Enzymatic Assembly of Diverse Lactone Structures: An Intramolecular C-H Functionalization Strategy.

Lactones are cyclic esters with extensive applications in materials science, medicinal chemistry, and the food and perfume industries. Nature's strategy for the synthesis of many lactones found in natural products always relies on a single type of retrosynthetic strategy, a C-O bond disconnection. Here, we describe a set of laboratory-engineered enzymes that use a new-to-nature C-C bond-forming strategy to assemble diverse lactone structures. These engineered "carbene transferases" catalyze intramolecular carbene insertions into benzylic or allylic C-H bonds, which allow for the synthesis of lactones with different ring sizes and ring scaffolds from simple starting materials. Starting from a serine-ligated cytochrome P450 variant previously engineered for other carbene-transfer activities, directed evolution generated a variant P411-LAS-5247, which exhibits a high activity for constructing a five-membered ε-lactone, lactam, and cyclic ketone products (up to 5600 total turnovers (TTN) and >99% enantiomeric excess (ee)). Further engineering led to variants P411-LAS-5249 and P411-LAS-5264, which deliver six-membered δ-lactones and seven-membered ε-lactones, respectively, overcoming the thermodynamically unfavorable ring strain associated with these products compared to the γ-lactones. This new carbene-transfer activity was further extended to the synthesis of complex lactone scaffolds based on fused, bridged, and spiro rings. The enzymatic platform developed here complements natural biosynthetic strategies for lactone assembly and expands the structural diversity of lactones accessible through C-H functionalization.

Directed evolution of P411 enzymes for amination of inert C-H bonds.

Functionalizing inert C-H bonds selectively is a formidable task due to their strong bond energy and the difficulty of distinguishing chemically similar C-H bonds. While enzymatic oxygenation of C-H bonds is ubiquitous and well established, there is currently no known natural enzymatic process for direct nitrogen insertion. Instead, nature typically relies on pre-oxidized compounds for nitrogen incorporation. Direct biocatalytic C-H amination methods developed in the last few years are only selective for activated C-H bonds that contain specific groups such as benzylic, allylic, or propargylic groups. However, we recently used directed evolution to generate cytochrome P411 enzymes (engineered P450 enzymes with axial ligand mutation from cysteine to serine) that directly aminate inert C-H bonds with high site-, diastereo-, and enantioselectivity. Using these enzymes, we demonstrated the regiodivergent desymmetrization of methylcyclohexane, among other reactions. This chapter provides a comprehensive account of the experimental protocols used to evolve P411s for aminating unactivated C-H bonds. These methods are illustrative and can be adapted for other directed enzyme evolution campaigns.

Enzymatic Nitrogen Incorporation Using Hydroxylamine.

Hydroxylamine-derived reagents have enabled versatile nitrene transfer reactions for introducing nitrogen-containing functionalities in small-molecule catalysis, as well as biocatalysis. These reagents, however, result in a poor atom economy and stoichiometric organic waste. Activating hydroxylamine (NH2OH) for nitrene transfer offers a low-cost and sustainable route to amine synthesis, since water is the sole byproduct. Despite its presence in nature, hydroxylamine is not known to be used for enzymatic nitrogen incorporation in biosynthesis. Here, we report an engineered heme enzyme that can utilize hydroxylammonium chloride, an inexpensive commodity chemical, for nitrene transfer. Directed evolution of Pyrobaculum arsenaticum protoglobin generated efficient enzymes for benzylic C-H primary amination and styrene aminohydroxylation. Mechanistic studies supported a stepwise radical pathway involving rate-limiting hydrogen atom transfer. This unprecedented activity is a useful addition to the "nitrene transferase" repertoire and hints at possible future discovery of natural enzymes that use hydroxylamine for amination chemistry.

Development of Nonclassical Photoprecursors for Rh2 Nitrenes.

Characterization of reactive intermediates in C-H functionalization is challenging due to the fleeting lifetimes of these species. Synthetic photochemistry provides a strategy to generate post-turnover-limiting-step intermediates in catalysis under cryogenic conditions that enable characterization. We have a long-standing interest in the structure and reactivity of Rh2 nitrene intermediates, which are implicated as transient intermediates in Rh2-catalyzed C-H amination. Previously, we demonstrated that Rh2 complexes bearing organic azide ligands can serve as solid-state and in crystallo photoprecursors in the synthesis of transient Rh2 nitrenoids. Complementary solution-phase experiments have not been available due to the weak binding of most organic azides to Rh2 complexes. Furthermore, the volatility of the N2 that is evolved during in crystallo nitrene synthesis from these precursors has prevented the in crystallo observation of C-H functionalization from lattice-confined nitrenes. Motivated by these challenges, here we describe the synthesis and photochemistry of nonclassical nitrene precursors based on sulfilimine ligands. Sulfilimines bind to Rh2 carboxylate complexes more tightly than the corresponding azides, which has enabled the full solid-state and solution-phase characterization of these new complexes. The higher binding affinity of sulfilimine ligands as compared with organic azides has enabled both solution-phase and solid-state nitrene photochemistry. Cryogenic photochemical studies of Rh2 sulfilimine complexes confined within polystyrene thin films demonstrate that sulfilimine photochemistry can be accomplished at low temperature but that C-H amination is rapid at temperatures compatible with N═S photoactivation. The potential of these structures to serve as platforms for multistep in crystallo cascades is discussed.

Enantio- and Diastereoenriched Enzymatic Synthesis of 1,2,3-Polysubstituted Cyclopropanes from (Z/E)-Trisubstituted Enol Acetates.

In nature and synthetic chemistry, stereoselective [2 + 1] cyclopropanation is the most prevalent strategy for the synthesis of chiral cyclopropanes, a class of key pharmacophores in pharmaceuticals and bioactive natural products. One of the most extensively studied reactions in the organic chemist's arsenal, stereoselective [2 + 1] cyclopropanation, largely relies on the use of stereodefined olefins, which can require elaborate laboratory synthesis or tedious separation to ensure high stereoselectivity. Here, we report engineered hemoproteins derived from a bacterial cytochrome P450 that catalyze the synthesis of chiral 1,2,3-polysubstituted cyclopropanes, regardless of the stereopurity of the olefin substrates used. Cytochrome P450BM3 variant P411-INC-5185 exclusively converts (Z)-enol acetates to enantio- and diastereoenriched cyclopropanes and in the model reaction delivers a leftover (E)-enol acetate with 98% stereopurity, using whole Escherichia coli cells. P411-INC-5185 was further engineered with a single mutation to enable the biotransformation of (E)-enol acetates to α-branched ketones with high levels of enantioselectivity while simultaneously catalyzing the cyclopropanation of (Z)-enol acetates with excellent activities and selectivities. We conducted docking studies and molecular dynamics simulations to understand how active-site residues distinguish between the substrate isomers and enable the enzyme to perform these distinct transformations with such high selectivities. Computational studies suggest the observed enantio- and diastereoselectivities are achieved through a stepwise pathway. These biotransformations streamline the synthesis of chiral 1,2,3-polysubstituted cyclopropanes from readily available mixtures of (Z/E)-olefins, adding a new dimension to classical cyclopropanation methods.

Enantio- and Diastereoenriched Enzymatic Synthesis of 1,2,3-Polysubstituted Cyclopropanes from (Z/E)-Trisubstituted Enol Acetates.

In nature and synthetic chemistry, stereoselective [2+1] cyclopropanation is the most prevalent strategy for the synthesis of chiral cyclopropanes, a class of key pharmacophores in pharmaceuticals and bioactive natural products. One of the most extensively studied reactions in the organic chemist's arsenal, stereoselective [2+1] cyclopropanation, largely relies on the use of stereodefined olefins, which require elaborate laboratory synthesis or tedious separation to ensure high stereoselectivity. Here we report engineered hemoproteins derived from a bacterial cytochrome P450 that catalyze the synthesis of chiral 1,2,3-polysubstituted cyclopropanes, regardless of the stereopurity of the olefin substrates used. Cytochrome P450 BM3 variant IC-G3 exclusively converts ( Z )-enol acetates to enantio- and diastereoenriched cyclopropanes and in our model reaction delivers a leftover ( E )-enol acetate with 98% stereopurity, using whole Escherichia coli cells. IC-G3 was further engineered with a single mutation to enable the biotransformation of ( E )-enol acetates to α -branched ketones with high levels of enantioselectivity while simultaneously catalyzing the cyclopropanation of ( Z )-enol acetates with excellent activities and selectivities. We conducted docking studies and molecular dynamics simulations to understand how active-site residues distinguish between the substrate isomers and enable the enzyme to perform these distinct transformations with such high selectivities. Computational studies suggest the observed enantio- and diastereoselectivities are achieved through a stepwise pathway. These biotransformations streamline the synthesis of chiral 1,2,3-polysubstituted cyclopropanes from readily available mixtures of ( Z/E )-olefins, adding a new dimension to classical cyclopropanation methods.

Protoglobin-Catalyzed Formation of cis-Trifluoromethyl-Substituted Cyclopropanes by Carbene Transfer.

Trifluoromethyl-substituted cyclopropanes (CF3 -CPAs) constitute an important class of compounds for drug discovery. While several methods have been developed for synthesis of trans-CF3 -CPAs, stereoselective production of corresponding cis-diastereomers remains a formidable challenge. We report a biocatalyst for diastereo- and enantio-selective synthesis of cis-CF3 -CPAs with activity on a variety of alkenes. We found that an engineered protoglobin from Aeropyrnum pernix (ApePgb) can catalyze this unusual reaction at preparative scale with low-to-excellent yield (6-55 %) and enantioselectivity (17-99 % ee), depending on the substrate. Computational studies revealed that the steric environment in the active site of the protoglobin forced iron-carbenoid and substrates to adopt a pro-cis near-attack conformation. This work demonstrates the capability of enzyme catalysts to tackle challenging chemistry problems and provides a powerful means to expand the structural diversity of CF3 -CPAs for drug discovery.

New Additions to the Arsenal of Biocatalysts for Noncanonical Amino Acid Synthesis.

Noncanonical amino acids (ncAAs) merge the conformational behavior and native interactions of proteinogenic amino acids with nonnative chemical motifs and have proven invaluable in developing modern therapeutics. This blending of native and nonnative characteristics has resulted in essential drugs like nirmatrelvir, which comprises three ncAAs and is used to treat COVID-19. Enzymes are appearing prominently in recent syntheses of ncAAs, where they demonstrate impressive control over the stereocenters and functional groups found therein. Here we review recent efforts to expand the biocatalyst arsenal for synthesizing ncAAs with natural enzymes. We also discuss how new-to-nature enzymes can contribute to this effort by catalyzing reactions inspired by the vast repertoire of chemical catalysis and acting on substrates that would otherwise not be used in synthesizing ncAAs. Abiotic enzyme-catalyzed reactions exploit the selectivity afforded by a macromolecular catalyst to access molecules not available to natural enzymes and perhaps not even chemical catalysis.

Enzymatic Nitrogen Insertion into Unactivated C-H Bonds.

Selective functionalization of aliphatic C-H bonds, ubiquitous in molecular structures, could allow ready access to diverse chemical products. While enzymatic oxygenation of C-H bonds is well established, the analogous enzymatic nitrogen functionalization is still unknown; nature is reliant on preoxidized compounds for nitrogen incorporation. Likewise, synthetic methods for selective nitrogen derivatization of unbiased C-H bonds remain elusive. In this work, new-to-nature heme-containing nitrene transferases were used as starting points for the directed evolution of enzymes to selectively aminate and amidate unactivated C(sp3)-H sites. The desymmetrization of methyl- and ethylcyclohexane with divergent site selectivity is offered as demonstration. The evolved enzymes in these lineages are highly promiscuous and show activity toward a wide array of substrates, providing a foundation for further evolution of nitrene transferase function. Computational studies and kinetic isotope effects (KIEs) are consistent with a stepwise radical pathway involving an irreversible, enantiodetermining hydrogen atom transfer (HAT), followed by a lower-barrier diastereoselectivity-determining radical rebound step. In-enzyme molecular dynamics (MD) simulations reveal a predominantly hydrophobic pocket with favorable dispersion interactions with the substrate. By offering a direct path from saturated precursors, these enzymes present a new biochemical logic for accessing nitrogen-containing compounds.

Nitrene Photochemistry of Manganese N-Haloamides*.

Manganese complexes supported by macrocyclic tetrapyrrole ligands represent an important platform for nitrene transfer catalysis and have been applied to both C-H amination and olefin aziridination catalysis. The reactivity of the transient high-valent Mn nitrenoids that mediate these processes renders characterization of these species challenging. Here we report the synthesis and nitrene transfer photochemistry of a family of MnIII N-haloamide complexes. The S=2 N-haloamide complexes are characterized by 1 H NMR, UV-vis, IR, high-frequency and -field EPR (HFEPR) spectroscopies, and single-crystal X-ray diffraction. Photolysis of these complexes results in the formal transfer of a nitrene equivalent to both C-H bonds, such as the α-C-H bonds of tetrahydrofuran, and olefinic substrates, such as styrene, to afford aminated and aziridinated products, respectively. Low-temperature spectroscopy and analysis of kinetic isotope effects for C-H amination indicate halogen-dependent photoreactivity: Photolysis of N-chloroamides proceeds via initial cleavage of the Mn-N bond to generate MnII and amidyl radical intermediates; in contrast, photolysis of N-iodoamides proceeds via N-I cleavage to generate a MnIV nitrenoid (i.e., {MnNR}7 species). These results establish N-haloamide ligands as viable precursors in the photosynthesis of metal nitrenes and highlight the power of ligand design to provide access to reactive intermediates in group-transfer catalysis.

Dual Polymerization Pathway for Polyolefin-Polar Block Copolymer Synthesis via MILRad: Mechanism and Scope.

This work explores the mechanism whereby a cationic diimine Pd(II) complex combines coordination insertion and radical polymerization to form polyolefin-polar block copolymers. The initial requirement involves the insertion of a single acrylate monomer into the Pd(II)-polyolefin intermediates, which generate a stable polymeric chelate through a chain-walking mechanism. This thermodynamically stable chelate was also found to be photochemically inactive, and a unique mechanism was discovered which allows for radical polymerization. Rate-determining opening of the chelate by an ancillary ligand followed by additional chain walking allows the metal to migrate to the α-carbon of the acrylate moiety. Ultimately, the molecular parameters necessary for blue-light-triggered Pd-C bond homolysis from this α-carbon to form a carbon-centered macroradical species were established. This intermediate is understood to initiate free radical polymerization of acrylic monomers, thereby facilitating block copolymer synthesis from a single Pd(II) complex. Key intermediates were isolated and comprehensively characterized through exhaustive analytical methods which detail the mechanism while confirming the structural integrity of the polyolefin-polar blocks. Chain walking combined with blue-light irradiation functions as the mechanistic switch from coordination insertion to radical polymerization. On the basis of these discoveries, robust di- and triblock copolymer syntheses have been demonstrated with olefins (ethylene and 1-hexene) which produce amorphous or crystalline blocks and acrylics (methyl acrylate, ethyl acrylate, n-butyl acrylate, and methyl methacrylate) in broad molecular weight ranges and compositions, yielding AB diblocks and BAB triblocks.

In Crystallo Snapshots of Rh2-Catalyzed C-H Amination.

While X-ray crystallography routinely provides structural characterization of kinetically stable pre-catalysts and intermediates, elucidation of the structures of transient reactive intermediates, which are intimately engaged in bond-breaking and -making during catalysis, is generally not possible. Here, we demonstrate in crystallo synthesis of Rh2 nitrenoids that participate in catalytic C-H amination, and we characterize these transient intermediates as triplet adducts of Rh2. Further, we observe the impact of coordinating substrate, which is present in excess during catalysis, on the structure of transient Rh2 nitrenoids. By providing structural characterization of authentic C-H functionalization intermediates, and not kinetically stabilized model complexes, these experiments provide the opportunity to define critical structure-activity relationships.

C-H Amination Mediated by Cobalt Organoazide Adducts and the Corresponding Cobalt Nitrenoid Intermediates.

Treatment of (ArL)CoBr (ArL = 5-mesityl-1,9-(2,4,6-Ph3C6H2)dipyrrin) with a stoichiometric amount of 1-azido-4-(tert-butyl)benzene N3(C6H4-p-tBu) furnished the corresponding four-coordinate organoazide-bound complex (ArL)CoBr(N3(C6H4-p-tBu)). Spectroscopic and structural characterization of the complex indicated redox innocent ligation of the organoazide. Slow expulsion of dinitrogen (N2) was observed at room temperature to afford a ligand functionalized product via a [3 + 2] annulation, which can be mediated by a high-valent nitrene intermediate such as a CoIII iminyl (ArL)CoBr(•N(C6H4-p-tBu)) or CoIV imido (ArL)CoBr(N(C6H4-p-tBu)) complex. The presence of the proposed intermediate and its viability as a nitrene group transfer reagent are supported by intermolecular C-H amination and aziridination reactivities. Unlike (ArL)CoBr(N3(C6H4-p-tBu)), a series of alkyl azide-bound CoII analogues expel N2 only above 60 °C, affording paramagnetic intermediates that convert to the corresponding Co-imine complexes via α-H-atom abstraction. The corresponding N2-released structures were observed via single-crystal-to-crystal transformation, suggesting formation of a Co-nitrenoid intermediate in solid-state. Alternatively, the alkyl azide-bound congeners supported by a more sterically accessible dipyrrinato scaffold tBuL (tBuL = 5-mesityl-(1,9-di-tert-butyl)dipyrrin) facilitate intramolecular 1,3-dipolar cycloaddition as well as C-H amination to furnish 1,2,3-dihydrotriazole and substituted pyrrolidine products, respectively. For the C-H amination, we observe that the temperature required for azide activation varies depending on the presence of weak C-H bonds, suggesting that the alkyl azide adducts serve as viable species for C-H amination when the C-H bonds are (1) proximal to the azide moiety and (2) sufficiently weak to be activated.

Kinetic versus thermodynamic metalation enables synthesis of isostructural homo- and heterometallic trinuclear clusters.

Temperature-dependent metalation of the new hexadentate ligand (tris(5-(pyridin-2-yl)-1H-pyrrol-2-yl)methane; H3TPM) enables the selective synthesis of both mononuclear (i.e. Na(THF)4[Fe(TPM)], kinetic product) and trinuclear (i.e. Fe3(TPM)2, thermodynamic product) complexes. Exposure of Na(THF)4[Fe(TPM)] to FeCl2 or ZnCl2 triggers cluster expansion to generate homo- or heterometallic trinuclear complexes, respectively. The developed approach enables systematic variation of ion content in isostructural metal clusters via programmed assembly.

Characterization of a Reactive Rh2 Nitrenoid by Crystalline Matrix Isolation.

The fleeting lifetimes of reactive intermediates in C-H functionalization chemistry often prevent their direct characterization. For example, the critical nitrenoid intermediates that mediate Rh2-catalyzed C-H amination have eluded characterization for more than 40 years. In the absence of structural characterization of these species, methodological development is often computationally guided. Here we report the first X-ray crystal structure of a reactive Rh2 nitrenoid, enabled by N2 elimination from an organic azide ligand within a single-crystal matrix. The resulting high-resolution structure displays metrical parameters consistent with a triplet nitrene complex of Rh2. The demonstration of facile access to reactive metal nitrenoids within a crystalline matrix provides a platform for structural characterization of the transient species at the heart of C-H functionalization.

Observation of a Photogenerated Rh2 Nitrenoid Intermediate in C-H Amination.

Rh2-catalyzed C-H amination is a powerful method for nitrogenating organic molecules. While Rh2 nitrenoids are often invoked as reactive intermediates in these reactions, the exquisite reactivity and fleeting lifetime of these species has precluded their observation. Here, we report the photogeneration of a transient Rh2 nitrenoid that participates in C-H amination. The developed approach to Rh2 nitrenoids, based on photochemical cleavage of N-Cl bonds in N-chloroamido ligands, has enabled characterization of a reactive Rh2 nitrenoid by mass spectrometry and transient absorption spectroscopy. We anticipate that photogeneration of metal nitrenoids will contribute to the development of C-H amination catalysis by providing tools to directly study the structures of these critical intermediates.

Probing Substrate Diffusion in Interstitial MOF Chemistry with Kinetic Isotope Effects.

Metal-organic frameworks (MOFs) have garnered substantial interest as platforms for site-isolated catalysis. Efficient diffusion of small-molecule substrates to interstitial lattice-confined catalyst sites is critical to leveraging unique opportunities of these materials as catalysts. Understanding the rates of substrate diffusion in MOFs is challenging, and few in situ chemical tools are available to evaluate substrate diffusion during interstitial MOF chemistry. Herein, we demonstrate nitrogen atom transfer (NAT) from a lattice-confined Ru2 nitride to toluene to generate benzylamine. We use the comparison of the intramolecular deuterium kinetic isotope effect (KIE), determined for amination of a partially deuterated substrate, with the intermolecular KIE, determined by competitive amination of a mixture of perdeuterated and undeuterated substrates, to establish the relative rates of substrate diffusion and interstitial chemistry. We anticipate that the developed KIE-based experiments will contribute to the development of porous materials for group-transfer catalysis.

Direct Characterization of a Reactive Lattice-Confined Ru2 Nitride by Photocrystallography.

Reactive metal-ligand (M-L) multiply bonded complexes are ubiquitous intermediates in redox catalysis and have thus been long-standing targets of synthetic chemistry. The intrinsic reactivity of mid-to-late M-L multiply bonded complexes renders these structures challenging to isolate and structurally characterize. Although synthetic tuning of the ancillary ligand field can stabilize M-L multiply bonded complexes and result in isolable complexes, these efforts inevitably attenuate the reactivity of the M-L multiple bond. Here, we report the first direct characterization of a reactive Ru2 nitride intermediate by photocrystallography. Photogeneration of reactive M-L multiple bonds within crystalline matrices supports direct characterization of these critical intermediates without synthetic derivatization.