Regiodivergent Radical Termination for Intermolecular Biocatalytic C-C Bond Formation.

Radical hydrofunctionalizations of electronically unbiased dienes are challenging to render regioselective, because the products are nearly identical in energy. Here, we report two engineered FMN-dependent "ene"-reductases (EREDs) that catalyze regiodivergent hydroalkylations of cyclic and linear dienes. While previous studies focused exclusively on the stereoselectivity of alkene hydroalkylation, this work highlights that EREDs can control the regioselectivity of hydrogen atom transfer, providing a method for selectively preparing constitutional isomers that would be challenging to prepare using traditional synthetic methods. Engineering the ERED from Gluconabacter sp. (GluER) furnished a variant that favors the γ,δ-unsaturated ketone, while an engineered variant from a commercial ERED panel favors the δ,ε-unsaturated ketone. The effect of beneficial mutations has been investigated using substrate docking studies and the mechanism probed by isotope labeling experiments. A variety of α-bromo ketones can be coupled with cyclic and linear dienes. These interesting building blocks can also be further modified to generate difficult-to-access heterocyclic compounds.

Biocatalysis using Thermostable Cytochrome P450 Enzymes in Bacterial Membranes - Comparison of Metabolic Pathways with Human Liver Microsomes and Recombinant Human Enzymes.

Detailed structural characterization of small molecule metabolites is desirable during all stages of drug development, and often relies on the synthesis of metabolite standards. However, introducing structural changes into already complex, highly functionalized small molecules both regio- and stereo-selectively can be challenging using purely chemical approaches, introducing delays into the drug pipeline. An alternative is to use the cytochrome P450 enzymes (P450s) that produce the metabolites in vivo, taking advantage of the enzyme's inherently chiral active site to achieve regio- and stereoselectivity. Importantly, biotransformations are more sustainable: they proceed under mild conditions and avoid environmentally damaging solvents and transition metal catalysts. Recombinant enzymes avoid the need to use animal liver microsomes. However, native enzymes must be stabilized to work for extended periods or at elevated temperatures, and stabilizing mutations can alter catalytic activity. Here we assessed a set of novel, thermostable P450s in bacterial membranes, a format analogous to liver microsomes, for their ability to metabolize drugs through various pathways and compared them to human liver microsomes. Collectively, the thermostable P450s could replicate the metabolic pathways seen with human liver microsomes, including bioactivation to protein-reactive intermediates. Novel metabolites were found, suggesting the possibility of obtaining metabolites not produced by human or rodent liver microsomes. Importantly, no alteration in assay conditions from standard protocols for microsomal incubations was necessary. Thus, such bacterial membranes represent an analogous metabolite generation system to liver microsomes in terms of metabolites produced and ease of use, but which provides access to more diversity of metabolite structures. SIGNIFICANCE STATEMENT: In drug development it is often chemically challenging, to synthesize authentic metabolites of drug candidates for structural identification and evaluation of activity and safety. Biosynthesis using microsomes or recombinant human enzymes is confounded by the instability of the enzymes. Here we show that thermostable ancestral cytochrome P450 enzymes derived from P450 families responsible for human drug metabolism offer advantages over the native human forms in being more robust and over microbial enzymes in faithfully reflecting human drug metabolism.

Phytochemicals in Drug Discovery-A Confluence of Tradition and Innovation.

Phytochemicals have a long and successful history in drug discovery. With recent advancements in analytical techniques and methodologies, discovering bioactive leads from natural compounds has become easier. Computational techniques like molecular docking, QSAR modelling and machine learning, and network pharmacology are among the most promising new tools that allow researchers to make predictions concerning natural products' potential targets, thereby guiding experimental validation efforts. Additionally, approaches like LC-MS or LC-NMR speed up compound identification by streamlining analytical processes. Integrating structural and computational biology aids in lead identification, thus providing invaluable information to understand how phytochemicals interact with potential targets in the body. An emerging computational approach is machine learning involving QSAR modelling and deep neural networks that interrelate phytochemical properties with diverse physiological activities such as antimicrobial or anticancer effects.

The Impact of Metagenomics on Biocatalysis.

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.

Engineering Biocatalysts for the C-H Activation of Fatty Acids by Ancestral Sequence Reconstruction.

Selective, one-step C-H activation of fatty acids from biomass is an attractive concept in sustainable chemistry. Biocatalysis has shown promise for generating high-value hydroxy acids, but to date enzyme discovery has relied on laborious screening and produced limited hits, which predominantly oxidise the subterminal positions of fatty acids. Herein we show that ancestral sequence reconstruction (ASR) is an effective tool to explore the sequence-activity landscape of a family of multidomain, self-sufficient P450 monooxygenases. We resurrected 11 catalytically active CYP116B ancestors, each with a unique regioselectivity fingerprint that varied from subterminal in the older ancestors to mid-chain in the lineage leading to the extant, P450-TT. In lineages leading to extant enzymes in thermophiles, thermostability increased from ancestral to extant forms, as expected if thermophily had arisen de novo. Our studies show that ASR can be applied to multidomain enzymes to develop active, self-sufficient monooxygenases as regioselective biocatalysts for fatty acid hydroxylation.

Biocatalysis in Drug Design: Engineered Reductive Aminases (RedAms) Are Used to Access Chiral Building Blocks with Multiple Stereocenters.

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.

Interrogation of an Enzyme Library Reveals the Catalytic Plasticity of Naturally Evolved [4+2] Cyclases.

Stereoselective carbon-carbon bond forming reactions are quintessential transformations in organic synthesis. One example is the Diels-Alder reaction, a [4+2] cycloaddition between a conjugated diene and a dienophile to form cyclohexenes. The development of biocatalysts for this reaction is paramount for unlocking sustainable routes to a plethora of important molecules. To obtain a comprehensive understanding of naturally evolved [4+2] cyclases, and to identify hitherto uncharacterised biocatalysts for this reaction, we constructed a library comprising forty-five enzymes with reported or predicted [4+2] cycloaddition activity. Thirty-one library members were successfully produced in recombinant form. In vitro assays employing a synthetic substrate incorporating a diene and a dienophile revealed broad-ranging cycloaddition activity amongst these polypeptides. The hypothetical protein Cyc15 was found to catalyse an intramolecular cycloaddition to generate a novel spirotetronate. The crystal structure of this enzyme, along with docking studies, establishes the basis for stereoselectivity in Cyc15, as compared to other spirotetronate cyclases.

A Universal, Continuous Assay for SAM-dependent Methyltransferases.

Enzyme-catalyzed late-stage functionalization (LSF), such as methylation of drug molecules and lead structures, enables direct access to more potent active pharmaceutical ingredients (API). S-adenosyl-l-methionine-dependent methyltransferases (MTs) can play a key role in the development of new APIs, as they catalyze the chemo- and regioselective methylation of O-, N-, S- and C-atoms, being superior to traditional chemical routes. To identify suitable MTs, we developed a continuous fluorescence-based, high-throughput assay for SAM-dependent methyltransferases, which facilitates screening using E. coli cell lysates. This assay involves two enzymatic steps for the conversion of S-adenosyl-l-homocysteine into H2 S to result in a selective fluorescence readout via reduction of an azidocoumarin sulfide probe. Investigation of two O-MTs and an N-MT confirmed that this assay is suitable for the determination of methyltransferase activity in E. coli cell lysates.

The Role of Cytochrome†P450 AbyV in the Final Stages of Abyssomicin†C Biosynthesis.

Abyssomicin C and its atropisomer are potent inhibitors of bacterial folate metabolism. They possess complex polycyclic structures, and their biosynthesis has been shown to involve several unusual enzymatic transformations. Using a combination of synthesis and in vitro assays we reveal that AbyV, a cytochrome P450 enzyme from the aby gene cluster, catalyses a key late-stage epoxidation required for the installation of the characteristic ether-bridged core of abyssomicin C. The X-ray crystal structure of AbyV has been determined, which in combination with molecular dynamics simulations provides a structural framework for our functional data. This work demonstrates the power of combining selective carbon-13 labelling with NMR spectroscopy as a sensitive tool to interrogate enzyme-catalysed reactions in vitro with no need for purification.

Exploring the enzyme-catalyzed synthesis of isotope labeled cyclopropanes.

Cyclopropanes are commonly employed structural moieties in drug design since their incorporation is often associated with increased target affinity, improved metabolic stability, and increased rigidity to access bioactive conformations. Robust chemical cyclopropanation procedures have been developed which proceed with high yield and broad substrate scope, and have been applied to labeled substrates. Recently, engineered enzymes have been shown to perform cyclopropanations with remarkable diastereoselectivity and enantioselectivity, but this biocatalytic approach has not been applied to labeled substrates to date. In this study, the use of enzyme catalysis for the synthesis of labeled cyclopropanes was investigated. Two readily available enzymes, a modified CYP450 enzyme and a modified Aeropyrum pernix protoglobin, were investigated for the cyclopropanation of a variety of substituted styrenes. For this biocatalytic transformation, the enzymes required the use of ethyl diazoacetate. Due to the highly energetic nature of this molecule, alternatives were investigated. The final optimized cyclopropanation was successfully demonstrated using n-hexyl diazoacetate, resulting in moderate to high enantiomeric excess. The optimized procedure was used to generate labeled cyclopropanes from 13 C-glycine, forming all four labeled stereoisomers of phosphodiesterase type-IV inhibitor, MK0952. These reactions provide a convenient and effective biocatalytic route to stereoselective 13 C-labeled cyclopropanes and serve as a proof-of-concept for generating stereoselective labeled cyclopropanes.

Oxalate Oxidase for In Situ H2 O2 -Generation in Unspecific Peroxygenase-Catalysed Drug Oxyfunctionalisations.

H2 O2 -driven enzymes are of great interest for industrial biotransformations. Herein, we show for the first time that oxalate oxidase (OXO) is an efficient in situ source of H2 O2 for one of these biocatalysts, which is known as unspecific peroxygenase (UPO). OXO is reasonably robust, produces only CO2 as a by-product and uses oxalate as a cheap sacrificial electron donor. UPO has significant potential as an industrial catalyst for selective C-H oxyfunctionalisations, as we confirm herein by testing a diverse drug panel using miniaturised high-throughput assays and mass spectrometry. 33 out of 64 drugs were converted in 5 µL-scale reactions by the UPO with OXO (conversion >70 % for 11 drugs). Furthermore, oxidation of the drug tolmetin was achieved on a 50 mg scale (TONUPO 25 664) with 84 % yield, which was further improved via enzyme immobilization. This one-pot approach ensures adequate H2 O2 levels, enabling rapid access to industrially relevant molecules that are difficult to obtain by other routes.

Enzymatic Late-Stage Modifications: Better Late Than Never.

Enzyme catalysis is gaining increasing importance in synthetic chemistry. Nowadays, the growing number of biocatalysts accessible by means of bioinformatics and enzyme engineering opens up an immense variety of selective reactions. Biocatalysis especially provides excellent opportunities for late-stage modification often superior to conventional de novo synthesis. Enzymes have proven to be useful for direct introduction of functional groups into complex scaffolds, as well as for rapid diversification of compound libraries. Particularly important and highly topical are enzyme-catalysed oxyfunctionalisations, halogenations, methylations, reductions, and amide bond formations due to the high prevalence of these motifs in pharmaceuticals. This Review gives an overview of the strengths and limitations of enzymatic late-stage modifications using native and engineered enzymes in synthesis while focusing on important examples in drug development.

Qualification of impurities based on metabolite data.

Regulatory Guidance documents ICH Q3A (R2) and ICH Q3B (R2) state that "impurities that are also significant metabolites present in animal and/or human studies are generally considered qualified". However, no guidance is provided regarding data requirements for qualification, nor is a definition of the term "significant metabolite" provided. An opportunity is provided to define those categories and potentially avoid separate toxicity studies to qualify impurities. This can reduce cost, animal use and time, and avoid delays in drug development progression. If the concentration or amount of a metabolite, in animals or human, is similar to that of the known, structurally identical impurity (arising from the administered test material), the qualification of the impurity on the grounds of it also being a metabolite is justified. We propose two complementary approaches to support conclusions to this effect: 1) demonstrate that the impurity is formed by metabolism in animals and/or man, based preferably on plasma exposures or, alternatively, amounts excreted in urine, and, where appropriate, 2) show that animal exposure to (or amount of) the impurity/metabolite is equal or greater in animals than in humans. An important factor of both assessments is the maximum theoretical concentration (or amount) (MTC or MTA) of the impurity/metabolite achievable from the administered dose and recommendations on the estimation of the MTC and MTA are presented.

Metabolism of Strained Rings: Glutathione S-transferase-Catalyzed Formation of a Glutathione-Conjugated Spiro-azetidine without Prior Bioactivation.

AZD1979 [(3-(4-(2-oxa-6-azaspiro[3.3]heptan-6-ylmethyl)phenoxy)azetidin-1-yl)(5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-yl)methanone] is a melanin-concentrating hormone receptor 1 antagonist designed for the treatment of obesity. In this study, metabolite profiles of AZD1979 in human hepatocytes revealed a series of glutathione-related metabolites, including the glutathionyl, cysteinyl, cysteinylglycinyl, and mercapturic acid conjugates. The formation of these metabolites was not inhibited by coincubation with the cytochrome P450 (P450) inhibitor 1-aminobenzotriazole. In efforts to identify the mechanistic features of this pathway, investigations were performed to characterize the structure of the glutathionyl conjugate M12 of AZD1979 and to identify the enzyme system catalyzing its formation. Studies with various human liver subcellular fractions established that the formation of M12 was NAD(P)H-independent and proceeded in cytosol and S9 fractions but not in microsomal or mitochondrial fractions. The formation of M12 was inhibited by ethacrynic acid, an inhibitor of glutathione S-transferases (GSTs). Several human recombinant GSTs, including GSTA1, A2-2, M1a, M2-2, T1-1, and GST from human placenta, were incubated with AZD1979. All GSTs tested catalyzed the formation of M12, with GSTA2-2 being the most efficient. Metabolite M12 was purified from rat liver S9 incubations and its structure elucidated by NMR. These results establish that M12 is the product of the GST-catalyzed glutathione attack on the carbon atom α to the nitrogen atom of the strained spiro-azetidinyl moiety to give, after ring opening, the corresponding amino-thioether conjugate product, a direct conjugation pathway that occurs without the prior substrate bioactivation by P450. SIGNIFICANCE STATEMENT: The investigated compound, AZD1979, contains a 6-substituted-2-oxa-6-azaspiro[3.3]heptanyl derivative that is an example of strained heterocycles, including spiro-fused ring systems, that are widely used in synthetic organic chemistry. An unusual azetidinyl ring-opening reaction involving a nucleophilic attack by glutathione, which does not involve prior cytochrome P450-catalyzed bioactivation of the substrate and which is catalyzed by glutathione transferases, is reported. We propose a mechanism involving the protonated cyclic aminyl intermediate that undergoes nucleophilic attack by glutathione thiolate anion in this reaction, catalyzed by glutathione transferases.

Late-Stage Functionalization of Histidine in Unprotected Peptides.

The late-stage functionalization (LSF) of peptides represents a valuable strategy for the design of potent peptide pharmaceuticals by enabling rapid exploration of chemical diversity and offering novel opportunities for peptide conjugation. While the C(sp2 )-H activation of tryptophan (Trp) is well documented, the resurgence of radical chemistry is opening new avenues for the C-H functionalization of other aromatic side-chains. Herein, we report the first example of LSF at C2 of histidine (His) utilizing a broad scope of aliphatic sulfinate salts as radical precursors. In this work, the exquisite selectivity for histidine functionalization was demonstrated through the alkylation of complex unprotected peptides in aqueous media. Finally, this methodology was extended for the installation of a ketone handle, providing an unprecedented anchor for selective oxime/hydrazone conjugation at histidine.

Exploring PTDH-P450BM3 Variants for the Synthesis of Drug Metabolites.

The conversion of a series of pharmaceutical compounds was examined with three variants of cytochrome P450BM3 fused to phosphite dehydrogenase (PTDH) to enable cofactor recycling. Conditions for enzyme production were optimized, and the purified PTDH-P450BM3 variants were tested against 32 commercial drugs by using rapid UPLC-MS analysis. The sets of mutations (R47L/F87V/L188Q and R47L/F87V/L188Q/E267V/G415S) improved conversion for all compounds, and a variety of products were detected. Product analysis showed that reaction types included C-hydroxylation, N-oxidation, demethylation, and aromatization. Interestingly, enzymatic aromatization could occur independent of the addition of reducing coenzyme. These results identified new conversions catalyzed by P450BM3 variants and showed that a small set of mutations in the oxygenase domain could broaden both substrate range and reaction type.

Oxetane Substrates of Human Microsomal Epoxide Hydrolase.

Oxetanyl building blocks are increasingly used in drug discovery because of the improved drug-like properties they confer on drug candidates, yet little is currently known about their biotransformation. A series of oxetane-containing analogs was studied and we provide the first direct evidence of oxetane hydrolysis by human recombinant microsomal epoxide hydrolase (mEH). Incubations with human liver fractions and hepatocytes were performed with and without inhibitors of cytochrome P450 (P450), mEH and soluble epoxide hydrolase (sEH). Reaction dependence on NADPH was investigated in subcellular fractions. A full kinetic characterization of oxetane hydrolysis is presented, in both human liver microsomes and human recombinant mEH. In human liver fractions and hepatocytes, hydrolysis by mEH was the only oxetane ring-opening metabolic route, with no contribution from sEH or from cytochrome P450-catalyzed oxidation. Minimally altering the structural elements in the immediate vicinity of the oxetane can greatly modulate the efficiency of hydrolytic ring cleavage. In particular, higher pKa in the vicinity of the oxetane and an increased distance between the oxetane ring and the benzylic nitrogen improve reaction rate, which is further enhanced by the presence of methyl groups near or on the oxetane. This work defines oxetanes as the first nonepoxide class of substrates for human mEH, which was previously known to catalyze the hydrolytic ring opening of electrophilic and potentially toxic epoxide-containing drugs, drug metabolites, and exogenous organochemicals. These findings will be of value for the development of biologically active oxetanes and may be exploited for the biocatalytic generation of enantiomerically pure oxetanes and diols.

P450BM3 fused to phosphite dehydrogenase allows phosphite-driven selective oxidations.

To facilitate the wider application of the NADPH-dependent P450BM3, we fused the monooxygenase with a phosphite dehydrogenase (PTDH). The resulting monooxygenase-dehydrogenase fusion enzyme acts as a self-sufficient bifunctional catalyst, accepting phosphite as a cheap electron donor for the regeneration of NADPH.The well-expressed fusion enzyme was purified and analyzed in comparison to the parent enzymes. Using lauric acid as substrate for P450BM3, it was found that the fusion enzyme had similar substrate affinity and hydroxylation selectivity while it displayed a significantly higher activity than the non-fused monooxygenase. Phosphite-driven conversions of lauric acid at restricted NADPH concentrations confirmed multiple turnovers of the cofactor. Interestingly, both the fusion enzyme and the native P450BM3 displayed enzyme concentration dependent activity and the fused enzyme reached optimal activity at a lower enzyme concentration. This suggests that the fusion enzyme has an improved tendency to form functional oligomers.To explore the constructed phosphite-driven P450BM3 as a biocatalyst, conversions of the drug compounds omeprazole and rosiglitazone were performed. PTDH-P450BM3 driven by phosphite was found to be more efficient in terms of total turnover when compared with P450BM3 driven by NADPH. The results suggest that PTDH-P450BM3 is an attractive system for use in biocatalytic and drug metabolism studies.

Synthesis of 1ß-hydroxydeoxycholic acid in H-2 and unlabeled forms.

1ß-hydroxydeoxycholic acid in unlabeled and stable isotope labeled forms was required for use as a biomarker for Cytochrome P450 3A4/5 activity. A lengthy synthesis was undertaken to deliver the unlabeled compound and in the process, to develop a route to the deuterium labeled compound. The synthesis of the unlabeled compound was completed but in a very low yield. Concurrent with the synthetic approach, a biosynthetic route was pursued and this approach proved to be much more rapid and afforded the compound in both unlabeled and deuterium labeled forms in a 1-step oxidation from deoxycholic acid and [D4 ]deoxycholic acid, respectively.

CYP3A Specifically Catalyzes 1ß-Hydroxylation of Deoxycholic Acid: Characterization and Enzymatic Synthesis of a Potential Novel Urinary Biomarker for CYP3A Activity.

The endogenous bile acid metabolite 1ß-hydroxy-deoxycholic acid (1ß-OH-DCA) excreted in human urine may be used as a sensitive CYP3A biomarker in drug development reflecting in vivo CYP3A activity. An efficient and stereospecific enzymatic synthesis of 1ß-OH-DCA was developed using a Bacillus megaterium (BM3) cytochrome P450 (P450) mutant, and its structure was confirmed by nuclear magnetic resonance (NMR) spectroscopy. A [(2)H4]-labeled analog of 1ß-OH-DCA was also prepared. The major hydroxylated metabolite of deoxycholic acid (DCA) in human liver microsomal incubations was identified as 1ß-OH-DCA by comparison with the synthesized reference analyzed by UPLC-HRMS. Its formation was strongly inhibited by CYP3A inhibitor ketoconazole. Screening of 21 recombinant human cytochrome P450 (P450) enzymes showed that, with the exception of extrahepatic CYP46A1, the most abundant liver P450 subfamily CYP3A, including CYP3A4, 3A5, and 3A7, specifically catalyzed 1ß-OH-DCA formation. This indicated that 1ß-hydroxylation of DCA may be a useful marker reaction for CYP3A activity in vitro. The metabolic pathways of DCA and 1ß-OH-DCA in human hepatocytes were predominantly via glycine and, to a lesser extent, via taurine and sulfate conjugation. The potential utility of 1ß-hydroxylation of DCA as a urinary CYP3A biomarker was illustrated by comparing the ratio of 1ß-OH-DCA:DCA in a pooled spot urine sample from six healthy control subjects to a sample from one patient treated with carbamazepine, a potent CYP3A inducer; 1ß-OH-DCA:DCA was considerably higher in the patient versus controls (ratio 2.8 vs. 0.4). Our results highlight the potential of 1ß-OH-DCA as a urinary biomarker in clinical CYP3A DDI studies.