Stereodivergent Synthesis of Pyridyl Cyclopropanes via Enzymatic Activation of Pyridotriazoles.

Hemoproteins have recently emerged as powerful biocatalysts for new-to-nature carbene transfer reactions. Despite this progress, these strategies have remained largely limited to diazo-based carbene precursor reagents. Here, we report the development of a biocatalytic strategy for the stereoselective construction of pyridine-functionalized cyclopropanes via the hemoprotein-mediated activation of pyridotriazoles (PyTz) as stable and readily accessible carbene sources. This method enables the asymmetric cyclopropanation of a variety of olefins, including electron-rich and electrodeficient ones, with high activity, high stereoselectivity, and enantiodivergent selectivity, providing access to mono- and diarylcyclopropanes that incorporate a pyridine moiety and thus two structural motifs of high value in medicinal chemistry. Mechanistic studies reveal a multifaceted role of 7-halogen substitution in the pyridotriazole reagent toward favoring multiple catalytic steps in the transformation. This work provides the first example of asymmetric olefin cyclopropanation with pyridotriazoles, paving the way to the exploitation of these attractive and versatile reagents for enzyme-catalyzed carbene-mediated reactions.

Chemoenzymatic synthesis of optically active α-cyclopropyl-pyruvates and cyclobutenoates via enzyme-catalyzed carbene transfer with diazopyruvate.

Cyclopropanes are recurrent structural motifs in natural products and bioactive molecules. Recently, biocatalytic cyclopropanations have emerged as a powerful approach to access enantioenriched cyclopropanes, complementing chemocatalytic approaches developed over the last several decades. Here, we report the development of a first biocatalytic strategy for cyclopropanation using ethyl α-diazopyruvate as a novel enzyme-compatible carbene precursor. Using myoglobin variant Mb(H64V,V68G) as the biocatalyst, this method afforded the efficient synthesis of α-cyclopropylpyruvates in high diastereomeric ratios and enantiomeric excess (up to 99% ee). The ketoester moiety in the cyclopropane products can be used to synthesize diverse optically pure cyclopropane derivatives. Furthermore, the enzymatically obtained α-cyclopropylpyruvate products could be converted into enantiopure cyclobutenoates via a metal-free photochemical ring expansion without loss of optical activity.

Biocatalytic Strategy for the Highly Stereoselective Synthesis of Fluorinated Cyclopropanes.

Fluorinated cyclopropanes are highly desired pharmacophores in drug discovery owing to the rigid nature of the cyclopropane ring and the beneficial effects of C-F bonds on the pharmacokinetic properties, cell permeability, and metabolic stability of drug molecules. Herein a biocatalytic strategy for the stereoselective synthesis of mono-fluorinated and gem-difluoro cyclopropanes is reported though the use of engineered myoglobin-based catalysts. In particular, this system allows for a broad range of gem-difluoro alkenes to be cyclopropanated in the presence of diazoacetonitrile with excellent diastereo and enantiocontrol (up to 99 : 1 d.r. and 99 % e.e.), thereby enabling a transformation not currently accessible with chemocatalytic methods. The synthetic utility of the present approach is further exemplified through the gram-scale synthesis of a key gem-difluorinated cyclopropane intermediate useful for the preparation of fluorinated bioactive molecules.

Stereoselective Construction of ß-, γ-, and δ-Lactam Rings via Enzymatic C-H Amidation.

Lactam rings are found in many biologically active natural products and pharmaceuticals, including important classes of antibiotics. Methods for the asymmetric synthesis of these molecules are therefore highly desirable, particularly through the selective functionalization of unreactive aliphatic C-H bonds. Here we show the development of a strategy for the asymmetric synthesis of ß-, γ-, and δ-lactams via hemoprotein-catalysed intramolecular C-H amidation reaction with readily available dioxazolone reagents. Engineered myoglobin variants serve as excellent biocatalysts for this transformation yielding the desired lactam products in high yields, high enantioselectivity, and on preparative scale. Mechanistic and computational studies elucidate the nature of the C-H amination and enantiodetermining steps and provide insights into protein-mediated control of regioselectivity and stereoselectivity. Additionally, an alkaloid natural product and a drug molecule were synthesized chemoenzymatically in much fewer steps (7-8 vs. 11-12) than previously reported, further demonstrating the power of biosynthetic strategy for the preparation of complex bioactive molecules.

Biocatalytic strategy for the construction of sp3-rich polycyclic compounds from directed evolution and computational modelling.

Catalysis with engineered enzymes has provided more efficient routes for the production of active pharmaceutical agents. However, the potential of biocatalysis to assist in early-stage drug discovery campaigns remains largely untapped. In this study, we have developed a biocatalytic strategy for the construction of sp3-rich polycyclic compounds via the intramolecular cyclopropanation of benzothiophenes and related heterocycles. Two carbene transferases with complementary regioisomer selectivity were evolved to catalyse the stereoselective cyclization of benzothiophene substrates bearing diazo ester groups at the C2 or C3 position of the heterocycle. The detailed mechanisms of these reactions were elucidated by a combination of crystallographic and computational analyses. Leveraging these insights, the substrate scope of one of the biocatalysts could be expanded to include previously unreactive substrates, highlighting the value of integrating evolutionary and rational strategies to develop enzymes for new-to-nature transformations. The molecular scaffolds accessed here feature a combination of three-dimensional and stereochemical complexity with 'rule-of-three' properties, which should make them highly valuable for fragment-based drug discovery campaigns.

Mechanistic manifold in a hemoprotein-catalyzed cyclopropanation reaction with diazoketone.

Hemoproteins have recently emerged as promising biocatalysts for new-to-nature carbene transfer reactions. However, mechanistic understanding of the interplay between productive and unproductive pathways in these processes is limited. Using spectroscopic, structural, and computational methods, we investigate the mechanism of a myoglobin-catalyzed cyclopropanation reaction with diazoketones. These studies shed light on the nature and kinetics of key catalytic steps in this reaction, including the formation of an early heme-bound diazo complex intermediate, the rate-determining nature of carbene formation, and the cyclopropanation mechanism. Our analyses further reveal the existence of a complex mechanistic manifold for this reaction that includes a competing pathway resulting in the formation of an N-bound carbene adduct of the heme cofactor, which was isolated and characterized by X-ray crystallography, UV-Vis, and Mössbauer spectroscopy. This species can regenerate the active biocatalyst, constituting a non-productive, yet non-destructive detour from the main catalytic cycle. These findings offer a valuable framework for both mechanistic analysis and design of hemoprotein-catalyzed carbene transfer reactions.

Cellular Validation of a Chemically Improved Inhibitor Identifies Monoubiquitination on OTUB2.

Ubiquitin thioesterase OTUB2, a cysteine protease from the ovarian tumor (OTU) deubiquitinase superfamily, is often overexpressed during tumor progression and metastasis. Development of OTUB2 inhibitors is therefore believed to be therapeutically important, yet potent and selective small-molecule inhibitors targeting OTUB2 are scarce. Here, we describe the development of an improved OTUB2 inhibitor, LN5P45, comprising a chloroacethydrazide moiety that covalently reacts to the active-site cysteine residue. LN5P45 shows outstanding target engagement and proteome-wide selectivity in living cells. Importantly, LN5P45 as well as other OTUB2 inhibitors strongly induce monoubiquitination of OTUB2 on lysine 31. We present a route to future OTUB2-related therapeutics and have shown that the OTUB2 inhibitor developed in this study can help to uncover new aspects of the related biology and open new questions regarding the understanding of OTUB2 regulation at the post-translational modification level.

Stereoselective Construction of ß-, γ-, and δ-Lactam Rings via Enzymatic C-H Amidation.

Lactam rings are found in many biologically active natural products and pharmaceuticals, including important classes of antibiotics. Given their widespread presence in bioactive molecules, methods for the asymmetric synthesis of these molecules, in particular through the selective functionalization of ubiquitous yet unreactive aliphatic C-H bonds, are highly desirable. In this study, we report the development of a novel strategy for the asymmetric synthesis of 4-, 5-, and 6-membered lactams via an unprecedented hemoprotein-catalyzed intramolecular C-H amidation reaction with readily available dioxazolone reagents. Engineered myoglobin variants serve as excellent biocatalysts for this transformation producing an array of ß-, γ-, and δ-lactam molecules in high yields, with high enantioselectivity, and on preparative scale. Mechanistic and computational studies elucidate the nature of the C-H amination and enantiodetermining steps in these reactions and provide insights into protein-mediated control of regioselectivity and stereoselectivity. Using this system, it was possible to accomplish the chemoenzymatic total synthesis of an alkaloid natural product and a drug molecule in much fewer steps (7-8 vs. 11-12) than previously possible, which showcases the power of this biosynthetic strategy toward enabling the preparation of complex bioactive molecules.

Dehaloperoxidase Catalyzed Stereoselective Synthesis of Cyclopropanol Esters.

Chiral cyclopropanols are highly desirable building blocks for medicinal chemistry, but the stereoselective synthesis of these molecules remains challenging. Here, a novel strategy is reported for the diastereo- and enantioselective synthesis of cyclopropanol derivatives via the biocatalytic asymmetric cyclopropanation of vinyl esters with ethyl diazoacetate (EDA). A dehaloperoxidase enzyme from Amphitrite ornata was repurposed to catalyze this challenging cyclopropanation reaction, and its activity and stereoselectivity were optimized via protein engineering. Using this system, a broad range of electron-deficient vinyl esters were efficiently converted to the desired cyclopropanation products with up to 99.5:0.5 diastereomeric and enantiomeric ratios. In addition, the engineered dehaloperoxidase-based biocatalyst is able to catalyze a variety of other abiological carbene transfer reactions, including N-H/S-H carbene insertion with EDA as well as cyclopropanation with diazoacetonitrile, thus adding to the multifunctionality of this enzyme and defining it as a valuable new scaffold for the development of novel carbene transferases.

Enantioselective Single and Dual α-C-H Bond Functionalization of Cyclic Amines via Enzymatic Carbene Transfer.

Cyclic amines are ubiquitous structural motifs found in pharmaceuticals and biologically active natural products, making methods for their elaboration via direct C-H functionalization of considerable synthetic value. Herein, we report the development of an iron-based biocatalytic strategy for enantioselective α-C-H functionalization of pyrrolidines and other saturated N-heterocycles via a carbene transfer reaction with diazoacetone. Currently unreported for organometallic catalysts, this transformation can be accomplished in high yields, high catalytic activity, and high stereoselectivity (up to 99:1 e.r. and 20,350 TON) using engineered variants of cytochrome P450 CYP119 from Sulfolobus solfataricus. This methodology was further extended to enable enantioselective α-C-H functionalization in the presence of ethyl diazoacetate as carbene donor (up to 96:4 e.r. and 18,270 TON), and the two strategies were combined to achieve a one-pot as well as a tandem dual C-H functionalization of a cyclic amine substrate with enzyme-controlled diastereo- and enantiodivergent selectivity. This biocatalytic approach is amenable to gram-scale synthesis and can be applied to drug scaffolds for late-stage C-H functionalization. This work provides an efficient and tunable method for direct asymmetric α-C-H functionalization of saturated N-heterocycles, which should offer new opportunities for the synthesis, discovery, and optimization of bioactive molecules.

Highly stereoselective and enantiodivergent synthesis of cyclopropylphosphonates with engineered carbene transferases.

Organophosphonate compounds have represented a rich source of biologically active compounds, including enzyme inhibitors, antibiotics, and antimalarial agents. Here, we report the development of a highly stereoselective strategy for olefin cyclopropanation in the presence of a phosphonyl diazo reagent as carbene precursor. In combination with a 'substrate walking' protein engineering strategy, two sets of efficient and enantiodivergent myoglobin-based biocatalysts were developed for the synthesis of both (1R,2S) and (1S,2R) enantiomeric forms of the desired cyclopropylphosphonate ester products. This methodology enables the efficient transformation of a broad range of vinylarene substrates at a preparative scale (i.e. gram scale) with up to 99% de and ee. Mechanistic studies provide insights into factors that contribute to make this reaction inherently more challenging than hemoprotein-catalyzed olefin cyclopropanation with ethyl diazoacetate investigated previously. This work expands the range of synthetically useful, enzyme-catalyzed transformations and paves the way to the development of metalloprotein catalysts for abiological carbene transfer reactions involving non-canonical carbene donor reagents.

Tuning Enzyme Thermostability via Computationally Guided Covalent Stapling and Structural Basis of Enhanced Stabilization.

Enhancing the thermostability of enzymes without impacting their catalytic function represents an important yet challenging goal in protein engineering and biocatalysis. We recently introduced a novel method for enzyme thermostabilization that relies on the computationally guided installation of genetically encoded thioether "staples" into a protein via cysteine alkylation with the noncanonical amino acid O-2-bromoethyl tyrosine (O2beY). Here, we demonstrate the functionality of an expanded set of electrophilic amino acids featuring chloroacetamido, acrylamido, and vinylsulfonamido side-chain groups for protein stapling using this strategy. Using a myoglobin-based cyclopropanase as a model enzyme, our studies show that covalent stapling with p-chloroacetamido-phenylalanine (pCaaF) provides higher stapling efficiency and enhanced stability (thermodynamic and kinetic) compared to the other stapled variants and the parent protein. Interestingly, molecular simulations of conformational flexibility of the cross-links show that the pCaaF staple allows fewer energetically feasible conformers than the other staples, and this property may be a broader indicator of stability enhancement. Using this strategy, pCaaF-stapled variants with significantly enhanced stability against thermal denaturation (ΔTm' = +27 °C) and temperature-induced heme loss (ΔT50 = +30 °C) were obtained while maintaining high levels of catalytic activity and stereoselectivity. Crystallographic analyses of singly and doubly stapled variants provide key insights into the structural basis for stabilization, which includes both direct interactions of the staples with protein residues and indirect interactions through adjacent residues involved in heme binding. This work expands the toolbox of protein stapling strategies available for protein stabilization.

Enantioselective Synthesis of α-Trifluoromethyl Amines via Biocatalytic N-H Bond Insertion with Acceptor-Acceptor Carbene Donors.

The biocatalytic toolbox has recently been expanded to include enzyme-catalyzed carbene transfer reactions not occurring in Nature. Herein, we report the development of a biocatalytic strategy for the synthesis of enantioenriched α-trifluoromethyl amines through an asymmetric N-H carbene insertion reaction catalyzed by engineered variants of cytochrome c552 from Hydrogenobacter thermophilus. Using a combination of protein and substrate engineering, this metalloprotein scaffold was redesigned to enable the synthesis of chiral α-trifluoromethyl amino esters with up to >99% yield and 95:5 er using benzyl 2-diazotrifluoropropanoate as the carbene donor. When the diazo reagent was varied, the enantioselectivity of the enzyme could be inverted to produce the opposite enantiomers of these products with up to 99.5:0.5 er. This methodology is applicable to a broad range of aryl amine substrates, and it can be leveraged to obtain chemoenzymatic access to enantioenriched ß-trifluoromethyl-ß-amino alcohols and halides. Computational analyses provide insights into the interplay of protein- and reagent-mediated control on the enantioselectivity of this reaction. This work introduces the first example of a biocatalytic N-H carbenoid insertion with an acceptor-acceptor carbene donor, and it offers a biocatalytic solution for the enantioselective synthesis of α-trifluoromethylated amines as valuable synthons for medicinal chemistry and the synthesis of bioactive molecules.

Nanoparticle-Mediated Delivery of Micheliolide Analogs to Eliminate Leukemic Stem Cells in the Bone Marrow.

Micheliolide (MCL) is a naturally occurring sesquiterpene lactone that selectively targets leukemic stem cells (LSCs), which persist after conventional chemotherapy for myeloid leukemias, leading to disease relapse. To overcome modest MCL cytotoxicity, analogs with ≈two-threefold greater cytotoxicity against LSCs are synthesized via late-stage chemoenzymatic C-H functionalization. To enhance bone marrow delivery, MCL analogs are entrapped within bone-targeted polymeric nanoparticles (NPs). Robust drug loading capacities of up to 20% (mg drug mg-1 NP) are obtained, with release dominated by analog hydrophobicity. NPs loaded with a hydrolytically stable analog are tested in a leukemic mouse model. Median survival improved by 13% and bone marrow LSCs are decreased 34-fold following NPMCL treatments versus controls. Additionally, selective leukemic cell and LSC cytotoxicity of the treatment versus normal hematopoietic cells is observed. Overall, these studies demonstrate that MCL-based antileukemic agents combined with bone-targeted NPs offer a promising strategy for eradicating LSCs.

Engineered Myoglobin Catalysts for Asymmetric Intermolecular Cyclopropanation Reactions.

Biocatalysis has covered an increasingly important role in the synthesis and manufacturing of pharmaceuticals and other high value compounds. In the interest of expanding the range of synthetically useful reactions accessible via biocatalysts, our group has explored the potential and application of engineered myoglobins for 'abiological' carbene transfer catalysis. These transformations provide a direct route for the construction of new carbon-carbon and carbon-heteroatom bonds, including the synthesis of cyclopropane rings, which are key motifs and pharmacophores in many drugs and bioactive natural products. In this award article, we survey the progress made by our group toward the development of myoglobin-based catalysts for asymmetric intermolecular cyclopropanation reactions. The high stereoselectivity exhibited by these biocatalysts in these reactions, combined with their broad substrate scope, scalability, and robustness to high substrate loading and organic co-solvents, contribute to make these systems particularly useful for chemical synthesis and biocatalysis at the preparative scale. Extension of the scope of biocatalytic carbene transfer reactions to include different classes of carbene donor reagents has created new opportunities for the asymmetric synthesis of functionalized cyclopropanes. Furthermore, the integration of myoglobin-catalyzed stereoselective cyclopropanations with chemical diversification of the enzymatic products has furnished attractive chemoenzymatic strategies to access a diverse range of optically active cyclopropane scaffolds of high value for drug discovery, medicinal chemistry, and the synthesis of natural products.

MOrPH-PhD: A Phage Display System for the Functional Selection of Genetically Encoded Macrocyclic Peptides.

Macrocyclic peptides represent promising scaffolds for targeting biomolecules with high affinity and selectivity, making methods for the diversification and functional selection of these macrocycles highly valuable for drug discovery purposes. We recently reported a novel phage display platform (called MOrPH-PhD) for the creation and functional exploration of combinatorial libraries of genetically encoded cyclic peptides. In this system, spontaneous, posttranslational peptide cyclization by means of a cysteine-reactive non-canonical amino acid is integrated with M13 bacteriophage display, enabling the creation of genetically encoded macrocyclic peptide libraries displayed on phage particles. Using this system, it is possible to rapidly generate and screen large libraries of phage-displayed macrocyclic peptides (up to 108 to 1010 members) in order to identify high-affinity binders of a target protein of interest. Herein, we describe step-by-step protocols for the production of MOrPH-PhD libraries, the screening of these libraries against an immobilized protein target, and the isolation and characterization of functional macrocyclic peptides from these genetically encoded libraries.

Cyclic peptides with a distinct arginine-fork motif recognize the HIV trans-activation response RNA in vitro and in cells.

RNA represents a potential target for new antiviral therapies, which are urgently needed to address public health threats such as the human immunodeficiency virus (HIV). We showed previously that the interaction between the viral Tat protein and the HIV-1 trans-activation response (TAR) RNA was blocked by TB-CP-6.9a. This cyclic peptide was derived from a TAR-binding loop that emerged during lab evolution of a TAR-binding protein (TBP) family. Here we synthesized and characterized a next-generation, cyclic-peptide library based on the TBP scaffold. We sought to identify conserved RNA-binding interactions and the influence of cyclization linkers on RNA binding and antiviral activity. A diverse group of cyclization linkers, encompassing disulfide bonds to bicyclic aromatic staples, was used to restrain the cyclic peptide geometry. Thermodynamic profiling revealed specific arginine-rich sequences with low to submicromolar affinity driven by enthalpic and entropic contributions. The best compounds exhibited no appreciable off-target binding to related molecules, such as BIV TAR and human 7SK RNAs. A specific arginine-to-lysine change in the highest affinity cyclic peptide reduced TAR binding by tenfold, suggesting that TBP-derived cyclic peptides use an arginine-fork motif to recognize the TAR major groove while differentiating the mode of binding from other TAR-targeting molecules. Finally, we showed that HIV infectivity in cell culture was reduced in the presence of cyclic peptides constrained by methylene or naphthalene-based linkers. Our findings provide insight into the molecular determinants required for HIV-1 TAR recognition and antiviral activity. These findings are broadly relevant to the development of antivirals that target RNA molecules.

Engineered and Artificial Metalloenzymes for Selective C-H Functionalization.

The direct functionalization of C-H bonds constitutes a powerful strategy to construct and diversify organic molecules. However, controlling the chemo- and site-selectivity of this transformation in particularly complex molecular settings represents a significant challenge. Metalloenzymes are ideal platforms for achieving catalyst-controlled selective C-H bond functionalization as their reactivities can be tuned by protein engineering and/or redesign of their cofactor environment. In this review, we highlight recent progress in the development of engineered and artificial metalloenzymes for C-H functionalization, with a focus on biocatalytic strategies for selective C-H oxyfunctionalization and halogenation as well as C-H amination and C-H carbene insertion via abiological nitrene and carbene transfer chemistries. Engineered heme- and non-heme iron dependent enzymes have emerged as promising scaffolds for executing these transformations with high chemo-, regio- and stereocontrol as well as tunable selectivity. These emerging systems and methodologies have expanded the toolbox of sustainable strategies for organic synthesis and created new opportunities for the generation of chiral building blocks, the late-stage C-H functionalization of complex molecules, and the total synthesis of natural products.

Comprehensive Structure-Activity Profiling of Micheliolide and its Targeted Proteome in Leukemia Cells via Probe-Guided Late-Stage C-H Functionalization.

The plant-derived sesquiterpene lactone micheliolide was recently found to possess promising antileukemic activity, including the ability to target and kill leukemia stem cells. Efforts toward improving the biological activity of micheliolide and investigating its mechanism of action have been hindered by the paucity of preexisting functional groups amenable for late-stage derivatization of this molecule. Here, we report the implementation of a probe-based P450 fingerprinting strategy to rapidly evolve engineered P450 catalysts useful for the regio- and stereoselective hydroxylation of micheliolide at two previously inaccessible aliphatic positions in this complex natural product. Via P450-mediated chemoenzymatic synthesis, a broad panel of novel micheliolide analogs could thus be obtained to gain structure-activity insights into the effect of C2, C4, and C14 substitutions on the antileukemic activity of micheliolide, ultimately leading to the discovery of "micheliologs" with improved potency against acute myelogenic leukemia cells. These late-stage C-H functionalization routes could be further leveraged to generate a panel of affinity probes for conducting a comprehensive analysis of the protein targeting profile of micheliolide in leukemia cells via chemical proteomics analyses. These studies introduce new micheliolide-based antileukemic agents and shed new light onto the biomolecular targets and mechanism of action of micheliolide in leukemia cells. More broadly, this work showcases the value of the present P450-mediated C-H functionalization strategy for streamlining the late-stage diversification and elucidation of the biomolecular targets of a complex bioactive molecule.