Initial Rate Measurements Research Articles

Titration and quantification of open and closed Lewis acid sites in Sn-Beta zeolites that catalyze glucose isomerization

Methods to quantify framework Lewis acidic Sn4+ sites in zeolite Beta (Sn-Beta) with four Lewis base titrants (pyridine, deuterated acetonitrile, n-propylamine, ammonia) were developed using infrared (IR) spectroscopy or temperature programmed desorption (TPD). Integrated molar extinction coefficients (E, cmμmol−1) were measured for IR bands of pyridine adsorbed to Lewis acidic Sn sites (E(1450cm−1)=1.42±0.30) and of CD3CN adsorbed to open (E(2316cm−1)=1.04±0.22) and closed (E(2308cm−1)=2.04±0.43) Sn sites, and differ from analogous E values for Lewis acidic Al sites by up to 3.6×. TPD of Sn-Beta samples after saturation with NH3 or n-propylamine (NPA) and purging to remove physisorbed species also enabled quantification of Lewis acid sites, which is seldom performed despite analogous methods that use such titrants to quantify Brønsted sites in solid acids. These four Lewis bases titrated Lewis acidic Sn sites with equimolar stoichiometry and counted similar numbers of Sn sites on low-defect Sn-Beta samples synthesized in fluoride media (Sn-Beta-F) with high Sn content (Si/Sn <150). NPA binding on residual silanol defect sites, however, caused overestimation of Sn sites by TPD on Sn-Beta-F samples with low Sn content (Si/Sn >175) or high defect Sn-Beta samples prepared via post-synthetic insertion of Sn atoms into framework vacancy defects of dealuminated Beta zeolites (Sn-Beta-OH). Molar ratios of open-to-closed Sn sites varied widely (0.29–1.64) among the eight Sn-Beta samples studied here. Open Sn sites have been proposed as the dominant active sites for aqueous-phase glucose–fructose isomerization via intramolecular 1,2-hydride shift, consistent with successive poisoning of Sn-Beta samples with pyridine prior to measurement of initial glucose isomerization rates that suppressed reactivity at pyridine uptakes similar to the number of open Sn sites counted ex-situ by CD3CN. Measured first-order glucose isomerization rate constants in water (per open Sn, 373K), which reflect free energy differences between isomerization transition states and two coordinated water molecules at Sn sites, were ∼50× higher on hydrophobic Sn-Beta-F than on hydrophilic Sn-Beta-OH zeolites. The characterization methods reported herein enable normalization of initial glucose isomerization turnover rates on Sn-Beta zeolites by their number of open Sn sites, as required prior to interpreting the catalytic consequences of structural heterogeneities introduced by differences in sample preparation or treatment history.

Coupling carboxylic acid reductase to inorganic pyrophosphatase enhances cell-free in vitro aldehyde biosynthesis

Carboxylic acid reductases (CARs) have been harnessed in metabolic pathways to produce aldehydes in engineered organisms. However, desired aldehyde products inhibit cell growth and limit product titers currently achievable from fermentative processes. Aldehyde toxicity can be entirely circumvented by performing aldehyde biosynthesis in non-cellular systems. Use of purified CARs for preparative-scale aldehyde synthesis has been limited by in vitro turnover of model CARs, such as CarNi from Nocardia iowensis, despite robust conversion of substrates associated with expression in heterologous hosts such as E. coli and yeast. In this study, we report that in vitro activity of CarNi is inhibited by formation of the co-product pyrophosphate, and that pairing of an inorganic pyrophosphatase (PpaEc) with CarNi substantially improves the rate and yield of aldehyde biosynthesis. We demonstrate that, in the presence of PpaEc, Michaelis–Menten kinetic models based on initial rate measurements accurately predict CarNi kinetics within an in vitro pathway over longer timescales. We rationalize our novel observations for CarNi by examining previously posed arguments for pyrophosphate hydrolysis made in the context of other adenylate-forming enzymes. Overall, our findings may aid in increasing adoption of CARs for cell-free in vitro aldehyde biosynthetic processes.

Kinetics of acetophenone reduction to (R)-1-phenylethanol by a whole-cell Pichia capsulata biocatalyst

(R)-1-phenylethanol is an important substance in fragrance and flavor industry. In this work, the reduction of acetophenone to (R)-1-phenylethanol in an aqueous medium was examined using Pichia capsulata as a whole-cell biocatalyst. Progress curve and initial rate measurements were used to obtain kinetic data. The experiments were carried out at pH 5, temperature of 25 °C, and in the presence of glucose to maintain in vivo regeneration of NADH. A model of the reversible reaction kinetics considering the substrate inhibition of the forward reaction was developed. Five kinetic parameters of this model were determined by a simultaneous fit of a reaction rate dependence on substrate concentration and 18 substrate and product concentration progress curves with very good accuracy. Equilibrium constant of the reaction and equilibrium conversion of acetophenone to (R)-1-phenylethanol were 13.7 and 93%, respectively.

Whispering gallery mode resonators for rapid label-free biosensing in small volume droplets.

Rapid biosensing requires fast mass transport of the analyte to the surface of the sensing element. To optimize analysis times, both mass transport in solution and the geometry and size of the sensing element need to be considered. Small dielectric spheres, tens of microns in diameter, can act as label-free biosensors using whispering gallery mode (WGM) resonances. WGM resonances are sensitive to the effective refractive index, which changes upon analyte binding to recognition sites on functionalized resonators. The spherical geometry and tens of microns diameter of these resonators provides an efficient target for sensing while their compact size enables detection in limited volumes. Here, we explore conditions leading to rapid analyte detection using WGM resonators as label-free sensors in 10 μL sample droplets. Droplet evaporation leads to potentially useful convective mixing, but also limits the time over which analysis can be completed. We show that active droplet mixing combined with initial binding rate measurements is required for accurate nanomolar protein quantification within the first minute following injection.

On the Relationship between the Rate of Salivary Flow and Salivary Fluoride Clearance

The amount of fluoride retained in the mouth following the application of dentifrices, mouthwashes, etc. may be important in determining their anticaries efficacy. In this study we investigated the relationship between the salivary flow rate and salivary fluoride clearance. Ten adults tested six mouthrinses, consisting of aqueous sodium fluoride solutions (0.013, 0.026 mol/l) with and without added sodium chloride (1.28 mol/l) or sucrose (0.44 mol/l), in a randomised order. Prior to each test, subjects swallowed, rinsed for 2 min with 2 ml water and then expectorated into a preweighed container to obtain a measure of initial saliva flow rate. Next, the procedure was repeated using one of the test rinses. Finally, samples of unstimulated whole saliva were collected for up to 3 h after each mouthrinse application and analysed for fluoride. Salivary fluoride concentrations were significantly lower after application of mouthrinses that contained either sucrose or NaCl, both of which compounds markedly enhanced salivary flow, than after the use of corresponding mouthrinses without any additive. Area under the salivary fluoride clearance curve (AUC) values were inversely correlated with salivary flow rate on an individual basis (p < 0.01). The observed behaviour could not be completely attributed to treatment dilution by saliva at the time of application.

Employment of Pedo-transfer functions for predicting initial and final infiltration rates using horton model and soil readily available characteristics

Field measurements of initial and final infiltration rates, two important soil characteristics in soil water management, are costly and time consuming. Research efforts are on for determining them by using the Horton model. Pedo-transfer functions (PTFs) were applied for predicting initial and final infiltration rates, and empirical coefficient k of the model using readily available characteristics (RACs) of the soil. Several RACs of the soil besides the soil infiltration curves were determined in 135 different points of East Azarbayjan Province, Iran. The infiltration rates of the model could be predicted reasonably well using the applied PTFs. Applied PTFs resulted in accurate prediction of initial and final infiltration rates of the model with adjusted R2 of 0.798 to 0.872 and 0.639 to 0.715, respectively. There was however, low accuracy for the empirical coefficient of the model.

Kinetic characterization, optimum conditions for catalysis and substrate preference of secretory phospholipase A2 from Glycine max in model membrane systems.

Two secretory phospholipase A2 (sPLA2s) from Glycine max, GmsPLA2-IXA-1 and GmsPLA2-XIB-2, have been purified as recombinant proteins and the activity was evaluated in order to obtain the optimum conditions for catalysis using mixed micelles and lipid monolayers as substrate. Both sPLA2s showed a maximum enzyme activity at pH 7 and a requirement of Ca(2+) in the micromolar range. These parameters were similar to those found for animal sPLA2s but a surprising optimum temperature for catalysis at 60 °C was observed. The effect of negative interfacial charges on the hydrolysis of organized substrates was evaluated through initial rate measurements using short chain phospholipids with different head groups. The enzymes showed subtle differences in the specificity for phospholipids with different head groups (DLPC, DLPG, DLPE, DLPA) in presence or absence of NaCl. Both recombinant enzymes showed lower activity toward anionic phospholipids and a preference for the zwitterionic ones. The values of the apparent kinetic parameters (Vmax and KM) demonstrated that these enzymes have more affinity for phosphatidylcholine compared with phosphatidylglycerol, in contrast with the results observed for pancreatic sPLA2. A hopping mode of catalysis was proposed for the action of these sPLA2 on mixed phospholipid/triton micelles. On the other hand, Langmuir-monolayers assays indicated an optimum lateral surface pressure for activity in between 13 and 16 mN/m for both recombinant enzymes.

Heterogeneous Kinetic Study for Esterification of Acetic Acid with Ethanol

The esterification of acetic acid with ethanol has been investigated in the presence of a low cost ion exchange resin mainly used in the water treatment industry namely Trilite SCR-B as a heterogeneous catalyst. Internal mass transfer limitations have proven to be negligible using analysis based on Thieles modulus. The results of the experimental runs done by varying stirrer speeds of the reactor showed the absence of external mass transfer limitations. The kinetic experimental results were correlated with the heterogeneous kinetic models Langmuir-Hinshelwood (LH), Eley-Rideal (ER) and pseudo homogeneous kinetic model. The LH model was found to be correlating the experimental results better than the other two models. Reaction and adsorption constants were further evaluated within the temperature range between 335K and 355K, with 1:1 ethanol to acetic moles ratio under a catalyst loading of 50g/L. The study results of adsorption of both water and ethanol on to the cation exchange resin surface were found to be exothermic under the tested conditions. Furthermore, effect of concentration of each reactant on the reaction rate was examined using initial reaction rate measurements. ENGINEER, Vol. 47, No. 01, pp. 9-15, 2014

New Insight into the Residual Rate of Borosilicate Glasses: Effect of S/V and Glass Composition

We report and discuss initial and residual rate measurements performed on 6 borosilicate glasses to highlight (i) some glass composition effects (namely Zn and Fe), (ii) the role of dissolved silica, and (iii) the importance of the glass‐surface‐area‐to‐solution‐volume (S/V) ratio. We show that there is no apparent relation between the initial and the residual rate. As an example, the well‐known positive effect of Zn on short‐term glass dissolution becomes adverse under residual rate conditions. Dissolved silica as well as S/V strongly impact the transient rate drop regime, but have no or minor effects on the magnitude of the residual rate of the SON68 glass. Increasing S/V is indeed a good mean to explore higher reaction progresses. Additional simulations performed with the GRAAL model show that, in the case of SON68 glass altered in deionized water, the residual rate continuously diminishes despite the formation of secondary crystalline phases. This result can be used to derive conservative rate values. More generally, it is shown that the residual rate can fluctuate depending on the relative importance of two coupled mechanisms: the formation of a passivating layer and the transformation of this layer into more stable compounds.

Molybdenum versus Tungsten for the Epoxidation of Cyclooctene Catalyzed by [Cp*2M2O5

AbstractThe catalytic activity of [Cp*2M2O5] (M = Mo, W; Cp* = pentamethylcyclopentadienyl) for the homogeneous epoxidation of a solution of cyclooctene in MeCN/toluene follows the order Mo &gt;&gt; W when using tert‐butyl hydroperoxide (TBHP)/decane as oxidant, in contrast to the inverse order (W &gt;&gt; Mo) when using aqueous H2O2 as oxidant. The catalytic activity for the Mo system strongly depends on the solvent used to deliver the oxidant (TBHP/decane &gt;&gt; TBHP/H2O). The low activity of the W system is also decreased when using TBHP/water in place of TBHP/decane. For both metals, H2O2/H2O is a better oxidant than TBHP/H2O. However, whereas the Mo‐based catalyst is much more active for the TBHP/decane epoxidation in spite of the lower TBHP oxidizing power (TBHP/decane &gt; H2O2/H2O &gt; TBHP/H2O), the W‐based system is much more active for the H2O2/H2O epoxidation in spite of the negative effect of water (H2O2/H2O &gt; TBHP/decane &gt; TBHP/H2O). The kinetic profile of the TBHP/decane epoxidation process is affected by product inhibition. Initial rate measurements show that the rate law is first order with respect to substrate and has saturation behavior with respect to the oxidant.

Adenine Binding Mode Is a Key Factor in Triggering the Early Release of NADH in Coenzyme A-dependent Methylmalonate Semialdehyde Dehydrogenase

Structural dynamics associated with cofactor binding have been shown to play key roles in the catalytic mechanism of hydrolytic NAD(P)-dependent aldehyde dehydrogenases (ALDH). By contrast, no information is available for their CoA-dependent counterparts. We present here the first crystal structure of a CoA-dependent ALDH. The structure of the methylmalonate semialdehyde dehydrogenase (MSDH) from Bacillus subtilis in binary complex with NAD(+) shows that, in contrast to what is observed for hydrolytic ALDHs, the nicotinamide ring is well defined in the electron density due to direct and H(2)O-mediated hydrogen bonds with the carboxamide. The structure also reveals that a conformational isomerization of the NMNH is possible in MSDH, as shown for hydrolytic ALDHs. Finally, the adenine ring is substantially more solvent-exposed, a result that could be explained by the presence of a Val residue at position 229 in helix α(F) that reduces the depth of the binding pocket and the absence of Gly-225 at the N-terminal end of helix α(F). Substitution of glycine for Val-229 and/or insertion of a glycine residue at position 225 resulted in a significant decrease of the rate constant associated with the dissociation of NADH from the NADH/thioacylenzyme complex, thus demonstrating that the weaker stabilization of the adenine ring is a key factor in triggering the early NADH release in the MSDH-catalyzed reaction. This study provides for the first time structural insights into the mechanism whereby the cofactor binding mode is responsible at least in part for the different kinetic behaviors of the hydrolytic and CoA-dependent ALDHs.

Kinetic assay of D-Penicillamine in pure and pharmaceutical formulations based on ligand substitution reaction

A kinetic analytical method using uncatalyzed ligand substitution reaction and spectrophotometeric monitoring for the quantification of D-pencillamine (D-PA) i.e. 2-amino-3-mercapto-3-methylbutanoic acid or β,β-dimethylcysteine or 3-mercaptovaline or 3-mercapto-D-valine in pure as well as dosage forms has been validated and applied. The method is based on the uncatalyzed ligand substitution reaction between [Fe(CN)5(H2O)]3− and D-PA which produces the complex [Fe(CN)5(D-PA)]3−. The substituted complex [Fe(CN)5(D-PA)]3− shows an absorption maximum at 421nm. The proposed method shows good linear dynamic range of 14.92–149.21μgmL−1. By measuring the fixed time absorbance as a measure of initial rate of the complex formation during the course of the reaction at this wavelength, D-PA can be determined in the range 14.92–149.21μgmL−1. The recoveries of D-PA from various samples spiked at the usage level have been quantitative. The limit of quantification (3σ) was determined as 5.16, 3.68 and 3.13μgmL−1 of D-PA corresponding to the fixed time of 2, 3 and 5min respectively. The common excipients used as additives in pharmaceuticals did not interfere in the proposed method. The method was successfully employed for the analysis of D-PA content in commercial pharmaceutical preparations (tablets) and revealed quantities almost equal to those measured using the standard method, and demonstrated good accuracy and precision. The proposed method is rapid, simple, accurate and precise without the need for authentic analyte standards. It could therefore be used as an alternative to the quantification of D-PA in various samples.

Wood preservative properties of Zn and Mn containing solution obtained by battery recycling process

Wood preservative properties of a solution of ZnSO4 and MnSO4 (Zn/Mn molar ratio: 2∶1), obtained by a process for household battery recycling were studied. Its effect on the growth and enzymatic activity of wood rotting basidiomycetes (Coriolus versicolor, Phanerochaete chrysosporium, Pleurotus ostreatus, Ganoderma resinaceum, Punctularia atropurpurascens, Pycnoporus sanguineous, Inonotus sp, Gymnopilus spectabilis and Gloeophyllum trabeum) was analysed, based on measurements of initial growth rate, minimum inhibitory growth and decolourisation inhibiting concentration. The study solution inhibited mycelial growth and extracellular ligninolytic activity at concentrations between 0·5 and 2 mM Zn2+, depending on species. The above results were compared with the effect of solutions of pure sulphates. Study solution inhibited mycelial growth at a lower concentration than that of pure sulphates. Wood preservative effect of the study solution was demonstrated in tests using wood substrate (small blocks of Pinus taeda L. and Eucalyptus grandis), finding a minimum toxic value equivalent to 3% Zn2+.

Development and Characterization of a Portable Fibre Optic Dosimeter

A portable fibre optic dosimeter has been developed that uses transparent fluroperovskite dosimeters at the end of a multimode polymer fibre. It uses two stimulation sources that take advantage of the bimodal trap distribution so that the dose rate and cumulative dose can be monitored. Photoluminescence (PL), optically stimulated luminescence (OSL) and radioluminescence (RL) measurements were made on nearly tissue equivalent NaMgF3:Eu2+, NaMgF3:Mn2+, and high sensitivity RbMgF3:Eu2+ and CsCdF3:Mn2+. Initial dose rate and dose measurements were made using our fibre dosimeter during and after exposure to 60 keV gamma rays.

The photolysis of flash-frozen dilute hydrogen peroxide solutions

[1] Hydrogen peroxide (HOOH) in ice and snow is an important chemical tracer for the oxidative capacity of past atmospheres. A previous study on the photolysis of HOOH used ice samples made from solutions frozen over the course of minutes, conditions that should result in HOOH present in quasi-liquid layers (QLLs). However, since HOOH in natural snows and ices can also be present in the ice matrix, in this work we studied HOOH photolysis in ice samples prepared by flash freezing very dilute aqueous solutions, to keep HOOH in the ice matrix. In addition, rather than following HOOH photolysis by using an organic probe to scavenge photoformed OH, here we measured changes in HOOH concentrations. Based on initial rates of HOOH photolysis, our resulting quantum yields for OH formation, Φ(OH), in ice range from approximately 0.25 to 0.43 between 238 and 265 K, respectively. These values are up to 50% smaller than previously published values from the QLL HOOH experiments, indicating that HOOH in the ice matrix undergoes slower photolysis. Further, during our experiments the HOOH photolysis rate constant sometimes shows a biphasic behavior, with slower photolysis after several hours of illumination. Using these final rate constants of HOOH decay we derive a lower bound, temperature-independent value for Φ(OH) of 0.29(±0.10). Based on Φ(OH) values from our initial rate measurements, we calculate HOOH lifetimes in polar environments as large as 30 days at the summer solstice, which is too short to allow burial of HOOH beneath the photic zone in the snowpack.

Initial rates technique as a procedure to predict the anaerobic digester operation

In this study a novel and practical procedure was developed, that involves: initial methane production rate measurement in batch tests, kinetic parameters determination and modeling application in a continuous digester. The procedure was evaluated with three experimental conditions: raw sludge as substrate incubated at 35°C and 55°C and thermal pretreated sludge incubated at 35°C. The initial specific methane production rate was fitted with the Monod type equation in order to calculate the kinetic parameters. The values obtained for the maximum specific methane production rate were 0.043, 0.143 and 0.052gCH4gVSI−1d−1 for each experimental condition, aforementioned. The substantial increment of this parameter at thermophilic condition shows the differences in the specific maximum growth rate between thermophilic and mesophilic populations. The affinity constant values were 3.842, 4.790 and 4.623gL−1 for each experimental condition; however, a significant uncertainty was obtained due to some identification problems. A preliminary validation of the procedure was applied for predicting the operation of a continuous digester treating raw sewage sludge. The overall behavior of the system was represented by the model, although it slightly underestimates the experimental values, by approximately 20%. The results achieved, indicate that the procedure may be used as a tool in a real scale operation; however, further research must be performed.

RNA-directed packaging of enzymes within virus-like particles.

RNA-directed packaging of enzymes within virus-like particles.

Catalytic Reactions of the Homogentisate Prenyl Transferase Involved in Plastoquinone-9 Biosynthesis

Homogentisate solanesyl transferase (HST) catalyzes the prenylation and decarboxylation of homogentisate to form 2-methyl-6-solanesyl-1,4-benzoquinol, the first intermediate in plastoquinone-9 biosynthesis. In vitro, HST from Spinacia oleracea L., Arabidopsis thaliana, and Chlamydomonas reinhardtii were all found to use not only solanesyl diphosphate but also short chain prenyl diphosphates of 10-20 carbon atoms as prenyl donors. Surprisingly, with these donors, prenyl transfer was largely decoupled from decarboxylation, and thus the major products were 6-prenyl-1,4-benzoquinol-2-methylcarboxylates rather than the expected 2-methyl-6-prenyl-1,4-benzoquinols. The 6-prenyl-1,4-benzoquinol-2-methylcarboxylates were not substrates for HST-catalyzed decarboxylation, and the enzyme kinetics associated with forming these products appeared quite distinct from those for 2-methyl-6-prenyl-1,4-benzoquinol formation in respect of catalytic rate, substrate K(m) value, and the pattern of inhibition by haloxydine, a molecule that appeared to act as a dead end mimic of homogentisate. These observations were reconciled into a simple model for the HST mechanism. Here, prenyl diphosphate binds to HST to form at least two alternative complexes that go on to react differently with homogentisate and prenylate it either with or without it first being decarboxylated. It is supposed that solanesyl diphosphate binds tightly and preferentially in the mode that compels prenylation with decarboxylation.

Mechanistic Investigation of Chiral Phosphoric Acid Catalyzed Asymmetric Baeyer–Villiger Reaction of 3‐Substituted Cyclobutanones with H2O2 as the Oxidant

The mechanism of the chiral phosphoric acid catalyzed Baeyer-Villiger (B-V) reaction of cyclobutanones with hydrogen peroxide was investigated by using a combination of experimental and theoretical methods. Of the two pathways that have been proposed for the present reaction, the pathway involving a peroxyphosphate intermediate is not viable. The reaction progress kinetic analysis indicates that the reaction is partially inhibited by the gamma-lactone product. Initial rate measurements suggest that the reaction follows Michaelis-Menten-type kinetics consistent with a bifunctional mechanism in which the catalyst is actively involved in both carbonyl addition and the subsequent rearrangement steps through hydrogen-bonding interactions with the reactants or the intermediate. High-level quantum chemical calculations strongly support a two-step concerted mechanism in which the phosphoric acid activates the reactants or the intermediate in a synergistic manner through partial proton transfer. The catalyst simultaneously acts as a general acid, by increasing the electrophilicity of the carbonyl carbon, increases the nucleophilicity of hydrogen peroxide as a Lewis base in the addition step, and facilitates the dissociation of the OH group from the Criegee intermediate in the rearrangement step. The overall reaction is highly exothermic, and the rearrangement of the Criegee intermediate is the rate-determining step. The observed reactivity of this catalytic B-V reaction also results, in part, from the ring strain in cyclobutanones. The sense of chiral induction is rationalized by the analysis of the relative energies of the competing diastereomeric transition states, in which the steric repulsion between the 3-substituent of the cyclobutanone and the 3- and 3'-substituents of the catalyst, as well as the entropy and solvent effects, are found to be critically important.

Oxidation of alcohols using supported gold and gold–palladium nanoparticles

A key discovery in the last two decades has been the realisation that gold, when prepared as supported nanoparticles, is exceptionally effective as an oxidation catalyst, particularly for the oxidation of carbon monoxide at sub-ambient temperature but also for a number of organic reactions of synthetic significance. To some extent this observation is counterintuitive since extended gold surfaces do not chemisorb oxygen, nor do they corrode. For some oxidation reactions, the catalytic activity is markedly enhanced by the addition of palladium. This paper is concerned with recent advances in understanding the mechanism of catalysis by gold–palladium alloy nanoparticles of one such organic reaction, the oxidation of alcohols to the corresponding carbonyl compounds by molecular oxygen. We report detailed reaction studies using a high activity catalyst prepared by sol-immobilisation on a titania support. Using solvent-free conditions, benzyl alcohol is oxidised primarily to benzaldehyde but small amounts of toluene are also formed. The origin of these products is explored using initial rate measurements, deuterium labelling and kinetic isotope effects, and by the study of substituent effects. The effect of changing the nature of the catalyst support is also briefly examined. On the basis of all the results, we consider that we have evidence for multiple reaction pathways in this heterogeneous system. We put forward general mechanisms for the overall processes and describe confirmatory experiments in support of these, and we suggest possible reaction intermediates involved in the heterogeneously catalysed reaction.