High-resolution NMR Spectroscopy Research Articles

4-Amino-3-(1-{[amino(3-methyl-2-oxido-1,2,5-oxadiazol-4-yl)methylene]hydrazinylidene}ethyl)-1,2,5-oxadiazole 2-Oxide

Functionally substituted 1,2,5-oxadiazole 2-oxides (furoxans) are important pharmaceutical scaffolds used for the preparation of various pharmacologically active substances. Furoxans bearing hydrazone functionality are considered as promising drug candidates for the treatment of neglected diseases. However, pharmacologically oriented hydrazones derived from (furoxanyl)amidrazones and acetylfuroxans have remained unknown so far. In this communication, a simple method for the synthesis of 4-amino-3-(1-{[amino(3-methyl-2-oxido-1,2,5-oxadiazol-4-yl)methylene]hydrazinylidene}ethyl)-1,2,5-oxadiazole 2-oxide is described. The structure of the synthesized compound was established by elemental analysis, high-resolution mass spectrometry, 1H, 13C NMR and IR spectroscopy.

Rapid identification of short oligonucleotide impurities using lithium adduct consolidated MALDI-TOF mass spectrometry

Nucleic acids are an increasingly popular platform for the development of pharmaceuticals to treat and prevent multiple diseases including those where traditional small molecule and protein-based drug development efforts have failed. Short oligonucleotide therapeutics, which consist of antisense oligonucleotides (ASOs) and short interfering ribonucleic acids (siRNAs), are prepared by solid phase chemical synthesis, which can generate various impuriies that have the potential to lower drug safety and efficacy. Drug substance-related impurities can be especially difficult to identify and characterize. To address this short coming, here we apply lithium adduct consolidation with matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) to afford a simplified, quick, and facile method for identification of a known drug substance impurity, isobutyryl (iBu) groups arising from incomplete deprotection during solid phase synthesis. We further employ high-resolution nuclear magnetic resonance (NMR) spectroscopy to confirm assessment of the iBu impurity. This lithium adduction consolidation method should find general applicability for routine quality assessment of synthetic oligonucleotides in both academic and industrial laboratories.

Calcium Binds to Transthyretin with Low Affinity.

The plasma protein transthyretin (TTR), a transporter for thyroid hormones and retinol in plasma and cerebrospinal fluid, is responsible for the second most common type of systemic (ATTR) amyloidosis either in its wild type form or as a result of destabilizing genetic mutations that increase its aggregation propensity. The association between free calcium ions (Ca2+) and TTR is still debated, although recent work seems to suggest that calcium induces structural destabilization of TTR and promotes its aggregation at non-physiological low pH in vitro. We apply high-resolution NMR spectroscopy to investigate calcium binding to TTR showing the formation of labile interactions, which leave the native structure of TTR substantially unaltered. The effect of calcium binding on TTR-enhanced aggregation is also assessed at physiological pH through the mechano-enzymatic mechanism. Our results indicate that, even if the binding is weak, about 7% of TTR is likely to be Ca2+-bound in vivo and therefore more aggregation prone as we have shown that this interaction is able to increase the protein susceptibility to the proteolytic cleavage that leads to aggregation at physiological pH. These events, even if involving a minority of circulating TTR, may be relevant for ATTR, a pathology that takes several decades to develop.

A Randomized Trial of ω-3 Fatty Acid Supplementation and Circulating Lipoprotein Subclasses in Healthy Older Adults

Omega-3 (n-3) PUFAs are recognized for triglyceride-lowering effects in people with dyslipidemia, but it remains unclear if n-3-PUFA intake influences lipoprotein profiles in older adults without hypertriglyceridemia. The objective was to determine the effect of n-3-PUFA supplementation on plasma lipoprotein subfractions in healthy older men and women in the absence of cardiovascular disease (CVD) or hypertriglyceridemia. This was a secondary analysis and considered exploratory. Thirty young (20-35 y old) and 54 older (65-85 y old) men and women were enrolled in the study. Fasting plasma samples were collected. After baseline sample collection, 44 older adults were randomly assigned to receive either n-3-PUFA ethyl esters (3.9g/d) or placebo (corn oil) for 6 mo. Pre- and postintervention plasma samples were used for quantitative lipoprotein subclass analysis using high-resolution proton NMR spectroscopy. The number of large, least-dense LDL particles decreased 17%-18% with n-3 PUFAs compared with placebo (<1% change; P<0.01). The number of small, dense LDL particles increased 26%-44% with n-3 PUFAs compared with placebo (∼11% decrease; P<0.01). The cholesterol content of large HDL particles increased by 32% with n-3 PUFAs and by 2% in placebo (P<0.01). The cholesterol content of small HDL particles decreased by 23% with n-3 PUFAs and by 2% in placebo (P<0.01). Despite increasing abundance of small, dense LDL particles that are associated with CVD risk, n-3 PUFAs reduced total triglycerides, maintained HDL, reduced systolic blood pressure, and shifted the HDL particle distribution toward a favorable cardioprotective profile in healthy older adults without dyslipidemia. This study suggests potential benefits of n-3-PUFA supplementation to lipoprotein profiles in healthy older adults without dyslipidemia, which should be considered when weighing the potential health benefits against the cost and ecological impact of widespread use of n-3-PUFA supplements.This trial was registered at clinicaltrials.gov as NCT03350906.

Electrochemical Depolymerization of Lignin in a Biomass-based Solvent.

Breaking down lignin into smaller units is the key to generate high value‐added products. Nevertheless, dissolving this complex plant polyphenol in an environment‐friendly way is often a challenge. Levulinic acid, which is formed during the hydrothermal processing of lignocellulosic biomass, has been shown to efficiently dissolve lignin. Herein, levulinic acid was evaluated as a medium for the reductive electrochemical depolymerization of the lignin macromolecule. Copper was chosen as the electrocatalyst due to the economic feasibility and low activity towards the hydrogen evolution reaction. After depolymerization, high‐resolution mass spectrometry and nuclear magnetic resonance spectroscopy revealed lignin‐derived monomers and dimers. A predominance of aryl ether and phenolic groups was observed. Depolymerized lignin was further evaluated as an anti‐corrosion coating, revealing enhancements on the electrochemical stability of the metal. Via a simple depolymerization process of biomass waste in a biomass‐based solvent, a straightforward approach to produce high value‐added compounds or tailored biobased materials was demonstrated.

NMR-Based Metabolomics Identify Metabolic Change in Spleen of Idiopathic Thrombocytopenic Purpura Patients.

Idiopathic thrombocytopenic purpura (ITP) is a common hematological disease and the abnormal platelet destruction in the spleen is a critical pathological mechanism for ITP. However, the metabolomic change in the spleen caused by ITP is still unclear. In the present study, the metabolomic information of 18 ITP and 20 normal spleen samples were detected by using 1H high-resolution magic angle spinning NMR spectroscopy (1H MAS NMR). Compared with normal spleen, the concentrations of acetate, alanine, glutamine, glycerol, isoleucine, lysine, valine, phenylalanine, leucine, and methanol in ITP spleen tissue were elevated and 3-hydroxybutyric acid, ascorbate, asparagine, ethanol, glycogen, low-density lipoprotein, malonate, myo-inositol, glycerophosphocholine, pyroglutamate, and taurine were decreased. Amino acids metabolic pathways, such as branched-chain amino acids pathway, were identified as the main involved pathways based on enrichment analysis. The decrease in taurine level in the spleen was the most obvious metabolic signature involving ITP with high sensitivity and specificity to distinguish the spleen of ITP from the normal (CI: 0.825–0.982). Notably, the level of taurine in the spleen was negatively correlated with the efficacy of splenectomy (r = 0.622, p = 0.006). Collectively, the data from our study revealed previously unknown ITP-related metabolomic changes in the spleen and found a potential diagnostic and efficacy-predictive biomarker for ITP treatment.

N-[3-(Chloromethyl)-1,2-benzisoxazol-5-yl]acetamide

Functionally substituted 1,2-benzisoxazoles are very important and promising heterocycles with various pharmacological activities. Benzoxazoles containing reactive 3-chloromethyl and 5-amino groups are practically unexplored derivatives in this series. In this communication, the simple method for the synthesis of N-[3-(chloromethyl)-1,2-benzisoxazol-5-yl]acetamide which is an interesting precursor for the preparation of a series of 3,5-disubstituted benzoxazoles was described. The structure of the synthesized compound was established by elemental analysis, high-resolution mass spectrometry, 1H, 13C NMR and IR spectroscopy, and mass spectrometry.

Multiphase Drug Distribution and Exchange in Oil-in-Water Nanoemulsion Revealed by High-Resolution 19F qNMR.

An oil-in-water (o/w) nanoemulsion (NE), composed of oil globules, stabilized by a surfactant, and dispersed in an aqueous phase, is increasingly developed in complex drug formulation. Kinetically stable NEs are used to formulate hydrophobic drugs and typically provide higher dosage strengths and better content uniformity. However, little is known accurately about drug distribution in its multiphase solution, especially for the possible drug presence in the surfactant (s) phase, the interface layer between the dispersed oil (o) and the continuous water (w) phases. Here, high-resolution 19F quantitative NMR spectroscopy was applied directly and noninvasively on an o/w NE drug product containing difluprednate (DFPN). The well-resolved 19F peaks of DFPN depended on the shielding molecules in each phase, which revealed mass-balanced DFPN distribution in multiple phases of (w), (s), and (o) of NE globules at a quantity of 1.8 ± 0.1, 35 ± 2, and 59 ± 3% per labeled content, respectively. Furthermore, the dilution-dependent 19F peak line broadening and shift suggested a millisecond dynamic exchange between the NE and the less-noticed smaller but thermodynamically stable microemulsion (ME) globules in NE solution. The high-resolution NMR result revealed that the drug availability could be quickly achieved using an o/w NE formulation because of the drug multiphase distribution and the ME-assisted fast drug exchange among globules.

Recent advances on the stability of dietary polyphenols

Recent advances on the stability of dietary polyphenols

New chromone analog and pyrrole alkaloid produced by Penicillium sclerotiorum and their antibacterial activity

A new chromone analog (1) and a new pyrrole alkaloid (2), together with four known compounds, were isolated from the endophytic fungus Penicillium sclerotiorum MPT-250 obtained from the stems of Taxus wallichiana var. chinensis (Pilger) Florin. The structural elucidation of these metabolites was performed by high-resolution mass spectrometry and NMR spectroscopy. Compounds 1 and 5 exhibited significant antibacterial activity against carbapenems-resistant Pseudomonas aeruginosa and multidrug-resistant Enterococcus faecium with an minimum inhibitory concentration (MIC) value of 3.13 μg/ml respectively.

5,5′-Thiobis(4-chloro-3H-1,2-dithiole-3-thione)

3H-1,2-Dithiole-3-thiones are important compounds with many types of significant pharmacological activity. Although many derivatives of this class have been described in the literature, their thioethers have not previously been obtained. In this communication, it is shown that the reaction of 4,5-dichloro-3H-1,2-dithiole-3-thione with potassium isocyanate unexpectedly gave 5,5′-thiobis(4-chloro-3H-1,2-dithiole-3-thione). The structure of the synthesized compound was established by elemental analysis, high-resolution mass spectrometry, 13C NMR and IR spectroscopy, and mass spectrometry.

Metabolomic analysis of endometrial cancer by high-resolution magic angle spinning NMR spectroscopy.

To analyze endometrial metabolite profiles between patients with endometrial cancer and controls. Seventeen (17) women with endometrium cancer and 18 controls were enrolled in this study. 1H HR-MAS (High Resolution-Magic Angle Spinning) NMR (Nuclear Magnetic Resonance) spectroscopy data obtained from endometrial tissue samples of patients with endometrial cancer and control group were analyzed with bioinformatics methods. Principal component analysis (PCA) and the partial least squares discriminant analysis (PLS-DA) score plots obtained with the multivariate statistical analysis of pre-processed spectral data shows a separation between the samples from patients with endometrial cancer and controls. Analysis results suggest that the levels of lactate, glucose, o-phosphoethanolamine, choline, glycerophosphocholine, phosphocholine, leucine, isoleucine, valine, glutamate, glutamine, n-acetyltyrosine, methionine, taurine, alanine, aspartate and phenylalanine are increased in patients with endometrial cancer compared to the controls. The metabolomics signature of patients with endometrial cancer is different from that of benign endometrial tissue.

High-Resolution Magic-Angle Spinning NMR Spectroscopy for Evaluation of Cell Shielding by Virucidal Composites Based on Biogenic Silver Nanoparticles, Flexible Cellulose Nanofibers and Graphene Oxide.

Antiviral and non-toxic effects of silver nanoparticles onto in vitro cells infected with coronavirus were evaluated in this study using High-Resolution Magic-Angle Spinning Nuclear Magnetic Resonance (HR-MAS NMR) spectroscopy. Silver nanoparticles were designed and synthesized using an orange flavonoid—hesperetin (HST)—for reduction of silver(I) and stabilization of as obtained nanoparticles. The bio-inspired process is a simple, clean, and sustainable way to synthesize biogenic silver nanoparticles (AgNP@HST) with diameters of ∼20 nm and low zeta potential (−40 mV), with great colloidal stability monitored for 2 years. The nanoparticles were used for the fabrication of two types of antiviral materials: colloids (AgNP@HST spray) and 3D flexible nanostructured composites. The composites, decorated with AgNP@HST (0.05 mmol L−1), were made using cellulose nanofibers (CNF) obtained from orange peel and graphene oxide (GO), being denominated CNF@GO@AgNP@HST. Both materials showed high virucidal activity against coronaviruses in cell infection in vitro models and successfully inhibited the viral activity in cells. HR-MAS 1H-NMR technique was used for determining nanomaterials’ effects on living cells and their influences on metabolic pathways, as well as to study viral effects on cells. It was proven that none of the manufactured materials showed toxicity towards the intact cells used. Furthermore, viral infection was reverted when cells, infected with the coronavirus, were treated using the as-fabricated nanomaterials. These significant results open possibilities for antiviral application of 3D flexible nanostructured composite such as packaging papers and filters for facial masks, while the colloidal AgNP@HST spray can be used for disinfecting surfaces, as well as a nasal, mouth, and eye spray.

Temporal profile of serum metabolites and inflammation following closed head injury in rats is associated with HPA axis hyperactivity.

Closed head injury (CHI) causes neurological disability along with systemic alterations that can activate neuro-endocrine response through hypothalamic-pituitary-adrenal (HPA) axis activation. A dysregulated HPA axis function can lead to relocation of energy substrates and alteration in metabolic pathways and inflammation at the systemic level. Assessment of time-dependent changes in serum metabolites and inflammation after both mild and moderate CHI. Along with this, serum corticosterone levels and hypothalamic microglial response were observed. Rats underwent mild and moderate weight-drop injury and their serum and hypothalamus were assessed at acute, sub-acute and chronic timepoints. Changes in serum metabolomics were determined using high resolution NMR spectroscopy. Serum inflammatory cytokine, corticosterone levels and hypothalamic microglia were assessed at all timepoints. Metabolites including lactate, choline and branched chain amino acids were found as the classifiers that helped distinguish between control and injured rats during acute, sub-acute and chronic timepoints. While, increased αglucose: βglucose and TMAO: choline ratios after acute and sub-acute timepoints of mild injury differentiated from moderate injured rats. The injured rats also showed distinct inflammatory profile where IL-1β and TNF-α levels were upregulated in moderate injured rats while IL-10 levels were downregulated in mild injured rats. Furthermore, injury specific alterations in serum metabolic and immunologic profile were found to be associated with hyperactive HPA axis, with consistent increase in serum corticosterone concentration post injury. The hypothalamic microglia showed a characteristic activated de-ramified cellular morphology in both mild and moderate injured rats. The study suggests that HPA axis hyperactivity along with hypothalamic microglial activation led to temporal changes in the systemic metabolism and inflammation. These time dependent changes in the metabolite profile of rats can further strengthen the knowledge of diagnostic markers and help distinguish injury related outcomes after TBI.

Cembranoids From the Leaves of Nicotiana tabacum and Their Neuroprotective Activities

A new cembrane-type diterpenoid (1) and 9 known cembranoids (2-10) were isolated from the leaves of Nicotiana tabacum. The structure of the new metabolite was confirmed by extensive analyses of their spectroscopic data, including high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), 1D- and 2D nuclear magnetic resonance (NMR) spectroscopy, and infrared spectroscopy (IR). All of the isolated compounds were tested for their protective effects against oxygen-glucose deprivation (OGD)-induced neurotoxicity in SH-SY5Y cells. The new compound 1 exhibited a moderate protective effect against OGD-induced neurotoxicity at 10 μM.

Magnetic Resonance Imaging (MRI) and MR Spectroscopic Methods in Understanding Breast Cancer Biology and Metabolism.

A common malignancy that affects women is breast cancer. It is the second leading cause of cancer-related death among women. Metabolic reprogramming occurs during cancer growth, invasion, and metastases. Functional magnetic resonance (MR) methods comprising an array of techniques have shown potential for illustrating physiological and molecular processes changes before anatomical manifestations on conventional MR imaging. Among these, in vivo proton (1H) MR spectroscopy (MRS) is widely used for differentiating breast malignancy from benign diseases by measuring elevated choline-containing compounds. Further, the use of hyperpolarized 13C and 31P MRS enhanced the understanding of glucose and phospholipid metabolism. The metabolic profiling of an array of biological specimens (intact tissues, tissue extracts, and various biofluids such as blood, urine, nipple aspirates, and fine needle aspirates) can also be investigated through in vitro high-resolution NMR spectroscopy and high-resolution magic angle spectroscopy (HRMAS). Such studies can provide information on more metabolites than what is seen by in vivo MRS, thus providing a deeper insight into cancer biology and metabolism. The analysis of a large number of NMR spectral data sets through multivariate statistical methods classified the tumor sub-types. It showed enormous potential in the development of new therapeutic approaches. Recently, multiparametric MRI approaches were found to be helpful in elucidating the pathophysiology of cancer by quantifying structural, vasculature, diffusion, perfusion, and metabolic abnormalities in vivo. This review focuses on the applications of NMR, MRS, and MRI methods in understanding breast cancer biology and in the diagnosis and therapeutic monitoring of breast cancer.

Magic angle spinning dynamic nuclear polarization solid-state NMR spectroscopy of γ-irradiated molecular organic solids

In the past 15 years, magic angle spinning (MAS) dynamic nuclear polarization (DNP) has emerged as a method to increase the sensitivity of high-resolution solid-state NMR spectroscopy experiments. Recently, γ-irradiation has been used to generate significant concentrations of homogeneously distributed free radicals in a variety of solids, including quartz, glucose, and cellulose. Both γ-irradiated quartz and glucose previously showed significant MAS DNP enhancements. Here, γ-irradiation is applied to twelve small organic molecules to test the applicability of γ-irradiation as a general method of creating stable free radicals for MAS DNP experiments on organic solids and pharmaceuticals. Radical concentrations in the range of 0.25 ​mM–10 ​mM were observed in irradiated glucose, histidine, malic acid, and malonic acid, and significant 1H DNP enhancements of 32, 130, 19, and 11 were obtained, respectively, as measured by 1H→13C CPMAS experiments. However, concentrations of free radicals below 0.05 ​mM were generally observed in organic molecules containing aromatic rings, preventing sizeable DNP enhancements. DNP sensitivity gains for several of the irradiated compounds exceed that which can be obtained with the relayed DNP approach that uses exogeneous polarizing agent solutions and impregnation procedures. In several cases, significant 1H DNP enhancements were realized at room temperature. This study demonstrates that in many cases γ-irradiation is a viable alternative to addition of stable exogenous radicals for DNP experiments on organic solids.

Chemical shielding and electric field gradient tensors of disodium 5′-adenosine triphosphate trihydrate determined by solid-state NMR spectroscopy and ab initio calculations

The tri-phosphate and sodium ion environments in well crystallized disodium 5′adenosine triphosphate trihydrate (ATP•3H2O) are investigated using high-resolution solid-state NMR spectroscopy and ab initio calculations. The six inequivalent phosphorus sites resolved in 31P MAS spectra have been assigned on the basis of experimentally obtained results and theoretical ab initio quantum chemical calculations of 31P chemical shielding tensors with increased numerical accuracy. Aided by 31P-31P dipolar connectivity established in DQ-SQ correlation experiment and DFT calculations carried out at the B3LYP/(aug-cc-pDVZ(3s,2p,1d), 6–31G(d,p)) level, phosphorus resonance assignments in MAS spectra have been made and the 31P chemical shielding anisotropies and tensor orientations in the molecule-fixed frame have been determined. 31P shielding tensors are found to be oriented with the most shielded direction nearly perpendicular to the O-P-O plane involving the two adjoining oxygens. The complete resolution of the four sodium sites has been achieved from 3Q-MAS experiments performed at 7.05 T and this has enabled 23Na quadrupole parameters to be determined experimentally and compared with theoretical calculations. Besides aiding a partial resonance assignment of the 23Na 3Q-MAS spectrum, our HF/6–311++G(2d,2p) calculations of 23Na EFG tensors show that for two sodium sites which are tightly coordinated the orientation of the EFG tensor is distinct. These exhibit unique directions along which the field gradient is the smallest and largest.

Identification of Pigmented Substances in Black Honey from Leucosceptrum canum: Novel Quinonoids Contribute to Honey Color.

Leucosceptrum canum (Lamiaceae) honey (LCH) is a rare and high-value honey with an unusual dark brown color. Its pigments may be related to the purported health benefits of the honey and could serve as indicators of authentic LCH. Here, we purified the pigmented substances contributing to LCH's color and identified them using high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy. The major pigmented substances were 1-[4-(3-carboxy-propylamino)-3,6-dioxo-cyclohexa-1,4-dienyl]-pyrrolidine-2-carboxylic acid (GPBQ), 1-[4-(3-carboxy-propylamino)-3,6-dioxo-cyclohexa-1,4-dienyl]-pyrrolidine-2-carboxylic acid. (GAPBQ), and 2,5-di-(N-prolyl)-para-benzoquinone (DPBQ). According to UHPLC-DAD data recorded at 370 nm, GPBQ, GAPBQ, and DPBQ contents ranged from 2.58 to 5.13, 2.93 to 4.96, and 4.26 to 9.22 mg/kg, respectively. These amino acid-quinone conjugates, found here for the first time, are pigments that can serve as markers to control the quality of LCH.

Interfacing Liquid State Hyperpolarization Methods with NMR Instrumentation

Advances in liquid state hyperpolarization methods have enabled new applications of high-resolution NMR spectroscopy. Utilizing strong signal enhancements from hyperpolarization allows performing NMR spectroscopy at low concentration, or with high time resolution. Making use of the high, but rapidly decaying hyperpolarization in the liquid state requires new techniques to interface hyperpolarization equipment with liquid state NMR spectrometers. This article highlights rapid injection, high resolution NMR spectroscopy with hyperpolarization produced by the techniques of dissolution dynamic nuclear polarization (D-DNP) and para-hydrogen induced polarization (PHIP). These are popular, albeit not the only methods to produce high polarization levels for liquid samples. Gas and liquid driven sample injection techniques are compatible with both of these hyperpolarization methods. The rapid sample injection techniques are combined with adapted NMR experiments working in a single, or small number of scans. They expand the application of liquid state hyperpolarization to spins with comparably short relaxation times, provide enhanced control over sample conditions, and allow for mixing experiments to study reactions in real time.