Carbon Isotope Labeling Research Articles

Efficient palladium-catalyzed electrocarboxylation enables late-stage carbon isotope labelling

Carbon isotope labelling of bioactive molecules is essential for accessing the pharmacokinetic and pharmacodynamic properties of new drug entities. Aryl carboxylic acids represent an important class of structural motifs ubiquitous in pharmaceutically active molecules and are ideal targets for the installation of a radioactive tag employing isotopically labelled CO2. However, direct isotope incorporation via the reported catalytic reductive carboxylation (CRC) of aryl electrophiles relies on excess CO2, which is incompatible with carbon-14 isotope incorporation. Furthermore, the application of some CRC reactions for late-stage carboxylation is limited because of the low tolerance of molecular complexity by the catalysts. Herein, we report the development of a practical and affordable Pd-catalysed electrocarboxylation setup. This approach enables the use of near-stoichiometric 14CO2 generated from the primary carbon-14 source Ba14CO3, facilitating late-stage and single-step carbon-14 labelling of pharmaceuticals and representative precursors. The proposed isotope-labelling protocol holds significant promise for immediate impact on drug development programmes.

A metal-catalysed functional group metathesis approach to the carbon isotope labelling of carboxylic acids.

The distribution, metabolism and ultimate fate of molecules within the body is central to the activity of pharmaceuticals. However, the introduction of radioisotopes into the metabolically stable carbon sites on drugs to probe these features typically requires toxic, radioactive gases such as [14C]CO and [14C]CO2. Here we describe an approach to directly carbon-label carboxylic-acid-containing pharmaceuticals via a metal-catalysed functional group exchange reaction, forming 14C-labelled carboxylic-acid-containing drugs without radioactive gases, in one pot, using an easily available and handled carboxylic acid 14C source. To enable this process, a functional group metathesis of carbon-carbon covalent bonds in acid chloride functionalities is developed, exploiting the ability of nickel catalysts to both reversibly activate carbon-chloride bonds and exchange functionalities between organic molecules. The drug development applicability is illustrated by the direct incorporation of the 14C label or 13C label into an array of complex aryl, alkyl, vinyl and heterocyclic carboxylic acid drugs or drug candidates without gases or a special apparatus, at ambient conditions and without loss of the radiolabel.

Light-Driven Metabolic Pathways in Non-Photosynthetic Biohybrid Bacteria.

Biomanufacturing via microorganisms relies on carbon substrates for molecular feedstocks and a source of energy to carry out enzymatic reactions. This creates metabolic bottlenecks and lowers the efficiency for substrate conversion. Nanoparticle biohybridization with proteins and whole cell surfaces can bypass the need for redox cofactor regeneration for improved secondary metabolite production in a non-specific manner. Here we propose using nanobiohybrid organisms (Nanorgs), intracellular protein-nanoparticle hybrids formed through the spontaneous coupling of core-shell quantum dots (QDs) with histidine-tagged enzymes in non-photosynthetic bacteria, for light-mediated control of bacterial metabolism. This proved to eliminate metabolic constrictions and replace glucose with light as the source of energy in Escherichia coli, with an increase in growth by 1.7-fold in 75 % reduced nutrient media. Metabolomic tracking through carbon isotope labeling confirmed flux shunting through targeted pathways, with accumulation of metabolites downstream of respective targets. Finally, application of Nanorgs with the Ehrlich pathway improved isobutanol titers/yield by 3.9-fold in 75 % less sugar from E. coli strains with no genetic alterations. These results demonstrate the promise of Nanorgs for metabolic engineering and low-cost biomanufacturing.

Spatially resolved CO2 carbon stable isotope analyses at the microscale using Raman spectroscopy

Measuring the carbon stable isotope ratio (13C/12C, expressed as δ13CCO2) in geogenic CO2 fluids is a crucial geochemical tool for studying Earth's degassing. Carbon stable isotope analysis is traditionally performed by bulk mass spectrometry. Although Raman spectroscopy distinguishes 12CO2 and 13CO2 isotopologue bands in spectra, using this technique to determine CO2 isotopic signature has been challenging. Here, we report on in-situ non-destructive analyses of the C stable isotopic composition of CO2, applying a novel high-resolution Raman configuration on 42 high-density CO2 fluid inclusions in mantle rocks from the Lake Tana region (Ethiopia) and El Hierro (Canary Islands). We collected two sets of three spectra with different acquisition times at high spectral resolution in each fluid inclusion. Among the 84 sets of spectra, 58 were characterised by integrated 13CO2/12CO2 band area ratios with reproducibility better than 4‰. Our results demonstrate the determination of δ13CCO2 by Raman spectroscopy in individual fluid inclusions with an error better than 2.5 ‰, which satisfactorily matches bulk mass spectrometry analyses in the same rock samples, supporting the accuracy of the measurements. We thus show that Raman Spectroscopy can provide a fundamental methodology for non-destructive, site-specific, and spatially resolved carbon isotope labelling at the microscale.

A Japan-UK Collaboration in Organic Synthesis.

This invited Team Profile was created by Shuo Wang, Igor Larrosa, Hideki Yorimitsu and Greg Perry. They recently published an article on carboxylic acid salts as dual-function reagents for carboxylation and carbon isotope labeling. This project brought together researchers based in Japan and the UK, and demonstrates how scientists with different cultural backgrounds can work together to build powerful results. "Carboxylic Acid Salts as Dual-Function Reagents for Carboxylation and Carbon Isotope Labeling", S. Wang, I. Larrosa, H. Yorimitsu, G. J. P. Perry, Angew. Chem. Int. Ed. 2023, e202218371.

A Japan–UK Collaboration in Organic Synthesis

AbstractThis invited Team Profile was created by Shuo Wang, Igor Larrosa, Hideki Yorimitsu and Greg Perry. They recently published an article on carboxylic acid salts as dual‐function reagents for carboxylation and carbon isotope labeling. This project brought together researchers based in Japan and the UK, and demonstrates how scientists with different cultural backgrounds can work together to build powerful results. “Carboxylic Acid Salts as Dual‐Function Reagents for Carboxylation and Carbon Isotope Labeling”, S. Wang, I. Larrosa, H. Yorimitsu, G. J. P. Perry, Angew. Chem. Int. Ed. 2023, e202218371.

Carboxylic Acid Salts as Dual‐Function Reagents for Carboxylation and Carbon Isotope Labeling

AbstractThe potassium salts of carboxylic acids are developed as efficient carboxylating agents through CO2 exchange. We describe these carboxylates as dual‐function reagents because they function as a combined source of CO2 and base/metalating agent. By using the salt of a commercially available carboxylic acid, this protocol overcomes difficulties when using CO2 gas or organometallic reagents, such as pressurized containers or strictly inert conditions. The reaction proceeds under mild conditions, does not require transition metals or other additives, and shows broad substrate scope. Through the preparation of several biologically important molecules, we show how this strategy provides an opportunity for isotope labeling with low equivalents of labeled CO2.

Carboxylic Acid Salts as Dual-Function Reagents for Carboxylation and Carbon Isotope Labeling.

The potassium salts of carboxylic acids are developed as efficient carboxylating agents through CO2 exchange. We describe these carboxylates as dual-function reagents because they function as a combined source of CO2 and base/metalating agent. By using the salt of a commercially available carboxylic acid, this protocol overcomes difficulties when using CO2 gas or organometallic reagents, such as pressurized containers or strictly inert conditions. The reaction proceeds under mild conditions, does not require transition metals or other additives, and shows broad substrate scope. Through the preparation of several biologically important molecules, we show how this strategy provides an opportunity for isotope labeling with low equivalents of labeled CO2 .

Spotlight on the Winners of the DCO‐SCF 2021 Awards – An Ever Valuable Collaboration with EurJOC

Spotlight on the Winners of the DCO‐SCF 2021 Awards – An Ever Valuable Collaboration with EurJOC

Stochastic simulation algorithm for isotope-based dynamic flux analysis

Carbon isotope labeling method is a standard metabolic engineering tool for flux quantification in living cells. To cope with the high dimensionality of isotope labeling systems, diverse algorithms have been developed to reduce the number of variables or operations in metabolic flux analysis (MFA), but lacks generalizability to non-stationary metabolic conditions. In this study, we present a stochastic simulation algorithm (SSA) derived from the chemical master equation of the isotope labeling system. This algorithm allows to compute the time evolution of isotopomer concentrations in non-stationary conditions, with the valuable property that computational time does not scale with the number of isotopomers. The efficiency and limitations of the algorithm is benchmarked for the forward and inverse problems of 13C-DMFA in the pentose phosphate pathways, and is compared with EMU-based methods for NMFA and MFA including the central carbon metabolism. Overall, SSA constitutes an alternative class to deterministic approaches for metabolic flux analysis that is well adapted to comprehensive dataset including parallel labeling experiments, and whose limitations associated to the sampling size can be overcome by using Monte Carlo sampling approaches.

Biodegradation of poly(butylene succinate) in soil laboratory incubations assessed by stable carbon isotope labelling

Using biodegradable instead of conventional plastics in agricultural applications promises to help overcome plastic pollution of agricultural soils. However, analytical limitations impede our understanding of plastic biodegradation in soils. Utilizing stable carbon isotope (13C-)labelled poly(butylene succinate) (PBS), a synthetic polyester, we herein present an analytical approach to continuously quantify PBS mineralization to 13CO2 during soil incubations and, thereafter, to determine non-mineralized PBS-derived 13C remaining in the soil. We demonstrate extensive PBS mineralization (65 % of added 13C) and a closed mass balance on PBS−13C over 425 days of incubation. Extraction of residual PBS from soils combined with kinetic modeling of the biodegradation data and results from monomer (i.e., butanediol and succinate) mineralization experiments suggest that PBS hydrolytic breakdown controlled the overall PBS biodegradation rate. Beyond PBS biodegradation in soil, the presented methodology is broadly applicable to investigate biodegradation of other biodegradable polymers in various receiving environments.

Parallel Screening with 14C‐Labeled Carbon Dioxide: De‐risking the Staudinger‐Aza‐Wittig Reaction**

AbstractCarbon isotope labeling is a useful technology for tracking the fate of organic compounds in the environment and in living organisms. In this context, the development of robust and general methodologies amenable to the direct functionalization of CO2 remains a significant task. In this communication, a de‐risking approach was developed to evaluate the robustness of the Staudinger aza‐Wittig sequence for carbon isotope labeling. This technology is based on [14C]CO2 screening that allowed to investigate the tolerance of the procedure with most representative heterocycles and functional groups found in FDA approved drugs.

Late-Stage Carbon-14 Labeling and Isotope Exchange: Emerging Opportunities and Future Challenges.

Carbon-14 (14C) is a gold standard technology routinely utilized in pharmaceutical and agrochemical industries for tracking synthetic organic molecules and providing their metabolic and safety profiles. While the state of the art has been dominated for decades by traditional multistep synthetic approaches, the recent emergence of late-stage carbon isotope labeling has provided new avenues to rapidly access carbon-14-labeled biologically relevant compounds. In particular, the development of carbon isotope exchange has represented a fundamental paradigm change, opening the way to unexplored synthetic transformations. In this Perspective, we discuss the recent developments in the field with a critical assessment of the literature. We subsequently discuss research directions and future challenges within this rapidly evolving field.

Quantitative Carbon Isotope Labeling of Carboxylic Acids

Key words alkoxycarbonylation - carbon isotope exchange - carboxylic acids - ex-situ CO generation - nickel catalysis - photocatalysis

Charge distribution in turbostratic few-layer graphene studied by carbon isotope labeling

Elucidating layer-resolved charge distribution in van der Waals stacked crystals is crucial for electronic applications based on 2D materials. Here, we use CVD-grown few-layer graphene (FLG) labeled by carbon isotopes as a prototype, in which Raman features of two isotope components identify distinct layers. Electrical transfer characteristics of graphene field-effect transistors (GFETs) are employed to quantitatively calibrate the correspondence of Fermi level and G-phonon frequency that shifts with the charge-carrier concentration (n) variation. The isotope-assisted spectroscopy reveals previously unprobed characteristics that the n value of both top and bottom layers in FLG is close rather than following an exponential decay law. This negligible gradient likely benefits from the π−π interaction that favors the interlayer diffusion of charges. In addition, each extra layer reduces the degree of charge exchange at the graphene/dopant interface. These results have important implications for FLG nanoelectronics and provide a robust framework by which one can further investigate the critical properties of other 2D systems.

Nickel-Mediated Alkoxycarbonylation for Complete Carbon Isotope Replacement.

Many commercial drugs, as well as upcoming pharmaceutically active compounds in the pipeline, display aliphatic carboxylic acids or derivatives thereof as key structural entities. Synthetic methods for rapidly accessing isotopologues of such compounds are highly relevant for undertaking critical pharmacological studies. In this paper, we disclose a direct synthetic route allowing for full carbon isotope replacement via a nickel-mediated alkoxycarbonylation. Employing a nickelII pincer complex ([(N2N)Ni-Cl]) in combination with carbon-13 labeled CO, alkyl iodide, sodium methoxide, photocatalyst, and blue LED light, it was possible to generate the corresponding isotopically labeled aliphatic carboxylates in good yields. Furthermore, the developed methodology was applied to the carbon isotope substitution of several pharmaceutically active compounds, whereby complete carbon-13 labeling was successfully accomplished. It was initially proposed that the carboxylation step would proceed via the in situ formation of a nickellacarboxylate, generated by CO insertion into the Ni-alkoxide bond. However, preliminary mechanistic investigations suggest an alternative pathway involving attack of an open shell species generated from the alkyl halide to a metal ligated CO to generate an acyl NiIII species. Subsequent reductive elimination involving the alkoxide eventually leads to carboxylate formation. An excess of the alkoxide was essential for obtaining a high yield of the product. In general, the presented methodology provides a simple and convenient setup for the synthesis and carbon isotope labeling of aliphatic carboxylates, while providing new insights about the reactivity of the N2N nickel pincer complex applied.

Effects of combined tannic acid/fluoride on sulfur transformations and methanogenic pathways in swine manure.

Livestock manure emits reduced sulfur compounds and methane, which affect nature and the climate. These gases are efficiently mitigated by addition of a tannic acid-sodium fluoride combination inhibitor (TA-NaF), and to some extent by acidification. In this paper, TA-NaF treatment was performed on swine manure to study the treatment influence on methanogenic pathways and sulfur transformation pathways in various laboratory experiments. Stable carbon isotope labeling revealed that both untreated and TA-NaF treated swine manures were dominated by hydrogenotrophic methanogenesis. However, in supplementary experiments in wastewater sludge, TA-NaF clearly inhibited acetoclastic methanogenesis, whereas acidification inhibited hydrogenotrophic methanogenesis. In swine manure, TA-NaF inhibited s-amino acid catabolism to a larger extent than sulfate reduction. Conversely, acidification reduced sulfate reduction activity more than s-amino acid degradation. TA-NaF treatment had no significant effect on methanogenic community structure, which was surprising considering clear effects on isotope ratios of methane and carbon dioxide. Halophile sulfate reducers adapted well to TA-NaF treatment, but the community change also depended on temperature. The combined experimental work resulted in a proposed inhibition scheme for sulfur transformations and methanogenic pathways as affected by TA-NaF and acidification in swine manure and in other inocula.

Investigation on the Stoichiometry of Carbon Dioxide in Isotope-Exchange Reactions with Phenylacetic Acids

AbstractThe functionalization of carbon dioxide (CO2) as a C1 building block has attracted enormous attention. Carboxylation reactions, in particular, are of major interest for applications in isotope labeling. Due to the inexpensive nature of CO2, information about its stoichiometric use is generally unavailable in the literature. Because of the rarity and limited availability of CO2 isotopomers, this parameter is of concern for applications in carbon-isotope labeling. We investigated the effects of the stoichiometry of labeled CO2 on carbon isotope exchange of phenyl­acetic acids. Both thermal and photocatalytic procedures were studied, providing insight into product outcome and isotope incorporation. Preliminary results on isotope-dilution effects of carbonate bases in photocatalytic carboxylation reactions have also been obtained.

Microaerobic growth-decoupled production of α-ketoglutarate and succinate from xylose in a one-pot process using Corynebacteriumglutamicum.

Lignocellulosic biomass is the most abundant raw material on earth. Its efficient use for novel bio-based materials is essential for an emerging bioeconomy. Possible building blocks for such materials are the key TCA-cycle intermediates α-ketoglutarate and succinate. These organic acids have a wide range of potential applications, particularly in use as monomers for established or novel biopolymers. Recently, Corynebacteriumglutamicum was successfully engineered and evolved towards an improved utilization of d-xylose via the Weimberg pathway, yielding the strain WMB2evo . The Weimberg pathway enables a carbon-efficient C5-to-C5 conversion of d-xylose to α-ketoglutarate and a shortcut route to succinate as co-product in a one-pot process. C.glutamicumWMB2evo was grown under dynamic microaerobic conditions on d-xylose, leading to the formation of comparably high amounts of succinate and only small amounts of α-ketoglutarate. Subsequent carbon isotope labeling experiments verified the targeted production route for both products in C.glutamicumWMB2evo . Fed-batch process development was initiated and the effect of oxygen supply and feeding strategy for a growth-decoupled co-production of α-ketoglutarate and succinate were studied in detail. The finally established fed-batch production process resulted in the formation of 78.4mmolL-1 (11.45gL-1 ) α-ketoglutarate and 96.2mmolL-1 (11.36gL-1 ) succinate. The developed one-pot process represents a promising approach for the combined supply of bio-based α-ketoglutarate and succinate. Future work will focus on tailor-made down-stream processing of both organic acids from the fermentation broth to enable their application as building blocks in chemical syntheses. Alternatively, direct conversion of one or both acids via whole-cell or cell-free enzymatic approaches can be envisioned; thus, extending the network of value chains starting from cheap and renewable d-xylose.

Unravelling the process of petroleum hydrocarbon biodegradation in different filter materials of constructed wetlands by stable isotope fractionation and labelling studies

Maintaining and supporting complete biodegradation during remediation of petroleum hydrocarbon contaminated groundwater in constructed wetlands is vital for the final destruction and removal of contaminants. We aimed to compare and gain insight into biodegradation and explore possible limitations in different filter materials (sand, sand amended with biochar, expanded clay). These filters were collected from constructed wetlands after two years of operation and batch experiments were conducted using two stable isotope techniques; (i) carbon isotope labelling of hexadecane and (ii) hydrogen isotope fractionation of decane. Both hydrocarbon compounds hexadecane and decane were biodegraded. The mineralization rate of hexadecane was higher in the sandy filter material (3.6 µg CO2 g−1 day−1) than in the expanded clay (1.0 µg CO2 g−1 day−1). The microbial community of the constructed wetland microcosms was dominated by Gram negative bacteria and fungi and was specific for the different filter materials while hexadecane was primarily anabolized by bacteria. Adsorption / desorption of petroleum hydrocarbons in expanded clay was observed, which might not hinder but delay biodegradation. Very few cases of hydrogen isotope fractionation were recorded in expanded clay and sand & biochar filters during decane biodegradation. In sand filters, decane was biodegraded more slowly and hydrogen isotope fractionation was visible. Still, the range of observed apparent kinetic hydrogen isotope effects (AKIEH = 1.072–1.500) and apparent decane biodegradation rates (k = − 0.017 to − 0.067 day−1) of the sand filter were low. To conclude, low biodegradation rates, small hydrogen isotope fractionation, zero order mineralization kinetics and lack of microbial biomass growth indicated that mass transfer controlled biodegradation.