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.

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.

Impact of Low-Temperature Water Exposure and Removal on Zeolite HY.

Aqueous-phase postsynthetic modifications of the industrially important Y-type zeolite are commonly used to change overall acid site concentrations, introduce stabilizing rare-earth cations, impart bifunctional character through metal cation exchange, and tailor the distribution of Brønsted and Lewis acid sites. Zeolite Y is known to undergo framework degradation in the presence of both vapor- and liquid-phase water at temperatures exceeding 100 °C, and rare-earth exchanged and stabilized HY catalysts are commonly used for fluidized catalytic cracking due to their increased hydrothermal resilience. Here, using detailed spectroscopy, crystallography, and flow-reactor experiments, we reveal unexpected decreases in Brønsted acid site (BAS) density for zeolite HY following exposure even to room-temperature liquid water. These data indicate that aqueous-phase ion-exchange procedures commonly used to modify zeolite Y are impacted by the liquid water and its removal, even when fractional heating rates and inert conditions much less severe than standard practice are used for catalyst dehydration. X-ray diffraction, thermogravimetric, and spectroscopic analyses reveal that the majority of framework degradation occurs during the removal of a strongly bound water fraction in HY, which does not form when NH4Y is immersed in liquid water and which leads to reduced acidity in HY even when dehydration conditions much milder than those typically practiced are employed. Na+-exchanged HY prepared via room-temperature aqueous dissolution demonstrates that Brønsted acid sites are lost in excess of the theoretical maximum that is possible from sodium titration. The structural impact of low-temperature aqueous-phase ion-exchange methods complicates the interpretation of subsequent data and likely explains the wide variation in reported acid site concentrations and catalytic activity of HY zeolites with high-Al content.

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.

17O Labeling Reveals Paired Active Sites in Zeolite Catalysts.

Current needs for extending zeolite catalysts beyond traditional gas-phase hydrocarbon chemistry demand detailed characterization of active site structures, distributions, and hydrothermal impacts. A broad suite of homonuclear and heteronuclear NMR correlation experiments on dehydrated H-ZSM-5 catalysts with isotopically enriched 17O frameworks reveals that at least two types of paired active sites exist, the amount of which depends on the population of fully framework-coordinated tetrahedral Al (Al(IV)-1) and partially framework-coordinated tetrahedral Al (Al(IV)-2) sites, both of which can be denoted as (SiO)4-n-Al(OH)n. The relative amounts of Al(IV)-1 and Al(IV)-2 sites, and subsequent pairing, cannot be inferred from the catalyst Si/Al ratio, but depend on synthetic and postsynthetic modifications. Correlation experiments demonstrate that, on average, acidic hydroxyl groups from Al(IV)-1/Al(IV)-2 pairs are closer to one another than those from Al(IV)-1/Al(IV)-1 pairs, as supported by computational DFT calculations. Through-bond and through-space polarization transfer experiments exploiting 17O nuclei reveal a number of different acidic hydroxyl groups in varying Si/Al catalysts, the relative amounts of which change following postsynthetic modifications. Using room-temperature isotopic exchange methods, it was determined that 17O was homogeneously incorporated into the zeolite framework, while 17O → 27Al polarization transfer experiments demonstrated that 17O incorporation does not occur for extra-framework AlnOm species. Data from samples exposed to controlled hydrolysis indicates that nearest neighbor Al pairs in the framework are more susceptible to hydrolytic attack. The data reported here suggest that Al(IV)-1/Al(IV)-2 paired sites are synergistic sites leading to increased reactivity in both low- and high-temperature reactions. No evidence was found for paired framework/nonframework sites.

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.

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.

Structure and Catalytic Characterization of a Second Framework Al(IV) Site in Zeolite Catalysts Revealed by NMR at 35.2 T.

Ultrahigh field 27Al{1H} 2D correlation NMR experiments demonstrate that at least two framework Al(IV) sites with hydroxyl groups can exist in acidic zeolite catalysts in their dehydrated and catalytically active states. In addition to the known Al(IV) at the framework bridging acid site (BAS), a new site created by a second tetrahedral Al atom and its hydroxyl group protons in zeolite HZSM-5 is clearly resolved at 35.2 T field strengths, enabled by recently developed series-connected hybrid (SCH) magnet technology. Coupled with computational modeling, extensive 27Al MQMAS experiments at multiple field strengths, and 1H MAS NMR experiments, these data indicate that this second tetrahedrally coordinated Al site (denoted Al(IV)-2) experiences an increased chemical shift and unique quadrupolar parameters relative to the BAS in both dehydrated and hydrated states. These new experimental data, supported by computational and catalytic reaction work, indicate that the second site arises from partially bonded framework (SiO)4-n-Al(OH)n species that significantly increase catalyst reactivity in benzene hydride-transfer and n-hexane cracking reactions. Al(IV)-2 sites result either from framework crystallization defects or from incomplete postsynthetic hydrolysis of a framework Al, prior to the formation of extraframework Al. Populations of this second acidic proton site created by the Al(IV)-2 species are shown to be controlled via postsynthetic catalyst treatments, should be general to different catalyst structures, and significantly enhance catalyst reactivity in the cited probe reactions when they are present. The results herein communicate the highest magnetic field strength data on active zeolite catalyst structures to date and enable for the first time the detection of Al and H association on a dry HZSM-5 catalyst, i.e., under conditions representative of typical end-use processes.

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.

Identification of active sites on supported metal catalysts with carbon nanotube hydrogen highways.

Catalysts consisting of metal particles supported on reducible oxides exhibit promising activity and selectivity for a variety of current and emerging industrial processes. Enhanced catalytic activity can arise from direct contact between the support and the metal or from metal-induced promoter effects on the oxide. Discovering the source of enhanced catalytic activity and selectivity is challenging, with conflicting arguments often presented based on indirect evidence. Here, we separate the metal from the support by a controlled distance while maintaining the ability to promote defects via the use of carbon nanotube hydrogen highways. As illustrative cases, we use this approach to show that the selective transformation of furfural to methylfuran over Pd/TiO2 occurs at the Pd-TiO2 interface while anisole conversion to phenol and cresol over Cu/TiO2 is facilitated by exposed Ti3+ cations on the support. This approach can be used to clarify many conflicting arguments in the literature.

Enhancing the Acylation Activity of Acetic Acid by Formation of an Intermediate Aromatic Ester.

Acylation is an effective C-C bond-forming reaction to condense acetic acid and lignin-derived aromatic compounds into acetophenones, valuable precursors to fuels and chemicals. However, acetic acid is intrinsically an ineffective acylating agent. Here, we report that its acylation activity can be greatly enhanced by forming intermediate aromatic esters directly derived from acetic acid and phenolic compounds. Additionally, the acylation reaction was studied in the liquid phase over acid zeolites and was found to happen in two steps: 1) formation of an acylium ion and 2) C-C bond formation between the acylium ion and the aromatic substrate. Each of these steps may be rate-limiting, depending on the type of acylating agent and the aromatic substrate. Oxygen-containing substituents, such as -OH and -OCH3 , can activate aromatic substrates for step 2, with -OH> -OCH3 , whereas alkyl substituent -R cannot. At the same time, aromatic esters can rearrange to acetophenones by both an intramolecular pathway and, preferentially, an intermolecular one.

Direct carbon-carbon coupling of furanics with acetic acid over Brønsted zeolites.

Effective carbon-carbon coupling of acetic acid to form larger products while minimizing CO2 emissions is critical to achieving a step change in efficiency for the production of transportation fuels from sustainable biomass. We report the direct acylation of methylfuran with acetic acid in the presence of water, all of which can be readily produced from biomass. This direct coupling limits unwanted polymerization of furanics while producing acetyl methylfuran. Reaction kinetics and density functional theory calculations illustrate that the calculated apparent barrier for the dehydration of the acid to form surface acyl species is similar to the experimentally measured barrier, implying that this step plays a significant role in determining the net reaction rate. Water inhibits the overall rate, but selectivity to acylated products is not affected. We show that furanic species effectively stabilize the charge of the transition state, therefore lowering the overall activation barrier. These results demonstrate a promising new route to C-C bond-forming reactions for the production of higher-value products from biomass.

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.

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.

Multiwalled Carbon Nanotubes at the Interface of Pickering Emulsions.

Carbon nanotubes exhibit very unique properties in biphasic systems. Their interparticle attraction leads to reduced droplet coalescence rates and corresponding improvements in emulsion stability. Here we use covalent and noncovalent techniques to modify the hydrophilicity of multiwalled carbon nanotubes (MWCNTs) and study their resulting behavior at an oil-water interface. By using both paraffin wax/water and dodecane/water systems, the thickness of the layer of MWNTs at the interface and resulting emulsion stability are shown to vary significantly with the approach used to modify the MWNTs. Increased hydrophilicity of the MWNTs shifts the emulsions from water-in-oil to oil-in-water. The stability of the emulsion is found to correlate with the thickness of nanotubes populating the oil-water interface and relative strength of the carbon nanotube network. The addition of a surfactant decreases the thickness of nanotubes at the interface and enhances the overall interfacial area stabilized at the expense of increased droplet coalescence rates. To the best of our knowledge, this is the first time the interfacial thickness of modified carbon nanotubes has been quantified and correlated to emulsion stability.

Epitaxial Growth of ZSM-5@Silicalite-1: A Core-Shell Zeolite Designed with Passivated Surface Acidity.

The design of materials with spatially controlled chemical composition has potential advantages for wide-reaching applications that span energy to medicine. Here, we present a method for preparing a core-shell aluminosilicate zeolite with continuous translational symmetry of nanopores and an epitaxial shell of tunable thickness that passivates Brønsted acid sites associated with framework Al on exterior surfaces. For this study, we selected the commercially relevant MFI framework type and prepared core-shell particles consisting of an aluminosilicate core (ZSM-5) and a siliceous shell (silicalite-1). Transmission electron microscopy and gas adsorption studies confirmed that silicalite-1 forms an epitaxial layer on ZSM-5 crystals without blocking pore openings. Scanning electron microscopy and dynamic light scattering were used in combination to confirm that the shell thickness can be tailored with nanometer resolution (e.g., 5-20 nm). X-ray photoelectron spectroscopy and temperature-programmed desorption measurements revealed the presence of a siliceous shell, while probe reactions using molecules that were either too large or adequately sized to access MFI pores confirmed the uniform shell coverage. The synthesis of ZSM-5@silicalite-1 offers a pathway for tailoring the physicochemical properties of MFI-type materials, notably in the area of catalysis, where surface passivation can enhance product selectivity without sacrificing catalyst activity. The method described herein may prove to be a general platform for zeolite core-shell design with potentially broader applicability to other porous materials.

Decoupling HZSM-5 catalyst activity from deactivation during upgrading of pyrolysis oil vapors.

The independent evaluation of catalyst activity and stability during the catalytic pyrolysis of biomass is challenging because of the nature of the reaction system and rapid catalyst deactivation that force the use of excess catalyst. In this contribution we use a modified pyroprobe system in which pulses of pyrolysis vapors are converted over a series of HZSM-5 catalysts in a separate fixed-bed reactor controlled independently. Both the reactor-bed temperature and the Si/Al ratio of the zeolite are varied to evaluate catalyst activity and deactivation rates independently both on a constant surface area and constant acid site basis. Results show that there is an optimum catalyst-bed temperature for the production of aromatics, above which the production of light gases increases and that of aromatics decrease. Zeolites with lower Si/Al ratios give comparable initial rates for aromatics production, but far more rapid catalyst deactivation rates than those with higher Si/Al ratios.