Modular chemoenzymatic synthesis of ten fusicoccane diterpenoids.

Fusicoccane diterpenoids display intriguing biological activities, including the ability to act as modulators of 14-3-3 protein-protein interactions. However, their innate structural complexity and diverse oxygenation patterns present enormous synthetic challenges. Here we develop a modular chemoenzymatic approach that combines de novo skeletal construction and late-stage hybrid C-H oxidations to achieve the synthesis of ten complex fusicoccanes in 8-13 steps each. A convergent fragment coupling strategy allowed rapid access to a key tricyclic intermediate, which was subjected to chemical and enzymatic C-H oxidations to modularly prepare five oxidized family members. We also conceived a complementary biomimetic skeletal remodelling strategy to synthetically access five rearranged fusicoccanes with unusual bridgehead double bonds. This work may facilitate future investigation into the biological activities of the fusicoccanes and also inspire the implementation of similar hybrid strategies to provide family-level synthetic solutions to other natural product scaffolds.

Chemoenzymatic Synthesis of 4,5-Dihydroxyisoleucine Fragment of α-Amanitin.

The ability of α-amanitin to potently inhibit RNA polymerase II (RNAP II) has elicited further research into its use as a novel payload for antibody-drug conjugates. Despite this promise, the de novo synthesis of α-amanitin is still a major challenge as it possesses an unusual bicyclic octapeptide structure that contains several oxidized amino acids, most notably 4,5-dihydroxy-l-isoleucine. Here, we report a concise chemoenzymatic synthesis of this key amino acid residue, which features two regioselective and diastereoselective enzymatic C-H oxidations on l-isoleucine.

One-pot chemoenzymatic syntheses of non-canonical amino acids.

Despite their prevalent use in drug discovery and protein biochemistry, non-canonical amino acids are still challenging to synthesize through purely chemical means. In recent years, biocatalysis has emerged as a transformative paradigm for small-molecule synthesis. One strategy to further empower biocatalysis is to use it in combination with modern chemical reactions and take advantage of the strengths of each method to enable access to challenging structural motifs that were previously unattainable using each method alone. In this Mini-Review, we highlight several recent case studies that feature the synergistic use of chemical and enzymatic transformations in one pot to synthesize novel non-canonical amino acids. ONE-SENTENCE SUMMARY: This Mini-Review highlights several recent case studies that feature the synergistic use of chemical and enzymatic transformations in one pot to synthesize novel non-canonical amino acids.

Optical Control of Proteasomal Protein Degradation with a Photoswitchable Lipopeptide.

Photolipids have emerged as attractive tools for the optical control of lipid functions. They often contain an azobenzene photoswitch that imparts a cis double-bond upon irradiation. Herein, we present the application of photoswitching to a lipidated natural product, the potent proteasome inhibitor cepafungin I. Several azobenzene-containing lipids were attached to the cyclopeptide core, yielding photoswitchable derivatives. Most notably, PhotoCep4 exhibited a 10-fold higher cellular potency in its light-induced cis-form, matching the potency of natural cepafungin I. The length of the photolipid tail and distal positioning of the azobenzene photoswitch with respect to the macrocycle is critical for this activity. In a proteome-wide experiment, light-triggered PhotoCep4 modulation showed high overlap with constitutively active cepafungin I. The mode of action was studied using crystallography and revealed an identical binding of the cyclopeptide in comparison to cepafungin I, suggesting that differences in their cellular activity originate from switching the tail structure. The photopharmacological approach described herein could be applicable to many other natural products as lipid conjugation is common and often necessary for potent activity. Such lipids are often introduced late in synthetic routes, enabling facile chemical modifications.

Self-sufficient P450-reductase chimeras for biocatalysis.

In recent years, cytochromes P450 have emerged as powerful, versatile biocatalysts for the site-selective functionalization of small molecules. Catalyzing an impressive range of chemical transformations, these enzymes have been widely used to effect C-H oxidation, biaryl coupling, and carbon-heteroatom bond formation, among many other reactions. However, the majority of P450s are multi-protein systems that employ secondary redox partners in key steps of the catalytic cycle, which limits their broader applicability. In response, the discovery of self-sufficient P450s, such as P450BM3 and P450RhF, has provided a template for the construction of artificial, self-sufficient P450-reductase fusions. In this chapter, we describe a procedure for the design, assembly, and application of two engineered, self-sufficient P450s of Streptomyces origin via fusion with an exogenous reductase domain. In particular, we generated artificial chimeras of P450s PtmO5 and TleB by linking them covalently with the reductase domain of P450RhF. Upon verification of their activities, both enzymes were employed in preparative-scale biocatalytic reactions. This approach can feasibly be applied to any P450 of interest, thereby laying the groundwork for the production of self-sufficient P450s for diverse chemical applications.

Charting the Evolution of Chemoenzymatic Strategies in the Syntheses of Complex Natural Products.

Bolstered by recent advances in bioinformatics, genetics, and enzyme engineering, the field of chemoenzymatic synthesis has enjoyed a rapid increase in popularity and utility. This Perspective explores the integration of enzymes into multistep chemical syntheses, highlighting the unique potential of biocatalytic transformations to streamline the synthesis of complex natural products. In particular, we identify four primary conceptual approaches to chemoenzymatic synthesis and illustrate each with a number of landmark case studies. Future opportunities and challenges are also discussed.

Comprehensive Structure-Activity Relationship Studies of Cepafungin Enabled by Biocatalytic C-H Oxidations.

The cepafungins are a class of highly potent and selective eukaryotic proteasome inhibitor natural products with potential to treat refractory multiple myeloma and other cancers. The structure-activity relationship of the cepafungins is not fully understood. This Article chronicles the development of a chemoenzymatic approach to cepafungin I. A failed initial route involving derivatization of pipecolic acid prompted us to examine the biosynthetic pathway for the production of 4-hydroxylysine, which culminated in the development of a 9-step synthesis of cepafungin I. An alkyne-tagged analogue enabled chemoproteomic studies of cepafungin and comparison of its effects on global protein expression in human multiple myeloma cells to the clinical drug bortezomib. A preliminary series of analogues elucidated critical determinants of potency in proteasome inhibition. Herein we report the chemoenzymatic syntheses of 13 additional analogues of cepafungin I guided by a proteasome-bound crystal structure, 5 of which are more potent than the natural product. The lead analogue was found to have 7-fold greater proteasome ß5 subunit inhibitory activity and has been evaluated against several multiple myeloma and mantle cell lymphoma cell lines in comparison to the clinical drug bortezomib.

Engineering Catalytically Self-Sufficient P450s.

The P450 superfamily comprises some of the most powerful and versatile enzymes for the site-selective oxidation of small molecules. One of the main drawbacks for the applications of the P450s in biotechnology is that the majority of these enzymes is multicomponent in nature and requires the presence of suitable redox partners to support their functions. Nevertheless, the discovery of several self-sufficient P450s, namely those from Classes VII and VIII, has served as an inspiration for fusion approaches to generate chimeric P450 systems that are self-sufficient. In this Perspective, we highlight the domain organizations of the Class VII and Class VIII P450 systems, summarize recent case studies in the engineering of catalytically self-sufficient P450s based on these systems, and outline outstanding challenges in the field, along with several emerging technologies as potential solutions.

Finding Superior Biocatalysts via Homolog Screening.

Many enzymes possess high catalytic efficiency and selectivity that far surpass classical organic or organometallic catalysts. However, the initial starting enzyme for a given transformation does not always possess the right properties needed for broad utilization. Searching in genome/protein sequence libraries for homologs, aided with powerful bioinformatic tools developed in recent years, provides an avenue to identify superior biocatalysts. Herein, we highlight several case studies to illustrate the power of this concept. A brief discussion on its complementarity with contemporary approaches in protein engineering (such as directed evolution) and possible future developments is also provided.

Concise Chemoenzymatic Synthesis of Gedunin.

The limonoids have attracted significant attention from the synthetic community owing to their striking structural complexity and medicinal potential. Recent efforts notwithstanding, synthetic access to many intact or ring D-seco limonoids still remains elusive. Here, we report the first de novo synthesis of gedunin, a ring D-seco limonoid with HSP90 inhibitory activity, that proceeds in 13 steps. Two enabling features in our strategy are the application of modern catalytic transformations to set the key quaternary centers in the carbocyclic core and the use of biocatalytic oxidation at C3 to establish a chemical handle to access the A-ring enone motif. The strategy presented herein may provide an entry point to a wider range of oxidized limonoids.

Remote B-Ring Oxidation of Sclareol with an Engineered P450 Facilitates Divergent Access to Complex Terpenoids.

Though chiral pool synthesis is widely accepted as a powerful strategy in complex molecule synthesis, the effectiveness of the approach is intimately linked to the range of available chiral building blocks and the functional groups they possess. To date, there is still a pressing need for new remote functionalization methods that would allow the installation of useful chemical handles on these building blocks to enable a broader spectrum of synthetic manipulations. Herein, we report the engineering of a P450BM3 variant for the regioselective C-H oxidation of sclareol at C6. The synthetic utility of the resulting product was demonstrated in a formal synthesis of ansellone B, the first total synthesis of the 2,3-seco-labdane excolide B, and a model study toward (+)-pallavicinin.

A Chiral-Pool-Based Strategy to Access trans-syn-Fused Drimane Meroterpenoids: Chemoenzymatic Total Syntheses of Polysin, N-Acetyl-polyveoline and the Chrodrimanins.

trans-syn-Fused drimane meroterpenoids are unique natural products that arise from contra-thermodynamic polycyclizations of their polyene precursors. Herein we report the first total syntheses of four trans-syn-fused drimane meroterpenoids, namely polysin, N-acetyl-polyveoline, chrodrimanin C, and verruculide A, in 7-18 steps from sclareolide. The trans-syn-fused drimane unit is accessed through an efficient acid-mediated C9 epimerization of sclareolide. Subsequent applications of enzymatic C-H oxidation and contemporary annulation methodologies install the requisite C3 hydroxyl group and enable rapid generation of structural complexity to provide concise access to these natural products.

Regiodivergent Biocatalytic Hydroxylation of L-Glutamine Facilitated by Characterization of Non-Heme Dioxygenases from Non-Ribosomal Peptide Biosyntheses.

We report the functional characterization of two iron- and α-ketoglutarate-dependent dioxygenases that are capable of hydroxylating free-standing glutamine at its C3 and C4 position respectively. In particular, the C4 hydroxylase, Q4Ox, catalyzes the reaction with approximately 4,300 total turnover numbers, facilitating synthesis of a solid-phase compatible building block and stereochemical elucidation at the C4 position of the hydroxylated product. This work will enable the development of novel synthetic strategies to prepare useful glutamine derivatives and stimulate further discoveries of new amino acid hydroxylases with distinct substrate specificities.

Exploration of Iron- and a-Ketoglutarate-Dependent Dioxygenases as Practical Biocatalysts in Natural Product Synthesis.

Catalytic C─H oxidation is a powerful transformation with enormous promise to streamline access to complex molecules. In recent years, biocatalytic C─H oxidation strategies have received tremendous attention due to their potential to address unmet regio- and stereoselectivity challenges that are often encountered with the use of small-molecule-based catalysts. This Account provides an overview of recent contributions from our laboratory in this area, specifically in the use of iron- and α-ketoglutarate-dependent dioxygenases in the chemoenzymatic synthesis of complex natural products.

Concise Chemoenzymatic Synthesis of Fasamycin A.

We report the development of a chemoenzymatic approach toward fasamycin A, a halogenated naphthacenoid that exhibits activities against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis. The synthesis was accomplished in a convergent manner: two fragments were combined together in a Sammes annulation to afford a dimethylnaphthacenone system. Finally, an enzymatic halogenation was employed to introduce the requisite chlorine substituent of the natural product at a late stage.

Practical Enzymatic Production of Carbocycles.

The Pd-catalyzed carbon-carbon bond formation pioneered by Heck in 1969 has dominated medicinal chemistry development for the ensuing fifty years. As the demand for more complex three-dimensional active pharmaceuticals continues to increase, preparative enzyme-mediated assembly, by virtue of its exquisite selectivity and sustainable nature, is poised to provide a practical and affordable alternative for accessing such compounds. In this minireview, we summarize recent state-of-the-art developments in practical enzyme-mediated assembly of carbocycles. When appropriate, background information on the enzymatic transformation is provided and challenges and/or limitations are also highlighted.

Stereoselective Synthesis of ß-Branched Aromatic α-Amino Acids by Biocatalytic Dynamic Kinetic Resolution*.

ß-Branched noncanonical amino acids are valuable molecules in modern drug development efforts. However, they are still challenging to prepare due to the need to set multiple stereocenters in a stereoselective fashion, and contemporary methods for the synthesis of such compounds often rely on the use of rare-transition-metal catalysts with designer ligands. Herein, we report a highly diastereo- and enantioselective biocatalytic transamination method to prepare a broad range of aromatic ß-branched α-amino acids. Mechanistic studies show that the transformation proceeds through dynamic kinetic resolution that is unique to the optimal enzyme. To highlight its utility and practicality, the biocatalytic reaction was applied to the synthesis of several sp3 -rich cyclic fragments and the first total synthesis of jomthonic acid A.

Synthetic utility of oxygenases in site-selective terpenoid functionalization.

Terpenoids are one of the largest classes of natural products whose members possess a wide variety of biological activities. With several exceptions, scalable production of complex terpenoids with either purely biological or chemical methods still remains a major challenge. However, recent efforts to combine the two approaches in chemoenzymatic synthesis hold tremendous promise to address this challenge. Central to this paradigm is the development of useful biocatalytic methods, such as regioselective C-H oxidation, for terpene modifications. This review highlights recent applications of biocatalytic hydroxylation for site-selective modification of terpenoids.

Reinvigorating the Chiral Pool: Chemoenzymatic Approaches to Complex Peptides and Terpenoids.

Biocatalytic transformations that leverage the selectivity and efficiency of enzymes represent powerful tools for the construction of complex natural products. Enabled by innovations in genome mining, bioinformatics, and enzyme engineering, synthetic chemists are now more than ever able to develop and employ enzymes to solve outstanding chemical problems, one of which is the reliable and facile generation of stereochemistry within natural product scaffolds. In recognition of this unmet need, our group has sought to advance novel chemoenzymatic strategies to both expand and reinvigorate the chiral pool. Broadly defined, the chiral pool comprises cheap, enantiopure feedstock chemicals that serve as popular foundations for asymmetric total synthesis. Among these building blocks, amino acids and enantiopure terpenes, whose core structures can be mapped onto several classes of structurally and pharmaceutically intriguing natural products, are of particular interest to the synthetic community.In this Account, we summarize recent efforts from our group in leveraging biocatalytic transformations to expand the chiral pool, as well as efforts toward the efficient application of these transformations in natural products total synthesis, the ultimate testing ground for any novel methodology. First, we describe several examples of enzymatic generation of noncanonical amino acids as means to simplify the synthesis of peptide natural products. By extracting amino acid hydroxylases from native biosynthetic pathways, we obtain efficient access to hydroxylated variants of proline, lysine, arginine, and their derivatives. The newly installed hydroxyl moiety then becomes a chemical handle that can facilitate additional complexity generation, thereby expanding the pool of amino acid-derived building blocks available for peptide synthesis. Next, we present our efforts in enzymatic C-H oxidations of diverse terpene scaffolds, in which traditional chemistry can be combined with strategic applications of biocatalysis to selectively and efficiently derivatize several commercial terpenoid skeletons. The synergistic logic of this approach enables a small handful of synthetic intermediates to provide access to a plethora of terpenoid natural product families. Taken together, these findings demonstrate the advantages of applying enzymes in total synthesis in conjunction with established methodologies, as well as toward the expansion of the chiral pool to enable facile incorporation of stereochemistry during synthetic campaigns.

Modular Chemoenzymatic Synthesis of GE81112 B1 and Related Analogues Enables Elucidation of Its Key Pharmacophores.

The GE81112 complex has garnered much interest due to its broad antimicrobial properties and unique ability to inhibit bacterial translation initiation. Herein we report the use of a chemoenzymatic strategy to complete the first total synthesis of GE81112 B1. By pairing iron and α-ketoglutarate dependent hydroxylases found in GE81112 biosynthesis with traditional synthetic methodology, we were able to access the natural product in 11 steps (longest linear sequence). Following this strategy, 10 GE81112 B1 analogues were synthesized, allowing for identification of its key pharmacophores. A key feature of our medicinal chemistry effort is the incorporation of additional biocatalytic hydroxylations in modular analogue synthesis to rapidly enable exploration of relevant chemical space.