Bond Of Substrate Research Articles

Highly Selective and Catalytic Oxygenations of C−H and C=C Bonds by a Mononuclear Nonheme High‐Spin Iron(III)‐Alkylperoxo Species

AbstractThe reactivity of a mononuclear high‐spin iron(III)‐alkylperoxo intermediate [FeIII(t‐BuLUrea)(OOCm)(OH2)]2+(2), generated from [FeII(t‐BuLUrea)(H2O)(OTf)](OTf) (1) [t‐BuLUrea=1,1′‐(((pyridin‐2‐ylmethyl)azanediyl)bis(ethane‐2,1‐diyl))bis(3‐(tert‐butyl)urea), OTf=trifluoromethanesulfonate] with cumyl hydroperoxide (CmOOH), toward the C−H and C=C bonds of hydrocarbons is reported.2oxygenates the strong C−H bonds of aliphatic substrates with high chemo‐ and stereoselectivity in the presence of 2,6‐lutidine. While2itself is a sluggish oxidant, 2,6‐lutidine assists the heterolytic O−O bond cleavage of the metal‐bound alkylperoxo, giving rise to a reactive metal‐based oxidant. The roles of the urea groups on the supporting ligand, and of the base, in directing the selective and catalytic oxygenation of hydrocarbon substrates by2are discussed.

C-H Alkylation via Multisite-Proton-Coupled Electron Transfer of an Aliphatic C-H Bond.

The direct, site-selective alkylation of unactivated C(sp3)-H bonds in organic substrates is a long-standing goal in synthetic chemistry. General approaches to the activation of strong C-H bonds include radical-mediated processes involving highly reactive intermediates, such as heteroatom-centered radicals. Herein, we describe a catalytic, intermolecular C-H alkylation that circumvents such reactive species via a new elementary step for C-H cleavage involving multisite-proton-coupled electron transfer (multisite-PCET). Mechanistic studies indicate that the reaction is catalyzed by a noncovalent complex formed between an iridium(III) photocatalyst and a monobasic phosphate base. The C-H alkylation proceeds efficiently using diverse hydrocarbons and complex molecules as the limiting reagent and represents a new approach to the catalytic functionalization of unactivated C(sp3)-H bonds.

Evidence for distinct rate-limiting steps in the cleavage of alkenes by carotenoid cleavage dioxygenases

Carotenoid cleavage dioxygenases (CCDs) use a nonheme Fe(II) cofactor to split alkene bonds of carotenoid and stilbenoid substrates. The iron centers of CCDs are typically five-coordinate in their resting states, with solvent occupying an exchangeable site. The involvement of this iron-bound solvent in CCD catalysis has not been experimentally addressed, but computational studies suggest two possible roles. 1) Solvent dissociation provides a coordination site for O2, or 2) solvent remains bound to iron but changes its equilibrium position to allow O2 binding and potentially acts as a proton source. To test these predictions, we investigated isotope effects (H2O versus D2O) on two stilbenoid-cleaving CCDs, Novosphingobium aromaticivorans oxygenase 2 (NOV2) and Neurospora crassa carotenoid oxygenase 1 (CAO1), using piceatannol as a substrate. NOV2 exhibited an inverse isotope effect (kH/kD ∼ 0.6) in an air-saturated buffer, suggesting that solvent dissociates from iron during the catalytic cycle. By contrast, CAO1 displayed a normal isotope effect (kH/kD ∼ 1.7), suggesting proton transfer in the rate-limiting step. X-ray absorption spectroscopy on NOV2 and CAO1 indicated that the protonation states of the iron ligands are unchanged within pH 6.5-8.5 and that the Fe(II)-aquo bond is minimally altered by substrate binding. We pinpointed the origin of the differential kinetic behaviors of NOV2 and CAO1 to a single amino acid difference near the solvent-binding site of iron, and X-ray crystallography revealed that the substitution alters binding of diffusible ligands to the iron center. We conclude that solvent-iron dissociation and proton transfer are both associated with the CCD catalytic mechanism.

Effect of organic solvent on enzymatic degradation of cyclic PBS-based polymers by lipase N435

Poly(butylene succinate-co-cyclohexane dimethanol succinate) (P(BS-co-CHDMS)) and poly(butylene succinate-co-butanediol cyclohexanedicarboxylic acid) (P(BS-co-BCHDA)) were catalytically degraded by Candida antarctica lipase Novozyme 435 (N435) in CHCl3 and THF. The results indicated that the degradation rate was P(BS-co-CHDMS) > P(BS-co-BCHDA) > poly(butylene succinate) (PBS). The degradation rate of copolyesters was higher in CHCl3 than in THF, the highest degradation rate of 67% being obtained for P(BS-co-CHDMS). Hence, the CHCl3 solvent is more suitable for the enzyme-catalytic degradation of copolyesters, since the lipase can easier recognize the butylene succinate (BS-), (butanediol cyclohexanedicarboxylic acid) (BCA-), and (cyclohexane dimethanol succinate-type) (CMS-type) ester bonds in this solvent. Moreover, it can recognize the CMS-type ester bonds with a higher specificity than the (butanediol cyclohexanedicarboxylic acid type) (BCA-type) ester bonds. Molecular simulation results indicated that the structure of the lipase was stable in CHCl3 and THF. However, CHCl3 proved to be more suitable for a stable activity of the enzyme. The active pocket contains acyl-binding hydrophilic residues which are recognized by the substrate. The increase in the content of saturated cycles can increase the hydrophobicity of the substrate and thus, the amount of substrate bond to enzyme active site is increased, which facilitates the enzymatic degradation of copolyesters.

Mixed Electron–Proton Conductors Enable Spatial Separation of Bond Activation and Charge Transfer in Electrocatalysis

Electrochemical energy conversion requires electrodes that can simultaneously facilitate substrate bond activation and electron-proton charge transfer. Traditional electrodes co-localize both functions to a single solid|liquid interface even though each process is typically favored in a disparate reaction environment. Herein, we establish a strategy for spatially separating bond activation and charge transfer by exploiting mixed electron-proton conduction (MEPC) in an oxide membrane. Specifically, we interpose a MEPC WOx membrane between a Pt catalyst and aqueous electrolyte and show that this composite electrode is active for the hydrogen oxidation reaction (HOR). Consistent with H2 activation occurring at the gas|solid interface, the composite electrode displays HOR current densities over 8-fold larger than the diffusion-limited rate of HOR catalysis at a singular Pt|solution interface. The segregation of bond activation and charge separation steps also confers excellent tolerance to poisons and impurities introduced to the electrolyte. Mechanistic studies establish that H2 activation at the Pt|gas interface is coupled to the electron-proton charge separation at the WOx|solution interface via rapid H-diffusion in the bulk of the WOx. Consequently, the rate of HOR is principally controlled by the rate of H-spillover at the Pt|WOx boundary. Our results establish MEPC membrane electrodes as a platform for spatially separating the critical bond activation and charge transfer steps of electrocatalysis.

The right bond in the right place

Biocatalysis Enzymes excel at specificity because of their constrained active sites. With appropriate evolutionary pressure, they can be made to differentiate between similar substrates or between positions on a single substrate. Cho et al. used directed evolution to generate cytochrome P450 variants that target different C–H bonds in substrates, forming lactam rings of varying size (see the Perspective by Hepworth and Flitsch). The enzyme directs amidation to the desired position and simultaneously prevents other side reactions. Science , this issue p. [575][1]; see also p. [529][2] [1]: /lookup/doi/10.1126/science.aaw9068 [2]: /lookup/doi/10.1126/science.aax3335

RETRACTED: Site-selective enzymatic C‒H amidation for synthesis of diverse lactams.

A major challenge in carbon‒hydrogen (C‒H) bond functionalization is to have the catalyst control precisely where a reaction takes place. In this study, we report engineered cytochrome P450 enzymes that perform unprecedented enantioselective C‒H amidation reactions and control the site selectivity to divergently construct β-, γ-, and δ-lactams, completely overruling the inherent reactivities of the C‒H bonds. The enzymes, expressed in Escherichia coli cells, accomplish this abiological carbon‒nitrogen bond formation via reactive iron-bound carbonyl nitrenes generated from nature-inspired acyl-protected hydroxamate precursors. This transformation is exceptionally efficient (up to 1,020,000 total turnovers) and selective (up to 25:1 regioselectivity and 97%, please refer to compound 2v enantiomeric excess), and can be performed easily on preparative scale.

Reactivity of Re2O7 in aromatic solvents – Cleavage of a β-O-4 lignin model substrate by Lewis-acidic rhenium oxide nanoparticles

The nature of the active species in a dehydration reaction using the “catalyst” Re2O7 is investigated. During studies on the cleavage of the β-O-4 bond of a lignin model substrate, apparent “TOFs” of 240 h−1 and good selectivities of up to 73% for the sensitive product phenylacetaldehyde (compared to other applied (Lewis) acids) are observed. However, divergent kinetic data during the studies led to an in-depth investigation on the activation mechanism of Re2O7 in the applied apolar, aprotic medium. Here, it is shown, that, in fact, Re2O7 itself is completely inactive in the dehydration reaction, but forms even at low temperatures mixed valence rhenium nanoparticles, which promote the desired reaction. The nanoparticles are characterized by XPS, DLS and TEM. In order to discriminate whether either dissolved rhenium compounds or nanoparticles are the active species, 17O NMR studies and kinetic experiments are performed. An activation mechanism of rhenium heptoxide is suggested based on a careful analysis of byproducts under catalytic conditions and application of selected potential catalysts. Furthermore, an outlook is given, as these nanoparticles might participate in numerous other catalytic reactions.

Comparison of the moisture damage of bituminous binder coupled with glass and limestone substrate using pull-off test

In this paper, the moisture susceptibility of different bituminous binders with two substrates (glass and limestone) was investigated. To that end, the tensile strength of different combinations of bituminous binder–substrate bond was measured using a pull-off test. This test was adapted from the pneumatic adhesion tensile testing instrument (PATTI) test to improve repeatability. Samples were tested in dry condition and after a 7-day conditioning in hot water bath (60 °C). An analysis of variance (ANOVA) was performed on the test results. Overall, the results show that in dry condition, the pull-off strength is a function of the bituminous binder type rather than of the substrate type. After water conditioning, an increase in the pull-off strength was observed for the bituminous binder without polymers and coupled with glass substrate. This was associated with an increase in binder stiffness. For the limestone substrate, the effect of water conditioning was significant only for one type of binder.

Enzymatic Cascade Reactions in Biosynthesis.

Enzyme-mediated cascade reactions are widespread in biosynthesis. To facilitate comparison with the mechanistic categorizations of cascade reactions by synthetic chemists and delineate the common underlying chemistry, we discuss four types of enzymatic cascade reactions: those involving nucleophilic, electrophilic, pericyclic, and radical reactions. Two subtypes of enzymes that generate radical cascades exist at opposite ends of the oxygen abundance spectrum. Iron-based enzymes use O2 to generate high valent iron-oxo species to homolyze unactivated C-H bonds in substrates to initiate skeletal rearrangements. At anaerobic end, enzymes reversibly cleave S-adenosylmethionine (SAM) to generate the 5'-deoxyadenosyl radical as a powerful oxidant to initiate C-H bond homolysis in bound substrates. The latter enzymes are termed radical SAM enzymes. We categorize the former as "thwarted oxygenases".

Machine Learning Identifies Chemical Characteristics That Promote Enzyme Catalysis.

Despite tremendous progress in understanding and engineering enzymes, knowledge of how enzyme structures and their dynamics induce observed catalytic properties is incomplete, and capabilities to engineer enzymes fall far short of industrial needs. Here, we investigate the structural and dynamic drivers of enzyme catalysis for the rate-limiting step of the industrially important enzyme ketol-acid reductoisomerase (KARI) and identify a region of the conformational space of the bound enzyme–substrate complex that, when populated, leads to large increases in reactivity. We apply computational statistical mechanical methods that implement transition interface sampling to simulate the kinetics of the reaction and combine this with machine learning techniques from artificial intelligence to select features relevant to reactivity and to build predictive models for reactive trajectories. We find that conformational descriptors alone, without the need for dynamic ones, are sufficient to predict reactivity with greater than 85% accuracy (90% AUC). Key descriptors distinguishing reactive from almost-reactive trajectories quantify substrate conformation, substrate bond polarization, and metal coordination geometry and suggest their role in promoting substrate reactivity. Moreover, trajectories constrained to visit a region of the reactant well, separated from the rest by a simple hyperplane defined by ten conformational parameters, show increases in computed reactivity by many orders of magnitude. This study provides evidence for the existence of reactivity promoting regions within the conformational space of the enzyme–substrate complex and develops methodology for identifying and validating these particularly reactive regions of phase space. We suggest that identification of reactivity promoting regions and re-engineering enzymes to preferentially populate them may lead to significant rate enhancements.

A Mononuclear Nonheme Iron(IV)-Oxo Complex of a Substituted N4Py Ligand: Effect of Ligand Field on Oxygen Atom Transfer and C-H Bond Cleavage Reactivity.

A mononuclear iron(II) complex [FeII(N4PyMe2)(OTf)](OTf)(1), supported by a new pentadentate ligand, bis(6-methylpyridin-2-yl)- N, N-bis((pyridin-2-yl)methyl)methanamine (N4PyMe2), has been isolated and characterized. Introduction of methyl groups in the 6-position of two pyridine rings makes the N4PyMe2 a weaker field ligand compared to the parent N4Py ligand. Complex 1 is high-spin in the solid state and converts to [FeII(N4PyMe2)(CH3CN)](OTf)2 (1a) in acetonitrile solution. The iron(II) complex in acetonitrile displays temperature-dependent spin-crossover behavior over a wide range of temperature. In its reaction with m-CPBA or oxone in acetonitrile at -10 °C, the iron(II) complex converts to an iron(IV)-oxo species, [FeIV(O)(N4PyMe2)]2+ (2). Complex 2 exhibits the Mössbauer parameters δ = 0.05 mm/s and Δ EQ = 0.62 mm/s, typical of N-ligated S = 1 iron(IV)-oxo species. The iron(IV)-oxo complex has a half-life of only 14 min at 25 °C and is reactive toward oxygen-atom-transfer and hydrogen-atom-transfer (HAT) reactions. Compared to the parent complex [FeIV(O)(N4Py)]2+, 2 is more reactive in oxidizing thioanisole and oxygenates the C-H bonds of aliphatic substrates including that of cyclohexane. The enhanced reactivity of 2 toward cyclohexane results from the involvement of the S = 2 transition state in the HAT pathway and a lower triplet-quintet splitting compared to [FeIV(O)(N4Py)]2+, as supported by DFT calculations. The second-order rate constants for HAT by 2 is well correlated with the C-H bond dissociation energies of aliphatic substrates. Surprisingly, the slope of this correlation is different from that of [FeIV(O)(N4Py)]2+, and 2 is more reactive only in the case of strong C-H bonds (>86 kcal/mol), but less reactive in the case of weaker C-H bonds. Using oxone as the oxidant, the iron(II) complex displays catalytic oxidations of substrates with low activity but with good selectivity.

Selective biocatalytic hydroxylation of unactivated methylene C-H bonds in cyclic alkyl substrates.

The cytochrome P450 monooxygenase CYP101B1 from Novosphingobium aromaticivorans selectively hydroxylated methylene C-H bonds in cycloalkyl rings. Cycloketones and cycloalkyl esters containing C6, C8, C10 and C12 rings were oxidised with high selectively on the opposite side of the ring to the carbonyl substituent. Cyclodecanone was oxidised to oxabicycloundecanol derivatives in equilibrium with the hydroxycyclodecanones.

Functional Assays of Thiol Isomerase ERp5.

Endoplasmic reticulum protein 5 (ERp5) is a member of the thiol isomerase family of enzymes, whose prototype member is protein disulphide isomerase (PDI). Thiol isomerases catalyze reduction/oxidation (redox) reactions which lead to the cleavage, formation, or isomerization of disulphide bonds in protein substrates. Thiol isomerase reactions on protein disulphides are important for the correct folding of proteins in the endoplasmic reticulum and for the regulation of various protein functions in the extracellular space. Apart from the disulphide reactions, thiol isomerases assist protein folding by chaperone activity.The disulphide redox activity of ERp5 can be measured with functional assays involving artificial or natural substrates containing disulphide bonds. Herein we describe step-by-step assays of ERp5 reductase, isomerization, and de-nitrosylation activity. Disulphide reductase assays include insulin or di-eosin-GSSG as substrates whereas the isomerization assay includes RNase as substrate. The reduction of natural substrates, i.e., integrin αIIbβ3, can be detected using maleimide labels of free thiols and Western blotting. The biotin switch assay is used to measure the de-nitrosylation of S-nitrosylated substrates. These assays can measure the activity of purified ERp5 protein but can also be applied for the measurement of thiol isomerase activity in cellular samples.

A coordinatively unsaturated iridium complex with an unsymmetrical redox-active ligand: (spectro)electrochemical and reactivity studies.

Redox-active ligands, owing to their electron reservoir capability, are well suited for the generation of coordinatively unsaturated metal complexes. We present here iridium complexes with an unsymmetrically substituted o-phenylenediamine ligand. A coordinatively unsaturated, formally iridium(iii) complex with the fully reduced o-phenylenediamide (or o-diamidobenzene) ligand was isolated and structurally characterized. This coordinatively unsaturated metal complex undergoes methylation reactions with a CH3+ source to form a new species with an Ir-CH3 bond. The redox-active Ir-CH3 complex performs the activation of CDCl3. The same activation reaction was also tested for other haloforms. In all types of reactions, the masked coordination site at the metal center and the electron reservoir behavior of the redox-active ligand are used for reactivity. Furthermore, we show that the aforementioned iridium(iii) complex performs redox-induced dihydrogen activation. This activation process was used to catalytically transfer the electrons and protons of dihydrogen to a substrate molecule. Crystallographic, spectroscopic, electrochemical, spectroelectrochemical and DFT methods were used to elucidate the geometric and the electronic structures of the metal complex in the various redox forms and to probe the mechanism of the investigated reactions. We demonstrate here how the cooperative behavior between a catalytically active metal center and a redox non-innocent ligand can be utilized to perform substrate bond activation and transformation.

Distinguishing the differences in β-glycosylceramidase folds, dynamics, and actions informs therapeutic uses

Glycosyl hydrolases (GHs) are carbohydrate-active enzymes that hydrolyze a specific β-glycosidic bond in glycoconjugate substrates; β-glucosidases degrade glucosylceramide, a ubiquitous glycosphingolipid. GHs are grouped into structurally similar families that themselves can be grouped into clans. GH1, GH5, and GH30 glycosidases belong to clan A hydrolases with a catalytic (β/α)8 TIM barrel domain, whereas GH116 belongs to clan O with a catalytic (α/α)6 domain. In humans, GH abnormalities underlie metabolic diseases. The lysosomal enzyme glucocerebrosidase (family GH30), deficient in Gaucher disease and implicated in Parkinson disease etiology, and the cytosol-facing membrane-bound glucosylceramidase (family GH116) remove the terminal glucose from the ceramide lipid moiety. Here, we compare enzyme differences in fold, action, dynamics, and catalytic domain stabilization by binding site occupancy. We also explore other glycosidases with reported glycosylceramidase activity, including human cytosolic β-glucosidase, intestinal lactase-phlorizin hydrolase, and lysosomal galactosylceramidase. Last, we describe the successful translation of research to practice: recombinant glycosidases and glucosylceramide metabolism modulators are approved drug products (enzyme replacement therapies). Activity-based probes now facilitate the diagnosis of enzyme deficiency and screening for compounds that interact with the catalytic pocket of glycosidases. Future research may deepen the understanding of the functional variety of these enzymes and their therapeutic potential.

Structure–function analyses reveal the mechanism of the ARH3-dependent hydrolysis of ADP-ribosylation

ADP-ribosylation of proteins plays key roles in multiple biological processes, including DNA damage repair. Recent evidence suggests that serine is an important acceptor for ADP-ribosylation, and that serine ADP-ribosylation is hydrolyzed by ADP-ribosylhydrolase 3 (ARH3 or ADPRHL2). However, the structural details in ARH3-mediated hydrolysis remain elusive. Here, we determined the structure of ARH3 in a complex with ADP-ribose (ADPR). Our analyses revealed a group of acidic residues in ARH3 that keep two Mg2+ ions at the catalytic center for hydrolysis of Ser-linked ADP-ribosyl group. In particular, dynamic conformational changes involving Glu41 were observed in the catalytic center. Our observations suggest that Mg2+ ions together with Glu41 and water351 are likely to mediate the cleavage of the glycosidic bond in the serine-ADPR substrate. Moreover, we found that ADPR is buried in a groove and forms multiple hydrogen bonds with the main chain and side chains of ARH3 residues. On the basis of these structural findings, we used site-directed mutagenesis to examine the functional roles of key residues in the catalytic pocket of ARH3 in mediating the hydrolysis of ADP-ribosyl from serine and DNA damage repair. Moreover, we noted that ADPR recognition is essential for the recruitment of ARH3 to DNA lesions. Taken together, our study provides structural and functional insights into the molecular mechanism by which ARH3 hydrolyzes the ADP-ribosyl group from serine and contributes to DNA damage repair.

Comparative Nitrene-Transfer Chemistry to Olefinic Substrates Mediated by a Library of Anionic Mn(II) Triphenylamido-Amine Reagents and M(II) Congeners (M = Fe, Co, Ni) Favoring Aromatic over Aliphatic Alkenes

Selective amination of σ and π entities such as C–H and C═C bonds of substrates remains a challenging endeavor for current catalytic methodologies devoted to the synthesis of abundant nitrogen-containing chemicals. The present work addresses an approach toward discriminating aromatic over aliphatic alkenes in aziridination reactions, relying on the use of anionic metal reagents (M = Mn, Fe, Co, Ni) to attenuate reactivity in a metal-dependent manner. A family of MnII reagents bearing a triphenylamido-amine scaffold and various pendant arms has been synthesized and characterized by various techniques, including cyclic voltammetry. Aziridination of styrene by PhI═NTs in the presence of each MnII catalyst establishes a trend of increasing yield with increasing MnII/III anodic potential. The FeII, CoII, and NiII congeners of the highest-yielding MnII catalyst have been synthesized and explored in the aziridination of aromatic and aliphatic alkenes, exhibiting good to high yields with para-substituted styrenes...

Bioinspired Olefin cis-Dihydroxylation and Aliphatic C-H Bond Hydroxylation with Dioxygen Catalyzed by a Nonheme Iron Complex.

A mononuclear iron(II)-α-hydroxy acid complex [(TpPh,Me)FeII(benzilate)] (TpPh,Me = hydrotris(3-phenyl-5-methylpyrazol-1-yl)borate) of a facial tridentate ligand has been isolated and characterized to explore its catalytic efficiency for aerial oxidation of organic substrates. In the reaction between the iron(II)-benzilate complex and O2, the metal-coordinated benzilate is stoichiometrically converted to benzophenone with concomitant reduction of dioxygen on the iron center. Based on the results from interception experiments and labeling studies, different iron-oxygen oxidants are proposed to generate in situ in the reaction pathway depending upon the absence or presence of an external additive (such as protic acid or Lewis acid). The five-coordinate iron(II) complex catalytically cis-dihydroxylates olefins and oxygenates the C-H bonds of aliphatic substrates using O2 as the terminal oxidant. The iron(II) complex exhibits better catalytic activity in the presence of a Lewis acid.

Bracing copper for the catalytic oxidation of C–H bonds

A structural unit found in the active site of some copper proteins, the histidine brace, is comprised of an N-terminal histidine that chelates a single copper ion through its amino terminus NH2 and the π–N of its imidazole side chain. Coordination is completed by the τ-N of a further histidine side chain, to give an overall N3 T-shaped coordination at the copper ion. The histidine brace appears in several proteins, including lytic polysaccharide monooxygenases LPMOs and particulate methane monooxygenases pMMOs, both of which catalyse the oxidation of substrates with strong C–H bonds (bond dissociation enthalpies ~100 kcal mol–1). As such, the copper histidine brace is the focus of research aimed at understanding how nature catalyses the oxidation of unactivated C–H bonds. In this Perspective, we evaluate these studies, which further give bioinspired direction to coordination chemists in the design and preparation of small molecule copper oxidation catalysts. The histidine brace found in certain copper oxidases enables the oxidation of strong C–H bonds in organic substrates. This Perspective highlights and discusses the possible structural and electronic features of this motif and how these features underlie its role in challenging oxidative catalysis.