Cu(II) Promoted C(sp3 )-H Activation in Unactivated Cycloalkanes: Oxo-Alkylation of Styrenes to Synthesize ß-Disubstituted Ketones.

We report the Cu(II) catalyzed synthesis of ß-disubstituted ketones from styrene via oxo-alkylation with unactivated cycloalkanes as the alkylating agent in presence of tert-butylhydroperoxide (TBHP) and 1-methylimidazole as oxidant and base respectively. ß-disubstituted ketones are known to be synthesized by using either expensive Ru/Ir complexes, or low-cost metal complexes (e. g., Fe, Mn) with activated species like aldehyde, acid, alcohol, or phthalimide derivatives as the alkylating agent, however, use of unactivated cycloalkanes directly as the alkylating agent remains challenging. A wide range of aliphatic C-H substrates as well as various olefinic arenes and heteroarene (35 substrates including 14 new substrates) are well-tolerated in this method. Hammett analysis shed more light on the substitution effect in the olefinic part on the overall mechanism. Furthermore, the controlled experiments, kinetic isotope effect study, and theoretical calculations (DFT) enable us to gain deeper insight of mechanistic intricacies of this new simple and atom-economic methodology.

Gold(III) Auracycles Featuring C(sp3)-Au-C(sp2) Bonds: Synthesis and Mechanistic Insights into the Cycloauration Step.

The direct auration of arenes is a key step in numerous gold-catalyzed reactions. Although reported more than 100 years ago, understanding of its underlying mechanism has been hampered by the difficulties in the isolation of relevant intermediates given the propensity of gold(III) species to undergo reductive elimination. Here, we report the synthesis and isolation of a new family of intriguing zwitterionic [C(sp3)^C(sp2)]-auracyclopentanes, as well as of their alkyl-gold(III) precursors and demonstrate their value as mechanistic probes to study the C(sp2)-Au bond-forming event. Experimental investigations employing Kinetic Isotope Effects (KIE), Hammett plot, and Eyring analysis provided important insights into the formation of the auracycle. The data suggest a SEAr mechanism wherein the slowest step might be the π-coordination between the arene and the gold(III) center, en route to the Wheland intermediate. We also show that these auracyclopentanes can work as catalysts in several gold-promoted transformations.

Mechanistic Aspects on [3+2] Cycloaddition (32CA) Reactions of Azides to Nitroolefins: A Computational and Kinetic Study.

[3+2] cycloadditions of nitroolefins have emerged as a selective and catalyst-free alternative for the synthesis of 1,2,3-triazoles from azides. We describe mechanistic studies into the cycloaddition/rearomatization reaction sequence. DFT calculations revealed a rate-limiting cycloaddition step proceeding via an asynchronous TS with high kinetic selectivity for the 1,5-triazole. Kinetic studies reveal a second-order rate law, and 13 C kinetic isotopic effects at natural abundance were measured with a significant normal effect at the conjugated olefinic centers of 1.0158 and 1.0216 at the α and ß-carbons of ß-nitrostyrene. Distortion/interaction-activation strain and energy decomposition analyses revealed that the major regioisomeric pathway benefits from an earlier and less-distorted TS, while intermolecular interaction terms dominate the preference for 1,5- over 1,4-cycloadducts. In addition, the major regioisomer also has more favorable electrostatic and dispersion terms. Additionally, while static DFT calculations suggest a concerted but highly asynchronous Ei-type HNO2 elimination mechanism, quasiclassical direct-dynamics calculations reveal the existence of a dynamic intermediate.

Deuterium Isotope Effects on Acid-Base Equilibrium of Organic Compounds.

Deuterium isotope effects on acid-base equilibrium have been investigated using a combined path integral and free-energy perturbation simulation method. To understand the origin of the linear free-energy relationship of ΔpKa=pKaD2O-pKaH2O versus pKaH2O, we examined two theoretical models for computing the deuterium isotope effects. In Model 1, only the intrinsic isotope exchange effect of the acid itself in water was included by replacing the titratable protons with deuterons. Here, the dominant contribution is due to the difference in zero-point energy between the two isotopologues. In Model 2, the medium isotope effects are considered, in which the free energy change as a result of replacing H2O by D2O in solute-solvent hydrogen-bonding complexes is determined. Although the average ΔpKa change from Model 1 was found to be in reasonable agreement with the experimental average result, the pKaH2O dependence of the solvent isotope effects is absent. A linear free-energy relationship is obtained by including the medium effect in Model 2, and the main factor is due to solvent isotope effects in the anion-water complexes. The present study highlights the significant roles of both the intrinsic isotope exchange effect and the medium solvent isotope effect.

Kinetic isotope effects reveal early transition state of protein lysine methyltransferase SET8.

Protein lysine methyltransferases (PKMTs) catalyze the methylation of protein substrates, and their dysregulation has been linked to many diseases, including cancer. Accumulated evidence suggests that the reaction path of PKMT-catalyzed methylation consists of the formation of a cofactor(cosubstrate)-PKMT-substrate complex, lysine deprotonation through dynamic water channels, and a nucleophilic substitution (SN2) transition state for transmethylation. However, the molecular characters of the proposed process remain to be elucidated experimentally. Here we developed a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) method and corresponding mathematic matrix to determine precisely the ratios of isotopically methylated peptides. This approach may be generally applicable for examining the kinetic isotope effects (KIEs) of posttranslational modifying enzymes. Protein lysine methyltransferase SET8 is the sole PKMT to monomethylate histone 4 lysine 20 (H4K20) and its function has been implicated in normal cell cycle progression and cancer metastasis. We therefore implemented the MS-based method to measure KIEs and binding isotope effects (BIEs) of the cofactor S-adenosyl-l-methionine (SAM) for SET8-catalyzed H4K20 monomethylation. A primary intrinsic 13C KIE of 1.04, an inverse intrinsic α-secondary CD3 KIE of 0.90, and a small but statistically significant inverse CD3 BIE of 0.96, in combination with computational modeling, revealed that SET8-catalyzed methylation proceeds through an early, asymmetrical SN2 transition state with the C-N and C-S distances of 2.35-2.40 Å and 2.00-2.05 Å, respectively. This transition state is further supported by the KIEs, BIEs, and steady-state kinetics with the SAM analog Se-adenosyl-l-selenomethionine (SeAM) as a cofactor surrogate. The distinct transition states between protein methyltransferases present the opportunity to design selective transition-state analog inhibitors.

Mutation of a pH-modulating residue in a GH51 α-l-arabinofuranosidase leads to a severe reduction of the secondary hydrolysis of transfuranosylation products.

BACKGROUND: The development of enzyme-mediated glycosynthesis using glycoside hydrolases is still an inexact science, because the underlying molecular determinants of transglycosylation are not well understood. In the framework of this challenge, this study focused on the family GH51 α-l-arabinofuranosidase from Thermobacillus xylanilyticus, with the aim to understand why the mutation of position 344 provokes a significant modification of the transglycosylation/hydrolysis partition. METHODS: Detailed kinetic analysis (kcat, KM, pKa determination and time-course NMR kinetics) and saturation transfer difference nuclear magnetic resonance spectroscopy was employed to determine the synthetic and hydrolytic ability modification induced by the redundant N344 mutation disclosed in libraries from directed evolution. RESULTS: The mutants N344P and N344Y displayed crippled hydrolytic abilities, and thus procured improved transglycosylation yields. This behavior was correlated with an increased pKa of the catalytic nucleophile (E298), the pKa of the acid/base catalyst remaining unaffected. Finally, mutations at position 344 provoked a pH-dependent product inhibition phenomenon, which is likely to be the result of a significant modification of the proton sharing network in the mutants. CONCLUSIONS AND GENERAL SIGNIFICANCE: Using a combination of biochemical and biophysical methods, we have studied TxAbf-N344 mutants, thus revealing some fundamental details concerning pH modulation. Although these results concern a GH51 α-l-arabinofuranosidase, it is likely that the general lessons that can be drawn from them will be applicable to other glycoside hydrolases. Moreover, the effects of mutations at position 344 on the transglycosylation/hydrolysis partition provide clues as to how TxAbf can be further engineered to obtain an efficient transfuranosidase.

Water in Photosystem II: structural, functional and mechanistic considerations.

Water is clearly important for the functioning of Photosystem II (PSII). Apart from being the very substrate that needs to be transported in this water oxidation enzyme, water is also vital for the transport of protons to and from the catalytic center as well as other important co-factors and key residues in the enzyme. The latest crystal structural data of PSII have enabled detailed analyses of the location and possible function of water molecules in the enzyme. Significant progress has also been made recently in the investigation of channels and pathways through the protein complex. Through these studies, the mechanistic significance of water for PSII is becoming increasingly clear. An overview and discussion of key aspects of the current research on water in PSII is presented here. The role of water in three other systems (aquaporin, bacteriorhodopsin and cytochrome P450) is also outlined to illustrate further points concerning the central significance that water can have, and potential applications of these ideas for continued research on PSII. It is advocated that water be seen as an integral part of the protein and far from a mere solvent.

A hydrogen bond network in the active site of Anabaena ferredoxin-NADP(+) reductase modulates its catalytic efficiency.

Ferredoxin-nicotinamide-adenine dinucleotide phosphate (NADP(+)) reductase (FNR) catalyses the production of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) in photosynthetic organisms, where its flavin adenine dinucleotide (FAD) cofactor takes two electrons from two reduced ferredoxin (Fd) molecules in two sequential steps, and transfers them to NADP(+) in a single hydride transfer (HT) step. Despite the good knowledge of this catalytic machinery, additional roles can still be envisaged for already reported key residues, and new features are added to residues not previously identified as having a particular role in the mechanism. Here, we analyse for the first time the role of Ser59 in Anabaena FNR, a residue suggested by recent theoretical simulations as putatively involved in competent binding of the coenzyme in the active site by cooperating with Ser80. We show that Ser59 indirectly modulates the geometry of the active site, the interaction with substrates and the electronic properties of the isoalloxazine ring, and in consequence the electron transfer (ET) and HT processes. Additionally, we revise the role of Tyr79 and Ser80, previously investigated in homologous enzymes from plants. Our results probe that the active site of FNR is tuned by a H-bond network that involves the side-chains of these residues and that results to critical optimal substrate binding, exchange of electrons and, particularly, competent disposition of the C4n (hydride acceptor/donor) of the nicotinamide moiety of the coenzyme during the reversible HT event.

Spatial Confinement of Pt Nanoparticles in Carbon Nanotubes for Efficient and Selective H2 Evolution from Methanol.

H2 generation from methanol-water mixtures often requires high pressure and high temperature (200-300 °C). However, CO can be easily generated and poison the catalytic system under such high temperature. Therefore, it is highly desirable to develop the efficient catalytic systems for H2 production from methanol at room temperature, even at sub-zero temperatures. Herein, carbon nanotube-supported Pt nanocomposites are designed and synthesized as high-performance nano-catalysts, via stabilization of Pt nanoparticles onto carbon nanotube (CNT), for H2 production upon methanol dehydrogenation at sub-zero temperatures. Therein, the optimal Pt/CNT nanocomposite presents the superior catalytic performance in H2 production upon methanol dehydrogenation at the expense of B2(OH)4, with the TOF of 299.51 min-130 oC. Compared with other common carriers, Pt/CNT exhibited the highest catalytic performance in H2 production, emphasizing the critical role of CNT in methanol dehydrogenation. The confinement of Pt nanoparticles by CNTs is conducive to inhibiting the aggregation of Pt nanoparticles, thereby significantly increasing its catalytic performance and stability. The kinetic study, detailed mechanistic insights, and density functional theory (DFT) calculation confirm that the breaking of O─H bond of CH3OH is the rate-controlling step for methanol dehydrogenation, and both H atoms of H2 are supplied by methanol. Interestingly, H2 is also successfully produced from methanol dehydrogenation at -10 °C, which absolutely solves the freezing problem in the H2 evolution upon water-splitting reaction.

Sub-Millisecond Photoinduced Dynamics of Free and EL222-Bound FMN by Stimulated Raman and Visible Absorption Spectroscopies.

Time-resolved femtosecond-stimulated Raman spectroscopy (FSRS) provides valuable information on the structural dynamics of biomolecules. However, FSRS has been applied mainly up to the nanoseconds regime and above 700 cm-1, which covers only part of the spectrum of biologically relevant time scales and Raman shifts. Here we report on a broadband (~200-2200 cm-1) dual transient visible absorption (visTA)/FSRS set-up that can accommodate time delays from a few femtoseconds to several hundreds of microseconds after illumination with an actinic pump. The extended time scale and wavenumber range allowed us to monitor the complete excited-state dynamics of the biological chromophore flavin mononucleotide (FMN), both free in solution and embedded in two variants of the bacterial light-oxygen-voltage (LOV) photoreceptor EL222. The observed lifetimes and intermediate states (singlet, triplet, and adduct) are in agreement with previous time-resolved infrared spectroscopy experiments. Importantly, we found evidence for additional dynamical events, particularly upon analysis of the low-frequency Raman region below 1000 cm-1. We show that fs-to-sub-ms visTA/FSRS with a broad wavenumber range is a useful tool to characterize short-lived conformationally excited states in flavoproteins and potentially other light-responsive proteins.

A stepwise retro-imino-ene as a key step in the mechanism of allene formation via the Crabbé acetylene homologation.

The mechanism of the acetylene homologation procedure accidentally discovered and further developed by Crabbé and coworkers is unknown. Kinetic isotope effect (KIE) experiments, however, suggest that an intramolecular hydrogen shift is the key step of the transformation. In this work, we present a computational study of this mechanism. We found that the reaction proceeds via an unexpected stepwise retro-imino-ene rearrangement. This mechanism justifies the role of Cu(I) as a reaction catalyst and is also compatible with the KIE experiments reported.

Kinetic isotope effects and synthetic strategies for deuterated carbon-11 and fluorine-18 labelled PET radiopharmaceuticals.

The deuterium labelling of pharmaceuticals is a useful strategy for altering pharmacokinetic properties, particularly for improving metabolic resistance. The pharmacological effects of such metabolites are often assumed to be negligible during standard drug discovery and are factored in later at the clinical phases of development, where the risks and benefits of the treatment and side-effects can be wholly assessed. This paradigm does not translate to the discovery of radiopharmaceuticals, however, as the confounding effects of radiometabolites can inevitably show in preliminary positron emission tomography (PET) scans and thus complicate interpretation. Consequently, the formation of radiometabolites is crucial to take into consideration, compared to non-radioactive metabolites, and the application of deuterium labelling is a particularly attractive approach to minimise radiometabolite formation. Herein, we provide a comprehensive overview of the deuterated carbon-11 and fluorine-18 radiopharmaceuticals employed in PET imaging experiments. Specifically, we explore six categories of deuterated radiopharmaceuticals used to investigate the activities of monoamine oxygenase (MAO), choline, translocator protein (TSPO), vesicular monoamine transporter 2 (VMAT2), neurotransmission and the diagnosis of Alzheimer's disease; from which we derive four prominent deuteration strategies giving rise to a kinetic isotope effect (KIE) for reducing the rate of metabolism. Synthetic approaches for over thirty of these deuterated radiopharmaceuticals are discussed from the perspective of deuterium and radioisotope incorporation, alongside an evaluation of the deuterium labelling and radiolabelling efficacies across these independent studies. Clinical and manufacturing implications are also discussed to provide a more comprehensive overview of how deuterated radiopharmaceuticals may be introduced to routine practice.

Insights Into the Known 13C Depletion of Methane-Contribution of the Kinetic Isotope Effects on the Serine Hydroxymethyltransferase Reaction.

We determined the kinetic isotope effect on the serine hydroxymethyltransferase reaction (SHMT), which provides important C1 metabolites that are essential for the biosynthesis of DNA bases, O-methyl groups of lignin and methane. An isotope effect on the SHMT reaction was suggested being responsible for the well-known isotopic depletion of methane. Using the cytosolic SHMT from pig liver, we measured the natural carbon isotope ratios of both atoms involved in the bond splitting by chemical degradation of the remaining serine before and after partial turnover. The kinetic isotope effect 13(VMax/Km) was 0.994 0.006 and 0.995 0.007 on position C-3 and C-2, respectively. The results indicated that the SHMT reaction does not contribute to the 13C depletion observed for methyl groups in natural products and methane. However, from the isotopic pattern of caffeine, isotope effects on the methionine synthetase reaction and on reactions forming Grignard compounds, the involved formation and fission of metal organic bonds are likely responsible for the observed general depletion of "activated" methyl groups. As metal organic bond formations in methyl transferases are also rate limiting in the formation of methane, they may likely be the origin of the known 13C depletion in methane.

Oscillatory Active-site Motions Correlate with Kinetic Isotope Effects in Formate Dehydrogenase.

Thermal motions of enzymes have been invoked to explain the temperature dependence of kinetic isotope effects (KIE) in enzyme-catalyzed hydride transfers. Formate dehydrogenase (FDH) from Candida boidinii exhibits a temperature independent KIE that becomes temperature dependent upon mutation of hydrophobic residues in the active site. Ternary complexes of FDH that mimic the transition state structure allow investigation of how these mutations influence active-site dynamics. A combination of X-ray crystallography, two-dimensional infrared (2D IR) spectroscopy, and molecular dynamic simulations characterize the structure and dynamics of the active site. FDH exhibits oscillatory frequency fluctuations on the picosecond timescale, and the amplitude of these fluctuations correlates with the temperature dependence of the KIE. Both the kinetic and dynamic phenomena can be reproduced computationally. These results provide experimental evidence for a connection between the temperature dependence of KIEs and motions of the active site in an enzyme-catalyzed reaction consistent with activated tunneling models of the hydride transfer reaction.

Measurement of Kinetic Isotope Effects by Continuously Monitoring Isotopologue Ratios Using NMR Spectroscopy.

Nuclear magnetic spectroscopic (NMR) methods are discussed for the measurement of heavy atom (13C, 18O, 15N) and secondary deuterium kinetic isotope effects. The discussion focuses primarily on the NMR methods that enable the measurement of quantitative spectra and not on methods to make labeled substrates. Two main techniques are considered: single-point determinations on natural abundance material and the continuous monitoring of isotopically enriched materials. The second method is described in more detail, and we include a discussion of the current state of instrumentation and computer programs for data acquisition and analysis.

Novel polyamine analogues: from substrates towards potential inhibitors of monoamine oxidases.

New polyamine derivatives 1-8, related to the previously reported N(1),N(12)-dibenzyldodecane-1,12-diamine (Bis-Bza-Diado) and N(1)-benzyl-spermine (BD6), have been synthesized and used as "probes" (potential substrates or inhibitors) of the human monoamine oxidases (MAO A and MAO B) and Vascular-Adhesion-protein -1 (VAP-1). Compound 8, the most effective inhibitor of the series, is characterized by a 12-methylene carbon chain ending with an isothiocyanate (ITC) group. Interestingly, it behaves as competitive inhibitor of MAO B and as irreversible inhibitor of MAO A. Compound 3, an asymmetric spermine analogue bearing a thiophene ring, acts as a reversible mixed inhibitor, selective for MAO B (K(IE) = 23 µM). Docking studies performed using the available Protein Data Bank (PDB) structures of MAO A and MAO B, suggested that the different mode of inhibition of 8 may be explained by the different binding poses of 8 into the active site cavities of the two MAO isoforms. The ε-amino group of Lys 305 of MAO A is proposed as possible target of the ITC group of the inhibitor. Further studies are in progress to confirm this hypothesis. These results indicate a potential use of the polyamine scaffold for the development of new MAO inhibitors for application in human pathologies involving these enzymes.