Studies on the mechanism of the peroxyoxalate chemiluminescence reaction : part 2, further identification of intermediates using 2D EXSY 13C nuclear magnetic resonance spectroscopy

Further consideration has been given to the reaction pathway of a model peroxyoxalate chemiluminescence system. Again utilising doubly labelled oxalyl chloride and anhydrous hydrogen peroxide, 2D EXSY ¹³C nuclear magnetic resonance (NMR) spectroscopy experiments allowed for the characterisation of unknown products and key intermediate species on the dark side of the peroxyoxalate chemiluminescence reaction. Exchange spectroscopy afforded elucidation of a scheme comprised of two distinct mechanistic pathways, one of which contributes to chemiluminescence. ¹³C NMR experiments carried out at varied reagent molar ratios demonstrated that excess amounts of hydrogen peroxide favoured formation of 1,2-dioxetanedione: the intermediate that, upon thermolysis, has been long thought to interact with a fluorophore to produce light.

Degradation of VOCs by phototcatalytic oxidation reactor using TiO2 pellets

Volatile Organic Compounds (VOCs) are air pollutants that come from burning fossil fuels and industrial emissions. They have potentially adverse health effects being carcinogenic and highly persistent in the environment. The use of photocatalytic oxidation to remove VOCs has the potential to be applied in indoor air quality improvement and industrial emission control. A fixed bed photocatalytic reactor was designed and built. UV black light lamps were installed in the reactor to provide a source of UV radiation. A non-film titania media as pellets were placed on the three fixed beds within the reactor. Toluene and acetone were used as indicators of VOCs during the experiment. With a flow rate of 12.75l/min, the oxidation efficiencies were obtained at four different concentrations of acetone laden gas streams ranging from 40ppm to 250ppm. It was found that the lower the acetone concentration of the untreated inlet gas, the higher the oxidation efficiency. The oxidation efficiency was in the range of 40–70% for various concentrations of untreated gases. Two concentrations of toluene laden gas stream were also tested using the same reactor. The oxidation efficiencies were found as 50% for 120ppm toluene gas and 45% for 300ppm toluene gas. It was found that the times required for toluene to reach oxidization equilibrium have been halved than for acetone gas stream. Other parameters such as flow rate and UV intensity were also altered to see their effects on the oxidation efficiency. A full spectrum scan was carried out using a Bio-rad Infrared spectrometer. It was found that the main components of the treated gas stream from the outlet of the reactor were CO2 and water along with small amount of untreated acetone. The suspected intermediates of aliphatic hydrocarbons and CO were found in very minimal amounts or undetectable. The research experiments supported that the TiO2 pellets can work effectively in a fixed bed photocatalytic reactor and achieve significant oxidation efficiencies for degradation of toluene and acetone as indicators of VOCs.

Enhanced degradation efficiency of toluene using titania/silica photocatalysis as a regeneration process

Three kinds of titania/silica pellets were prepared using the sol-gel method with surface areas of 50.4m² g-1, 421.1m².g^-1 and 89.1m².g^-1. An annular reactor was designed and built to determine the degradation efficiency of toluene and to investigate the relationship between the adsorption and desorption-photocatalytic processes. Surface area is an important factor influencing the adsorption-photocatalytic efficiency. Higher surface areas of pellets contribute to high rates of conversion of toluene. Un-reacted toluene and reaction intermediates accumulating on their surface deactivated the titania/silica catalyst. To overcome this problem, the adsorption and regeneration process were alternated in a dual reactor system. Connecting or disconnecting the toluene feed gas enabled one reactor to adsorb toluene, while the second reactor was regenerated by photocatalysis. Using UV irradiation and titania/silica pellets with high BET surface area (421.1 m².g^-1), the alternating adsorption/regeneration processes kept the degradation efficiency of toluene at 90% after 8 hours operation. By improving the adsorption-photocatalysis efficiency, and minimising the generation and accumulation of intermediate on the surface of pellets, the method extended catalyst life and maintained a high degradation efficiency of toluene.

Glycogen availability does not affect the TCA cycle or TAN pools during prolonged, fatiguing exercise

The hypothesis that fatigue during prolonged exercise arises from insufficient intramuscular glycogen, which limits tricarboxylic acid cycle (TCA) activity due to reduced TCA cycle intermediates (TCAI), was tested in this experiment. Seven endurance-trained men cycled at approximately 70% of peak O(2) uptake (Vo(2 peak)) until exhaustion with low (LG) or high (HG) preexercise intramuscular glycogen content. Muscle glycogen content was lower (P < 0.05) at fatigue than at rest in both trials. However, the increase in the sum of four measured TCAI (>70% of the total TCAI pool) from rest to 15 min of exercise was not different between trials, and TCAI content was similar after 103 +/- 15 min of exercise (2.62 +/- 0.31 and 2.59 +/- 0.28 mmol/kg dry wt for LG and HG, respectively), which was the point of volitional fatigue during LG. Subjects cycled for an additional 52 +/- 9 min during HG, and although glycogen was markedly reduced (P < 0.05) during this period, no further change in the TCAI pool was observed, thus demonstrating a clear dissociation between exercise duration and the size of the TCAI pool. Neither the total adenine nucleotide pool (TAN = ATP + ADP + AMP) nor IMP was altered compared with rest in either trial, whereas creatine phosphate levels were not different when values measured at fatigue were compared with those measured after 15 min of exercise. These data demonstrate that altered glycogen availability neither compromises TCAI pool expansion nor affects the TAN pool or creatine phosphate or IMP content during prolonged exercise to fatigue. Therefore, our data do not support the concept that a decrease in muscle TCAI during prolonged exercise in humans compromises aerobic energy provision or is the cause of fatigue.

5`-aminoimidazole-4-carboxyamide-ribonucleoside- activated glucose transport is not prevented by nitric oxide synthase inhibition in rat isolated skeletal muscle

1. The nucleoside intermediate 5'-aminoimidazole-4-carboxyamide-ribonucleoside (AICAR) activates skeletal muscle AMP-activated protein kinase (AMPK) and increases glucose uptake. The AMPK phosphorylates neuronal nitric oxide synthase (nNOS)µ in skeletal muscle fibres. There is evidence that both AMPK and nNOSµ may be involved in the regulation of contraction-stimulated glucose uptake.
2. We examined whether both AICAR- and contraction-stimulated glucose uptake were mediated by NOS in rat skeletal muscle.
3. Rat isolated epitrochlearis muscles were subjected in vitro to electrically stimulated contractions for 10 min and/or incubated in the presence or absence of AICAR (2 mmol/L) or the NOS inhibitor NG-monomethyl-l-arginine (l-NMMA; 100 µmol/L).
4. Muscle contraction significantly (P < 0.05) altered the metabolic profile of the muscle. In contrast, AICAR and l-NMMA had no effect on the metabolic profile of the muscle, except that AICAR increased muscle 5'-aminoimidazole-4-carboxyamide-ribonucleotide (ZMP) and AICAR content. Nitric oxide synthase inhibition caused a small but significant (P < 0.05) reduction in basal 3-O-methylglucose transport, which was observed in all treatments. 5'-Aminoimidazole-4-carboxyamide-ribonucleoside significantly increased (P < 0.05) glucose transport above basal, with NOS inhibition decreasing this slightly (increased by 209% above basal compared with 184% above basal with NOS inhibition). Contraction significantly increased glucose transport above basal, with NOS inhibition substantially reducing this (107% increase vs 31% increase). 5'-Aminoimidazole-4-carboxyamide-ribonucleoside plus contraction in combination were not additive on glucose transport.
5. These results suggest that NO plays a role in basal glucose uptake and may regulate contraction-stimulated glucose uptake. However, NOS/nitric oxide do not appear to be signalling intermediates in AICAR-stimulated skeletal muscle glucose uptake.

Removal of VOCs by photocatalysis process using adsorption enhanced TiO2–SiO2 catalyst

Volatile organic compounds (VOCs) exist widely in both the indoor and outdoor environment. The main contributing sources of VOCs are motor vehicle exhaust and solvent utilization. Some VOCs are toxic and carcinogenic to human health, such as benzene. In this study, TiO₂–SiO₂ based photocatalysts were synthesized using the sol–gel method, with high surface areas of 274.1–421.1 m²/g obtained. Two types of pellets were used as catalysts in a fixed-bed reactor installed with a UV black light lamp. Experiments were conducted to compare their efficiencies in degrading the VOCs. Toluene was used as the VOC indicator. When the toluene laden gas stream passed through the photocatalytic reactor, the removal efficiencies were determined using a FTIR multi-gas analyser, which was connected to the outlet of the reactor to analyse the toluene concentrations. As the TiO₂–SiO₂ pellets used have a high adsorption capacity, they had dual functions as a photocatalyst and adsorbent in the hybrid photocatalysis and adsorption system. The experiments demonstrated that the porous photocatalyst with very high adsorptive capacity enhanced the subsequent photocatalysis reactions and lead to a positive synergistic effect. The catalyst can be self-regenerated by photocatalytic oxidation of the adsorbed VOCs. When the UV irradiation and feeding gas is continuous, a destruction efficiency of about 25% was achieved over a period of 20 h. Once the system was designed and operated into adsorption/regeneration mode, a higher removal efficiency of about 55% was maintained. It was found that the catalyst pellets with a higher surface area (421 m²/g) achieved higher conversion efficiency (100%) for a longer period than those with a lower surface area. A full spectrum scan was carried out using a Bio-rad Infrared spectrometer, finding that the main components of the treated gas stream leaving the reactor, along with untreated toluene, were CO₂ and water. The suspected intermediates of aliphatic hydrocarbons and CO were found in minimal amounts or were non detectable. The kinetic rate constants were calculated from the experimental results, it appeared that the stronger adsorption capacity, i.e. larger specific surface area, the higher conversion efficiency would be achieved.

Drug bioactivation, covalent binding to target proteins and toxicity relevance

A number of therapeutic drugs with different structures and mechanisms of action have been reported to undergo metabolic activation by Phase I or Phase II drug-metabolizing enzymes. The bioactivation gives rise to reactive metabolites/intermediates, which readily confer covalent binding to various target proteins by nucleophilic substitution and/or Schiff's base mechanism. These drugs include analgesics (e.g., acetaminophen), antibacterial agents (e.g., sulfonamides and macrolide antibiotics), anticancer drugs (e.g., irinotecan), antiepileptic drugs (e.g., carbamazepine), anti-HIV agents (e.g., ritonavir), antipsychotics (e.g., clozapine), cardiovascular drugs (e.g., procainamide and hydralazine), immunosupressants (e.g., cyclosporine A), inhalational anesthetics (e.g., halothane), nonsteroidal anti-inflammatory drugs (NSAIDSs) (e.g., diclofenac), and steroids and their receptor modulators (e.g., estrogens and tamoxifen). Some herbal and dietary constituents are also bioactivated to reactive metabolites capable of binding covalently and inactivating cytochrome P450s (CYPs). A number of important target proteins of drugs have been identified by mass spectrometric techniques and proteomic approaches. The covalent binding and formation of drug-protein adducts are generally considered to be related to drug toxicity, and selective protein covalent binding by drug metabolites may lead to selective organ toxicity. However, the mechanisms involved in the protein adduct-induced toxicity are largely undefined, although it has been suggested that drug-protein adducts may cause toxicity either through impairing physiological functions of the modified proteins or through immune-mediated mechanisms. In addition, mechanism-based inhibition of CYPs may result in toxic drug-drug interactions. The clinical consequences of drug bioactivation and covalent binding to proteins are unpredictable, depending on many factors that are associated with the administered drugs and patients. Further studies using proteomic and genomic approaches with high throughput capacity are needed to identify the protein targetsof reactive drug metabolites, and to elucidate the structure-activity relationships of drug's covalent binding to proteins and their clinical outcomes.

Kinetic modelling for photosynthesis of hydrogen and Methane through catalytic reduction of carbon dioxide with water vapour

In a photocatalytic reduction process when products formed are not effectively desorbed, they could hinder the diffusion of intermediates on the surface of the catalyst, as well as increase the chance of collisions among the products, resulting photo-oxidation in a reserve reaction on the surface. This paper analyses a simple kinetic model incorporating the coupled effect of the adsorptive photocatalytic reduction and oxidation. The development is based on Langmuir–Hinshelwood mechanism to model the formation rates of hydrogen and methane through photocatalytic reduction of carbon dioxide with water vapour. Experimental data obtained from literatures have achieved a very good fit. Such model could aid as a tool for related areas of studies. A comparative study using the model developed, showed that product concentration in term of ppm would be an effective measurement of product yields through photocatalytic reduction of carbon dioxide with water vapour.

Synthetic studies towards anthraquinones

The oxidation of substituted phenols with phenyliodonium diacetate in methanol was found to afford 2,4-cyclohexadienones, 2,5-cyclohexadienones or mixtures of isomers depending on the substrate being oxidized. A reaction mechanism was proposed for this oxidation which involved an intermediate aryloxenium ion. A strong correlation was observed between the experimentally determined product ratios and the results predicted by calculation of the LUMO coefficients of the proposed intermediates, Annulation of these cyclohexadienones with the anion derived from cyanophthalide afforded substituted anthraquinones in high yields. The chemistry relating to the annulation of Michael acceptors with phthalide anions was comprehensively reviewed. A mild selective method for the oxidation of hydroquinones to quinones using dibenzoyl peroxide and base is presented. A general synthetic approach to C-glycosylanthraquinones was presented, based on the annulation of a C-glycosylcyclohexadienone with the anion derived from cyanophthalide, A suitable precursor to a C-glycosylcyclohexadienone, 2-(2’,3’,4’,6’-tetra-0-acetyl-|3-D-glucopyranosyl)benzyloxybenzene, was prepared via the reaction of benzoylbromoglucose with 2-benzyloxyphenylmagnesium bromide, A group of molecules were prepared by a Marschalk reaction between /ewcoquinizarin and aldehydo-sugsrs. These compounds are potential bioreductive alkylating agents in which molecular simplicity can be achieved without overly sacrificing DNA binding ability.

Steroidogenesis in cultured mammalian glial cells

A protocol for culturing mammalian type 1 astrocytic cells, using female post-natal rat cerebral cortical tissue, was established and refined for use in steroidogenic metabolic studies incorporating progestin radioisotopes. Cultures were characterised for homogeneity using standard morphological and immunostaining techniques. Qualitative and quantitative studies were conducted to characterise the progesterone (P) metabolic pathways present in astrocytes in vitro. Of particular interest was the formation of the P metabolite, 5á-pregnan-3á-ol-20-one (THP). THP is a GABA(A) receptor agonist, believed to play a vital role in neural functioning and CNS homeostasis. One aim of this study was to observe any modulatory effects selected neuroactive ligands have on the conversion of P into THP, in an attempt to link astrocytic steroidogenesis with neuronal control. In qualitative studies, chromatographic procedures were used to establish the progestin profile of cerebral cortical astrocytes. Tritiated P, DHP (5á-pregnan-3,20-dione) and THP incurbates were preliminary fractionated by either normal phase (NP) or reverse phase (RP) high performance liquid chromatography (HPLC). The radiometabolites associated with each fraction were further chromatographed, before and/or after chemical derivatistation, by the aforemention HPLC procedures and thin layer chromatography (TLC). Steroid radiometabolites were tentatively identified by comparing their chromatographic mobility with authentic steroids. The identity of the main putative 5á-reduced P metabolities, DHP, THP and 5á-pregnan-3á,20á-diol (20áOH-THP) were further confirmed by isotopic dilution analysis. Their conclusive identification, along with the tentative identification of 20á-hydroxypreg-4-en-3-one (20áOH-P) and 20á-hydroxy-5á-pregnan-3-one (20áOH-DHP), verify the localisation of 5á-reductase, 3á-hydroxy steroif oxidoreductase (HSOR), and 20á-HSOR activity in the cultured astrocytes utilised in this study programme. Other minor metabolites detected were tentatively identified, including 5á-pregnan-3á,21-diol-20-one (THDoc), indicating the presence of 21-hydroxylase enzymatic activity. THDoc, like THP, is a GABA(A) receptor agonist. The chemical and physical characterisation of several yet unidentified progestin metabolites, associated with a highly polar RP HPLC fraction (designated RP peak 1*), indicate the presence of one or more extra hydroxylase enzymes. Quantitative analysis included a preliminary study. In this study, the percentage yields of radiometabolites formed in cultures incubated with increasing substrate concentrations of (3)H-P for 24 hours were determined. At the lower concentrations examined (ie 0.5 to 50nM), the metabolites associated with the polar RP HPLC fraction (RP peak 1*) collectively have the highest percentage yield. They are subsequently considered metabolic end products of degradative catabolic P pathways. The percentage yield of THP peaks in the medium concentration ranges (ie 5 to 500nM), whereas DHP remains fairly static at a low level with increasing concentration. Both DHP and THP are considered metabolic pathway intermediates. The percentage yield of 20áOH-THP continues to increase with increasing concentration over 5nM, superseding THP approaching the highest concentration examined (5000nM). This indicated the formation of 20áOH-THP does not occur entirely via THP. 20áOH-THP also possibly serves as the direct intermediate in the formation of the main radiometabolites associated with RP peak 1*. A time/yield study incorporating incubation times from one to 24 hours was also conducted. The full array of radiometabolites (individually or in groups) formed in astrocyte cultures incubated with 50nM tritiated P, DHP of THP, were assayed. Cultures were observed to rapidly convert any DHP into THP, showing astrocytic 3á-HSOR activity is very high. The study also showed 5á-reduction (ie the conversation of P into DHP) is the rate limiting reaction in the two step conversion of P into THP. 5á-Reduction also appears to be a rate limiting step in the formation of 20á-hydroxylated metabolites in astrocytes. Cultures incubated with the tritiated 5á-reduced pregnanes from one to four hours form greater quantities to 20á-hydroxylated radiometabolites compared to cultures incubated with (3)H-P. The time yield/studies also provided further evidence the unidentified polar radiometabolites associated with RP peak 1* are metabolic end products. For the P and DHP incubates, the collective formation of the aforementioned polar radiometabolites initially lags behind the formation of THP. As the formation of the latter begins to plateau with increasing time between four to 24 hours, the net yield of radiometabolites associated with RP peak 1* continues to rise. The time/yield studies also indicate 5á-reduction and perhaps 3á-hydroxylation are pre-requisite steps in the formation of the polar metabolites. Cultures incubated with the 5á-reduced progestins from one to four hours form higher yields of the radiometabolites associated with RP peak 1* compared to cultures incubated with P as substrate. The net yields of the radiometabolites associated with RP peak 1* for cultures incubated with THP were substantially higher compared to cultures incubated with DHP after equivalent times. The effect selected neuroligands have on the yield of radiometabolites formed by cultured astrocytes incubated with 50nM (3)H-P was also examined. Dibutyryl cyclic adenosine monophosphate (DBcAMP), not actually a neuroligand per se, but an analog of the intracellular secondary messenger cAMP, was also utilised in these studies. The inhibitory neurotransmitter ă-amino-nbutyric acid (GABA), DBcAMP and isoproterenol (a â-adrenergic receptor agonist) all quickly induce a transient but substantial increase in 20á-HSOR activity in cultured astrocytes. Cultures pretreated with these three compounds (10, 20 and 1µM respectively) form substantially higher yields of 20á-hydroxylated metabolites, including 20áOH-THP (between 200 to 580% greater), when incubated with 50nM (3)H-P for one to four hours. These increases also coincide with increases in the net yield of metabolites formed (by 16 to 48%). The same pre-treated cultures form significantly lower yields of THP, by 25 to 41%, after one hour. This is most likely due to the increased metabolism of any formed THP into 20áOH-THP. Octopamine (an á-adrenergic agonist) only induces a slight increase in 20á-HSOR activity, having relatively little effect on the yield of 20áOH-THP formed. Pretreatment with octopamine induces a significant increase in the yield of THP for cultures incubated with (3)H-P for four hours (by 24%). The increase in THP formation appears to be due to an increase in 3á-HSOR activity, as judged by the concomitant drop in the yield of the 5á-reduced, 3-keto substrates. An increase in 5á-reductase activity cannot be excluded however. Isoproterenol appears to induce an increase in 5á-reductase activity as isoproterenol appears to induce an increase in 5á-reductase activity as isoproterenol one and four hour incubates have higher yields of DHP. This is in contrast to the other three incubates. After 12 hours, all incubates have higher yields of THP (15-30%).

Improved synthesis of commercially valuable opiates : development of reactions for industrial applications

Describes the synthesis of thebaine and noroxymorphone - key intermediates in the synthesis of opiate drugs. The design and optimisation of these synthetic pathways were developed keeping in mind the concerns of industry.

Mechanism of permanganate chemiluminescence

Spectroscopic and synthetic methods have been exploited to deduce the mechanism for acidic potassium permanganate chemiluminescence. We have employed electron paramagnetic resonance (EPR) spectroscopy with a continuous flow assembly to monitor the formation of radical intermediates in real time generated from substrate oxidation by manganese(VII). These transient species react with manganese(III) in solution to produce the  previously characterized manganese(II)* emission source. Using UV-vis, EPR, attenuated total reflection (ATR)-FTIR, and chemiluminescence spectroscopies, we have established that there are two distinct enhancement mechanisms that in combination afford a 50-fold increase in emission intensity when the reaction is conducted in the presence of phosphate oligomers. In addition to preventing disproportionation of the manganese(III) precursor, the phosphate oligomers form protective "cagelike” structures around the manganese(II)* emitter, thus preventing nonradiative relaxation pathways.

Evolutionary specialization of a tryptophan indole group for transition-state stabilization by eukaryotic transglutaminases

Covalent posttranslational protein modifications by eukaryotic transglutaminases proceed by a kinetic pathway of acylation and deacylation. Ammonia is released as the acylenzyme is formed, whereas the cross-linked product is released later in the deacylation step. Superposition of the active sites of transglutaminase type 2 (TG2) and the structurally related cysteine protease, papain, indicates that in the formation of tetrahedral intermediates, the backbone nitrogen of the catalytic Cys-277 and the N_Ɛ1 nitrogen of Trp-241 of TG2 could contribute to transition-state stabilization. The importance of this Trp-241 side chain was demonstrated by examining the kinetics of dansylcadaverine incorporation into a model peptide. Although substitution of the Trp-241 side chain with Ala or Gly had only a small effect on the Michaelis constant K_m (1.5-fold increase), it caused a >300-fold lowering of the catalytic rate constant k_cat. The wild-type and mutant TG2-catalyzed release of ammonia showed kinetics similar to the kinetics for the formation of cross-linked product, indicating that transitionstate stabilization in the acylation step was rate-limiting. In papain, a Gln residue is at the position of TG2-Trp-241. The conservation of Trp-241 in all eukaryotic transglutaminases and the finding that W241Q-TG2 had a much lower k_cat than wild-type enzyme suggest evolutionary specialization in the use of the indole group. This notion is further supported by the observation that transitionstate- stabilizing side chains of Tyr and His that operate in some serine and metalloproteases only partially substituted for Trp.

Real-time, in-situ, extinction spectroscopy studies on silver-nanoseed formation

In this paper, real-time extinction spectroscopy was employed to analyze in situ the fast formation process of silver seeds. The influencing factors for silver-seed formation, including the concentration of the reducer (NaBH 4), the amount of stabilizer (citrate), and the addition procedure of NaBH4, were evaluated. The configuration of the surface plasmon resonance (SPR) spectra of silver nanoparticles, which reflected the morphology of the produced silver seeds, was found to be distinctly affected by the NaBH4 concentration. Homogeneous silver nanoparticles were obtained when the added NaBH4 was lower in concentration than 2.00 mM. In contrast, higher concentrations of reducing agent (>3.00 mM NaBH4) resulted in uneven silver nanoparticles. Mie theory was applied to clarify the correlation of the SPR and the size of the silver seeds. Repeated additions of a small amount of the reducing agent could increase the monodispersity and isotropy of silver seeds. Real-time extinction spectroscopy is a convenient technique that achieves the in-situ and nondestructive measurement of intermediates in the formation process of silver nanoseeds.