A kinase-cGAS cascade to synthesize a therapeutic STING activator.

The introduction of molecular complexity in an atom- and step-efficient manner remains an outstanding goal in modern synthetic chemistry. Artificial biosynthetic pathways are uniquely able to address this challenge by using enzymes to carry out multiple synthetic steps simultaneously or in a one-pot sequence1-3. Conducting biosynthesis ex vivo further broadens its applicability by avoiding cross-talk with cellular metabolism and enabling the redesign of key biosynthetic pathways through the use of non-natural cofactors and synthetic reagents4,5. Here we describe the discovery and construction of an enzymatic cascade to MK-1454, a highly potent stimulator of interferon genes (STING) activator under study as an immuno-oncology therapeutic6,7 (ClinicalTrials.gov study NCT04220866 ). From two non-natural nucleotide monothiophosphates, MK-1454 is assembled diastereoselectively in a one-pot cascade, in which two thiotriphosphate nucleotides are simultaneously generated biocatalytically, followed by coupling and cyclization catalysed by an engineered animal cyclic guanosine-adenosine synthase (cGAS). For the thiotriphosphate synthesis, three kinase enzymes were engineered to develop a non-natural cofactor recycling system in which one thiotriphosphate serves as a cofactor in its own synthesis. This study demonstrates the substantial capacity that currently exists to use biosynthetic approaches to discover and manufacture complex, non-natural molecules.

Engineering Hydroxylase Activity, Selectivity, and Stability for a Scalable Concise Synthesis of a Key Intermediate to Belzutifan.

Biocatalytic oxidations are an emerging technology for selective C-H bond activation. While promising for a range of selective oxidations, practical use of enzymes catalyzing aerobic hydroxylation is presently limited by their substrate scope and stability under industrially relevant conditions. Here, we report the engineering and practical application of a non-heme iron and α-ketoglutarate-dependent dioxygenase for the direct stereo- and regio-selective hydroxylation of a non-native fluoroindanone en route to the oncology treatment belzutifan, replacing a five-step chemical synthesis with a direct enantioselective hydroxylation. Mechanistic studies indicated that formation of the desired product was limited by enzyme stability and product overoxidation, with these properties subsequently improved by directed evolution, yielding a biocatalyst capable of >15,000 total turnovers. Highlighting the industrial utility of this biocatalyst, the high-yielding, green, and efficient oxidation was demonstrated at kilogram scale for the synthesis of belzutifan.

Diverse Catalytic Reactions for the Stereoselective Synthesis of Cyclic Dinucleotide MK-1454.

As practitioners of organic chemistry strive to deliver efficient syntheses of the most complex natural products and drug candidates, further innovations in synthetic strategies are required to facilitate their efficient construction. These aspirational breakthroughs often go hand-in-hand with considerable reductions in cost and environmental impact. Enzyme-catalyzed reactions have become an impressive and necessary tool that offers benefits such as increased selectivity and waste limitation. These benefits are amplified when enzymatic processes are conducted in a cascade in combination with novel bond-forming strategies. In this article, we report a highly diastereoselective synthesis of MK-1454, a potent agonist of the stimulator of interferon gene (STING) signaling pathway. The synthesis begins with the asymmetric construction of two fluoride-bearing deoxynucleotides. The routes were designed for maximum convergency and selectivity, relying on the same benign electrophilic fluorinating reagent. From these complex subunits, four enzymes are used to construct the two bridging thiophosphates in a highly selective, high yielding cascade process. Critical to the success of this reaction was a thorough understanding of the role transition metals play in bond formation.

Hemoprotein-Catalyzed Cyclopropanation En Route to the Chiral Cyclopropanol Fragment of Grazoprevir.

Reactions that were once the exclusive province of synthetic catalysts can increasingly be addressed using biocatalysis. Through discovery of unnatural enzyme reactions, biochemists have significantly expanded the reach of enzymatic catalysis to include carbene transfer chemistries including olefin cyclopropanation. Here we describe hemoprotein cyclopropanation catalysts derived from thermophilic bacterial globins that react with diazoacetone and an unactivated olefin substrate to furnish a cyclopropyl ketone, a previously unreported reaction for enzyme catalysts. We further demonstrate that the resulting cyclopropyl ketone can be converted to a key cyclopropanol intermediate that occurs en route to the anti-hepatitis C drug grazoprevir.

Molecular basis for the broad substrate selectivity of a peptide prenyltransferase.

The cyanobactin prenyltransferases catalyze a series of known or unprecedented reactions on millions of different substrates, with no easily observable recognition motif and exquisite regioselectivity. Here we define the basis of broad substrate tolerance for the otherwise uncharacterized TruF family. We determined the structures of the Tyr-prenylating enzyme PagF, in complex with an isoprenoid donor analog and a panel of linear and macrocyclic peptide substrates. Unexpectedly, the structures reveal a truncated barrel fold, wherein binding of large peptide substrates is necessary to complete a solvent-exposed hydrophobic pocket to form the catalytically competent active site. Kinetic, mutational, chemical, and computational analyses revealed the structural basis of selectivity, showing a small motif within peptide substrates that is sufficient for recognition by the enzyme. Attaching this 2-residue motif to two random peptides results in their isoprenylation by PagF, demonstrating utility as a general biocatalytic platform for modifications on any peptide substrate.

Metabolic model for diversity-generating biosynthesis.

A conventional metabolic pathway leads to a specific product. In stark contrast, there are diversity-generating metabolic pathways that naturally produce different chemicals, sometimes of great diversity. We demonstrate that for one such pathway, tru, each ensuing metabolic step is slower, in parallel with the increasing potential chemical divergence generated as the pathway proceeds. Intermediates are long lived and accumulate progressively, in contrast with conventional metabolic pathways, in which the first step is rate-limiting and metabolic intermediates are short-lived. Understanding these fundamental differences enables several different practical applications, such as combinatorial biosynthesis, some of which we demonstrate here. We propose that these principles may provide a unifying framework underlying diversity-generating metabolism in many different biosynthetic pathways.

General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH.

To date, efforts to switch the cofactor specificity of oxidoreductases from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) have been made on a case-by-case basis with varying degrees of success. Here we present a straightforward recipe for altering the cofactor specificity of a class of NADPH-dependent oxidoreductases, the ketol-acid reductoisomerases (KARIs). Combining previous results for an engineered NADH-dependent variant of Escherichia coli KARI with available KARI crystal structures and a comprehensive KARI-sequence alignment, we identified key cofactor specificity determinants and used this information to construct five KARIs with reversed cofactor preference. Additional directed evolution generated two enzymes having NADH-dependent catalytic efficiencies that are greater than the wild-type enzymes with NADPH. High-resolution structures of a wild-type/variant pair reveal the molecular basis of the cofactor switch.

Structural Adaptability Facilitates Histidine Heme Ligation in a Cytochrome P450.

Almost all known members of the cytochrome P450 (CYP) superfamily conserve a key cysteine residue that coordinates the heme iron. Although mutation of this residue abolishes monooxygenase activity, recent work has shown that mutation to either serine or histidine unlocks non-natural carbene- and nitrene-transfer activities. Here we present the first crystal structure of a histidine-ligated P450. The T213A/C317H variant of the thermostable CYP119 from Sulfolobus acidocaldarius maintains heme iron coordination through the introduced ligand, an interaction that is accompanied by large changes in the overall protein structure. We also find that the axial cysteine C317 may be substituted with any other amino acid without abrogating folding and heme cofactor incorporation. Several of the axial mutants display unusual spectral features, suggesting that they have active sites with unique steric and electronic properties. These novel, highly stable enzyme active sites will be fruitful starting points for investigations of non-natural P450 catalysis and mechanisms.

Enzyme-controlled nitrogen-atom transfer enables regiodivergent C-H amination.

We recently demonstrated that variants of cytochrome P450BM3 (CYP102A1) catalyze the insertion of nitrogen species into benzylic C-H bonds to form new C-N bonds. An outstanding challenge in the field of C-H amination is catalyst-controlled regioselectivity. Here, we report two engineered variants of P450BM3 that provide divergent regioselectivity for C-H amination-one favoring amination of benzylic C-H bonds and the other favoring homo-benzylic C-H bonds. The two variants provide nearly identical kinetic isotope effect values (2.8-3.0), suggesting that C-H abstraction is rate-limiting. The 2.66-Å crystal structure of the most active enzyme suggests that the engineered active site can preorganize the substrate for reactivity. We hypothesize that the enzyme controls regioselectivity through localization of a single C-H bond close to the iron nitrenoid.

Enantioselective imidation of sulfides via enzyme-catalyzed intermolecular nitrogen-atom transfer.

Engineering enzymes with novel reaction modes promises to expand the applications of biocatalysis in chemical synthesis and will enhance our understanding of how enzymes acquire new functions. The insertion of nitrogen-containing functional groups into unactivated C-H bonds is not catalyzed by known enzymes but was recently demonstrated using engineered variants of cytochrome P450BM3 (CYP102A1) from Bacillus megaterium. Here, we extend this novel P450-catalyzed reaction to include intermolecular insertion of nitrogen into thioethers to form sulfimides. An examination of the reactivity of different P450BM3 variants toward a range of substrates demonstrates that electronic properties of the substrates are important in this novel enzyme-catalyzed reaction. Moreover, amino acid substitutions have a large effect on the rate and stereoselectivity of sulfimidation, demonstrating that the protein plays a key role in determining reactivity and selectivity. These results provide a stepping stone for engineering more complex nitrogen-atom-transfer reactions in P450 enzymes and developing a more comprehensive biocatalytic repertoire.

Non-natural olefin cyclopropanation catalyzed by diverse cytochrome P450s and other hemoproteins.

Recent work has shown that engineered variants of cytochrome P450BM3 (CYP102A1) efficiently catalyze non-natural reactions, including carbene and nitrene transfer reactions. Given the broad substrate range of natural P450 enzymes, we set out to explore if this diversity could be leveraged to generate a broad panel of new catalysts for olefin cyclopropanation (i.e., carbene transfer). Here, we took a step towards this goal by characterizing the carbene transfer activities of four new wild-type P450s that have different native substrates. All four were active and exhibited a range of product selectivities in the model reaction: cyclopropanation of styrene by using ethyl diazoacetate (EDA). Previous work on P450BM3 demonstrated that mutation of the axial coordinating cysteine, universally conserved among P450 enzymes, to a serine residue, increased activity for this non-natural reaction. The equivalent mutation in the selected P450s was found to activate carbene transfer chemistry both in vitro and in vivo. Furthermore, serum albumins complexed with hemin were also found to be efficient in vitro cyclopropanation catalysts.

Structural, functional, and spectroscopic characterization of the substrate scope of the novel nitrating cytochrome P450 TxtE.

A novel cytochrome P450 enzyme, TxtE, was recently shown to catalyze the direct aromatic nitration of L-tryptophan. This unique chemistry inspired us to ask whether TxtE could serve as a platform for engineering new nitration biocatalysts to replace current harsh synthetic methods. As a first step toward this goal, and to better understand the wild-type enzyme, we obtained high-resolution structures of TxtE in its substrate-free and substrate-bound forms. We also screened a library of substrate analogues for spectroscopic indicators of binding and for production of nitrated products. From these results, we found that the wild-type enzyme accepts moderate decoration of the indole ring, but the amino acid moiety is crucial for binding and correct positioning of the substrate and therefore less amenable to modification. A nitrogen atom is essential for catalysis, and a carbonyl must be present to recruit the αB'1 helix of the protein to seal the binding pocket.

Origin and variation of tunicate secondary metabolites.

Ascidians (tunicates) are rich sources of structurally elegant, pharmaceutically potent secondary metabolites and, more recently, potential biofuels. It has been demonstrated that some of these compounds are made by symbiotic bacteria and not by the animals themselves, and for a few other compounds evidence exists supporting a symbiotic origin. In didemnid ascidians, compounds are highly variable even in apparently identical animals. Recently, we have explained this variation at the genomic and metagenomic levels and have applied the basic scientific findings to drug discovery and development. This review discusses what is currently known about the origin and variation of symbiotically derived metabolites in ascidians, focusing on the family Didemnidae, where most research has occurred. Applications of our basic studies are also described.

Quantitation and speciation of residual protein within active pharmaceutical ingredients using image analysis with SDS-PAGE.

Recent advances in biocatalysis and directed enzyme evolution has led to a variety of enzymatically-driven, elegant processes for active pharmaceutical ingredient (API) production. For biocatalytic processes, quantitation of any residual protein within a given API is of great importance to ensure process robustness and quality, pure pharmaceutical products. Typical analytical methods for analyzing residual enzymes within an API, such as enzyme-linked immunosorbent assays (ELISA), colorimetric assays, and liquid chromatographic techniques, are limited for determining only the concentration of known proteins and require harsh solvents with high API levels for analysis. For the first time, total residual protein content in a small molecule API was quantitated using image analysis applied to SDS-PAGE. Herein, a proposed methodology for residual protein detection, quantitation, and size-based speciation is presented, in which an orthogonal technique is offered to traditional analysis methods, such as ELISA. Results indicate that our application of the analytical methodology is able to reliably quantitate both protein standards and the total residual protein present within a final API, with good agreement as compared to traditional ELISA results. Further, speciation of the residual protein within the API provides key information concerning the individual residual proteins present, including their molecular weight, which can lead to improved process development efforts for residual protein rejection and control. This analytical methodology thus offers an alternative tool for easily identifying, quantitating, and speciating residual protein content in the presence of small molecule APIs, with potential for wide applicability across industry for biocatalytic or directed enzyme evolution efforts within process development.

Enzymatic basis of ribosomal peptide prenylation in cyanobacteria.

The enzymatic basis of ribosomal peptide natural product prenylation has not been reported. Here, we characterize a prenyltransferase, LynF, from the TruF enzyme family. LynF is the first characterized representative of the TruF protein family, which is responsible for both reverse- and forward-O-prenylation of tyrosine, serine, and threonine in cyclic peptides known as cyanobactins. We show that LynF reverse O-prenylates tyrosine in macrocyclic peptides. Based upon these results, we propose that the TruF family prenylates mature cyclic peptides, from which the leader sequence and other enzyme recognition elements have been excised. This differs from the common model of ribosomal peptide biosynthesis, in which a leader sequence is required to direct post-translational modifications. In addition, we find that reverse O-prenylated tyrosine derivatives undergo a facile Claisen rearrangement at 'physiological' temperature in aqueous buffers, leading to forward C-prenylated products. Although the Claisen rearrangement route to natural products has been chemically anticipated for at least 40 years, it has not been demonstrated as a route to prenylated natural products. Here, we show that the Claisen rearrangement drives phenolic C-prenylation in at least one case, suggesting that this route should be reconsidered as a mechanism for the biosynthesis of prenylated phenolic compounds.

Engineered Ribosyl-1-Kinase Enables Concise Synthesis of Molnupiravir, an Antiviral for COVID-19.

Molnupiravir (MK-4482) is an investigational antiviral agent that is under development for the treatment of COVID-19. Given the potential high demand and urgency for this compound, it was critical to develop a short and sustainable synthesis from simple raw materials that would minimize the time needed to manufacture and supply molnupiravir. The route reported here is enabled through the invention of a novel biocatalytic cascade featuring an engineered ribosyl-1-kinase and uridine phosphorylase. These engineered enzymes were deployed with a pyruvate-oxidase-enabled phosphate recycling strategy. Compared to the initial route, this synthesis of molnupiravir is 70% shorter and approximately 7-fold higher yielding. Looking forward, the biocatalytic approach to molnupiravir outlined here is anticipated to have broad applications for streamlining the synthesis of nucleosides in general.

Insights into heterocyclization from two highly similar enzymes.

The cyanobactin biosynthetic pathways pat and tru, isolated from metagenomes of marine animals, lead to diverse natural products containing heterocycles derived from Cys, Ser, and Thr. Previous work has shown that PatD and TruD are extremely broad-substrate heterocyclase enzymes. These enzymes are virtually identical in their N-terminal putative catalytic domains, but only approximately 77% identical in their C-terminal putative substrate-binding domains. Here, we show that these differences allow the enzymes to control regioselectivity of posttranslational modifications, helping to control product chemistry in this hypervariable family of marine natural products.

Circular logic: nonribosomal peptide-like macrocyclization with a ribosomal peptide catalyst.

A protease from ribosomal peptide biosynthesis macrocyclizes diverse substrates, including those resembling nonribosomal peptide and hybrid polyketide-peptide products. The proposed mechanism is analogous to thioesterase-catalyzed chemistry, but the substrates are amide bonds rather than thioesters.

Marine molecular machines: heterocyclization in cyanobactin biosynthesis.

Natural products that contain amino-acid-derived (Cys, Ser, Thr) heterocycles are ubiquitous in nature, yet key aspects of their biosynthesis remain undefined. Cyanobactins are heterocyclic ribosomal peptide natural products from cyanobacteria, including symbiotic bacteria living with marine ascidians. In contrast to other ribosomal peptide heterocyclases that have been studied, the cyanobactin heterocyclase is a single protein that does not require an oxidase enzyme. Using this simplifying condition, we provide new evidence to support the hypothesis that these enzymes are molecular machines that use ATP in a product binding or orientation cycle. Further, we show that both protease inhibitors and ATP analogues inhibit heterocyclization and define the order of biochemical steps in the cyanobactin biosynthetic pathway. The cyanobactin pathway enzymes, PatD and TruD, are thiazoline and oxazoline synthetases.