Isotope Labeling Measurements Research Articles

Palladium-anchored calix[4]arene-derived porous organic polymer towards efficient hydrolytic cleavage of carbon disulfide

The release of carbon disulfide can have adverse effects on our environment and human health. The stability of carbon disulfide and the slow kinetics of hydrolysis can make it challenging to achieve efficient and practical cleavage of the CS bonds. Herein, a calix[4]arene-based porous organic polymer (CPOP-1) is innovatively synthesized through an optimized polycondensation reaction using C-Methylcalix[4]resorcinarene and hexafluoro-hexaazatriphenylene as monomers. Subsequently, palladium-induced calix[4]arene-based porous organic polymer was also synthesized via strong Pd−N coordination bonds to construct the metal-induced porous catalyst (CPOP-2). The polymeric catalyst active center [Pd2+(N^N)(NO3-)2] demonstrated outstanding catalytic hydrolysis performance (11.14 μmol g-1 h-1) in 10.5 h which is significantly enhanced by ca.13.2 times as compared to reported mononuclear Bpy-Pd(NO3)2, and 7.07 times than model trinuclear complex catalyst HATN-Pd-1, respectively. The control experiments revealed that POP catalysts showcased robust stability, prolonged effectiveness, and feasible recyclability during the hydrolytic cleavage of carbon disulfide at room temperature in aqueous solutions. Furthermore, the coordination environment of [Pd2+(N^N)] was validated through XPS, EXAFS, and isotope labeling measurements, and the hydrolysis cleavage products were confirmed e. g. CO2, sulfide, and protons. More importantly, a reaction mechanism was formulated coupled with theoretical calculations, and simulations. The proposed mechanism involves sequential OH- nucleophilic attacks on the carbon atoms of insert-coordinated CS2 and COS, leading to the cleavage of double CS bonds and the formation of CO bonds. The concurrent dissociation of the C−S bond and liberation of CO2 result in an intermediate structure characterized by [(N^N)Pd2+](SH-)2. This intermediate motif serves as the source of the thermodynamic driving force for the reaction.

Enzyme-Inspired Ligand Engineering of Gold Nanoclusters for Electrocatalytic Microenvironment Manipulation.

Natural enzymes intricately regulate substrate accessibility through specific amino acid sequences and folded structures at their active sites. Achieving such precise control over the microenvironment has proven to be challenging in nanocatalysis, especially in the realm of ligand-stabilized metal nanoparticles. Here, we use atomically precise metal nanoclusters (NCs) as model catalysts to demonstrate an effective ligand engineering strategy to control the local concentration of CO2 on the surface of gold (Au) NCs during electrocatalytic CO2 reduction reactions (CO2RR). The precise incorporation of two 2-thiouracil-5-carboxylic acid (TCA) ligands within the pocket-like cavity of [Au25(pMBA)18]- NCs (pMBA = para-mercaptobenzoic acid) leads to a substantial acceleration in the reaction kinetics of CO2RR. This enhancement is attributed to a more favorable microenvironment in proximity to the active site for CO2, facilitated by supramolecular interactions between the nucleophilic Nδ- of the pyrimidine ring of the TCA ligand and the electrophilic Cδ+ of CO2. A comprehensive investigation employing absorption spectroscopy, mass spectrometry, isotopic labeling measurements, electrochemical analyses, and quantum chemical computation highlights the pivotal role of local CO2 enrichment in enhancing the activity and selectivity of TCA-modified Au25 NCs for CO2RR. Notably, a high Faradaic efficiency of 98.6% toward CO has been achieved. The surface engineering approach and catalytic fundamentals elucidated in this study provide a systematic foundation for the molecular-level design of metal-based electrocatalysts.

Highly Selective Hydrogenation of Unsaturated Aldehydes in Aqueous Phase.

Chemoselective hydrogenation of carbonyl in unsaturated aldehydes is a significant process in the chemical industry, in which the development of aqueous-phase reaction systems as a substitution to organic ones is challenging. Herein, we report Ir atomic cluster catalysts anchored onto WO3-x nanorods via a reduction treatment at various temperatures (denoted as Ir/WOx-T, T = 200, 300, 400, and 500 °C), which accelerates the chemoselective hydrogenation of carbonyl groups in aqueous solutions. The optimal catalyst Ir/WOx-300 exhibits exceptional activity (TOF value: 1313.7 min-1) and chemoselectivity toward cinnamaldehyde (CAL) hydrogenation to cinnamyl alcohol (COL) (yield: ∼98.0%) in water medium, which is, to the best of our knowledge, the highest level compared with previously reported heterogeneous catalysts in liquid-phase reaction. Ac-HAADF-STEM, XAFS, and XPS verify the formation of interface structure (Irδ+-Ov-W5+ (0 ≤ δ ≤ 4); Ov denotes oxygen vacancy) induced by metal-support interaction and the largest concentration of interfacial Ir (Irδ+) in Ir/WOx-300. In situ studies (Raman, FT-IR), isotopic labeling measurements combined with DFT calculations substantiate that the hydrogenation of the C=O group consists of two pathways: water-mediated hydrogenation (predominant) and direct hydrogenation via H2 dissociation (secondary). In the former case, W5+-Ov site accelerates the activation adsorption of H2O, while Ir0 site facilitates the H-H bond cleavage of H2 and Irδ+ promotes the CAL adsorption. H2O molecule, as the source of hydrogen species, participates directly in the hydrogenation of the carbonyl group through a hydrogen-bonded network, with a largely reduced energy barrier relative to the H2 dissociation path. This work demonstrates a green catalytic route that breaks the activity-selectivity trade-off toward the selective hydrogenation of unsaturated aldehydes, which shows great potential in heterogeneous catalysis.

Boosting the H2 Production Efficiency via Photocatalytic Organic Reforming: The Role of Additional Hole Scavenging System

The simultaneous photocatalytic H2 evolution with environmental remediation over semiconducting metal oxides is a fascinating process for sustainable fuel production. However, most of the previously reported photocatalytic reforming showed nonstoichiometric amounts of the evolved H2 when organic substrates were used. To explain the reasons for this phenomenon, a careful analysis of the products and intermediates in gas and aqueous phases upon the photocatalytic hydrogen evolution from oxalic acid using Pt/TiO2 was performed. A quadrupole mass spectrometer (QMS) was used for the continuous flow monitoring of the evolved gases, while high performance ion chromatography (HPIC), isotopic labeling, and electron paramagnetic resonance (EPR) were employed to understand the reactions in the solution. The entire consumption of oxalic acid led to a ~30% lower H2 amount than theoretically expected. Due to the contribution of the photo-Kolbe reaction mechanism, a tiny amount of formic acid was produced then disappeared shortly after the complete consumption of oxalic acid. Nevertheless, a much lower concentration of formic acid was generated compared to the nonstoichiometric difference between the formed H2 and the consumed oxalic acid. Isotopic labeling measurements showed that the evolved H2, HD, and/or D2 matched those of the solvent; however, using D2O decreased the reaction rate. Interestingly, the presence of KI as an additional hole scavenger with oxalic acid had a considerable impact on the reaction mechanism, and thus the hydrogen yield, as indicated by the QMS and the EPR measurements. The added KI promoted H2 evolution to reach the theoretically predictable amount and inhibited the formation of intermediates without affecting the oxalic acid degradation rate. The proposed mechanism, by which KI boosts the photocatalytic performance, is of great importance in enhancing the overall energy efficiency for hydrogen production via photocatalytic organic reforming.

High-resolution 13C metabolic flux analysis.

Precise quantification of metabolic pathway fluxes in biological systems is of major importance in guiding efforts in metabolic engineering, biotechnology, microbiology, human health, and cell culture. 13C metabolic flux analysis (13C-MFA) is the predominant technique used for determining intracellular fluxes. Here, we present a protocol for 13C-MFA that incorporates recent advances in parallel labeling experiments, isotopic labeling measurements, and statistical analysis, as well as best practices developed through decades of experience. Experimental design to ensure that fluxes are estimated with the highest precision is an integral part of the protocol. The protocol is based on growing microbes in two (or more) parallel cultures with 13C-labeled glucose tracers, followed by gas chromatography-mass spectrometry (GC-MS) measurements of isotopic labeling of protein-bound amino acids, glycogen-bound glucose, and RNA-bound ribose. Fluxes are then estimated using software for 13C-MFA, such as Metran, followed by comprehensive statistical analysis to determine the goodness of fit and calculate confidence intervals of fluxes. The presented protocol can be completed in 4 d and quantifies metabolic fluxes with a standard deviation of ≤2%, a substantial improvement over previous implementations. The presented protocol is exemplified using an Escherichia coli ΔtpiA case study with full supporting data, providing a hands-on opportunity to step through a complex troubleshooting scenario. Although applications to prokaryotic microbial systems are emphasized, this protocol can be easily adjusted for application to eukaryotic organisms.

Fragmentation of Dicarboxylic and Tricarboxylic Acids in the Krebs Cycle Using GC-EI-MS and GC-EI-MS/MS

Isotope labeling measurements using mass spectrometry can provide informative insights on the metabolic systems of various organisms. The detailed identification of carbon positions included in the fragment ions of dicarboxylic and tricarboxylic acids in central carbon metabolism is needed for precise interpretation of the metabolic states. In this study, fragment ions containing the carbon backbone cleavage of dicarboxylic and tricarboxylic in the Krebs cycle were investigated by using gas chromatography (GC)-electron ionization (EI)-MS and GC-EI-MS/MS. The positions of decarboxylation in the dicarboxylic and tricarboxylic acids were successfully identified by analyses using position-specific 13C-labeled standards prepared by in vitro enzymatic reactions. For example, carboxyl groups of C1 and C6 of trimethylsilyl (TMS)- and tert-butyldimethylsilyl (TBDMS)-derivatized malic and citric acids were primarily cleaved by EI. MS/MS analyses were also performed, and fragment ions of TBDMS-citric and α-ketoglutaric acids (αKG) with the loss of two carboxyl groups in collision-induced dissociation (CID) were observed.

Assessing the Impact of the Nutrient Microenvironment on the Metabolism of Effector CD8+ T Cells.

Immune cell function is tightly regulated by cellular metabolism, which in turn is strongly linked to the nutrient availability in the microenvironment surrounding the cells. This link is critical for effector CD8+ T cells which, after activation, must migrate from nutrient-rich environments into nutrient-scarce regions such as the tumor microenvironment. Assessing how nutrient availability modulates the metabolism of effector CD8+ T cells is thus key for understanding how harsh environments may impair their proliferation and effector function. Here, we describe an approach to systematically study the impact of the nutrient microenvironment on the metabolism of effector CD8+ T cells, based on performing stable 13C isotope labeling measurements on in vitro-differentiated murine effector CD8+ T cells.

Metabolic flux analysis using 13C peptide label measurements

¹³C metabolic flux analysis (MFA) has become the experimental method of choice to investigate the cellular metabolism of microbes, cell cultures and plant seeds. Conventional steady-state MFA utilizes isotopic labeling measurements of amino acids obtained from protein hydrolysates. To retain spatial information in conventional steady-state MFA, tissues or subcellular fractions must be dissected or biochemically purified. In contrast, peptides retain their identity in complex protein extracts, and may therefore be associated with a specific time of expression, tissue type and subcellular compartment. To enable 'single-sample' spatially and temporally resolved steady-state flux analysis, we investigated the suitability of peptide mass distributions (PMDs) as an alternative to amino acid label measurements. PMDs are the discrete convolution of the mass distributions of the constituent amino acids of a peptide. We investigated the requirements for the unique deconvolution of PMDs into amino acid mass distributions (AAMDs), the influence of peptide sequence length on parameter sensitivity, and how AAMD and flux estimates that are determined through deconvolution compare to estimates from a conventional GC-MS measurement-based approach. Deconvolution of PMDs of the storage protein β-conglycinin of soybean (Glycine max) resulted in good AAMD and flux estimates if fluxes were directly fitted to PMDs. Unconstrained deconvolution resulted in inferior AAMD and flux estimates. PMD measurements do not include amino acid backbone fragments, which increase the information content in GC-MS-derived analyses. Nonetheless, the resulting flux maps were of comparable quality due to the precision of Orbitrap quantification and the larger number of peptide measurements.

Mapping photoautotrophic metabolism with isotopically nonstationary 13C flux analysis

Understanding in vivo regulation of photoautotrophic metabolism is important for identifying strategies to improve photosynthetic efficiency or re-route carbon fluxes to desirable end products. We have developed an approach to reconstruct comprehensive flux maps of photoautotrophic metabolism by computational analysis of dynamic isotope labeling measurements and have applied it to determine metabolic pathway fluxes in the cyanobacterium Synechocystis sp. PCC6803. Comparison to a theoretically predicted flux map revealed inefficiencies in photosynthesis due to oxidative pentose phosphate pathway and malic enzyme activity, despite negligible photorespiration. This approach has potential to fill important gaps in our understanding of how carbon and energy flows are systemically regulated in cyanobacteria, plants, and algae.

Identifying Static and Kinetic Lipid Phenotypes by High Resolution UPLC–MS: Unraveling Diet-Induced Changes in Lipid Homeostasis by Coupling Metabolomics and Fluxomics

A novel method to differentiate diet-induced alterations in plasma lipid phenotypes "static (concentration of lipids) and kinetic (endogenous production, e.g., denovo lipogenesis)" was employed. C57Bl6 mice were randomized into 2 groups and fed either a high-carbohydrate, low-fat (HC) or a carbohydrate-free, high-fat diet (HF) diet for 13 days; D(2)O was administered via intraperitoneal injection and then adding D(2)O to the drinking water for 96 h. Principal component analysis (PCA) revealed differences in the plasma lipid content, for example, triglycerides (TG) 50:2, 50:3, and 52:2 were up-regulated in mice fed the HC diet, whereas TG 52:4, 52:1, 54:5, 54:3, 54:4, and 54:2 were higher in animals fed the HF diet. However, although the fractional contribution of synthesis was ~10-fold lower in HF vs HC fed mice, changes in TG concentration were not entirely mediated by altered de novo lipogenesis. In addition, the ability to couple isotope labeling measurements with PCA analyses revealed cases where there were no differences in the concentration of a compound but its source was substantially altered. In summary, this strategy determined (i) the presence/absence of differences in concentration and (ii) the contribution of different pathways and synthesis that could affect lipid biology in a mouse model respectively.

Mechanistic Studies on the Copper‐Catalyzed Hydrosilylation of Ketones

AbstractThe copper‐catalyzed asymmetric hydrosilylation of ketones is an efficient method for the synthesis of chiral enantiopure secondary alcohols. Herein, we present a detailed computational study (DFT/B3LYP) of the copper(I)‐catalyzed reaction. In particular, the two transition states involved in the catalytic cycle have been determined. The insertion of the ketone into the Cu–H bond was found to have a lower activation barrier than the reaction of the copper alkoxy intermediate with the silane, which regenerates Cu–H along with the silyl ether product. Our findings also reveal the importance of the copper hydride dimer in controlling the reactivity toward the ketone. The conclusions are supported by experimental mechanistic investigations including kinetic studies, kinetic isotope effect, and isotope labeling measurements.

A transient isotopic labeling methodology for 13C metabolic flux analysis of photoautotrophic microorganisms

Metabolic flux analysis is increasingly recognized as an integral component of systems biology. However, techniques for experimental measurement of system-wide metabolic fluxes in purely photoautotrophic systems (growing on CO 2 as the sole carbon source) have not yet been developed due to the unique problems posed by such systems. In this paper, we demonstrate that an approach that balances positional isotopic distributions transiently is the only route to obtaining system-wide metabolic flux maps for purely autotrophic metabolism. The outlined transient 13C-MFA methodology enables measurement of fluxes at a metabolic steady-state, while following changes in 13C-labeling patterns of metabolic intermediates as a function of time, in response to a step-change in 13C-label input. We use mathematical modeling of the transient isotopic labeling patterns of central intermediates to assess various experimental requirements for photoautotrophic MFA. This includes the need for intracellular metabolite concentration measurements and isotopic labeling measurements as a function of time. We also discuss photobioreactor design and operation in order to measure fluxes under precise environmental conditions. The transient MFA technique can be used to measure and compare fluxes under different conditions of light intensity, nitrogen sources or compare strains with various mutations or gene deletions and additions.

Aqueous Transformation of a Metal Diformate to a Metal Dihydride Carbonyl Complex Accompanied by H2 Evolution from the Formato Ligands

The rhodium diformate complex [RhIII(tacn)(HCOO)2](OTf) (2, tacn = 1,4,7-triazacyclononane, OTf- = CF3SO3-) reacts with HCOONa at pH 6−7 at 80 °C in water to form a rhodium dihydride CO complex, [RhIII(tacn)(H)2(CO)](OTf) (5), with H2 evolution from the formato ligands of 2. This is the first example of H2 evolution from hydrogen atoms of formato ligands of a metal diformate complex. Complex 2 was synthesized from the reaction of a rhodium aqua complex, [RhIII(tacn)(H2O)3](OTf)3 (1), with HCOONa in water. Recrystallization of 2 with HCOONa in water gave crystals of a rhodium triformate complex, [RhIII(tacn)(HCOO)3] (3), whose structure was unequivocally determined by X-ray analysis. The key intermediate in the transformation from 2 to 5 is a rhodium hydride formate complex, [RhIII(tacn)H(HCOO)](OTf) (4), which was obtained by heating of an aqueous solution of the rhodium diformate complex 2 with 10 equiv of dimethyl sulfoxide (DMSO) at pH 6−7 in water. Complex 4 was well characterized by isotopic labeling measurements of 1H NMR, electrospray ionization mass spectrometry (ESI-MS), and GC.

Connective Tissue Metabolism in Bone and Cartilage of Adjuvant Arthritic Rat

The metabolism of connective tissue matrix components such as glycosaminoglycans and glycoproteins was investigated in normal as well as pathological tissues of bone and cartilage associated with adjuvant arthritis using rat as animal model of the disease. The inflammatory process of adjuvant arthritis was induced in rats with the inoculation of Freund's adjuvant containing heat killed Mycobacterium tuberculosis suspended in paraffin oil. The changes in the metabolism of matrix components in bone and cartilage were examined using radioactive isotopic labeling measurements during the acute as well as chronic phases of arthritic disease. The glycosaminoglycans were fractionated into sulfated and non-sulfated glycosaminoglycans by chemical and enzymic modifications. The biosynthesis of sulfated glycosaminoglycans was evaluated using radioactive labeled (35S)-sulfate. Alterations were demonstrated in the metabolism of connective tissue in the bone and cartilage tissues of arthritic rat. The results obtained showed an increased incorporation of radioactive sulfate in specimens of bone and cartilage during the process of adjuvant arthritis. The contents of sulfated as well as non-sulfated glycosaminoglycans were found to be increased in both the tissues of arthritic rat. Similarly, the amount of total glycosaminoglycans was also found to be increased significantly in the diseased tissues. In addition, various components of tissue glycoproteins such as fucose, sialic acid and total hexose were found to be elevated in insoluble fractions of bone and cartilage during the diseased state. The effects of experimentally induced adjuvant arthritis on the connective tissue were discussed in the light of changes taking place in the metabolism of glycosaminoglycans and glycoproteins in bone and cartilage of arthritic rat.