Nature-Inspired Radical Pyridoxal-Mediated C-C Bond Formation.

Pyridoxal-5'-phosphate (PLP) and derivatives of this cofactor enable a plethora of reactions in both enzyme-mediated and free-in-solution transformations. With few exceptions in each category, such chemistry has predominantly involved two-electron processes. This sometimes poses a significant challenge for using PLP to build tetrasubstituted carbon centers, especially when the reaction is reversible. The ability to access radical pathways is paramount to broadening the scope of reactions catalyzed by this coenzyme. In this study, we demonstrate the ability to access a radical PLP-based intermediate and engage this radical intermediate in a number of C-C bond-forming reactions. By selection of an appropriate oxidant, single-electron oxidation of the quinonoid intermediate can be achieved, which can subsequently be applied to C-C bond-forming reactions. Through this radical reaction pathway, we synthesized a series of α-tertiary amino acids and esters to investigate the substrate scope and identify nonproductive reaction pathways. Beyond the amino acid model system, we demonstrate that other classes of amine substrates can be applied in this reaction and that a range of small molecule reagents can serve as coupling partners to the semiquinone radical. We anticipate that this versatile semiquinone radical species will be central to the development of a range of novel reactions.

Substrate-Selective Catalysis Enabled Synthesis of Azaphilone Natural Products.

Achieving substrate-selectivity is a central element of nature's approach to synthesis. By relying on the ability of a catalyst to discriminate between components in a mixture, control can be exerted over which molecules will move forward in a synthesis. This approach can be powerful when realized but can be challenging to duplicate in the laboratory. In this work, substrate-selective catalysis is leveraged to discriminate between two intermediates that exist in equilibrium, subsequently directing the final cyclization to arrive at either the linear or angular tricyclic core common to subsets of azaphilone natural products. By using a flavin-dependent monooxygenase (FDMO) in sequence with an acyl transferase (AT), the conversion of several orcinaldehyde substrates directly to the corresponding linear tricyclic azaphilones in a single reaction vessel was achieved. Further, mechanistic studies support that a substrate equilibrium together with enzyme substrate selectivity play an import role in the selectivity of the final cyclization step. Using this strategy, five azaphilone natural products were synthesized for the first time as well as a number of unnatural derivatives thereof.

Biocatalytic Stereoselective Oxidation of 2-Arylindoles.

3-Hydroxyindolenines can be used to access several structural motifs that are featured in natural products and pharmaceutical compounds, yet the chemical synthesis of 3-hydroxyindolenines is complicated by overoxidation, rearrangements, and complex product mixtures. The selectivity possible in enzymatic reactions can overcome these challenges and deliver enantioenriched products. Herein, we present the development of an asymmetric biocatalytic oxidation of 2-arylindole substrates aided by a curated library of flavin-dependent monooxygenases (FDMOs) sampled from an ancestral sequence space, a sequence similarity network, and a deep-learning-based latent space model. From this library of FDMOs, a previously uncharacterized enzyme, Champase, from the Valley fever fungus, Coccidioides immitis strain RS, was found to stereoselectively catalyze the oxidation of a variety of substituted indole substrates. The promiscuity of this enzyme is showcased by the oxidation of a wide variety of substituted 2-arylindoles to afford the respective 3-hydroxyindolenine products in moderate to excellent yields and up to 95:5 er.

Simulating Electron Transfer Reactions in Solution: Radical-Polar Crossover.

Single-electron transfer (SET) promotes a wide variety of interesting chemical transformations, but modeling of SET requires a careful treatment of electronic and solvent effects to give meaningful insight. Therefore, a combined constrained density functional theory and molecular mechanics (CDFT/MM) tool is introduced specifically for SET-initiated reactions. Mechanisms for two radical-polar crossover reactions involving the organic electron donors tetrakis(dimethylamino)ethylene (TDAE) and tetrathiafulvalene (TTF) were studied with the new tool. An unexpected tertiary radical intermediate within the TDAE system was identified, relationships between kinetics and substitution in the TTF system were explained, and the impact of the solvent environments on the TDAE and TTF reactions were examined. The results highlight the need for including solvent dynamics when quantifying SET kinetics and thermodynamics, as a free energy difference of >20 kcal/mol was observed. Overall, the new method informs mechanistic analysis of SET-initiated reactions and therefore is envisioned to be useful for studying reactions in the condensed phase.

High-Throughput Liquid Chromatographic Analysis Using a Segmented Flow Injector with a 1 s Cycle Time.

High-throughput screening (HTS) workflows are revolutionizing many fields, including drug discovery, reaction discovery and optimization, diagnostics, sensing, and enzyme engineering. Liquid chromatography (LC) is commonly deployed during HTS to reduce matrix effects, distinguish isomers, and preconcentrate prior to detection, but LC separation time often limits throughput. Although subsecond LC separations have been demonstrated, they are rarely utilized during HTS due to limitations associated with the speed of common autosamplers. In this work, these limits are overcome by utilizing droplet microfluidics for sample introduction. In the method, a train of samples segmented by air are continuously pumped into the inlet of an LC injection valve that is actuated once each sample fills the sample loop. Coupled with 2.1 mm diameter × 5 mm long columns packed with 2.7 µm superficially porous C18 particles operated at 5 mL/min, the injector enabled separation of 3 components at 1 s/sample and analysis of a 96-well plate in 1.6 min with <2% peak area relative standard deviation. Analyte-dependent carryover was minimized by including wash droplets composed of organic solvent in between sample droplets. High-throughput LC coupled with mass spectrometric detection using the segmented flow injector was applied to a screen of inhibitors of a cytochrome P450-catalyzed hydroxylation reaction. Measurements of the reaction substrate and product concentrations made using fast LC with the segmented flow injector correlated well with measurements made using a more conventional, 3 min LC method. These results demonstrate the potential for droplet microfluidics to be used for sample introduction during high-throughput LC analysis.

Enabling Broader Adoption of Biocatalysis in Organic Chemistry.

Biocatalysis is becoming an increasingly impactful method in contemporary synthetic chemistry for target molecule synthesis. The selectivity imparted by enzymes has been leveraged to complete previously intractable chemical transformations and improve synthetic routes toward complex molecules. However, the implementation of biocatalysis in mainstream organic chemistry has been gradual to this point. This is partly due to a set of historical and technological barriers that have prevented chemists from using biocatalysis as a synthetic tool with utility that parallels alternative modes of catalysis. In this Perspective, we discuss these barriers and how they have hindered the adoption of enzyme catalysts into synthetic strategies. We also summarize tools and resources that already enable organic chemists to use biocatalysts. Furthermore, we discuss ways to further lower the barriers for the adoption of biocatalysis by the broader synthetic organic chemistry community through the dissemination of resources, demystifying biocatalytic reactions, and increasing collaboration across the field.

Stereodynamic Strategies to Induce and Enrich Chirality of Atropisomers at a Late Stage.

Enantiomers, where chirality arises from restricted rotation around a single bond, are atropisomers. Due to the unique nature of the origins of their chirality, synthetic strategies to access these compounds in an enantioselective manner differ from those used to prepare enantioenriched compounds containing point chirality arising from an unsymmetrically substituted carbon center. In particular stereodynamic transformations, such as dynamic kinetic resolutions, thermodynamic dynamic resolutions, and deracemizations, which rely on the ability to racemize or interconvert enantiomers, are a promising set of transformations to prepare optically pure compounds in the late stage of a synthetic sequence. Translation of these synthetic approaches from compounds with point chirality to atropisomers requires an expanded toolbox for epimerization/racemization and provides an opportunity to develop a new conceptual framework for the enantioselective synthesis of these compounds.

Deciphering the evolution of flavin-dependent monooxygenase stereoselectivity using ancestral sequence reconstruction.

Controlling the selectivity of a reaction is critical for target-oriented synthesis. Accessing complementary selectivity profiles enables divergent synthetic strategies, but is challenging to achieve in biocatalytic reactions given enzymes' innate preferences of a single selectivity. Thus, it is critical to understand the structural features that control selectivity in biocatalytic reactions to achieve tunable selectivity. Here, we investigate the structural features that control the stereoselectivity in an oxidative dearomatization reaction that is key to making azaphilone natural products. Crystal structures of enantiocomplementary biocatalysts guided the development of multiple hypotheses centered on the structural features that control the stereochemical outcome of the reaction; however, in many cases, direct substitutions of active site residues in natural proteins led to inactive enzymes. Ancestral sequence reconstruction (ASR) and resurrection were employed as an alternative strategy to probe the impact of each residue on the stereochemical outcome of the dearomatization reaction. These studies suggest that two mechanisms are active in controlling the stereochemical outcome of the oxidative dearomatization reaction: one involving multiple active site residues in AzaH and the other dominated by a single Phe to Tyr switch in TropB and AfoD. Moreover, this study suggests that the flavin-dependent monooxygenases (FDMOs) adopt simple and flexible strategies to control stereoselectivity, which has led to stereocomplementary azaphilone natural products produced by fungi. This paradigm of combining ASR and resurrection with mutational and computational studies showcases sets of tools for understanding enzyme mechanisms and provides a solid foundation for future protein engineering efforts.

Chemoenzymatic Synthesis of (+)-Xyloketal B.

Xyloketal B is a pentacyclic fungal marine natural product that has shown potential for the treatment of diseases such as Alzheimer's disease and atherosclerosis. Herein, we describe the first asymmetric synthesis of this natural product, which relies on a chemoenzymatic strategy. This approach leverages a biocatalytic benzylic hydroxylation to access to an ortho-quinone methide intermediate which is captured in a [4 + 2] cycloaddition to stereoselectively yield a key cyclic ketal intermediate enroute to (+)-xyloketal B. The relative configuration of this intermediate was rapidly confirmed as the desired stereoisomer using MicroED. To complete the synthesis, a second ortho-quinone methide was accessed through a reductive approach, ultimately leading to the stereoselective synthesis of (+)-xyloketal B.

Development of a P450 Fusion Enzyme for Biaryl Coupling in Yeast.

Despite the diverse and potent bioactivities displayed by axially chiral biaryl natural products, their application in drug discovery is limited by restricted access to these complex molecular scaffolds. In particular, fundamental challenges remain in controlling the site- and atroposelectivity in biaryl coupling reactions. In contrast, Nature has a wealth of biosynthetic enzymes that catalyze biaryl coupling reactions with catalyst-controlled selectivity. In particular, a growing subset of fungal P450s have been identified to catalyze site- and atroposelective biaryl couplings. Herein, we optimize a whole-cell biocatalytic platform in Pichia pastoris to synthesize biaryl molecules through the recombinant production of the fungal P450 KtnC. Moreover, engineering redox self-sufficient fusion enzymes further improves the efficiency of the system. Altogether, this work provides a platform for biaryl coupling reactions in yeast that can be applied to engineering a currently underexplored pool of fungal P450s into selective biocatalysts for the synthesis of complex biaryl compounds.

Understanding and Circumventing the Requirement for Native Thioester Substrates for α-Oxoamine Synthase Reactions.

Many enzyme classes require thioester electrophiles such as acyl-carrier proteins and acyl-coenzyme A substrates. For in vitro applications, these substrates can render these chemical transformations impractical. To address this challenge, we have investigated the mechanism of coenzyme A in gating catalysis of one α-oxoamine synthase, SxtA AOS. Through investigating the reactivity of SxtA AOS and corresponding enzyme variants against a panel of substrates and coenzyme A mimics, we determined that activity is gated through the binding of the pantetheine arm and a phosphate group that hydrogen bonds to residue Lys154 that is predicted by an AlphaFold2 model to be located in a tunnel leading to the active site. To provide an economical solution for preparative-scale reactions, in situ transthioesterification was used with pantetheine and simple thioester substrate precursors, resulting in productive reactions. These findings outline a strategy for employing ACP- and CoA-dependent enzymes that are inaccessible through other means without the need for cost-prohibitive coenzyme A or carrier protein-activated substrates.

Structural Basis for Control of Methylation Extent in Polyketide Synthase Metal-Dependent C-Methyltransferases.

Installation of methyl groups can significantly improve the binding of small-molecule drugs to protein targets; however, site-selective methylation often presents a significant synthetic challenge. Metal- and S-adenosyl-methionine (SAM)-dependent methyltransferases (MTs) in natural-product biosynthetic pathways are powerful enzymatic tools for selective or chemically challenging C-methylation reactions. Each of these MTs selectively catalyzes one or two methyl transfer reactions. Crystal structures and biochemical assays of the Mn2+-dependent monomethyltransferase from the saxitoxin biosynthetic pathway (SxtA MT) revealed the structural basis for control of methylation extent. The SxtA monomethyltransferase was converted to a dimethyltransferase by modification of the metal binding site, addition of an active site base, and an amino acid substitution to provide space in the substrate pocket for two methyl substituents. A reciprocal change converted a related dimethyltransferase into a monomethyltransferase, supporting our hypothesis that steric hindrance can prevent a second methylation event. A novel understanding of MTs will accelerate the development of MT-based catalysts and MT engineering for use in small-molecule synthesis.

The Transformative Power of Biocatalysis in Convergent Synthesis.

Achieving convergent synthetic strategies has long been a gold standard in constructing complex molecular skeletons, allowing for the rapid generation of complexity in comparatively streamlined synthetic routes. Traditionally, biocatalysis has not played a prominent role in convergent laboratory synthesis, with the application of biocatalysts in convergent strategies primarily limited to the synthesis of chiral fragments. Although the use of enzymes to enable convergent synthetic approaches is relatively new and emerging, combining the efficiency of convergent transformations with the selectivity achievable through biocatalysis creates new opportunities for efficient synthetic strategies. This Perspective provides an overview of recent developments in biocatalytic strategies for convergent transformations and offers insights into the advantages of these methods compared to their small molecule-based counterparts.

Biocatalytic oxidative cross-coupling reactions for biaryl bond formation.

Biaryl compounds, with two connected aromatic rings, are found across medicine, materials science and asymmetric catalysis1,2. The necessity of joining arene building blocks to access these valuable compounds has inspired several approaches for biaryl bond formation and challenged chemists to develop increasingly concise and robust methods for this task3. Oxidative coupling of two C-H bonds offers an efficient strategy for the formation of a biaryl C-C bond; however, fundamental challenges remain in controlling the reactivity and selectivity for uniting a given pair of substrates4,5. Biocatalytic oxidative cross-coupling reactions have the potential to overcome limitations inherent to numerous small-molecule-mediated methods by providing a paradigm with catalyst-controlled selectivity6. Here we disclose a strategy for biocatalytic cross-coupling through oxidative C-C bond formation using cytochrome P450 enzymes. We demonstrate the ability to catalyse cross-coupling reactions on a panel of phenolic substrates using natural P450 catalysts. Moreover, we engineer a P450 to possess the desired reactivity, site selectivity and atroposelectivity by transforming a low-yielding, unselective reaction into a highly efficient and selective process. This streamlined method for constructing sterically hindered biaryl bonds provides a programmable platform for assembling molecules with catalyst-controlled reactivity and selectivity.

Design principles for site-selective hydroxylation by a Rieske oxygenase.

Rieske oxygenases exploit the reactivity of iron to perform chemically challenging C-H bond functionalization reactions. Thus far, only a handful of Rieske oxygenases have been structurally characterized and remarkably little information exists regarding how these enzymes use a common architecture and set of metallocenters to facilitate a diverse range of reactions. Herein, we detail how two Rieske oxygenases SxtT and GxtA use different protein regions to influence the site-selectivity of their catalyzed monohydroxylation reactions. We present high resolution crystal structures of SxtT and GxtA with the native ß-saxitoxinol and saxitoxin substrates bound in addition to a Xenon-pressurized structure of GxtA that reveals the location of a substrate access tunnel to the active site. Ultimately, this structural information allowed for the identification of six residues distributed between three regions of SxtT that together control the selectivity of the C-H hydroxylation event. Substitution of these residues produces a SxtT variant that is fully adapted to exhibit the non-native site-selectivity and substrate scope of GxtA. Importantly, we also found that these selectivity regions are conserved in other structurally characterized Rieske oxygenases, providing a framework for predictively repurposing and manipulating Rieske oxygenases as biocatalysts.

State-of-the-Art Biocatalysis.

The use of enzyme-mediated reactions has transcended ancient food production to the laboratory synthesis of complex molecules. This evolution has been accelerated by developments in sequencing and DNA synthesis technology, bioinformatic and protein engineering tools, and the increasingly interdisciplinary nature of scientific research. Biocatalysis has become an indispensable tool applied in academic and industrial spheres, enabling synthetic strategies that leverage the exquisite selectivity of enzymes to access target molecules. In this Outlook, we outline the technological advances that have led to the field's current state. Integration of biocatalysis into mainstream synthetic chemistry hinges on increased access to well-characterized enzymes and the permeation of biocatalysis into retrosynthetic logic. Ultimately, we anticipate that biocatalysis is poised to enable the synthesis of increasingly complex molecules at new levels of efficiency and throughput.

Chemoenzymatic Total Synthesis of Natural Products.

The total synthesis of structurally complex natural products has challenged and inspired generations of chemists and remains an exciting area of active research. Despite their history as privileged bioactivity-rich scaffolds, the use of natural products in drug discovery has waned. This shift is driven by their relatively low abundance hindering isolation from natural sources and the challenges presented by their synthesis. Recent developments in biocatalysis have resulted in the application of enzymes for the construction of complex molecules. From the inception of the Narayan lab in 2015, we have focused on harnessing the exquisite selectivity of enzymes alongside contemporary small molecule-based approaches to enable concise chemoenzymatic routes to natural products.We have focused on enzymes from various families that perform selective oxidation reactions. For example, we have targeted xyloketal natural products through a strategy that relies on a chemo- and site-selective biocatalytic hydroxylation. Members of the xyloketal family are characterized by polycyclic ketal cores and demonstrate potent neurological activity. We envisioned assembling a representative xyloketal natural product (xyloketal D) involving a biocatalytically generated ortho-quinone methide intermediate. The non-heme iron (NHI) dependent monooxygenase ClaD was used to perform the benzylic hydroxylation of a resorcinol precursor, the product of which can undergo spontaneous loss of water to form an ortho-quinone methide under mild conditions. This intermediate was trapped using a chiral dienophile to complete the total synthesis of xyloketal D.A second class of biocatalytic oxidation that we have employed in synthesis is the hydroxylative dearomatization of resorcinol compounds using flavin-dependent monooxygenases (FDMOs). We anticipated that the catalyst-controlled site- and stereoselectivity of FDMOs would enable the total synthesis of azaphilone natural products. Azaphilones are bioactive compounds characterized by a pyranoquinone bicyclic core and a fully substituted chiral carbon atom. We leveraged the stereodivergent reactivity of FDMOs AzaH and AfoD to achieve the enantioselective synthesis of trichoflectin enantiomers, deflectin 1a, and lunatoic acid. We also leveraged FDMOs to construct tropolone and sorbicillinoid natural products. Tropolones are a structurally diverse class of bioactive molecules characterized by an aromatic cycloheptatriene core bearing an α-hydroxyketone moiety. We developed a two-step biocatalytic cascade to the tropolone natural product stipitatic aldehyde using the FDMO TropB and a NHI monooxygenase TropC. The FDMO SorbC obtained from the sorbicillin biosynthetic pathway was used in the concise total synthesis of a urea sorbicillinoid natural product.Our long-standing interest in using enzymes to carry out C-H hydroxylation reactions has also been channeled for the late-stage diversification of complex scaffolds. For example, we have used Rieske oxygenases to hydroxylate the tricyclic core common to paralytic shellfish toxins. The systemic toxicity of these compounds can be reduced by adding hydroxyl and sulfate groups, which improves their properties and potential as therapeutic agents. The enzymes SxtT, GxtA, SxtN, and SxtSUL were used to carry out selective C-H hydroxylation and O-sulfation in saxitoxin and related structures. We conclude this Account with a discussion of existing challenges in biocatalysis and ways we can currently address them.

Scalable biocatalytic C-H oxyfunctionalization reactions.

Catalytic C-H oxyfunctionalization reactions have garnered significant attention in recent years with their ability to streamline synthetic routes toward complex molecules. Consequently, there have been significant strides in the design and development of catalysts that enable diversification through C-H functionalization reactions. Enzymatic C-H oxygenation reactions are often complementary to small molecule based synthetic approaches, providing a powerful tool when deployable on preparative-scale. This review highlights key advances in scalable biocatalytic C-H oxyfunctionalization reactions developed within the past decade.

Structural basis for divergent C-H hydroxylation selectivity in two Rieske oxygenases.

Biocatalysts that perform C-H hydroxylation exhibit exceptional substrate specificity and site-selectivity, often through the use of high valent oxidants to activate these inert bonds. Rieske oxygenases are examples of enzymes with the ability to perform precise mono- or dioxygenation reactions on a variety of substrates. Understanding the structural features of Rieske oxygenases responsible for control over selectivity is essential to enable the development of this class of enzymes for biocatalytic applications. Decades of research has illuminated the critical features common to Rieske oxygenases, however, structural information for enzymes that functionalize diverse scaffolds is limited. Here, we report the structures of two Rieske monooxygenases involved in the biosynthesis of paralytic shellfish toxins (PSTs), SxtT and GxtA, adding to the short list of structurally characterized Rieske oxygenases. Based on these structures, substrate-bound structures, and mutagenesis experiments, we implicate specific residues in substrate positioning and the divergent reaction selectivity observed in these two enzymes.

Synthetic utility of one-pot chemoenzymatic reaction sequences.

In recent years, there has been a rapid and sustained increase in the development and use of one-pot chemoenzymatic reaction processes for the efficient synthesis of high-value molecules. This strategy can provide a number of advantages over traditional synthetic methods, including high levels of selectivity in reactions, mild and sustainable reaction conditions, and the ability to rapidly build molecular complexity in a single reaction vessel. Here, we present several examples of chemoenzymatic one-pot reaction sequences that demonstrate the diversity of transformations that can be incorporated in these processes.