformation of THQs into the corresponding aromatic quinoline derivatives, while N-cyclopropyl-N-alkylanilines were converted into 2-quinolone compounds through a horseradish peroxidase (HRP)-catalyzed annulation/aromatization reaction followed by Fe-mediated oxidation.
ABSTRACT
Herein, we report a one-pot, chemoenzymatic process for the synthesis of enantioenriched C(1)-allylated tetrahydroisoquinolines. This transformation couples a monoamine oxidase (MAO-N)-catalyzed oxidation with a metal catalyzed allylboration, followed by a biocatalytic deracemization to afford allylic amine derivatives in both high yields and good to high enantiomeric excess. The cascade is operationally simple, with all components added at the start of the reaction and can be used to generate key building blocks for further elaboration.
ABSTRACT
The synthesis of structurally diverse amines is of fundamental significance in the pharmaceutical industry due to the ubiquitous presence of amine motifs in biologically active molecules. Biocatalytic reductive amination for amine production has attracted great interest owing to its synthetic advantages. Herein, we report the direct synthesis of a wide range of sterically demanding secondary amines, including several important active pharmaceutical ingredients and pharmaceutical intermediates, via reductive amination of carbonyl substrates and bulky amine nucleophiles employing imine reductases. Key to success for this route is the identification of an imine reductase from Penicillium camemberti with unusual substrate specificity and its further engineering, which empowered the accommodation of a broad range of sterically demanding amine nucleophiles encompassing linear alkyl and (hetero)aromatic (oxy)alkyl substituents and the formation of final amine products with up to >99% conversion. The practical utility of the biocatalytic route has been demonstrated by its application in the preparative synthesis of the anti-hyperparathyroidism drug cinacalcet.
ABSTRACT
The case for a renewed focus on Nature in drug discovery is reviewed; not in terms of natural product screening, but how and why biomimetic molecules, especially those produced by natural processes, should deliver in the age of artificial intelligence and screening of vast collections both in vitro and in silico. The declining natural product-likeness of licensed drugs and the consequent physicochemical implications of this trend in the context of current practices are noted. To arrest these trends, the logic of seeking new bioactive agents with enhanced natural mimicry is considered; notably that molecules constructed by proteins (enzymes) are more likely to interact with other proteins (e.g., targets and transporters), a notion validated by natural products. Nature's finite number of building blocks and their interactions necessarily reduce potential numbers of structures, yet these enable expansion of chemical space with their inherent diversity of physical characteristics, pertinent to property-based design. The feasible variations on natural motifs are considered and expanded to encompass pseudo-natural products, leading to the further logical step of harnessing bioprocessing routes to access them. Together, these offer opportunities for enhancing natural mimicry, thereby bringing innovation to drug synthesis exploiting the characteristics of natural recognition processes. The potential for computational guidance to help identifying binding commonalities in the route map is a logical opportunity to enable the design of tailored molecules, with a focus on "organic/biological" rather than purely "synthetic" structures. The design and synthesis of prototype structures should pay dividends in the disposition and efficacy of the molecules, while inherently enabling greener and more sustainable manufacturing techniques.
ABSTRACT
The development of efficient and sustainable methods for the synthesis of nitrogen heterocycles is an important goal for the chemical industry. In particular, substituted chiral piperidines are prominent targets due to their prevalence in medicinally relevant compounds and their precursors. A potential biocatalytic approach to the synthesis of this privileged scaffold would be the asymmetric dearomatization of readily assembled activated pyridines. However, nature is yet to yield a suitable biocatalyst specifically for this reaction. Here, by combining chemical synthesis and biocatalysis, we present a general chemo-enzymatic approach for the asymmetric dearomatization of activated pyridines for the preparation of substituted piperidines with precise stereochemistry. The key step involves a stereoselective one-pot amine oxidase/ene imine reductase cascade to convert N-substituted tetrahydropyridines to stereo-defined 3- and 3,4-substituted piperidines. This chemo-enzymatic approach has proved useful for key transformations in the syntheses of antipsychotic drugs Preclamol and OSU-6162, as well as for the preparation of two important intermediates in synthetic routes of the ovarian cancer monotherapeutic Niraparib.
Subject(s)
Piperidines , Pyridines , Pyridines/chemistry , Stereoisomerism , Catalysis , Piperidines/chemistry , Imines/chemistryABSTRACT
Amino-polyols represent attractive chemical building blocks but can be challenging to synthesize because of the high density of asymmetric functionalities and the need for extensive protecting-group strategies. Here we present a three-component strategy for the stereoselective enzymatic synthesis of amino-diols and amino-polyols using a diverse set of prochiral aldehydes, hydroxy ketones, and amines as starting materials. We were able to combine biocatalytic aldol reactions, using variants of d-fructose-6-phosphate aldolase (FSA), with reductive aminations catalyzed by IRED-259, identified from a metagenomic library. A two-step process, without the need for intermediate isolation, was developed to avoid cross-reactivity of the carbonyl components. Stereoselective formation of the 2R,3R,4R enantiomers of amino-polyols was observed and confirmed by X-ray crystallography.
ABSTRACT
The unique ability of the 'electrochemical leaf' (e-Leaf) to drive and control nanoconfined enzyme cascades bidirectionally, while directly monitoring their rate in real-time as electrical current, is exploited to achieve deracemisation and stereoinversion of secondary alcohols using a single electrode in one pot. Two alcohol dehydrogenase enzymes with opposing enantioselectivities, from Thermoanaerobacter ethanolicus (selective for S) and Lactobacillus kefir (selective for R) are driven bidirectionally via coupling to the fast and quasi-reversible interconversion of NADP+/NADPH catalysed by ferredoxin NADP+ reductase - all enzymes being co-entrapped in a nanoporous indium tin oxide electrode. Activity of the Lactobacillus kefir enzyme depends on the binding of a non-catalytic Mg2+, allowing it to be switched off after an oxidative half-cycle, by adding EDTA - the S-selective enzyme, with a tightly-bound Zn2+, remaining fully active. Racemate â S or R â S conversions are thus achieved in high yield with unprecedented ease.
Subject(s)
Alcohol Dehydrogenase , Ferredoxins , Alcohol Dehydrogenase/metabolism , Edetic Acid , Electrochemistry , Ferredoxin-NADP Reductase/metabolism , Lactobacillus , NADP/metabolismABSTRACT
The importance of energized nanoconfinement for facilitating the study and execution of enzyme cascades that feature multiple exchangeable cofactors is demonstrated by experiments with carboxylic acid reductase (CAR), an enzyme that requires both NADPH and ATP during a single catalytic cycle. Conversion of cinnamic acid to cinnamaldehyde by a package of four enzymes loaded into and trapped in the random nanopores of an indium tin oxide (ITO) electrode is driven and monitored through the simultaneous delivery of electrical and chemical energy. The electrical energy is transduced by ferredoxin NADP+ reductase, which undergoes rapid, direct electron exchange with ITO and regenerates NADP(H). The chemical energy provided by phosphoenolpyruvate, a fuel contained in the bulk solution, is cotransduced by adenylate kinase and pyruvate kinase, which efficiently convert the AMP product back into ATP that is required for the next cycle. The use of the two-kinase system allows the recycling process to be dissected to evaluate the separate roles of AMP removal and ATP supply during presteady-state and steady-state catalysis.
ABSTRACT
N-alkanoyl-N-methylglucamides (MEGAs) are non-toxic surfactants widely used as commercial ingredients, but more sustainable syntheses towards these compounds are highly desirable. Here, we present a biocatalytic route towards MEGAs and analogues using a truncated carboxylic acid reductase construct tailored for amide bond formation (CARmm-A). CARmm-A is capable of selective amide bond formation without the competing esterification reaction observed in lipase catalysed reactions. A kinase was implemented to regenerate ATP from polyphosphate and by thorough reaction optimisation using design of experiments, the amine concentration needed for amidation was significantly reduced. The wide substrate scope of CARmm-A was exemplified by the synthesis of 24 commercially relevant amides, including selected examples on a preparative scale. This work establishes acyl-phosphate mediated chemistry as a highly selective strategy for biocatalytic amide bond formation in the presence of multiple competing alcohol functionalities.
Subject(s)
Amines , Surface-Active Agents , Amides/chemistry , Amines/chemistry , Biocatalysis , Lipase/metabolismABSTRACT
Allylic amines are a versatile class of synthetic precursors of many valuable nitrogen-containing organic compounds, including pharmaceuticals. Enzymatic allylic amination methods provide a sustainable route to these compounds but are often restricted to allylic primary amines. We report a biocatalytic system for the reductive N-allylation of primary and secondary amines, using biomass-derivable cinnamic acids. The two-step one-pot system comprises an initial carboxylate reduction step catalyzed by a carboxylic acid reductase to generate the corresponding α,ß-unsaturated aldehyde in situ. This is followed by reductive amination of the aldehyde catalyzed by a bacterial reductive aminase pIR23 or BacRedAm to yield the corresponding allylic amine. We exploited pIR23, a prototype bacterial reductive aminase, self-sufficient in catalyzing formal reductive amination of α,ß-unsaturated aldehydes with various amines, generating a broad range of secondary and tertiary amines accessed in up to 94% conversion under mild reaction conditions. Analysis of products isolated from preparative reactions demonstrated that only selective hydrogenation of the C=N bond had occurred, preserving the adjacent alkene moiety. This process represents an environmentally benign and sustainable approach for the synthesis of secondary and tertiary allylic amine frameworks, using renewable allylating reagents and avoiding harsh reaction conditions. The selectivity of the system ensures that bis-allylation of the alkylamines and (over)reduction of the alkene moiety are avoided.
ABSTRACT
Chiral amine diastereomers are ubiquitous in pharmaceuticals and agrochemicals1, yet their preparation often relies on low-efficiency multi-step synthesis2. These valuable compounds must be manufactured asymmetrically, as their biochemical properties can differ based on the chirality of the molecule. Herein we characterize a multifunctional biocatalyst for amine synthesis, which operates using a mechanism that is, to our knowledge, previously unreported. This enzyme (EneIRED), identified within a metagenomic imine reductase (IRED) collection3 and originating from an unclassified Pseudomonas species, possesses an unusual active site architecture that facilitates amine-activated conjugate alkene reduction followed by reductive amination. This enzyme can couple a broad selection of α,ß-unsaturated carbonyls with amines for the efficient preparation of chiral amine diastereomers bearing up to three stereocentres. Mechanistic and structural studies have been carried out to delineate the order of individual steps catalysed by EneIRED, which have led to a proposal for the overall catalytic cycle. This work shows that the IRED family can serve as a platform for facilitating the discovery of further enzymatic activities for application in synthetic biology and organic synthesis.
Subject(s)
Amines , Oxidoreductases , Amination , Amines/chemistry , Biocatalysis , Imines/chemistry , Oxidoreductases/genetics , Oxidoreductases/metabolism , StereoisomerismABSTRACT
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.