13C Nuclei Research Articles

Overcoming the Challenges of Hyperpolarizing Substrates with Parahydrogen‐Induced Polarization in an MRI System

Hyperpolarization of 13C nuclei in biomolecules and their administration as imaging agents enables in‐vivo monitoring of metabolism. This approach has demonstrated potential for deriving imaging biomarkers for cancer detection, differentiation, and therapy efficacy assessment. The in situ generation of polarized substrates using a permanent addition of parahydrogen to an unsaturated precursor inside the bore of an MRI system used for subsequent imaging circumvents the need for a dedicated external polarizer. This approach reduces polarization loss associated with sample transfer, minimizes hardware requirements and cost, and results in reduced spatial requirements. However, performing INEPT‐like pulsed sequences for heteronuclear spin‐order transfer in the bore of an MRI system is challenged by poor uniformity of static and excitation magnetic field and molecular convection during the polarization transfer. Therefore, here we characterize these effects, implement a robust modification to the pulse sequence, and measure experimentally the polarization improvement upon modification of the sequence. After rigorous optimization of the parameters, we obtained a 13C polarization of 44.5% for 50 mM of the 1‐13C site of ethyl acetate‐d6. Our parahydrogen‐induced polarization approach enhances the accessibility to hyperpolarized MRI, circumventing the need for an external polarizer.

Carbon-13 Hyperpolarization of α-Ketocarboxylates with Parahydrogen in Reversible Exchange.

Signal Amplification by Reversible Exchange (SABRE) is a relatively simple and fast hyperpolarization technique that has been used to hyperpolarize the α-ketocarboxylate pyruvate, a central metabolite and the leading hyperpolarized MRI contrast agent. In this work, we show that SABRE can readily be extended to hyperpolarize 13C nuclei at natural abundance on many other α-ketocarboxylates. Hyperpolarization is observed and optimized on pyruvate (P13C=17%) and 2-oxobutyrate (P13C=25%) with alkyl chains in the R-group, oxaloacetate (P13C=11%) and alpha-ketoglutarate (P13C=13%) with carboxylate moieties in the R group, and phenylpyruvate (P13C=2%) and phenylglyoxylate (P13C=2%) with phenyl rings in the R-group. New catalytically active SABRE binding motifs of the substrates to the hyperpolarization transfer catalyst-particularly for oxaloacetate-are observed. We experimentally explore the connection between temperature and exchange rates for all of these SABRE systems and develop a theoretical kinetic model, which is used to fit the hyperpolarization build-up and decay during SABRE activity.

Green synthesis, characterization, antioxidant, anti-fungal, antibacterial activities of a new 1-isopropyl-3,5-di-2-tolyl-1,3,5-triazacyclohexane

This work presents the green synthesis of a novel of 1- isopropyl- 3, 5- di-2-tolyl- 1, 3, 5-triazacyclohexane in good yield. It is made without a solvent, energy, and a catalyst as a green technique via a one-pot multi component condensation reaction between one equivalent of isopropylamine, two equivalents of o-toluidine, and three equivalents of formaline. The compound's structure has been determined using several spectroscopic methods, including thin-layer chromatography (TLC), ultraviolet-visible spectroscopy (UV-Vis), infrared spectroscopy (IR), and nuclear magnetic resonance spectroscopy (NMR) for both 1H and 13C nuclei. Additionally, the compound 'smelting point was determined. Subsequently, the molecule was evaluated for its antioxidant and biological activities. The antioxidant activity exhibited good performance in both the ABTS and Phenanthrolinetests. The antimicrobial activity of this novel compound was tested against four bacterialstrains, including two Gram-positive bacteria (Staphylococcus aurous ATCC 25923 and Bacillus cereus ATCC 10876) and two Gram-negative bacteria (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853), as well as two fungal strains (Fusarium oxysporum and Alternariaalternata)using the agar diffusion method and the direct contact method, respectively. The obtained results exhibited a high level of antibacterial and antifungal activity.

Development of a fully automated workstation for conducting routine SABRE hyperpolarization

SABRE is emerging as a fast, simple and low-cost hyperpolarization method because of its ability to regenerate enhanced NMR signals. Generally, SABRE hyperpolarization has been performed predominantly manually, leading to variations in reproducibility and efficiency. Recent advances in SABRE include the development of automated shuttling systems to address previous inconsistencies. However, the operational complexity of such systems and the challenges of integration with existing workflows hinder their widespread adoption. This work presents a fully automated lab workstation based on a benchtop NMR spectrometer, specifically designed to facilitate SABRE of different nuclei across different polarization fields. We demonstrated the capability of this system through a series of routine SABRE experimental protocols, including consecutive SABRE hyperpolarization with high reproducibility (average standard deviation of 1.03%), optimization polarization of 13C nuclei respect to the polarization transfer field, and measurement of polarization buildup rate or decay time across a wide range of magnetic fields. Furthermore, we have iteratively optimized the durations for pulsed SABRE-SHEATH 13C pyruvate. The constructed SABRE workstation offers full automation, high reproducibility, and functional diversification, making it a practical tool for conducting routine SABRE hyperpolarization experiments. It provides a robust platform for high-throughput and reliable SABRE and X-SABRE hyperpolarization studies.

Synergistic effect of CaCO3 particles and porous carbon towards the removal of Zn2+ ions in aqueous solutions

This work describes the preparation of an adsorbent containing CaCO3 particles dispersed in activated carbon and its use for the removal of Zn2+ ions from aqueous solutions. The combination of the large porosity of the activated carbon support with the easy ion exchange occurring at the surface of the carbonate particles led to the achievement of fast adsorption kinetics and excellent ion removal capacity for the composite adsorbent. The material was synthesized by wet impregnation of the porous carbon support with an aqueous solution of Ca(NO3)2, followed by evaporation of the solvent with subsequent heat treatment in an inert atmosphere. X-ray diffraction results demonstrated the presence of CaCO3 crystals in the material, but with broad and low intensity diffraction peaks, corresponding to an average crystallite size of 20 nm; solid-state 13C NMR spectroscopy confirmed the presence of this phase by means of the identification of the signal relative to the 13C nuclei of the CO32− group at ∼170 ppm. The carbon-based adsorbent containing the CaCO3 particles showed excellent ability to remove Zn2+ ions from aqueous solutions, with essentially all ions removed after 30 min of contact time. In addition, the kinetic analysis data were properly fitted using the pseudo-second order model, pointing to the occurrence of chemisorption as the rate limiting of the process. The equilibrium adsorption isotherms were correctly described by the Langmuir model, indicating that the adsorption takes place with the formation of a monolayer on the adsorbent surface. The results evidenced a synergistic effect of the porous carbon and the CaCO3 particles regarding the Zn2+ ions removal, with a fast adsorption kinetics at the initial stage of adsorption attributed to the high availability of the dispersed CaCO3 particles on the carbon surface.

Synthesis and Characterization of Symmetrical N-Heterocyclic Carbene Copper(II) Complexes-An Investigation of the Influence of Pyridinyl Substituents.

Three new tridentate copper(II) N-heterocyclic carbene (NHC) complexes have been obtained and characterized with symmetrical C-4 substitutions on their pendent pyridine rings. Substitutions including methyl (Me), methoxy (OMe), and chloro (Cl) groups, which extend the library pincer Cu-NHC complexes under investigation, modify the impact of pyridinyl basicity on NCN pincer complexes. Both ligand precursors and copper(II) complexes are characterized using a range of techniques, including nuclear magnetic resonance (NMR) spectroscopy for 1H, 13C, 31P, and 19F nuclei, electrospray ionization mass spectrometry (ESI-MS), X-ray crystallography, cyclic voltammetry, and UV-Vis spectroscopy. The pyridine substitutions lead to minimal changes to bond lengths and angles in the X-ray crystal structures of these related complexes; there is a pronounced impact on the electrochemical behavior of both the ligand precursors and copper complexes in the solution. The substitution in the pyridinyl units of these complexes show an impact on the catalytic reactivity of these complexes as applied to a model C-N bond-forming reaction (CEL cross-coupling) under well-established conditions; however, this observation does not correlate to the expected change in basicity in these ligands.

The Application of Hierarchical Cluster Analysis to Lignins Classification Based on Data of High-Resolution NMR and Solid-State NMR Spectra on 13C Nuclei

The Application of Hierarchical Cluster Analysis to Lignins Classification Based on Data of High-Resolution NMR and Solid-State NMR Spectra on 13C Nuclei

Hyperpolarization of 15N‐Pyridinium by Using Parahydrogen Enables Access to Reactive Oxygen Sensors and Pilot In Vivo Studies

AbstractMagnetic resonance with hyperpolarized contrast agents is one of the most powerful and noninvasive imaging platforms capable for investigating in vivo metabolism. While most of the utilized hyperpolarized agents are based on 13C nuclei, a milestone advance in this area is the emergence of 15N hyperpolarized contrast agents. Currently, the reported 15N hyperpolarized agents mainly utilize the dissolution dynamic nuclear polarization (d‐DNP) protocol. The parahydrogen enhanced 15N probes have proven to be elusive and have been tested almost exclusively in organic solvents. Herein, we designed a reaction based reactive oxygen sensor 15N‐boronobenzyl‐2‐styrylpyridinium (15N‐BBSP) which can be hyperpolarized with para‐hydrogen. Reactive oxygen species plays a vital role as one of the essential intracellular signalling molecules. Disturbance of the H2O2 level usually represents a hallmark of pathophysiological conditions. This H2O2 probe exhibited rapid responsiveness toward H2O2 and offered spectrally resolvable chemical shifts. We also provide strategies to bring the newly developed probe from the organic reaction solution into a biocompatible injection buffer and demonstrate the feasibility of in vivo 15N signal detection. The present work manifests its great potential not only for reaction based reactive sensing probes but also promises to serve as a platform to develop other contrast agents.

Hyperpolarization of 15N-Pyridinium by Using Parahydrogen Enables Access to Reactive Oxygen Sensors and Pilot In Vivo Studies.

Magnetic resonance with hyperpolarized contrast agents is one of the most powerful and noninvasive imaging platforms capable for investigating in vivo metabolism. While most of the utilized hyperpolarized agents are based on 13C nuclei, a milestone advance in this area is the emergence of 15N hyperpolarized contrast agents. Currently, the reported 15N hyperpolarized agents mainly utilize the dissolution dynamic nuclear polarization (d-DNP) protocol. The parahydrogen enhanced 15N probes have proven to be elusive and have been tested almost exclusively in organic solvents. Herein, we designed a reaction based reactive oxygen sensor 15N-boronobenzyl-2-styrylpyridinium (15N-BBSP) which can be hyperpolarized with para-hydrogen. Reactive oxygen species plays a vital role as one of the essential intracellular signalling molecules. Disturbance of the H2O2 level usually represents a hallmark of pathophysiological conditions. This H2O2 probe exhibited rapid responsiveness toward H2O2 and offered spectrally resolvable chemical shifts. We also provide strategies to bring the newly developed probe from the organic reaction solution into a biocompatible injection buffer and demonstrate the feasibility of in vivo 15N signal detection. The present work manifests its great potential not only for reaction based reactive sensing probes but also promises to serve as a platform to develop other contrast agents.

Selective correlations between aliphatic 13C nuclei in protein solid-state NMR

Solid-state nuclear magnetic resonance (NMR) is a potent tool for studying the structures and dynamics of insoluble proteins. It starts with signal assignment through multi-dimensional correlation experiments, where the aliphatic 13Cα-13Cβ correlation is indispensable for identifying specific residues. However, developing efficient methods for achieving this correlation is a challenge in solid-state NMR. We present a simple band-selective zero-quantum (ZQ) recoupling method, named POST-C4161 (PC4), which enhances 13Cα-13Cβ correlations under moderate magic-angle spinning (MAS) conditions. PC4 requires minimal 13C radio-frequency (RF) field and proton decoupling, exhibits high stability against RF variations, and achieves superior efficiency. Comparative tests on various samples, including the formyl-Met-Leu-Phe (fMLF) tripeptide, microcrystalline β1 immunoglobulin binding domain of protein G (GB1), and membrane protein of mechanosensitive channel of large conductance from Methanosarcina acetivorans (MaMscL), demonstrate that PC4 selectively enhances 13Cα-13Cβ correlations by up to 50 % while suppressing unwanted correlations, as compared to the popular dipolar-assisted rotational resonance (DARR). It has addressed the long-standing need for selective 13C–13C correlation methods. We anticipate that this simple but efficient PC4 method will have immediate applications in structural biology by solid-state NMR.

Cross-correlated relaxation in the NMR of near-equivalent spin pairs: Longitudinal relaxation and long-lived singlet order.

The evolution of nuclear spin state populations is investigated for the case of a 13C2-labeled triyne in solution, for which the near-equivalent coupled pairs of 13C nuclei experience cross-correlated relaxation mechanisms. Inversion-recovery experiments reveal different recovery curves for the main peak amplitudes, especially when the conversion of population imbalances to observable coherences is induced by a radio frequency pulse with a small flip angle. Measurements are performed over a range of magnetic fields by using a sample shuttle apparatus. In some cases, the time constant TS for decay of nuclear singlet order is more than 100 times larger than the time constant T1 for the equilibration of longitudinal magnetization. The results are interpreted by a theoretical model incorporating cross-correlated relaxation mechanisms, anisotropic rotational diffusion, and an external random magnetic field. A Lindbladian formalism is used to describe the dissipative dynamics of the spin system in an environment of finite temperature. Good agreement is achieved between theory and experiment.

Efficient Parahydrogen-Induced 13C Hyperpolarization on a Microfluidic Device.

We show the direct production and detection of 13C-hyperpolarized fumarate by parahydrogen-induced polarization (PHIP) in a microfluidic lab-on-a-chip (LoC) device and achieve 8.5% 13C polarization. This is the first demonstration of 13C-hyperpolarization of a metabolite by PHIP in a microfluidic device. LoC technology allows the culture of mammalian cells in a highly controlled environment, providing an important tool for the life sciences. In-situ preparation of hyperpolarized metabolites greatly enhances the ability to quantify metabolic processes in such systems by microfluidic NMR. PHIP of 1H nuclei has been successfully implemented in microfluidic systems, with mass sensitivities in the range of pmol/s. However, metabolic NMR requires high-yield production of hyperpolarized metabolites with longer spin life times than is possible with 1H. This can be achieved by transfer of the polarization onto 13C nuclei, which exhibit much longer T1 relaxation times. We report an improved microfluidic PHIP device, optimized using a finite element model, that enables the direct and efficient production of 13C-hyperpolarized fumarate.

A combined solid-state 1H, 13C, 17O NMR and periodic DFT study of hyperfine coupling tensors in paramagnetic copper(II) compounds

We report solid-state 1H, 13C, and 17O NMR determination of hyperfine coupling tensors (A-tensors) in several paramagnetic Cu(II) (d9, S = 1/2) complexes: trans-Cu(DL-Ala)2·H217O, Cu([1–13C]acetate)2·H2O, Cu([2–13C]acetate)2·H2O, and Cu(acetate)2·H217O. Using these new experimental results and some A-tensor data available in the literature for trans-Cu(L-Ala)2 and K2CuCl4·2H2O, we were able to examine the accuracy of A-tensor computation from a periodic DFT method implemented in the BAND program. We evaluated A-tensors on 1H (I = 1/2), 13C (I = 1/2), 14N (I = 1), 17O (I = 5/2), 39K (I = 3/2), 35Cl (I = 3/2), and 63Cu (I = 3/2) nuclei over a range spanning more than 3 orders of magnitude. We found that the BAND code can reproduce reasonably well the experimental results for both A-tensors and nuclear quadrupole coupling tensors.

A Squaramide-Based Organocatalyst as a Novel Versatile Chiral Solvating Agent for Carboxylic Acids.

A squaramide-based organocatalyst for asymmetric Michael reactions has been tested as a chiral solvating agent (CSA) for 26 carboxylic acids and camphorsulfonic acid, encompassing amino acid derivatives, mandelic acid, as well as some of its analogs, propionic acids like profens (ketoprofen and ibuprofen), butanoic acids and others. In many cases remarkably high enantiodifferentiations at 1H, 13C and 19F nuclei were observed. The interaction likely involves a proton transfer from the acidic substrates to the tertiary amine sites of the organocatalyst, thus allowing for pre-solubilization of the organocatalyst (when a chloroform solution of the substrate is employed) or the simultaneous solubilization of both the catalyst and the substrate. DOSY experiments were employed to evaluate whether the catalyst-substrate ionic adduct was a tight one or not. ROESY experiments were employed to investigate the role of the squaramide unit in the adduct formation. A mechanism of interaction was proposed in accordance with the literature data.

Model-independent Approach of the JUNO 8B Solar Neutrino Program

The physics potential of detecting 8B solar neutrinos will be exploited at the Jiangmen Underground Neutrino Observatory (JUNO), in a model-independent manner by using three distinct channels of the charged current (CC), neutral current (NC), and elastic scattering (ES) interactions. Due to the largest-ever mass of 13C nuclei in the liquid scintillator detectors and the expected low background level, 8B solar neutrinos are observable in the CC and NC interactions on 13C for the first time. By virtue of optimized event selections and muon veto strategies, backgrounds from the accidental coincidence, muon-induced isotopes, and external backgrounds can be greatly suppressed. Excellent signal-to-background ratios can be achieved in the CC, NC, and ES channels to guarantee the observation of the 8B solar neutrinos. From the sensitivity studies performed in this work, we show that JUNO, with 10 yr of data, can reach the 1σ precision levels of 5%, 8%, and 20% for the 8B neutrino flux, sin2θ12 , and Δm212 , respectively. Probing the details of both solar physics and neutrino physics would be unique and helpful. In addition, when combined with the Sudbury Neutrino Observatory measurement, the world's best precision of 3% is expected for the measurement of the 8B neutrino flux.

Squeezing formaldehyde into C60 fullerene

The cavity inside fullerene C60 provides a highly symmetric and inert environment for housing atoms and small molecules. Here we report the encapsulation of formaldehyde inside C60 by molecular surgery, yielding the supermolecular complex CH2O@C60, despite the 4.4 Å van der Waals length of CH2O exceeding the 3.7 Å internal diameter of C60. The presence of CH2O significantly reduces the cage HOMO-LUMO gap. Nuclear spin-spin couplings are observed between the fullerene host and the formaldehyde guest. The rapid spin-lattice relaxation of the formaldehyde 13C nuclei is attributed to a dominant spin-rotation mechanism. Despite being squeezed so tightly, the encapsulated formaldehyde molecules rotate freely about their long axes even at cryogenic temperatures, allowing observation of the ortho-to-para spin isomer conversion by infrared spectroscopy. The particle in a box nature of the system is demonstrated by the observation of two quantised translational modes in the cryogenic THz spectra.

Electronic Structures of Radical-Pair-Forming Cofactors in a Heliobacterial Reaction Center.

Photosynthetic reaction centers (RCs) are membrane proteins converting photonic excitations into electric gradients. The heliobacterial RCs (HbRCs) are assumed to be the precursors of all known RCs, making them a compelling subject for investigating structural and functional relationships. A comprehensive picture of the electronic structure of the HbRCs is still missing. In this work, the combination of selective isotope labelling of 13C and 15N nuclei and the utilization of photo-CIDNP MAS NMR (photochemically induced dynamic nuclear polarization magic-angle spinning nuclear magnetic resonance) allows for highly enhanced signals from the radical-pair-forming cofactors. The remarkable magnetic-field dependence of the solid-state photo-CIDNP effect allows for observation of positive signals of the electron donor cofactor at 4.7 T, which is interpreted in terms of a dominant contribution of the differential relaxation (DR) mechanism. Conversely, at 9.4 T, the emissive signals mainly originate from the electron acceptor, due to the strong activation of the three-spin mixing (TSM) mechanism. Consequently, we have utilized two-dimensional homonuclear photo-CIDNP MAS NMR at both 4.7 T and 9.4 T. These findings from experimental investigations are corroborated by calculations based on density functional theory (DFT). This allows us to present a comprehensive investigation of the electronic structure of the cofactors involved in electron transfer (ET).

Carbon-detected deuterium solid-state NMR rotating frame relaxation measurements for protein methyl groups under magic angle spinning

Deuterium rotating frame solid-state NMR relaxation measurements (2H R1ρ) are important tools in quantitative studies of molecular dynamics. We demonstrate how 2H to 13C cross-polarization (CP) approaches under 10–40 kHz magic angle spinning rates can be combined with the 2H R1ρ blocks to allow for extension of deuterium rotating frame relaxation studies to methyl groups in biomolecules. This extension permits detection on the 13C nuclei and, hence, for the achievement of site-specific resolution. The measurements are demonstrated using a nine-residue low complexity peptide with the sequence GGKGMGFGL, in which a single selective −13CD3 label is placed at the methionine residue. Carbon-detected measurements are compared with the deuterium direct-detection results, which allows for fine-tuning of experimental approaches. In particular, we show how the adiabatic respiration CP scheme and the double adiabatic sweep on the 2H and 13C channels can be combined with the 2H R1ρ relaxation rates measurement. Off-resonance 2H R1ρ measurements are investigated in addition to the on-resonance condition, as they extent the range of effective spin-locking field.

Measuring Hydroxyl Exchange Rates in Glycans Using a Synergistic Combination of Saturation Transfer and Relaxation Dispersion NMR.

Molecular recognition events mediated by glycans play pivotal roles in controlling the fate of diverse biological processes such as cellular communication and the immune response. The affinity of glycans for their target receptors is governed primarily by the hydrogen bonds formed by hydroxyl groups decorating the glycan surface. Hydroxyl exchange rate constants are therefore vital parameters that report on glycan structure and dynamics. Here we present a strategy for characterizing hydroxyl hydrogen/deuterium (H/D) exchange in glycans that employs a synergistic combination of 13C chemical exchange saturation transfer (CEST) and Carr-Purcell-Meiboom-Gill relaxation dispersion (CPMG) NMR methods. We show that the combination of CEST and CPMG experiments facilitates the sensitive detection of the small (∼0.1 ppm) two-bond deuterium isotope shift on a 13C nucleus when the attached hydroxyl group fluctuates between protonated and deuterated states. This shift is leveraged for measuring site-specific kinetic H/D exchange rate constants as well as thermodynamic free energies of isotope fractionation. The CEST and CPMG modules are integrated with a selective J-cross-polarization scheme that provides the flexibility for rapid characterization of H/D exchange at a specific hydroxyl site. Moreover, our approach enables the precise isothermal measurement of hydroxyl exchange rate constants without the need for cumbersome isotope labeling. The H/D exchange rate constants of three different glycans assessed using this method highlight its potential for detecting transient intra- and intermolecular hydrogen bonds. In addition, the trends in H/D exchange rate constants establish site-specific steric accessibility as a key determinant of solvent exchange dynamics in glycans.

Clustering in 13C

Measurement was conducted at the INFN-LNL, employing a Tandem accelerator to achieve a 95 MeV 14N beam focused on 10B target (201 µg/cm²) with the aim to explore the cluster structures of light carbon isotopes. The main focus of this report will be on the 13C nucleus. Data were collected with a six-telescope detection system that allowed for the observation of many-body exit channels. Here are presented some results of the data analysis for the excited states of 13C from reactions with two and three products in the exit channel. In the 10B(14N,11C)13C reaction, well-defined peaks were identified at 2.0, 3.7, 5.7, 7.4, 9.4, 11.7, 13.9, and 15.9 MeV. The states from ground state to 5.7 MeV are well understood and are used to correct the spectrum, while more states can contribute to the states at higher excitations and further investigation is needed. In the 10B(14N,11C9Be)4He channel, states were identified at 13.1, 13.9, 15.6, and 18.5 MeV. The experimental results show consistency with the previously published data, supporting theoretical models of clustering and molecular-like structures in 13C. Further analysis of other reaction channels and states will be presented in future publications, aiming to enhance our understanding of multi-center clustering in carbon isotopes.