Visible light absorbing iridium(III) complexes have been widely employed as photocatalysts in organic synthesis. Their catalytic reactivity and selectivity are generally optimized by modifying the structures of coordinated ligands to obtain desired photophysical properties. Artificial metalloenzymes (ArMs) can combine the unique features of both metal complexes and enzymes by incorporating a cofactor within a protein scaffold, which offers another strategy to improve the performance of metal catalysts. Herein, we describe a panel of Ir(III)-ArMs constructed by covalently embedding iridium(III) polypyridyl complexes into a prolyl oligopeptidase scaffold. A series of spectroscopic methods were used to examine how properties of the resulting ArMs are influenced by structural variation of the cyclometalated ligands and the protein scaffold. Visible light photocatalysis by these hybrid catalysts was also examined, leading to the finding that they catalyze inter/intra-molecular [2+2] photocycloaddition in aqueous solution and indicating that they can serve as new bio-photocatalysts for further exploration.

Phosphorylation plays important roles in biology by modulating the structure, reactivity, and biological function of a broad range of molecules. Biocatalytic phosphorylation has attracted attention from synthetic chemists due to its selectivity and mild reaction conditions using ATP as a phosphate donor. Given the potential synthetic utility of kinases with activity on small molecule substrates, we explored the activity of PsiK, the enzyme responsible for selective 4‐O‐phosphorylation of 4‐hydroxytryptamine or psilocin in psylocybin biosynthesis by Psilocybe cubensis. We find that PsiK has good activity on a range of substituted phenols and benzenediols beyond its native substrate, enabling preparative phosphorylation of different substrates, and substantially expands the substrate scope of biocatalytic phosphorylation. We also show that active site mutations can further expand substrate scope and improve site‐selectivity. This engineering effort was greatly expedited using DESI‐MS screening, which enabled analysis of 2,688 reactions in only 40 min. Finally, gram‐scale phosphorylation of a representative substrate was achieved with a turnover number over 10,000. Together, these results highlight the biocatalytic utility of PsiK and derivatives thereof for selective phosphorylation of phenols and benzenediols under mild conditions.

Diversification of structurally complex natural products remains a key challenge in the discovery of next-generation therapeutics. Premarineosin A, a potent and selective antimalarial natural product, is a promising yet underexplored scaffold due to limited availability and synthetic complexity. In this work, we overcome both barriers by coupling metabolic engineering with late-stage derivatization, enabling the first systematic exploration of the premarineosin A scaffold. Rational engineering of Streptomyces eitanensis , encoding a (-)-premarineosin A biosynthetic gene cluster, increased titers over 200-fold. Sustainable production of (-)-premarineosin A enabled a unique semi-synthetic and biocatalytic derivatization campaign. In this first structure-activity relationship study of premarineosin A, we accessed a suite of novel analogs, including a C12-brominated derivative with nanomolar potency (EC50 < 5 nM). This work establishes (-)-premarineosin A as a tractable and evolvable antimalarial scaffold, demonstrating how chemical biology approaches can unlock new structural and pharmacological space from complex microbial metabolites.

Despite the unique reactivity of vitamin B12 and its derivatives, B12-dependent enzymes remain underutilized in biocatalysis. In this study, we repurpose the B12-dependent transcription factor CarH to enable non-native radical cyclization reactions. An engineered variant of this enzyme, CarH*, catalyzes the formation γ- and δ-lactams via either redox-neutral or reductive ring closure with marked enhancement of reactivity and selectivity relative to the free B12 cofactor. CarH* also catalyzes an unusual spirocyclization via dearomatization of pendant arenes to produce bicyclic 1,3-diene products instead of 1,4-dienes provided by existing methods. These results and associated mechanistic studies highlight the importance of protein scaffolds for controlling the reactivity of B12 and expanding the synthetic utility of B12-dependent enzymes.

Single component flavin-dependent halogenases (FDHs) possesses both flavin reductase and FDH activity in a single enzyme. We recently reported that the single component FDH AetF catalyzes site-selective bromination and iodination of a variety of aromatic substrates and enantioselective bromolactonization and iodoetherification of styrenes bearing pendant carboxylic acid or alcohol substituents. Given this inherent reactivity and selectivity, we explored the utility of AetF as catalyst for alkene and alkyne C-H halogenation. We find that AetF catalyzes halogenation of a range of 1,1-disubstituted styrenes, often with high stereoselectivity. Despite the utility of haloalkenes for cross-coupling and other applications, accessing these compounds in a stereoselective manner typically requires functional group interconversion processes, and selective halogenation of 1,1’-disubstituted olefins remains rare. We also establish that AetF and homologues of this enzyme can halogenate terminal alkynes. Mutagenesis studies and deuterium kinetic isotope effects are used to support a mechanistic proposal involving covalent catalysis for halogenation of unactivated alkynes by AetF homologues. These findings expand the scope of FDH catalysis and continue to show the unique utility of single component FDHs for biocatalysis.

Bioelectrocatalysis involves the use of biomaterials to catalyze redox reactions at an electrode. Biocatalysis offers improved selectivity and typically involves milder reaction conditions, while electrocatalysis can involve the use of renewable energy as an electron source. Cobalamin (B12) dependent enzymes catalyze C-C and C-N bond forming reactions via radical mechanisms through the formation of a Co(III)-alkyl intermediate. Homolysis of the Co(III)-C bond leads to radical coupling with various substrates to form the product. However, these alkyl radicals can react through a variety of mechanistic pathways, leading to a mixture of products. Controlling the alkyl radical intermediate would lead to improved reaction selectivity. Here, we report on CarH*, an engineered variant of a B12 dependent photoreceptor protein, which catalyzes styrene C-H alkylation by trapping an alkyl radical intermediate in its protein scaffold. The reversible Co-alkyl bond homolysis protects the radical, allowing for selective olefin addition. However, to initiate this reactivity, the cobalt center of CarH* must be reduced through stoichiometric addition of the reducing agent titanium (III) citrate. Thus, we are developing a mediated electrochemical system to achieve this transformation without the use of a stoichiometric reductant and to investigate new reactivity in CarH* and similar variants. A diffusive redox mediator was found to deliver electrons to CarH* and drive alkyl radical formation. This methodology offers a more sustainable and general approach to electrochemical non-native biocatalysis and can be applied to discover new reactivity in B12 dependent enzymes.

Organohalogen compounds are extensively used as building blocks, intermediates, pharmaceuticals, and agrochemicals due to their unique chemical and biological properties. Installing halogen substituents, however, frequently requires functionalized starting materials and multistep functional group interconversion. Several classes of halogenases evolved in nature to enable halogenation of a different classes of substrates; for example, site-selective halogenation of electron rich aromatic compounds is catalyzed by flavin-dependent halogenases (FDHs). Mechanistic studies have shown that these enzymes use FADH2 to reduce O2 to water with concomitant oxidation of X- to HOX (X = Cl, Br, I). This species travels through a tunnel within the enzyme to access the FDH active site. Here, it is believed to interact with an active site lysine proximal to bound substrate, enabling electrophilic halogenation with selectivity imparted via molecular recognition, rather than directing groups or strong electronic activation. The unique selectivity of FDHs led to several early biocatalysis efforts, preparative halogenation was rare, and the hallmark catalyst-controlled selectivity of FDHs did not translate to non-native substrates. FDH engineering was limited to site-directed mutagenesis, which resulted in modest changes in site-selectivity or substrate preference. To address these limitations, we optimized expression conditions for the FDH RebH and its cognate flavin reductase (FRed), RebF. We then showed that RebH could be used for preparative halogenation of non-native substrates with catalyst-controlled selectivity. We reported the first examples in which the stability, substrate scope, and site selectivity of a FDH were improved to synthetically useful levels via directed evolution. X-ray crystal structures of evolved FDHs and reversion mutations showed that random mutations throughout the RebH structure were critical to achieving high levels of activity and selectivity on diverse aromatic substrates, and these data were used in combination with molecular dynamics simulations to develop predictive model for FDH selectivity. Finally, we used family wide genome mining to identify a diverse set of FDHs with novel substrate scope and complementary regioselectivity on large, three-dimensionally complex compounds. The diversity of our evolved and mined FDHs allowed us to pursue synthetic applications beyond simple aromatic halogenation. For example, we established that FDHs catalyze enantioselective reactions involving desymmetrization, atroposelective halogenation, and halocyclization. These results highlight the ability of FDH active sites to tolerate different substrate topologies. This utility was further expanded by our recent studies on the single component FDH/FRed, AetF. While we were initially drawn to AetF because it does not require a separate FRed, we found that it halogenates substrates that are not halogenated efficiently or at all by other FDHs and provides high enantioselectivity for reactions that could only be achieved using RebH variants after extensive mutagenesis. Perhaps most notably, AetF catalyzes site-selective aromatic iodination and enantioselective iodoetherification. Together, these studies highlight the origins of FDH engineering, the utility and limitations of the enzymes developed to date, and the promise of FDHs for an ever-expanding range of biocatalytic halogenation reactions.

Artificial metalloenzymes (ArMs) can combine the unique features of both metal complexes and enzymes by incorporating a cofactor within a protein scaffold. Herein, we describe a panel of ArMs constructed by covalently linking Ir(III) polypyridyl complexes into a prolyl oligopeptidase scaffold. Spectroscopic methods were used to examine how properties of the resulting ArMs are influenced by structural variation of the cyclometalated ligands and the protein scaffold. Visible light photocatalysis by these hybrid catalysts was also examined, leading to the finding that they catalyze inter/intra-molecular [2 + 2] photocycloaddition in aqueous solution. Low but reproducible enantioselectivity was observed using a cofactor that undergoes partial kinetic resolution upon bioconjugation within the ArM active site, showing the importance of scaffold/cofactor interactions for enabling selective ArM photocatalysis. Further evidence of the importance of cofactor/scaffold interactions was provided by analyzing native POP peptidase catalysis by the ArMs. Together, these studies show how Ir(III)-based ArMs constitute a promising starting point for ongoing studies to control the stereoselectivity of EnT reactions by engineering substrate binding/activation motifs in POP.

Fe(II)- and α-ketoglutarate-dependent halogenases and oxygenases can catalyze site-selective functionalization of C-H bonds via a variety of C-X bond forming reactions. Achieving high chemoselectivity for functionalization using non-native functional groups remains rare, however, particularly for non-native substrates. The current study shows that directed evolution can be used to engineer variants of an engineered dioxygenase, SadX, that address this challenge. Site-selective azidation of succinylated amino acids and a succinylated amine was achieved using variants with improved azidation yield and selectivity on a probe substrate as a result of mutations throughout the SadX structure. The installed azide group was reduced to a primary amine, and the succinyl group required for azidation was enzymatically cleaved to provide the corresponding amine. These results provide a promising starting point for evolving additional SadX variants with activity on structurally distinct substrates and for enabling enzymatic C-H functionalization with other non-native functional groups.

During efforts to explore the range of reactions catalyzed by the previously-engineered B12-dependent enzyme CarH*, we found that this enzyme catalyzes N-alkylation of aniline using ethyl diazoacetate but that B12 itself provides only trace conversion for this reaction. This observation suggested that the unique primary and second coordination sphere provided by CarH* accelerates non-native N-alkylation catalysis and that other B12-dependent proteins could provide further improvements in activity. We used a structural homology search to identify the corrinoid protein MtaC, which was reconstituted with B12 to generate a catalyst with significantly-improved aniline N-alkylation activity on a range of substrates. MtaC also displays remarkable thermal stability and organic solvent tolerance, remaining folded even in pure methanol. These results highlight how protein scaffolds can be used to impart new activity and other beneficial properties to metal catalysts and suggest that MtaC could serve as a useful platform for non-native B12 catalysis.

Despite the unique reactivity of vitamin B12 and its derivatives, B12-dependent enzymes remain underutilized in biocatalysis. In this study, we repurpose the B12-dependent transcription factor CarH to enable non-native radical cyclization reactions. An engineered variant of this enzyme, CarH*, catalyzes the formation γ- and δ-lactams via either redox-neutral or reductive ring closure with marked enhancement of reactivity and selectivity relative to the free B12 cofactor. CarH* also catalyzes an unusual spirocyclization via dearomatization of pendant arenes to produce bicyclic 1,3-diene products instead of 1,4-dienes provided by existing methods. These results and associated mechanistic studies highlight the importance of protein scaffolds for controlling the reactivity of B12 and expanding the synthetic utility of B12-dependent enzymes.

In nature, flavin dependent halogenases (FDHs) catalyze site-selective chlorination and bromination of aromatic natural products. This ability has led to extensive efforts to engineer FDHs for selective chlorination, bromination, and iodination of electron rich aromatic compounds. On the other hand, FDHs are unique among halogenases and haloperoxidases that exhibit catalyst-controlled site selectivity in that no examples of enantioselective FDH catalysis in natural product biosynthesis have been characterized. Over the past several years, our group has established that FDHs can catalyze enantioselective reactions involving desymmetrization, atroposelective halogenation, and halocyclization. Achieving high activity and selectivity for these reactions has required extensive mutagenesis and mitigation of problems resulting from hypohalous acid generated during FDH catalysis. The single-component flavin reductase/FDH AetF is unique among the wild type enzyme, we have studied in that it provides high activity and selectivity toward several asymmetric transformations. These results highlight the ability of FDH active sites to tolerate different substrate topologies and suggest that they could be useful for a broad range of oxidative halogenations.

The ability to site-selectively modify equivalent functional groups in a molecule has the potential to streamline syntheses and increase product yields by lowering step counts. Enzymes catalyze site-selective transformations throughout primary and secondary metabolism, but leveraging this capability for non-native substrates and reactions requires a detailed understanding of the potential and limitations of enzyme catalysis and how these bounds can be extended by protein engineering. In this review, we discuss representative examples of site-selective enzyme catalysis involving functional group manipulation and C-H bond functionalization. We include illustrative examples of native catalysis, but our focus is on cases involving non-native substrates and reactions often using engineered enzymes. We then discuss the use of these enzymes for chemoenzymatic transformations and target-oriented synthesis and conclude with a survey of tools and techniques that could expand the scope of non-native site-selective enzyme catalysis.

The design of allosteric regulation in proteins to dynamically control function is a challenge in synthetic biology. To address this challenge, we developed an integrated computational and experimental workflow to incorporate a metal-responsive chemical switch into proteins. Pairs of bipyridinylalanine (BpyAla) residues were genetically encoded into two structurally distinct enzymes, a serine protease and firefly luciferase, so that metal coordination would bias the conformations of these enzymes, leading to reversible control of activity. MD-simulations guided rational BpyAla placement, significantly reducing experimental workload, and cell-free protein synthesis coupled with high-throughput experimentation enabled rapid prototyping of variants. Ultimately, this strategy yielded enzymes with a robust 20-fold dynamic range in response to divalent metals over 24 on/off switches, demonstrating the potential of this approach. We envision that this strategy of genetically encoding chemical switches into enzymes will complement other protein engineering and synthetic biology efforts, enabling new opportunities for applications where precise regulation of protein function is critical.

Vitamin B12 derivatives catalyze a wide range of organic transformations, but B12-dependent enzymes are underutilized in biocatalysis relative to other metalloenzymes. In this study, we engineered a variant of the transcription factor CarH, called CarH*, that catalyzes styrene C-H alkylation with improved yield and selectivity relative to B12 itself. While the native function of CarH involves transcription regulation via AdoCbl Co(III)-carbon bond cleavage and β-hydride elimination to generate 4’,5’-didehydroadenosine, CarH*-catalyzed styrene alkylation proceeds via non-native oxidative addition and olefin addition coupled with a native-like β-hydride elimination. Mechanistic studies on this reaction echo findings from earlier studies on AdoCbl homolysis under strong cage conditions to suggest that CarH* can enable non-native radical chemistry with improved selectivity relative to B12 itself. These findings lay the groundwork for the development of B12-dependent enzymes as catalysts for a wide range of non-native transformations.

Of the different classes of halogenases characterized to date, flavin dependent halogenases (FDHs) are most commonly associated with site-selective halogenation of electron rich arenes and enol(ate) moieties in the biosynthesis of halogenated natural products. This capability has made them attractive biocatalysts, and extensive efforts have been devoted to both discovering and engineering these enzymes for different applications. We have established that engineered FDHs can catalyze different enantioselective halogenation processes, including halolactonization of simple alkenes with a tethered carboxylate nucleophile. In this study, we expand the scope of this reaction to include alcohol nucleophiles and a greater diversity of alkene substitution patterns to access a variety of chiral tetrahydrofurans. We also demonstrate that FDHs can be interfaced with ketoreductases to enable halocyclization using ketone substrates in one-pot cascade reactions and that the halocyclization products can undergo subsequent rearrangements to form novel hydroxylated and halogenated products. Together, these advances expand the utility of FDHs for enantio- and diastereoselective olefin functionalization.

Flavin-dependent halogenases (FDHs) catalyze selective halogenation of electron-rich aromatic compounds without the need for harsh oxidants required by conventional oxidative halogenation reactions. Predictive models for halogenase site selectivity could greatly improve their utility for chemical synthesis. Toward this end, we analyzed the structures and selectivity of three halogenase variants evolved to halogenate tryptamine with orthogonal selectivity. Crystal structures and reversion mutations revealed key residues involved in altering halogenase selectivity. Density functional theory calculations and molecular dynamics simulations are both consistent with hypohalous acid as the active halogenating species in FDH catalysis. This model was used to accurately predict the site selectivity of halogenase variants toward different synthetic substrates, providing a valuable tool for implementing halogenases in biocatalysis efforts.

Mononuclear non-heme Fe(II)- and -ketoglutarate dependent oxygenases (FeDOs) catalyze site-selective C-H hydroxylation. Variants of these enzymes in which a conserved Asp/Glu residue in the Fe(II)-binding facial triad is replaced by Ala/Gly can, in some cases, bind various anionic ligands and catalyze non-native chlorination and bromination reactions. In this study, we explore the binding of different anions to a FeDO facial triad variant, SadX, and the effects of that binding on HO• vs. X• rebound. We establish that chloride and bromide not only enable non-native halogenation reactions but that all anions investigated, including azide, cyanate, formate, and fluoride, significantly accelerate and influence the site selectivity of SadX hydroxylation catalysis. Azide and cyanate also lead to the formation of products resulting from N3•, NCO•, and OCN• rebound. While fluoride rebound is not observed, the rate acceleration provided by this ligand led us to calculate barriers for HO• and F• rebound from a putative Fe(III)(OH)(F) intermediate. These calculations suggest that the lack of fluorination is due to the relative barriers of the HO• and F• rebound transition states rather than an inaccessible barrier for F• rebound. Together, these results improve our understanding of FeDO facial triad variant tolerance of different anionic ligands, their ability to promote rebound involving those ligands, and inherent rebound preferences relative to HO• that will aid efforts to develop non-native catalysis using these enzymes.

In this study, we engineer a variant of the flavin-dependent halogenase RebH that catalyzes site-and atroposelective halogenation of 3-aryl-4(3H)-quinazolinones via kinetic or dynamic kinetic resolution. The required directed evolution uses a combination of random and site-saturation mutagenesis, substrate walking using two probe substrates, and a two-tiered screening approach involving the analysis of variant conversion and then enantioselectivity of improved variants. The resulting variant, 3-T, provides >99:1 e.r. for the (M)-atropisomer of the major brominated product, 25-fold improved conversion, and 91-fold improved site selectivity relative to the parent enzyme on the probe substrate used in the final rounds of evolution. This high activity and selectivity translate well to several additional substrates with varied steric and electronic properties. Computational modeling and docking simulations are used to rationalize the effects of key mutations on substrate binding. Given the range of substrates that have been used for atroposelective synthesis via electrophilic halogenation in the literature, these results suggest that flavin-dependent halogenases (FDHs) could find many additional applications for atroposelective catalysis. More broadly, this study highlights how RebH can be engineered to accept structurally diverse substrates that enable its use for enantioselective catalysis.