Enzymatic assembly of carbon-carbon bonds via iron-catalysed sp3 C-H functionalization.

Although abundant in organic molecules, carbon-hydrogen (C-H) bonds are typically considered unreactive and unavailable for chemical manipulation. Recent advances in C-H functionalization technology have begun to transform this logic, while emphasizing the importance of and challenges associated with selective alkylation at a sp3 carbon1,2. Here we describe iron-based catalysts for the enantio-, regio- and chemoselective intermolecular alkylation of sp3 C-H bonds through carbene C-H insertion. The catalysts, derived from a cytochrome P450 enzyme in which the native cysteine axial ligand has been substituted for serine (cytochrome P411), are fully genetically encoded and produced in bacteria, where they can be tuned by directed evolution for activity and selectivity. That these proteins activate iron, the most abundant transition metal, to perform this chemistry provides a desirable alternative to noble-metal catalysts, which have dominated the field of C-H functionalization1,2. The laboratory-evolved enzymes functionalize diverse substrates containing benzylic, allylic or α-amino C-H bonds with high turnover and excellent selectivity. Furthermore, they have enabled the development of concise routes to several natural products. The use of the native iron-haem cofactor of these enzymes to mediate sp3 C-H alkylation suggests that diverse haem proteins could serve as potential catalysts for this abiological transformation, and will facilitate the development of new enzymatic C-H functionalization reactions for applications in chemistry and synthetic biology.

Genetically programmed chiral organoborane synthesis.

Recent advances in enzyme engineering and design have expanded nature's catalytic repertoire to functions that are new to biology. However, only a subset of these engineered enzymes can function in living systems. Finding enzymatic pathways that form chemical bonds that are not found in biology is particularly difficult in the cellular environment, as this depends on the discovery not only of new enzyme activities, but also of reagents that are both sufficiently reactive for the desired transformation and stable in vivo. Here we report the discovery, evolution and generalization of a fully genetically encoded platform for producing chiral organoboranes in bacteria. Escherichia coli cells harbouring wild-type cytochrome c from Rhodothermus marinus (Rma cyt c) were found to form carbon-boron bonds in the presence of borane-Lewis base complexes, through carbene insertion into boron-hydrogen bonds. Directed evolution of Rma cyt c in the bacterial catalyst provided access to 16 novel chiral organoboranes. The catalyst is suitable for gram-scale biosynthesis, providing up to 15,300 turnovers, a turnover frequency of 6,100 h-1, a 99:1 enantiomeric ratio and 100% chemoselectivity. The enantiopreference of the biocatalyst could also be tuned to provide either enantiomer of the organoborane products. Evolved in the context of whole-cell catalysts, the proteins were more active in the whole-cell system than in purified forms. This study establishes a DNA-encoded and readily engineered bacterial platform for borylation; engineering can be accomplished at a pace that rivals the development of chemical synthetic methods, with the ability to achieve turnovers that are two orders of magnitude (over 400-fold) greater than those of known chiral catalysts for the same class of transformation. This tunable method for manipulating boron in cells could expand the scope of boron chemistry in living systems.

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.

High Throughput Screening with SAMDI Mass Spectrometry for Directed Evolution.

Advances in directed evolution have led to an exploration of new and important chemical transformations; however, many of these efforts still rely on the use of low-throughput chromatography-based screening methods. We present a high-throughput strategy for screening libraries of enzyme variants for improved activity. Unpurified reaction products are immobilized to a self-assembled monolayer and analyzed by mass spectrometry, allowing for direct evaluation of thousands of variants in under an hour. The method was demonstrated with libraries of randomly mutated cytochrome P411 variants to identify improved catalysts for C-H alkylation. The technique may be tailored to evolve enzymatic activity for a variety of transformations where higher throughput is needed.

Oxygen Activation and Radical Transformations in Heme Proteins and Metalloporphyrins.

As a result of the adaptation of life to an aerobic environment, nature has evolved a panoply of metalloproteins for oxidative metabolism and protection against reactive oxygen species. Despite the diverse structures and functions of these proteins, they share common mechanistic grounds. An open-shell transition metal like iron or copper is employed to interact with O2 and its derived intermediates such as hydrogen peroxide to afford a variety of metal-oxygen intermediates. These reactive intermediates, including metal-superoxo, -(hydro)peroxo, and high-valent metal-oxo species, are the basis for the various biological functions of O2-utilizing metalloproteins. Collectively, these processes are called oxygen activation. Much of our understanding of the reactivity of these reactive intermediates has come from the study of heme-containing proteins and related metalloporphyrin compounds. These studies not only have deepened our understanding of various functions of heme proteins, such as O2 storage and transport, degradation of reactive oxygen species, redox signaling, and biological oxygenation, etc., but also have driven the development of bioinorganic chemistry and biomimetic catalysis. In this review, we survey the range of O2 activation processes mediated by heme proteins and model compounds with a focus on recent progress in the characterization and reactivity of important iron-oxygen intermediates. Representative reactions initiated by these reactive intermediates as well as some context from prior decades will also be presented. We will discuss the fundamental mechanistic features of these transformations and delineate the underlying structural and electronic factors that contribute to the spectrum of reactivities that has been observed in nature as well as those that have been invented using these paradigms. Given the recent developments in biocatalysis for non-natural chemistries and the renaissance of radical chemistry in organic synthesis, we envision that new enzymatic and synthetic transformations will emerge based on the radical processes mediated by metalloproteins and their synthetic analogs.

Enantiodivergent α-Amino C-H Fluoroalkylation Catalyzed by Engineered Cytochrome P450s.

The introduction of fluoroalkyl groups into organic compounds can significantly alter pharmacological characteristics. One enabling but underexplored approach for the installation of fluoroalkyl groups is selective C( sp3)-H functionalization due to the ubiquity of C-H bonds in organic molecules. We have engineered heme enzymes that can insert fluoroalkyl carbene intermediates into α-amino C( sp3)-H bonds and enable enantiodivergent synthesis of fluoroalkyl-containing molecules. Using directed evolution, we engineered cytochrome P450 enzymes to catalyze this abiological reaction under mild conditions with total turnovers (TTN) up to 4070 and enantiomeric excess (ee) up to 99%. The iron-heme catalyst is fully genetically encoded and configurable by directed evolution so that just a few mutations to the enzyme completely inverted product enantioselectivity. These catalysts provide a powerful method for synthesis of chiral organofluorine molecules that is currently not possible with small-molecule catalysts.

The Enigmatic P450 Decarboxylase OleT Is Capable of, but Evolved To Frustrate, Oxygen Rebound Chemistry.

OleT is a cytochrome P450 enzyme that catalyzes the removal of carbon dioxide from variable chain length fatty acids to form 1-alkenes. In this work, we examine the binding and metabolic profile of OleT with shorter chain length (n ≤ 12) fatty acids that can form liquid transportation fuels. Transient kinetics and product analyses confirm that OleT capably activates hydrogen peroxide with shorter substrates to form the high-valent intermediate Compound I and largely performs C-C bond scission. However, the enzyme also produces fatty alcohol side products using the high-valent iron oxo chemistry commonly associated with insertion of oxygen into hydrocarbons. When presented with a short chain fatty acid that can initiate the formation of Compound I, OleT oxidizes the diagnostic probe molecules norcarane and methylcyclopropane in a manner that is reminiscent of reactions of many CYP hydroxylases with radical clock substrates. These data are consistent with a decarboxylation mechanism in which Compound I abstracts a substrate hydrogen atom in the initial step. Positioning of the incipient substrate radical is a crucial element in controlling the efficiency of activated OH rebound.

Alkyl Isocyanates via Manganese-Catalyzed C-H Activation for the Preparation of Substituted Ureas.

Organic isocyanates are versatile intermediates that provide access to a wide range of functionalities. In this work, we have developed the first synthetic method for preparing aliphatic isocyanates via direct C-H activation. This method proceeds efficiently at room temperature and can be applied to functionalize secondary, tertiary, and benzylic C-H bonds with good yields and functional group compatibility. Moreover, the isocyanate products can be readily converted to substituted ureas without isolation, demonstrating the synthetic potential of the method. To study the reaction mechanism, we have synthesized and characterized a rare MnIV-NCO intermediate and demonstrated its ability to transfer the isocyanate moiety to alkyl radicals. Using EPR spectroscopy, we have directly observed a MnIV intermediate under catalytic conditions. Isocyanation of celestolide with a chiral manganese salen catalyst followed by trapping with aniline afforded the urea product in 51% enantiomeric excess. This represents the only example of an asymmetric synthesis of an organic urea via C-H activation. When combined with our DFT calculations, these results clearly demonstrate that the C-NCO bond was formed through capture of a substrate radical by a MnIV-NCO intermediate.

Beyond ferryl-mediated hydroxylation: 40 years of the rebound mechanism and C-H activation.

Since our initial report in 1976, the oxygen rebound mechanism has become the consensus mechanistic feature for an expanding variety of enzymatic C-H functionalization reactions and small molecule biomimetic catalysts. For both the biotransformations and models, an initial hydrogen atom abstraction from the substrate (R-H) by high-valent iron-oxo species (Fen=O) generates a substrate radical and a reduced iron hydroxide, [Fen-1-OH ·R]. This caged radical pair then evolves on a complicated energy landscape through a number of reaction pathways, such as oxygen rebound to form R-OH, rebound to a non-oxygen atom affording R-X, electron transfer of the incipient radical to yield a carbocation, R+, desaturation to form olefins, and radical cage escape. These various flavors of the rebound process, often in competition with each other, give rise to the wide range of C-H functionalization reactions performed by iron-containing oxygenases. In this review, we first recount the history of radical rebound mechanisms, their general features, and key intermediates involved. We will discuss in detail the factors that affect the behavior of the initial caged radical pair and the lifetimes of the incipient substrate radicals. Several representative examples of enzymatic C-H transformations are selected to illustrate how the behaviors of the radical pair [Fen-1-OH ·R] determine the eventual reaction outcome. Finally, we discuss the powerful potential of "radical rebound" processes as a general paradigm for developing novel C-H functionalization reactions with synthetic, biomimetic catalysts. We envision that new chemistry will continue to arise by bridging enzymatic "radical rebound" with synthetic organic chemistry.

Manganese-catalyzed late-stage aliphatic C-H azidation.

We report a manganese-catalyzed aliphatic C-H azidation reaction that can efficiently convert secondary, tertiary, and benzylic C-H bonds to the corresponding azides. The method utilizes aqueous sodium azide solution as the azide source and can be performed under air. Besides its operational simplicity, the potential of this method for late-stage functionalization has been demonstrated by successful azidation of various bioactive molecules with yields up to 74%, including the important drugs pregabalin, memantine, and the antimalarial artemisinin. Azidation of celestolide with a chiral manganese salen catalyst afforded the azide product in 70% ee, representing a Mn-catalyzed enantioselective aliphatic C-H azidation reaction. Considering the versatile roles of organic azides in modern chemistry and the ubiquity of aliphatic C-H bonds in organic molecules, we envision that this Mn-azidation method will find wide application in organic synthesis, drug discovery, and chemical biology.

Targeted fluorination with the fluoride ion by manganese-catalyzed decarboxylation.

We describe the first catalytic decarboxylative fluorination reaction based on the nucleophilic fluoride ion. The reported method allows the facile replacement of various aliphatic carboxylic acid groups with fluorine. Moreover, the potential of this method for PET imaging has been demonstrated by the successful (18) F labeling of a variety of carboxylic acids with radiochemical conversions up to 50 %, representing a targeted decarboxylative (18) F labeling method with no-carrier-added [(18) F]fluoride. Mechanistic probes suggest that the reaction proceeds through the interaction of the manganese catalyst with iodine(III) carboxylates formed in situ from iodosylbenzene and the carboxylic acid substrates.

Late stage benzylic C-H fluorination with [¹8F]fluoride for PET imaging.

We describe the first late-stage (18)F labeling chemistry for aliphatic C-H bonds with no-carrier-added [(18)F]fluoride. The method uses Mn(salen)OTs as an F-transfer catalyst and enables the facile labeling of a variety of bioactive molecules and building blocks with radiochemical yields (RCY) ranging from 20% to 72% within 10 min without the need for preactivation of the labeling precursor. Notably, the catalyst itself can directly elute [(18)F]fluoride from an ion exchange cartridge with over 90% efficiency. Using this feature, the conventional and laborious dry-down step prior to reaction is circumvented, greatly simplifying the mechanics of this protocol and shortening the time for automated synthesis. Eight drug molecules, including COX, ACE, MAO, and PDE inhibitors, have been successfully [(18)F]-labeled in this way.

Radical fluorine transfer catalysed by an engineered nonheme iron enzyme.

Nonheme iron enzymes stand out as one of the most versatile biocatalysts for molecular functionalization. They facilitate a wide array of chemical transformations within biological processes, including hydroxylation, chlorination, epimerization, desaturation, cyclization, and more. Beyond their native biological functions, these enzymes possess substantial potential as powerful biocatalytic platforms for achieving abiological metal-catalyzed reactions, owing to their functional and structural diversity and high evolvability. To this end, our group has recently engineered a series of nonheme iron enzymes to employ non-natural radical-relay mechanisms for abiological radical transformations not previously known in biology. Notably, we have demonstrated that a nonheme iron enzyme, (S)-2-hydroxypropylphosphonate epoxidase from Streptomyces viridochromogenes (SvHppE), can be repurposed into an efficient and selective biocatalyst for radical fluorine transfer reactions. This marks the first known instance of a redox enzymatic process for C(sp3)F bond formation. This chapter outlines the detailed experimental protocol for engineering SvHPPE for fluorination reactions. Furthermore, the provided protocol could serve as a general guideline that might facilitate other engineering endeavors targeting nonheme iron enzymes for novel catalytic functions.

Precious metal clusters as fundamental agents in bioimaging usability.

Fluorescent nanomaterials (NMs) are widely used in imaging techniques in biomedical research. Especially in bioimaging systems, with the rapid development of imaging nanotechnology, precious metal clusters such as Au, Ag, and Cu NMs have emerged with different functional agents for biomedical applications. Compared with traditional fluorescent molecules, precious metal clusters have the advantages of high optical stability, easy regulation of shape and size, and multifunctionalization. In addition, NMs possess strong photoluminescent properties with good photostability, high release rate, and sub-nanometer size. They could be treated as fundamental agents in bioimaging usability. This review summarizes the recent advances in bioimaging utilization, it conveys that metal clusters refer to Au, Ag, and Cu fluorescent clusters and could provide a generalized overview of their full applications. It includes optical property measurement, precious metal clusters in bioimaging systems, and a rare earth element-doped heterogeneous structure illustrated in biomedical imaging with specific examples, that provide new and innovative ideas for fluorescent NMs in the field of bioimaging usability.

Parallel and competitive pathways for substrate desaturation, hydroxylation, and radical rearrangement by the non-heme diiron hydroxylase AlkB.

A purified and highly active form of the non-heme diiron hydroxylase AlkB was investigated using the diagnostic probe substrate norcarane. The reaction afforded C2 (26%) and C3 (43%) hydroxylation and desaturation products (31%). Initial C-H cleavage at C2 led to 7% C2 hydroxylation and 19% 3-hydroxymethylcyclohexene, a rearrangement product characteristic of a radical rearrangement pathway. A deuterated substrate analogue, 3,3,4,4-norcarane-d(4), afforded drastically reduced amounts of C3 alcohol (8%) and desaturation products (5%), while the radical rearranged alcohol was now the major product (65%). This change in product ratios indicates a large kinetic hydrogen isotope effect of ∼20 for both the C-H hydroxylation at C3 and the desaturation pathway, with all of the desaturation originating via hydrogen abstraction at C3 and not C2. The data indicate that AlkB reacts with norcarane via initial C-H hydrogen abstraction from C2 or C3 and that the three pathways, C3 hydroxylation, C3 desaturation, and C2 hydroxylation/radical rearrangement, are parallel and competitive. Thus, the incipient radical at C3 either reacts with the iron-oxo center to form an alcohol or proceeds along the desaturation pathway via a second H-abstraction to afford both 2-norcarene and 3-norcarene. Subsequent reactions of these norcarenes lead to detectable amounts of hydroxylation products and toluene. By contrast, the 2-norcaranyl radical intermediate leads to C2 hydroxylation and the diagnostic radical rearrangement, but this radical apparently does not afford desaturation products. The results indicate that C-H hydroxylation and desaturation follow analogous stepwise reaction channels via carbon radicals that diverge at the product-forming step.

Hydride dissociation energies of six-membered heterocyclic organic hydrides predicted by ONIOM-G4Method.

Hydride dissociation energy is of great importance in understanding the hydride-donating abilities of organic hydrides. Although the hydride dissociation energies of some organic hydrides have been experimentally measured, much less attention has been focused on the investigation of these quantities from the first principles of physics. Herein, we developed an ONIOM-G4 method and carefully benchmarked this new method against 48 experimental hydride dissociation energies of diverse bulky molecules. It was found that with the combined methods of the HF/6-31+G(d,p)//IEFPCM/Bondi1.15 solvation model, the ONIOM-G4 method can predict the hydride dissociation energies with an error bar of only 1.7 kcal/mol. With the newly developed ONIOM-G4 method, we then systematically studied the hydride dissociation energies of six categories of biologically and pharmaceutically important six-membered heterocyclic organic hydrides, namely, the organic hydrides containing 1,4-dihydropyridine, 1,4-dihydropyrazine, 1,4-oxazine, 1,4-thiazine, 4H-pyran, and 4H-thiopyran ring structures. An extensive hydride dissociation energy scale containing over 100 six-memebered heterocyclic organic hydrides has been established, which may find applications in both synthetic organic chemistry and mechanistic studies of various chemical or biological processes involving transferring of the hydride anion.

Directed evolution of nonheme iron enzymes to access abiological radical-relay C(sp3)-H azidation.

We report the reprogramming of nonheme iron enzymes to catalyze an abiological C(sp3)‒H azidation reaction through iron-catalyzed radical relay. This biocatalytic transformation uses amidyl radicals as hydrogen atom abstractors and Fe(III)‒N3 intermediates as radical trapping agents. We established a high-throughput screening platform based on click chemistry for rapid evolution of the catalytic performance of identified enzymes. The final optimized variants deliver a range of azidation products with up to 10,600 total turnovers and 93% enantiomeric excess. Given the prevalence of radical relay reactions in organic synthesis and the diversity of nonheme iron enzymes, we envision that this discovery will stimulate future development of metalloenzyme catalysts for synthetically useful transformations unexplored by natural evolution.

Theoretical study on acidities of (S)-proline amide derivatives in DMSO and its implications for organocatalysis.

The acidities (pK(a) values) of proline amide derivatives are of great importance for understanding the catalytic activity of proline-based organocatalysts. The development of new catalysts could also benefit from the systematic study of the pK(a) values of these compounds. However, only a few pK(a) values of the proline-based organocatalysts are currently available due to the difficulty in experimentally measurements. In this work, we set out to study the pK(a) values of various proline amide derivatives with theoretical calculations. Different theoretical methods were evaluated and the combined method, B3PW91/6-311++G(3df,2p)//B3LYP/6-31+G(d)//HF//CPCM/UA0, was found to be the best one in reproducing the pK(a) values of structurally unrelated amides and amide derivatives in DMSO. The MAD and RMSE of the newly developed theoretical model equal to 0.98 and 1.3 pK units, respectively. The method also enabled the systematically study on various structural effects on pK(a) values of proline amide derivatives, such as the ZE-isomerization, remote substitution, and alpha-substitution effects, for the first time. The pK(a) values of a series of chiral amides were also studied in this work. Finally, we applied the theoretical method to predict a large number of proline-based organocatalysts and established an extensive acidity scale of the compounds.

A theoretical study on C-COOH homolytic bond dissociation enthalpies.

The knowledge of C-COOH homolytic bond dissociation enthalpies (BDEs) is of great importance in understanding various chemical and biochemical processes involving the decarboxylation reaction. In the present study, the density functional theory (DFT method), B3P86/6-311++G(2df,2p)//B3LYP/6-31+G(d), is found to be reliable to predict the C-COOH BDE of various structurally unrelated carboxylic acids. The mean absolute deviation (MAD) and root-mean-square deviation (rmsd) of this optimal method are equal to 2.0 and 2.5 kcal/mol, respectively. With the authorized theoretical protocol in hand, an extensive C-COOH BDE scale containing over 100 carboxylic acids has been established. The availability of this body of data enabled a detailed investigation of remote substituent effect on four types of carboxylic acids, including para-substituted benzoic acid, beta-substituted cis-propenoic acid, beta-substituted trans-propenoic acid, and substituted propiolic acid. Also with the C-COOH BDE data obtained in this work, an excellent linear relationship has been found between the C-COOH BDE of carboxylic acids and the C-H BDE of their hydrocarbon analogues. After comparing the energy barrier of the Pd-catalyzed decarboxylation reaction (DeltaG(decarboxylation)++) with the related C-COOH BDE, a negative correlation between the DeltaG(decarboxylation)++ and the C-COOH BDE was found.