De novo design of luciferases using deep learning.

De novo enzyme design has sought to introduce active sites and substrate-binding pockets that are predicted to catalyse a reaction of interest into geometrically compatible native scaffolds1,2, but has been limited by a lack of suitable protein structures and the complexity of native protein sequence-structure relationships. Here we describe a deep-learning-based 'family-wide hallucination' approach that generates large numbers of idealized protein structures containing diverse pocket shapes and designed sequences that encode them. We use these scaffolds to design artificial luciferases that selectively catalyse the oxidative chemiluminescence of the synthetic luciferin substrates diphenylterazine3 and 2-deoxycoelenterazine. The designed active sites position an arginine guanidinium group adjacent to an anion that develops during the reaction in a binding pocket with high shape complementarity. For both luciferin substrates, we obtain designed luciferases with high selectivity; the most active of these is a small (13.9 kDa) and thermostable (with a melting temperature higher than 95 °C) enzyme that has a catalytic efficiency on diphenylterazine (kcat/Km = 106 M-1 s-1) comparable to that of native luciferases, but a much higher substrate specificity. The creation of highly active and specific biocatalysts from scratch with broad applications in biomedicine is a key milestone for computational enzyme design, and our approach should enable generation of a wide range of luciferases and other enzymes.

Molecular editing of aza-arene C-H bonds by distance, geometry and chirality.

Direct molecular editing of heteroarene carbon-hydrogen (C-H) bonds through consecutive selective C-H functionalization has the potential to grant rapid access into diverse chemical spaces, which is a valuable but often challenging venture to achieve in medicinal chemistry1. In contrast to electronically biased heterocyclic C-H bonds2-9, remote benzocyclic C-H bonds on bicyclic aza-arenes are especially difficult to differentiate because of the lack of intrinsic steric/electronic biases10-12. Here we report two conceptually distinct directing templates that enable the modular differentiation and functionalization of adjacent remote (C6 versus C7) and positionally similar (C3 versus C7) positions on bicyclic aza-arenes through careful modulation of distance, geometry and previously unconsidered chirality in template design. This strategy enables direct C-H olefination, alkynylation and allylation at adjacent C6 and C7 positions of quinolines in the presence of a competing C3 position that is spatially similar to C7. Notably, such site-selective, iterative and late-stage C-H editing of quinoline-containing pharmacophores can be performed in a modular fashion in different orders to suit bespoke synthetic applications. This Article, in combination with previously reported complementary methods, now fully establishes a unified late-stage 'molecular editing' strategy to directly modify bicyclic aza-arenes at any given site in different orders.

Cleaving arene rings for acyclic alkenylnitrile synthesis.

Synthetic chemistry is built around the formation of carbon-carbon bonds. However, the development of methods for selective carbon-carbon bond cleavage is a largely unmet challenge1-6. Such methods will have promising applications in synthesis, coal liquefaction, petroleum cracking, polymer degradation and biomass conversion. For example, aromatic rings are ubiquitous skeletal features in inert chemical feedstocks, but are inert to many reaction conditions owing to their aromaticity and low polarity. Over the past century, only a few methods under harsh conditions have achieved direct arene-ring modifications involving the cleavage of inert aromatic carbon-carbon bonds7,8, and arene-ring-cleavage reactions using stoichiometric transition-metal complexes or enzymes in bacteria are still limited9-11. Here we report a copper-catalysed selective arene-ring-opening reaction strategy. Our aerobic oxidative copper catalyst converts anilines, arylboronic acids, aryl azides, aryl halides, aryl triflates, aryl trimethylsiloxanes, aryl hydroxamic acids and aryl diazonium salts into alkenyl nitriles through selective carbon-carbon bond cleavage of arene rings. This chemistry was applied to the modification of polycyclic aromatics and the preparation of industrially important hexamethylenediamine and adipic acid derivatives. Several examples of the late-stage modification of complex molecules and fused ring compounds further support the potential broad utility of this methodology.

Intercepting fleeting cyclic allenes with asymmetric nickel catalysis.

Strained cyclic organic molecules, such as arynes, cyclic alkynes and cyclic allenes, have intrigued chemists for more than a century with their unusual structures and high chemical reactivity1. The considerable ring strain (30-50 kilocalories per mole)2,3 that characterizes these transient intermediates imparts high reactivity in many reactions, including cycloadditions and nucleophilic trappings, often generating structurally complex products4. Although strategies to control absolute stereochemistry in these reactions have been reported using stoichiometric chiral reagents5,6, catalytic asymmetric variants to generate enantioenriched products have remained difficult to achieve. Here we report the interception of racemic cyclic allene intermediates in a catalytic asymmetric reaction and provide evidence for two distinct mechanisms that control absolute stereochemistry in such transformations: kinetic differentiation of allene enantiomers and desymmetrization of intermediate π-allylnickel complexes. Computational studies implicate a catalytic mechanism involving initial kinetic differentiation of the cyclic allene enantiomers through stereoselective olefin insertion, loss of the resultant stereochemical information, and subsequent introduction of absolute stereochemistry through desymmetrization of an intermediate π-allylnickel complex. These results reveal reactivity that is available to cyclic allenes beyond the traditional cycloadditions and nucleophilic trappings previously reported, thus expanding the types of product accessible from this class of intermediates. Additionally, our computational studies suggest two potential strategies for stereocontrol in reactions of cyclic allenes. Combined, these results lay the foundation for the development of catalytic asymmetric reactions involving these classically avoided strained intermediates.

An enzymatic Alder-ene reaction.

An ongoing challenge in chemical research is to design catalysts that select the outcomes of the reactions of complex molecules. Chemists rely on organocatalysts or transition metal catalysts to control stereoselectivity, regioselectivity and periselectivity (selectivity among possible pericyclic reactions). Nature achieves these types of selectivity with a variety of enzymes such as the recently discovered pericyclases-a family of enzymes that catalyse pericyclic reactions1. Most characterized enzymatic pericyclic reactions have been cycloadditions, and it has been difficult to rationalize how the observed selectivities are achieved2-13. Here we report the discovery of two homologous groups of pericyclases that catalyse distinct reactions: one group catalyses an Alder-ene reaction that was, to our knowledge, previously unknown in biology; the second catalyses a stereoselective hetero-Diels-Alder reaction. Guided by computational studies, we have rationalized the observed differences in reactivities and designed mutant enzymes that reverse periselectivities from Alder-ene to hetero-Diels-Alder and vice versa. A combination of in vitro biochemical characterizations, computational studies, enzyme co-crystal structures, and mutational studies illustrate how high regioselectivity and periselectivity are achieved in nearly identical active sites.

Biochemical and structural characterization of a sphingomonad diarylpropane lyase for cofactorless deformylation.

Lignin valorization is being intensely pursued via tandem catalytic depolymerization and biological funneling to produce single products. In many lignin depolymerization processes, aromatic dimers and oligomers linked by carbon-carbon bonds remain intact, necessitating the development of enzymes capable of cleaving these compounds to monomers. Recently, the catabolism of erythro-1,2-diguaiacylpropane-1,3-diol (erythro-DGPD), a ring-opened lignin-derived ß-1 dimer, was reported in Novosphingobium aromaticivorans. The first enzyme in this pathway, LdpA (formerly LsdE), is a member of the nuclear transport factor 2 (NTF-2)-like structural superfamily that converts erythro-DGPD to lignostilbene through a heretofore unknown mechanism. In this study, we performed biochemical, structural, and mechanistic characterization of the N. aromaticivorans LdpA and another homolog identified in Sphingobium sp. SYK-6, for which activity was confirmed in vivo. For both enzymes, we first demonstrated that formaldehyde is the C1 reaction product, and we further demonstrated that both enantiomers of erythro-DGPD were transformed simultaneously, suggesting that LdpA, while diastereomerically specific, lacks enantioselectivity. We also show that LdpA is subject to a severe competitive product inhibition by lignostilbene. Three-dimensional structures of LdpA were determined using X-ray crystallography, including substrate-bound complexes, revealing several residues that were shown to be catalytically essential. We used density functional theory to validate a proposed mechanism that proceeds via dehydroxylation and formation of a quinone methide intermediate that serves as an electron sink for the ensuing deformylation. Overall, this study expands the range of chemistry catalyzed by the NTF-2-like protein family to a prevalent lignin dimer through a cofactorless deformylation reaction.

Origin of iodine preferential attack at sulfur in phosphorothioate and subsequent P-O or P-S bond dissociation.

Iodine-induced cleavage at phosphorothioate DNA (PT-DNA) is characterized by extremely high sensitivity (∼1 phosphorothioate link per 106 nucleotides), which has been used for detecting and sequencing PT-DNA in bacteria. Despite its foreseeable potential for wide applications, the cleavage mechanism at the PT-modified site has not been well established, and it remains unknown as to whether or not cleavage of the bridging P-O occurs at every PT-modified site. In this work, we conducted accurate ωB97X-D calculations and high-performance liquid chromatography-mass spectrometry to investigate the process of PT-DNA cleavage at the atomic and molecular levels. We have found that iodine chemoselectively binds to the sulfur atom of the phosphorothioate link via a strong halogen-chalcogen interaction (a type of halogen bond, with binding affinity as high as 14.9 kcal/mol) and thus triggers P-O bond cleavage via phosphotriester-like hydrolysis. Additionally, aside from cleavage of the bridging P-O bond, the downstream hydrolyses lead to unwanted P-S/P-O conversions and a loss of the phosphorothioate handle. The mechanism we outline helps to explain specific selectivity at the PT-modified site but also predicts the dynamic stoichiometry of P-S and P-O bond breaking. For instance, Tris is involved in the cascade derivation of S-iodo-phosphorothioate to S-amino-phosphorothioate, suppressing the S-iodo-phosphorothioate hydrolysis to a phosphate diester. However, hydrolysis of one-third of the Tris-O-grafting phosphotriester results in unwanted P-S/P-O conversions. Our study suggests that bacterial DNA phosphorothioation may more frequently occur than previous bioinformatic estimations have predicted from iodine-induced deep sequencing data.

Low-Temperature Mineralization of Fluorotelomers with Diverse Polar Head Groups.

Per- and polyfluoroalkyl substances (PFAS) are persistent environmental pollutants linked to harmful health effects. Currently employed PFAS destruction methods are energy-intensive and often produce shorter-chain and recalcitrant partially fluorinated byproducts. We report the mineralization of five fluorotelomer compounds via a base-mediated degradation using NaOH and mild temperatures (120 °C) in a mixture of DMSO:H2O (8:1 v/v). The studied fluorotelomers have varying polar head groups-carboxylic acids, sulfonic acids, alcohols, and phosphonic acids, which are the most common polar head groups used in commercial and industrial applications. The degradation intermediates and byproducts were characterized using 1H, 13C, and 19F NMR spectroscopy. Density functional theory computations at the M06-2X/6-311 + G(2d,p)-SMD-(DMSO) level were consistent with the observed intermediates and guided an overall mechanistic hypothesis. Degradation of each fluorotelomer occurs through a similar process, in which the nonfluorinated carbons and the first fluorinated carbon are cleaved from the remaining perfluoroalkyl fragment, which degrades through previously identified pathways. These findings provide important insight into PFAS degradation processes and suggest that PFAS containing at least one C-H bond within or adjacent to its fluoroalkyl chain can be degraded under these mild conditions. Many PFAS in current use as well as recalcitrant fluorinated byproducts generated from other PFAS degradation methods are candidates for this approach.

Iron Heme Enzyme-Catalyzed Cyclopropanations with Diazirines as Carbene Precursors: Computational Explorations of Diazirine Activation and Cyclopropanation Mechanism.

The mechanism of cyclopropanations with diazirines as air-stable and user-friendly alternatives to commonly employed diazo compounds within iron heme enzyme-catalyzed carbene transfer reactions has been studied by means of density functional theory (DFT) calculations of model systems, quantum mechanics/molecular mechanics (QM/MM) calculations, and molecular dynamics (MD) simulations of the iron carbene and the cyclopropanation transition state in the enzyme active site. The reaction is initiated by a direct diazirine-diazo isomerization occurring in the active site of the enzyme. In contrast, an isomerization mechanism proceeding via the formation of a free carbene intermediate in lieu of a direct, one-step isomerization process was observed for model systems. Subsequent reaction with benzyl acrylate takes place through stepwise C-C bond formation via a diradical intermediate, delivering the cyclopropane product. The origin of the observed diastereo- and enantioselectivity in the enzyme was investigated through MD simulations, which indicate a preferred formation of the cis-cyclopropane by steric control.

Photochemical Skeletal Editing of Pyridines to Bicyclic Pyrazolines and Pyrazoles.

We present an efficient one-pot photochemical skeletal editing protocol for the transformation of pyridines into diverse bicyclic pyrazolines and pyrazoles under mild conditions. The method requires no metals, photocatalysts, or additives and allows for the selective removal of specific carbon atoms from pyridines, allowing for unprecedented versatility. Our approach offers a convenient and efficient means for the late-stage modification of complex drug molecules by replacing the core pyridine skeleton. Moreover, we have successfully scaled up this procedure in stop-flow and flow-chemistry systems, showcasing its applicability to intricate transformations such as the Diels-Alder reaction, hydrogenation, [3 + 2] cycloaddition, and Heck reaction. Through control experiments and DFT calculations, we provide insights into the mechanistic underpinnings of this skeletal editing protocol.