Analysis Of Reaction Products Research Articles

TiO2 Photocatalyzed Oxidation of Drugs Studied by Laser Ablation Electrospray Ionization Mass Spectrometry

In drug discovery, it is important to identify phase I metabolic modifications as early as possible to screen for inactivation of drugs and/or activation of prodrugs. As the major class of reactions in phase I metabolism is oxidation reactions, oxidation of drugs with TiO2 photocatalysis can be used as a simple non-biological method to initially eliminate (pro)drug candidates with an undesired phase I oxidation metabolism. Analysis of reaction products is commonly achieved with mass spectrometry coupled to chromatography. However, sample throughput can be substantially increased by eliminating pretreatment steps and exploiting the potential of ambient ionization mass spectrometry (MS). Furthermore, online monitoring of reactions in a time-resolved way would identify sequential modification steps. Here, we introduce a novel (time-resolved) TiO2-photocatalysis laser ablation electrospray ionization (LAESI) MS method for the analysis of drug candidates. This method was proven to be compatible with both TiO2-coated glass slides as well as solutions containing suspended TiO2 nanoparticles, and the results were in excellent agreement with studies on biological oxidation of verapamil, buspirone, testosterone, andarine, and ostarine. Finally, a time-resolved LAESI MS setup was developed and initial results for verapamil showed excellent analytical stability for online photocatalyzed oxidation reactions within the set-up up to at least 1 h.Graphical

Analysis of Reaction Products After Ignition Process of 1-octanol/air Mixtures on a Hot Surface

1-octanol is considered an alternative to conventional diesel. In order to know the ignition parameters of 1-octanol, an experimental study of the explosive combustion of 1-octanol/air mixtures at ambient pressure and various initial temperatures was performed on a hot stainless-steel surface in a closed vessel. The ignition temperatures of 1-octanol/air mixtures at different initial conditions were determined. Using gas chromatography-mass spectrometry (GC-MS) and gas chromatography with flame ionisation detector (GC-FID) methods, the composition of the reaction products was investigated.

A study of thermal decomposition of bromoethane

The pyrolysis of bromoethane under dilute atmosphere and quasi-atmospheric pressure was studied at temperatures from 600 to 975 K using a fused silica jet stirred reactor (JSR) and an alumina tubular reactor (TR). In the JSR, a complete conversion was observed at a residence time of 2 s, a temperature of 975 K and an inlet mole fraction of bromoethane of 0.01. In the TR, the full conversion was observed from 925 K. In both reactors, the following species were quantified: methane, ethylene, acetylene, ethane, 1,3-butadiene, vinylacetylene and vinyl bromide. A new detailed kinetic model was developed and gave a good prediction for both reactors of the global reactivity and the major products. Flow rate and sensitivity analyses have been performed to better understand the important reaction pathways during bromoethane pyrolysis. They showed, in contrast to previous studies, that the radical mechanism is far from being negligible and that the sole analysis of reaction products can be misleading as the radical mechanism mainly lead to ethylene and HBr which are also the two products from the molecular decomposition reaction of bromoethane.

Ionic-Strength Effects on the Reactive Uptake of Ozone on Aqueous Pyruvic Acid: Implications for Air–Sea Ozone Deposition

A vertical wetted-wall flow-tube technique was used to explore the ionic strength effects at the air-water interface in mediating the sea-surface reaction between ozone (O3) and pyruvic acid (PA). The uptake coefficients of ozone on aqueous PA increase substantially with the concentrations of bromide (Br-) ions, clearly indicating that the dry deposition of ozone could be significantly enhanced due to the presence of carbonyl compounds such as PA at the bromide-rich sea surface. Based on the observed uptake coefficients, the estimated deposition velocity of ozone (100 ppb) for a nanomolar range of PA concentrations is ∼1 × 10-3 m s-1, which represents a significant contribution to the known deposition velocity of ozone at the sea surface. The analysis of reaction products by ultra-high-resolution Fourier transform-ion cyclotron resonance mass spectrometry suggests the formation of oligomers during both the dark and light-induced heterogeneous reactions between gaseous O3 and PA occurring at the surface of a dilute aqueous phase (representative of cloud droplets). The detected high-molecular-weight compounds are much more complex than the oligomeric species identified during the photolytic degradation of bulk aqueous PA alone.

Novel synthesis of lightweight geopolymer matrices from fly ash through carbonate-based activation

This paper describes a novel synthesis route for porous geopolymers based on carbonate-based activation of fly ash as the primary source material. The aqueous dissolution of sodium carbonate supplies NaOH which provides the alkaline medium for fly ash geopolymerization (along with externally supplied sodium silicate also, if needed). The CO2 released from the low temperature (∼100 °C) decomposition of carbonates formed as reaction products, acts as the pore-forming agent. The dosage of the alkaline agents is tailored to obtain a porous material with large enough porosity (∼50%), and sufficient mechanical strength (∼15 MPa) and thermal conductivity (∼0.3 W/m-K) so as to be used as structural/insulating elements in buildings. Detailed analysis of microstructure and reaction products reveals the presence of sodium carbonate crystals in the matrix, recrystallized from the initial carbonates added. Determination of CO2-equivalent emissions shows that these matrices, despite releasing some amount of CO2 in the pore-forming process, are more sustainable than similar matrices synthesized from portland cement or geopolymerized using same starting materials and other activating agents.

Members of the DUF231 Family are O-Acetyltransferases Catalyzing 2-O- and 3-O-Acetylation of Mannan

Mannans are hemicellulosic polysaccharides commonly found in the primary and secondary cell walls of land plants, and their mannosyl residues are often acetylated at O-2 and O-3. Currently, little is known about the genes responsible for the acetylation of mannans. In this report, we investigated the roles of a subgroup of DUF231 proteins including 11 from Arabidopsis thaliana and one from Amorphophallus konjac in mannan acetylation. Acetyltransferase activity assays of their recombinant proteins revealed that four Arabidopsis DUF231 proteins possessed an enzymatic activity capable of transferring acetyl groups from acetyl-CoA onto the mannohexaose acceptor, and thus were named mannan O-acetyltransferases (MOAT1, MOAT2, MOAT3 and MOAT4). Their close homolog from A. konjac (named AkMOAT1) also exhibited mannan O-acetyltransferase activity. Structural analysis of the MOAT-catalyzed reaction products demonstrated that these MOATs catalyzed 2-O- and 3-O-monoacetylation of mannosyl residues, an acetyl substitution pattern similar to that of Arabidopsis glucomannan. Site-directed mutagenesis showed that mutations of the conserved residues in the GDS and DXXH motifs of MOAT3 abolished its acetyltransferase activity, indicating the essential roles of these motifs in its activity. In addition, simultaneous RNA interference (RNAi) inhibition of the expression of the four Arabidopsis MOAT genes led to a drastic reduction in the degree of acetyl substitutions in glucomannan, further corroborating their role in glucomannan acetylation. Together, these results present the first lines of biochemical and genetic evidence demonstrating that these four Arabidopsis DUF231 members and their close A. konjac homolog are mannan O-acetyltransferases.

Xyloglucan O-acetyltransferases from Arabidopsis thaliana and Populus trichocarpa catalyze acetylation of fucosylated galactose residues on xyloglucan side chains.

AXY4/XGOAT1, AXY4L/XGOAT2 and PtrXGOATs are O-acetyltransferases acetylating fucosylated galactose residues on xyloglucan and AXY9 does not directly catalyze O-acetylation of xyloglucan but exhibits weak acetylesterase activity. Xyloglucan is a major hemicellulose that cross-links cellulose in the primary walls of dicot plants and the galactose (Gal) residues on its side chains can be mono- and di-O-acetylated. In Arabidopsis thaliana, mutations of three AXY (altered xyloglucan) genes, AXY4, AXY4L and AXY9, have previously been shown to cause a reduction in xyloglucan acetylation, but their biochemical functions remain to be investigated. In this report, we demonstrated that recombinant proteins of AXY4/XGOAT1 (xyloglucan O-acetyltransferase1), AXY4L/XGOAT2 and their close homologs from Populus trichocarpa, PtrXGOATs, displayed O-acetyltransferase activities transferring acetyl groups from acetyl CoA onto xyloglucan oligomers. Structural analysis of XGOAT-catalyzed reaction products revealed that XGOATs mediated predominantly 6-O-monoacetylation and a much lesser degree of 3-O and 4-O-monoacetylation and 4,6-di-O-acetylation of Gal residues on xyloglucan side chains. XGOATs appeared to preferentially acetylate fucosylated Gal residues with little activity toward non-fucosylated Gal residues. Mutations of the conserved amino acid residues in the GDS and DXXH motifs in AXY4/XGOAT1 resulted in a drastic reduction in its ability to transfer acetyl groups onto xyloglucan oligomers. In addition, although recombinant AXY9 was unable to transfer acetyl groups from acetyl CoA onto xyloglucan oligomers, it was catalytically active as demonstrated by its weak acetylesterase activity that was also exhibited by AXY4/XGOAT1 and AXY4L/XGOAT2. Furthermore, we showed that the AXY8 fucosidase was able to hydrolyze fucosyl residues from both non-acetylated and acetylated xyloglucan oligomers. These findings provide biochemical evidence that AXY4/XGOAT1, AXY4L/XGOAT2 and PtrXGOATs are xyloglucan O-acetyltransferases catalyzing acetyl transfer onto fucosylated Gal residues on xyloglucan side chains and the defucosylation of these acetylated side chains by apoplastic AXY8 generates side chains with acetylated, non-fucosylated Gal residues.

Development of macroporous Magnéli phase Ti4O7 ceramic materials: As an efficient anode for mineralization of poly- and perfluoroalkyl substances

Magnéli phase Ti4O7 ceramic materials having extensive interconnecting macropores with an average pore size of 2.6 μm and 21.6% porosity were fabricated using a high-temperature sintering method, and then tested as an anode in a batch mode for electrooxidative mineralization of environmentally persistent poly- and perfluoroalkyl substances (PFASs) such as perfluorooctanoate (PFOA) and perfluorooctanesulfonate (PFOS). Tests were also conducted with other “non-active” electrodes for comparison, including Ce-doped PbO2 and Ti/BDD electrodes. The results showed that the porous Ti4O7 ceramic anode exhibited greater PFOA/PFOS oxidation rates than those of the other electrodes. Only trace amounts of perfluorocarboxylic acid (PFCAs) with shortened chain lengths were observed as intermediates during PFOA mineralization. SO42− and F− were recovered as the mineralization products of PFOS electrooxidation, while no organofluorine compounds such as shorter-chain PFCAs were detected in the solution by high-resolution mass spectrometry (HRMS). Possible mechanisms for PFOA/PFOS mineralization over porous Ti4O7 ceramic electrode were proposed on the basis of reaction products analysis. In addition, the developed porous Ti4O7 ceramic anode was successfully applied to treatment of the still bottom liquid waste containing high concentrations of poly- and perfluoroalkyl substances (PFASs) as well as high chloride and organic content concentrations from the regeneration of ion-exchange resin that had been used for remediation of PFASs-impacted groundwater. The results illustrate the promise of the macroporous Magnéli phase Ti4O7 ceramic materials for electrochemical treatment of PFASs in water.

An Entropic Approach for Controlled Testing of Electrochemical Components

Custom-designed electrochemical test systems allow for specific measurements to be generated for isolated variables in advanced materials testing. A well designed first-principle system can give insight into more complex interactions often found in real-world devices. Often, such isolated test systems utilize sub-systems that draw upon external reactants for sustained testing and exhaust reaction products as a result. Practical limitations on delivery of purified feed streams and mediation of exhaust require system operation that can fail unexpectedly, often as a result of failure of the system itself rather than of the materials under investigation. A novel system for testing of zinc-air fuel cell cathodes is presented as an example system that allows for total internal flows of reactant and product chemical flows. Such systems allow for cyclic purification and analysis of reaction products and establishment of a device in which the only output of the testing is heat and reliable data. Examination of test system control of state properties and elimination of suspected system contaminants highlight the benefits of the developed self-contained flow systems. Insight into general design characteristics for robust electrochemical test systems are developed in light of the limitations of the presented entropy machines.

Rotating Disk Electrode – Online Electrochemical Mass Spectrometry for Oxygen Reduction Reaction on Pt Electrode Surfaces

Potential-dependent molecular behavior of dissolved O2 in the vicinity of a polycrystalline Pt electrode surface was studied in O2-saturated 0.1 M HClO4 solution using a newly developed online electrochemical mass spectrometry (OLEMS) with rotating disk electrode (RDE) method (RDE-OLEMS). A gas sampling tip was positioned at ~50 μm from the electrode surface; O2 concentration in the vicinity of the Pt electrode at open circuit potential (~0.8 V vs. RHE) depended on the disk rotation rates, and linear sweep voltammetry (LSV) of the Pt electrode showed diffusion-limiting currents in the RDE-OLEMS layout. Furthermore, simultaneously recorded RDE-OLEMS data for the potential-dependent O2 concentration were consistent with the LSV data recorded for a negative-going sweep. We have successfully applied the newly developed RDE-OLEMS method and it is effective for in-situ analysis of gaseous electrochemical reaction products and dissolved molecules in the vicinity of the electrode under electrochemical conditions.

Enhanced nitrate removal and high selectivity towards dinitrogen for groundwater remediation using biochar-supported nano zero-valent iron

We prepared nano zero-valent iron/biochar composites (nZVI/BC) to remove nitrate from groundwater with harmless products. Results show nZVI/BCs derived from different mass ratios of Fe(0) to biochar exhibited different nitrate removal efficiencies, and the mass ratio of 1:2 developed the nZVI/BC with optimum nitrate removal. The nZVI/BC maintained preferable nitrate removal (75.0%–97.0%) over a wide pH range 2–12. Nitrate removal amount declined due to serious corrosion and clogging of nZVI/BC’s surface when initial nitrate concentration exceeded 40 mg/L. The nitrate removal process followed a first-order kinetic reaction. As regards real groundwater, the nZVI/BC removed more nitrate than both pure Fe(0) nanoparticles and biochar. Reaction product analysis revealed 60.1% of removed nitrate selectively became dinitrogen. X-ray photoelectron spectroscopy measurements suggested that nitrate-oxidized Fe(0) resulted in significant increase of magnetite on the surface of nZVI/BC. The redox potential and pH of long duration in reaction changed around −210 mV and 8–9, respectively. These changes facilitated the selective reduction of nitrate to dinitrogen. We also proposed that biochar may provide favorable circumstances for nitrate reduction by directly mediating redox potential, pH and electron transfer, which establishes a possible mechanism for the enhanced removal and selective reduction of nitrate. Our study suggests that nZVI/BC would be a promising alternative for the remediation of nitrate-contaminated groundwater.

The Effects of Modifying the Catalytically Active Phase Through Ni Impregnation of the Cu-Ru Catalyst on Carbon Support in Nitrobenzene Hydrogenation

We report herein on the hydrogenation of nitrobenzene over 5%Cu-1%Ru/C and 5%Cu-1%Ru-8%Ni/C catalysts respectively, using a pilot laboratory installation and their performances in order to outline the effect of Ni addition in the Cu and Ru-based catalyst. The experiments were performed on beds of catalyst extrudate, with a volume of 40 cm3, for a range of temperatures from 45 to 75�C, pressures from 1 to 4 MPa, space velocities from 0.2-0.6h-1, feed ratio H2/C6H5NO2=10/1mol/mol. The performances were evaluated based on conversion values of nitrobenzene and the yields of the most significant reaction products, both of which were assessed through analyses of raw materials and reaction products by GC-MS. Although Ni is widely known as a common catalyst for hydrogenation, we have established the inhibitory effect of Ni addition on the Cu-Ru/C catalyst obtaining, in some cases lower total conversions, but having a potent effect on modifying the selectivity of reaction products, with a quantitatively varying effect depending on working pressure.

Gas Chromatography Columns Based on Functionalized Polymer for the Analysis of Catalytic Reaction Products

For the first time in this work, two methods of modification of poly(1-trimethylsilyl-1-propyne) (PTMSP) and the possibilities of its application for the determination of catalytic reaction products are described. Aging of PTMSP is determined by three mechanisms: physical, chemical and mechanical effects. Free volume decreases, texture characteristics change, and partial oxidation of the polymer takes place in the structure of PTMSP. The chromatographic behavior of PTMSP, which was used for 18 months, differs significantly from the properties of the freshly prepared polymer. Thermal stability and chemical inertness are the main requirements for chromatographic materials. Therefore, two methods were used to stabilize the aging process. The first method consisted of preparing a mixed phase by adding disubstituted polyacetylene poly(1-phenyl-1-propyne) (PPP) to PTMSP. The resulting sorbent Chromosorb Р NAW + 10 wt% (97% PTMSP + 3% PPP) is efficient for solving a wide range of chromatographic tasks such as simultaneous analysis of hydrocarbons and sulfur-containing inorganic gases, analysis of chlorine-substituted methanes in CCl4 and thiophene in benzene, separation of hydrocarbons and aromatic compounds. It should be noted, the low selectivity of separation of oxygen-containing components on this sorbent. A second method of PTMSP modification, namely the oxidation treatment with nitrous oxide, is proposed. It was shown that the polymer is oxidized by means of its C=C double bond with the formation of ketone, carbonyl and hydroxyl groups. The increase in number of those groups in the polymer changed the polarity of PTMSP. The change in polarity of polymer led to the selectivity of separations, including oxygen-containing substances. Examples of separations of aliphatic and aromatic hydrocarbons, oxygen- and halogen-containing substances are presented. Chromatographic characteristics of the sorbent Chromosorb P NAW + 10 wt% (97% PTMSP + 3% PPP) and PTMSP oxidized by nitrous oxide are stable in the temperature range from 30 to 220 °C for at least 1.5 years of continuous operation.

Special issue: Plasma and Liquids

Special issue: Plasma and Liquids

Chemical cross-linking in the structural analysis of protein assemblies.

For decades, chemical cross-linking of proteins has been an established method to study protein interaction partners. The chemical cross-linking approach has recently been revived by mass spectrometric analysis of the cross-linking reaction products. Chemical cross-linking and mass spectrometric analysis (CXMS) enables the identification of residues that are close in three-dimensional (3D) space but not necessarily close in primary sequence. Therefore, this approach provides medium resolution information to guide de novo structure prediction, protein interface mapping and protein complex model building. The robustness and compatibility of the CXMS approach with multiple biochemical methods have made it especially appealing for challenging systems with multiple biochemical compositions and conformation states. This review provides an overview of the CXMS approach, describing general procedures in sample processing, data acquisition and analysis. Selection of proper chemical cross-linking reagents, strategies for cross-linked peptide identification, and successful application of CXMS in structural characterization of proteins and protein complexes are discussed.

Synthesis of Aesculetin and Aesculin Glycosides Using Engineered Escherichia coli Expressing Neisseria polysaccharea Amylosucrase.

Because glycosylation of aesculetin and its 6-glucoside, aesculin, enhances their biological activities and physicochemical properties, whole-cell biotransformation and enzymatic synthesis methodologies using Neisseria polysaccharea amylosucrase were compared to determine the optimal production method for glycoside derivatives. High-performance liquid chromatography analysis of reaction products revealed two glycosylated products (AGG1 and AGG2) when aesculin was used as an acceptor, and three products (AG1, AG2, and AG3) when using aesculetin. The whole-cell biotransformation production yields of the major transfer products for each acceptor (AGG1 and AG1) were 85% and 25%, respectively, compared with 68% and 14% for enzymatic synthesis. These results indicate that whole-cell biotransformation is more efficient than enzymatic synthesis for the production of glycoside derivatives.

A group of Populus trichocarpa DUF231 proteins exhibit differential O-acetyltransferase activities toward xylan.

Wood represents the most abundant biomass produced by plants and one of its major components is acetyl xylan. Acetylation in xylan can occur at O-2 or O-3 of a xylosyl residue, at both O-2 and O-3 of a xylosyl residue, and at O-3 of a xylosyl residue substituted at O-2 with glucuronic acid. Acetyltransferases responsible for the regiospecific acetylation of xylan in tree species have not yet been characterized. Here we report the biochemical characterization of twelve Populus trichocarpa DUF231-containing proteins, named PtrXOATs, for their roles in the regiospecific acetylation of xylan. The PtrXOAT genes were found to be differentially expressed in Populus organs and among them, PtrXOAT1, PtrXOAT2, PtrXOAT9 and PtrXOAT10 exhibited the highest level of expression in stems undergoing wood formation. Activity assays of recombinant proteins demonstrated that all twelve PtrXOAT proteins were able to transfer acetyl groups from acetyl CoA onto a xylohexaose acceptor with PtrXOAT1, PtrXOAT2, PtrXOAT3, PtrXOAT11 and PtrXOAT12 having the highest activity. Structural analysis of the PtrXOAT-catalyzed reaction products using 1H NMR spectroscopy revealed that PtrXOAT1, PtrXAOT2 and PtrXOAT3 mediated 2-O- and 3-O-monoacetylation and 2,3-di-O-acetylation of xylosyl residues and PtrXOAT11 and PtrXOAT12 only catalyzed 2-O- and 3-O-monoacetylation of xylosyl residues. Of the twelve PtrXOATs, only PtrXOAT9 and PtrXOAT10 were capable of transferring acetyl groups onto the O-3 position of 2-O-glucuronic acid-substituted xylosyl residues. Furthermore, when expressed in the Arabidopsis eskimo1 mutant, PtrXOAT1, PtrXAOT2 and PtrXOAT3 were able to rescue the defects in xylan acetylation. Together, these results demonstrate that the twelve PtrXOATs are acetyltransferases with different roles in xylan acetylation in P. trichocarpa.

Protease of Stenotrophomonas sp. from Indonesian fermented food: gene cloning and analysis

Screening of proteolytic and fibrinolytic bacteria from Indonesian soy bean based fermented food Oncom revealed several potential isolates. Based on 16s rDNA gene analysis, one particular isolate with the highest proteolytic and fibrinolytic activity was identified as Stenotrophomonas sp. The protease gene was amplified to generate a 1749 bp Polymerase Chain Reaction product and BLAST analysis, revealed 90% homology with gene encoding protease enzyme from Stenotrophomonas maltophilia. The putative amino acid sequence indicated a serine protease enzyme with typical amino acid aspartate, histidine and serine in the catalytic triad. The gene was translated into a pre-pro-protein consisted of cleavage site on its N terminal and Pre-Peptidase Cterminal domain. Cloning of the protease gene in pET22b with Escherichia coli BL21 DE3 as the host showed that the gene was expressed as insoluble protein fraction. This is the first report for analysis of protease gene from food origin Stenotrophomonas sp.

Synthesis of aesculetin and aesculin glycosides using engineered Escherichia coli expressing Neisseria polysaccharea amylosucrase.

Because glycosylation of aesculetin and its 6-glucoside, aesculin, enhances their biological activities and physicochemical properties, whole-cell biotransformation and enzymatic synthesis methodologies using Neisseria polysaccharea amylosucrase were compared to determine the optimal production method for glycoside derivatives. High performance liquid chromatography analysis of reaction products revealed two glycosylated products (AGG1 and AGG2) when aesculin was used as an acceptor and three products (AG1, AG2, and AG3) when using aesculetin. The whole-cell biotransformation production yields of the major transfer products for each acceptor (AGG1 and AG1) were 85% and 25%, respectively, compared to 68% and 14% for enzymatic synthesis. These results indicate that whole-cell biotransformation is more efficient than enzymatic synthesis for the production of glycoside derivatives.

Product Studies and Mechanistic Analysis of the Reaction of Methylglyoxal with Deoxyguanosine.

Methylglyoxal (MG) is a highly reactive electrophile produced endogenously as a byproduct of glucose metabolism and protein catabolism and exogenously as a food contaminant. MG reacts spontaneously with proteins, lipids, and nucleic acids to form advanced glycation end products (AGEs), modifying or inhibiting their function. Protein AGEs are associated with pathological complications of diabetes, cancer, and neurodegenerative diseases, while the physiological impact of DNA, RNA, and lipid AGE formation is less well explored. Conflicting reports in the literature on the biologically significant DNA-AGE product distribution and mechanisms of formation prompted a re-examination of the reaction products of MG with dG, oligonucleotides, and plasmid DNA under varying conditions of MG:dG stoichiometry, pH, and reaction time. Major products identified using sequential mass fragmentation and authentic standards were N2-(1-carboxyethyl)-2'-dG (CEdG), N2-(1-carboxyethyl)-7-1-hydroxy-2-oxopropyl-dG (MG-CEdG), and 1,N2-(1,2-dihydroxy-2-methyl)ethano-2'-dG (cMG-dG). CEdG and MG-CEdG were observed in all DNA substrates, although cMG-dG was not detected to any significant extent in oligomeric or polymeric DNA. Product analyses of reactions under conditions of diminished water activity as well as results from H218O labeling indicated that MG hydration equilibria plays an important role in controlling product distribution. In contrast to previous reports, our data support independent mechanisms of formation of CEdG and cMG-dG, with the latter kinetic product undergoing reversible formation under physiological conditions.