Oxidative cyclization reagents reveal tryptophan cation-π interactions.

Methods for selective covalent modification of amino acids on proteins can enable a diverse array of applications, spanning probes and modulators of protein function to proteomics1-3. Owing to their high nucleophilicity, cysteine and lysine residues are the most common points of attachment for protein bioconjugation chemistry through acid-base reactivity3,4. Here we report a redox-based strategy for bioconjugation of tryptophan, the rarest amino acid, using oxaziridine reagents that mimic oxidative cyclization reactions in indole-based alkaloid biosynthetic pathways to achieve highly efficient and specific tryptophan labelling. We establish the broad use of this method, termed tryptophan chemical ligation by cyclization (Trp-CLiC), for selectively appending payloads to tryptophan residues on peptides and proteins with reaction rates that rival traditional click reactions and enabling global profiling of hyper-reactive tryptophan sites across whole proteomes. Notably, these reagents reveal a systematic map of tryptophan residues that participate in cation-π interactions, including functional sites that can regulate protein-mediated phase-separation processes.

A Transfer Hydrogenation Approach to Activity-Based Sensing of Formate in Living Cells.

Formate is a major reactive carbon species in one-carbon metabolism, where it serves as an endogenous precursor for amino acid and nucleic acid biosynthesis and a cellular source of NAD(P)H. On the other hand, aberrant elevations in cellular formate are connected to progression of serious diseases, including cancer and Alzheimer's disease. Traditional methods for formate detection in biological environments often rely on sample destruction or extensive processing, resulting in a loss of spatiotemporal information. To help address these limitations, here we present the design, synthesis, and biological evaluation of a first-generation activity-based sensing system for live-cell formate imaging that relies on iridium-mediated transfer hydrogenation chemistry. Formate facilitates an aldehyde-to-alcohol conversion on various fluorophore scaffolds to enable fluorescence detection of this one-carbon unit, including through a two-color ratiometric response with internal calibration. The resulting two-component probe system can detect changes in formate levels in living cells with a high selectivity over potentially competing biological analytes. Moreover, this activity-based sensing system can visualize changes in endogenous formate fluxes through alterations of one-carbon pathways in cell-based models of human colon cancer, presaging the potential utility of this chemical approach to probe the continuum between one-carbon metabolism and signaling in cancer and other diseases.

Distinct RNA N-demethylation pathways catalyzed by nonheme iron ALKBH5 and FTO enzymes enable regulation of formaldehyde release rates.

The AlkB family of nonheme Fe(II)/2-oxoglutarate-dependent oxygenases are essential regulators of RNA epigenetics by serving as erasers of one-carbon marks on RNA with release of formaldehyde (FA). Two major human AlkB family members, FTO and ALKBH5, both act as oxidative demethylases of N6-methyladenosine (m6A) but furnish different major products, N6-hydroxymethyladenosine (hm6A) and adenosine (A), respectively. Here we identify foundational mechanistic differences between FTO and ALKBH5 that promote these distinct biochemical outcomes. In contrast to FTO, which follows a traditional oxidative N-demethylation pathway to catalyze conversion of m6A to hm6A with subsequent slow release of A and FA, we find that ALKBH5 catalyzes a direct m6A-to-A transformation with rapid FA release. We identify a catalytic R130/K132/Y139 triad within ALKBH5 that facilitates release of FA via an unprecedented covalent-based demethylation mechanism with direct detection of a covalent intermediate. Importantly, a K132Q mutant furnishes an ALKBH5 enzyme with an m6A demethylation profile that resembles that of FTO, establishing the importance of this residue in the proposed covalent mechanism. Finally, we show that ALKBH5 is an endogenous source of FA in the cell by activity-based sensing of FA fluxes perturbed via ALKBH5 knockdown. This work provides a fundamental biochemical rationale for nonredundant roles of these RNA demethylases beyond different substrate preferences and cellular localization, where m6A demethylation by ALKBH5 versus FTO results in release of FA, an endogenous one-carbon unit but potential genotoxin, at different rates in living systems.

An Activity-Based Oxaziridine Platform for Identifying and Developing Covalent Ligands for Functional Allosteric Methionine Sites: Redox-Dependent Inhibition of Cyclin-Dependent Kinase 4.

Activity-based protein profiling (ABPP) is a versatile strategy for identifying and characterizing functional protein sites and compounds for therapeutic development. However, the vast majority of ABPP methods for covalent drug discovery target highly nucleophilic amino acids such as cysteine or lysine. Here, we report a methionine-directed ABPP platform using Redox-Activated Chemical Tagging (ReACT), which leverages a biomimetic oxidative ligation strategy for selective methionine modification. Application of ReACT to oncoprotein cyclin-dependent kinase 4 (CDK4) as a representative high-value drug target identified three new ligandable methionine sites. We then synthesized a methionine-targeting covalent ligand library bearing a diverse array of heterocyclic, heteroatom, and stereochemically rich substituents. ABPP screening of this focused library identified 1oxF11 as a covalent modifier of CDK4 at an allosteric M169 site. This compound inhibited kinase activity in a dose-dependent manner on purified protein and in breast cancer cells. Further investigation of 1oxF11 found prominent cation-π and H-bonding interactions stabilizing the binding of this fragment at the M169 site. Quantitative mass-spectrometry studies validated 1oxF11 ligation of CDK4 in breast cancer cell lysates. Further biochemical analyses revealed cross-talk between M169 oxidation and T172 phosphorylation, where M169 oxidation prevented phosphorylation of the activating T172 site on CDK4 and blocked cell cycle progression. By identifying a new mechanism for allosteric methionine redox regulation on CDK4 and developing a unique modality for its therapeutic intervention, this work showcases a generalizable platform that provides a starting point for engaging in broader chemoproteomics and protein ligand discovery efforts to find and target previously undruggable methionine sites.

Synthesis of (-)-Picrotoxinin by Late-Stage Strong Bond Activation.

We report a concise, stereocontrolled synthesis of the neurotoxic sesquiterpenoid (-)-picrotoxinin (1, PXN). The brevity of the route is due to regio- and stereoselective formation of the [4.3.0] bicyclic core by incorporation of a symmetrizing geminal dimethyl group at C5. Dimethylation then enables selective C-O bond formation in multiple intermediates. A series of strong bond (C-C and C-H) cleavages convert the C5 gem-dimethyl group to the C15 lactone of PXN.

Activity-Based Sensing: A Synthetic Methods Approach for Selective Molecular Imaging and Beyond.

Emerging from the origins of supramolecular chemistry and the development of selective chemical receptors that rely on lock-and-key binding, activity-based sensing (ABS)-which utilizes molecular reactivity rather than molecular recognition for analyte detection-has rapidly grown into a distinct field to investigate the production and regulation of chemical species that mediate biological signaling and stress pathways, particularly metal ions and small molecules. Chemical reactions exploit the diverse chemical reactivity of biological species to enable the development of selective and sensitive synthetic methods to decipher their contributions within complex living environments. The broad utility of this reaction-driven approach facilitates application to imaging platforms ranging from fluorescence, luminescence, photoacoustic, magnetic resonance, and positron emission tomography modalities. ABS methods are also being expanded to other fields, such as drug and materials discovery.

The High Chemofidelity of Metal-Catalyzed Hydrogen Atom Transfer.

The implementation of any chemical reaction in a structurally complex setting ( King , S. M. J. Org. Chem. 2014 , 79 , 8937 ) confronts structurally defined barriers: steric environment, functional group reactivity, product instability, and through-bond electronics. However, there are also practical barriers. Late-stage reactions conducted on small quantities of material are run inevitably at lower than optimal concentrations. Access to late-stage material limits extensive optimization. Impurities from past reactions can interfere, especially with catalytic reactions. Therefore, chemical reactions on which one can rely at the front lines of a complex synthesis campaign emerge from the crucible of total synthesis as robust, dependable, and widely applied. Trost conceptualized "chemoselectivity" as a reagent's selective reaction of one functional group or reactive site in preference to others ( Trost , B. M. Science 1983 , 219 , 245 ). Chemoselectivity and functional group tolerance can be evaluated quickly using robustness screens ( Collins , K. D. Nat. Chem. 2013 , 5 , 597 ). A reaction may also be characterized by its "chemofidelity", that is, its reliable reaction with a functional group in any molecular context. For example, ketone reduction by an electride (dissolving metal conditions) exhibits high chemofidelity but low chemoselectivity: it usually works, but many other functional groups are reduced at similar rates. Conversely, alkene coordination chemistry effected by π Lewis acids can exhibit high chemoselectivity ( Trost , B. M. Science 1983 , 219 , 245 ) but low chemofidelity: it can be highly selective for alkenes but sensitive to the substitution pattern ( Larionov , E. Chem. Commun. 2014 , 50 , 9816 ). In contrast, alkenes undergo reliable, robust, and diverse hydrogen atom transfer reactions from metal hydrides to generate carbon-centered radicals. Although there are many potential applications of this chemistry, its functional group tolerance, high rates, and ease of execution have led to its rapid deployment in complex synthesis campaigns. Its success derives from high chemofidelity, that is, its dependable reactivity in many molecular environments and with many alkene substitution patterns. Metal hydride H atom transfer (MHAT) reactions convert diverse, simple building blocks to more stereochemically and functionally dense products ( Crossley , S. W. M. Chem. Rev. 2016 , 116 , 8912 ). When hydrogen is returned to the metal, MHAT can be considered the radical equivalent of Brønsted acid catalysis-itself a broad reactivity paradigm. This Account summarizes our group's contributions to method development, reagent discovery, and mechanistic interrogation. Our earliest contribution to this area-a stepwise hydrogenation with high chemoselectivity and high chemofidelity-has found application to many problems. More recently, we reported the first examples of dual-catalytic cross-couplings that rely on the merger of MHAT cycles and nickel catalysis. With time, we anticipate that MHAT will become a staple of chemical synthesis.

Mn-, Fe-, and Co-Catalyzed Radical Hydrofunctionalizations of Olefins.

Cofactor-mimetic aerobic oxidation has conceptually merged with catalysis of syngas reactions to form a wide range of Markovnikov-selective olefin radical hydrofunctionalizations. We cover the development of the field and review contributions to reaction invention, mechanism, and application to complex molecule synthesis. We also provide a mechanistic framework for understanding this compendium of radical reactions.

Simple, chemoselective, catalytic olefin isomerization.

Catalytic amounts of Co(Sal(tBu,tBu))Cl and organosilane irreversibly isomerize terminal alkenes by one position. The same catalysts effect cycloisomerization of dienes and retrocycloisomerization of strained rings. Strong Lewis bases like amines and imidazoles, and labile functionalities like epoxides, are tolerated.

Simple, chemoselective hydrogenation with thermodynamic stereocontrol.

Few methods permit the hydrogenation of alkenes to a thermodynamically favored configuration when steric effects dictate the alternative trajectory of hydrogen delivery. Dissolving metal reduction achieves this control, but with extremely low functional group tolerance. Here we demonstrate a catalytic hydrogenation of alkenes that affords the thermodynamic alkane products with remarkably broad functional group compatibility and rapid reaction rates at standard temperature and pressure.

Synthesis of the Privileged 8-Arylmenthol Class by Radical Arylation of Isopulegol.

Hydrogen atom transfer (HAT) circumvents a disfavored Friedel-Crafts reaction in the derivatization of the inexpensive monoterpene isopulegol. A variety of readily prepared aryl and heteroaryl sulfonates undergo a formal hydroarylation to form 8-arylmenthols, privileged scaffolds for asymmetric synthesis, as typified by 8-phenylmenthol. High stereoselectivity is observed in related systems. This use of HAT significantly extends the chiral pool from the inexpensive monoterpene isopulegol.