High-resolution NMR Spectroscopy Research Articles

Challenges of analytical methods for the characterization of microsamples from David Alfaro Siqueiros mural painting.

This study presents the analysis of seven microsamples from Untitled Mural 3, painted by David Alfaro Siqueiros between 1964 and 1972. The mural has never been restored, therefore preserving the materials used by the artist. The microsamples were analyzed using a combination of techniques: optical microscopy (OM), scanning electron microscopy with energy-dispersive spectroscopy (SEM-EDS), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), micro-Fourier transform infrared spectroscopy (micro-FTIR) with reflection mode, and high-resolution nuclear magnetic resonance (NMR) spectroscopy. In this work, compounds common to those identified in the analysis of the contemporary mural Trazos de composición piramidal composition were identified, such as acrylic and nitrocellulose binders, titanium white and iron red oxide as pigments, and DEHP as a plasticizer. Unexpectedly, polyvinyl acetate (PVAc) and different nitrocellulose monomers were identified, in addition to chrome yellow instead of ochre, and a predominance of quartz, asbestos, and talc as fillers, instead of carbonates. The study confirmed that Siqueiros used multiple binders in successive layers in no apparent order, providing valuable insights into his artistic techniques. The research also proved an efficient methodology for studying very small and complex samples.

Distinct association of HRAS and KRAS with Mn(II) ion illustrated by paramagnetic NMR

Rat sarcoma virus oncogene (RAS) proteins are among crucial oncogenic proteins and are involved in several essential intracellular processes. The RAS protein has an intrinsic metal binding site for Mg2+, which is important for the conformational stability of the active site. Recently, it was reported that a second metal ion binding site, located further from the active site in HRAS (Harvey RAS homolog), binds Ca2+ with millimolar affinity. As one of the most abundant metal ions in cells, Mn2+ is a potential candidate for the second metal ion binding site in RAS proteins. Here, we examined the interaction of Mn2+ with HRAS and KRAS (Kirsten RAS homolog) using high resolution NMR spectroscopy. The NMR data showed that both the second metal ion binding site and the switch I and II regions bind Mn2+ in the RAS proteins. Furthermore, our paramagnetic NMR results disclosed the conformational differences in helix α3 and the following loop between HRAS and KRAS, accompanied by the association with metal ion binding. These results provide new insights into the interaction of RAS proteins and Mn2+ in the respective biological processes in cells.

NMR coupled with multivariate data analysis for monitoring the degradation of a formulated therapeutic monoclonal antibody.

The function of a protein is directly coupled to its higher-order structure (HOS). Deviations in this critical quality attribute (CQA) may be linked to a decrease in the efficacy and/or safety of the final therapeutic product. High-resolution nuclear magnetic resonance (NMR) spectroscopy has been recently highlighted for protein HOS characterization, thanks to its ability to capture small changes at the molecular and structural levels (primary, secondary, tertiary and quaternary). The present study was carried out to demonstrate the ability of NMR (1D 1H and 2D 1H-13C experiments) coupled with multivariate data analysis (PCA and PLS regression) to monitor the degradation of a formulated mAb-like protein and for the simultaneous quantification of related CQAs, i.e., potency, purity and impurities (aggregates and fragments). The results indicate that this approach could be applied to mitigate product quality risks during development, manufacturing and stability studies of mAb therapeutics.

High-resolution NMR spectroscopic approaches to quantify PET microplastics pollution in environmental freshwater samples

Reliable identification and precise quantification of microplastics pollution of the environment are essential prerequisites to comprehend the impact of microplastics on Earth’s ecosystems. In this study, we propose a workflow to examine polyethylene terephthalate (PET) contamination of environmental surface waters by applying high-resolution nuclear magnetic resonance (NMR) spectroscopic approaches. The detection of PET by high-resolution NMR spectroscopy enables the unambiguous identification and - at the same time - precise quantification at atomic resolution independent from the size of the particles obtained from surface waters. Monitoring the properties of translational diffusion and relaxation of PET chains present in the samples obtained from Lake Constance water by filtration (‘Manta trawls’), extraction and dissolving, hints towards a rather heterogeneous distribution in length of the PET chains. The workflow developed here achieved a limit of detection of 192.2 ng PET and a recovery rate of 88 ± 25% for PET microplastics that was spiked to the Manta trawls. The NMR driven analysis led to a concentration determination of 335 ± 200 ng PET per cubic meter of Lake Constance water. The workflow developed here offers not only a simple and reliable quantitative determination of the mass of PET in environmental samples independent of particle size but is additionally providing insights into the inherent polymeric features of PET, which are not accessible through other established methods of microplastics detection. Therefore, a broad application of the NMR spectroscopic approach presented here can be assumed.

The 1H HR-NMR Methods for the Evaluation of the Stability, Quality, Authenticity, and Shelf Life of Foods

1H High-Resolution Nuclear Magnetic Resonance (1H HR-NMR) spectroscopy is a powerful analytical methodology used in various fields, including food science. In the food science field, NMR combined with the principles of metabolomics can provide detailed information about a food’s molecular composition, structure, dynamics, and interactions within food matrices, making it invaluable for assessing changes during storage, processing, and shelf life. This entry aims to list the main applications of one-dimensional 1H HR-NMR methods in the field of food science, such as their use in the assessment of the stability, quality, authenticity, and shelf life of food samples. Several kinds of foods are taken into consideration to give a huge overview of the potentiality of the methods.

Localized Shims Enable Low-Field Simultaneous Multinuclear NMR Spectroscopy.

This Article introduces a smart localized shimset, integrated with a radiofrequency (RF) microsolenoid and optimized as a single unit to locally achieve high-resolution NMR spectroscopy, as a significant advancement toward achieving parallel NMR spectroscopy. The shimset, consisting exclusively of linear shims, demonstrates the capability to improve NMR line width from 84 to 4 Hz in a 1.05 T preclinical MRI scanner. This enhancement enables the resolution of j-couplings in the sample while requiring a total power of 545 mW. Furthermore, a novel method is presented for concurrently obtaining multinuclear NMR spectra using a single RF channel. This approach allows for the utilization of high-sensitivity NMR signals as a reference to correct field drift, facilitating signal averaging in a permanent magnet NMR scanner that is prone to significant temperature-dependent drift.

Chemical structures of polyketides and alkaloids isolated from a culture of fungus Curvularia moringae

Seven new polyketides [moringols I–VII (1–7)], a new alkaloid [moringamine I (8)], and seven known compounds (9–15) were isolated from the fungus Curvularia moringae JKYM-KR4. The planar chemical structures and relative configurations of the new compounds were elucidated by high-resolution mass spectrometry, 1D and 2D NMR spectroscopy, and DP4+ analysis using the calculated 13C NMR chemical shifts. For moringols I and II (1 and 2), the planar chemical structures and relative configurations were confirmed using X-ray crystallography. The absolute configurations of 1–6 and 8 were determined by ECD calculations. Among the isolated compounds, terpestacin (14) moderately inhibited the proliferation of HT-29 cells.

Exploring the cyclization of thiosemicarbazone to 1,3,4-thiadiazole: Synthesis, characterization and in-silico study

Breast cancer remains a significant health concern worldwide, necessitating the development of novel therapeutic strategies. In this study, we focus on the design, synthesis, and evaluation of isophorone-1,3,4-thiadiazoles as potential inhibitors targeting AKT1 and STAT3, crucial players in breast cancer progression. The synthesized compounds were rigorously characterized using High-Resolution Mass Spectrometry (HRMS) and Nuclear Magnetic Resonance (NMR) spectroscopy, encompassing both 1H and 13C spectra. Subsequently, Network pharmacology analysis facilitated for the identification of key genes and pathways associated with breast cancer, thereby identifying AKT1 and STAT3 as promising therapeutic targets for the designed compounds. Furthermore, the top two targets, AKT1 and STAT3, were chosen for molecular docking analyses to forecast the binding affinity of the ligands with the proteins, revealing favorable interactions between compounds 8a-c and these targets. Molecular dynamics was performed for 100 ns simulation to predict the stability of the compounds. Acceptable RMSD and RMSF values for amino acid residues indicated structural integrity and a stable protein-ligand interaction. Additionally, all designed compounds displayed favorable physicochemical properties and promising ADMET profiles. These 8a and 8c considered as promising leads for targeting AKT1 and STAT3.

Olive mill wastewater as a source of defense-promoting by-products against microbial pathogens

Olive oil is a core component of the Mediterranean diet known for its nutritional properties and health benefits. Olive industry is moving to novel extraction systems for higher oil yield and quality and for waste reduction, which is a relevant problem in the process due to its toxicity and high disposal costs. Multi-Phase Decanter (DMF) is a modern two-phase system performed without adding water during the process. Using DMF, a wet by-product indicated as pâté and consisting of the fruit pulp and vegetation water (VW) is recovered. The pâté has a high content of potentially bioactive molecules that may be exploited to promote plant resistance against microbial pathogens. In this work, to identify by/products of biological interest, the VW recovered from the pâté by centrifugation was subjected to fractionation by tangential-flow membrane filtration (TFMF), combining microfiltration (MF) and ultrafiltration (UF). High-resolution NMR spectroscopy indicated the presence of bioactive molecules such as flavonoids, hydroxytyrosol and oleuropein with known antimicrobial activity. High-Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) was performed to detect the presence of pectic oligosaccharides in the fractions, showing the enrichment, in the UF-concentrate fraction, of oligogalacturonides (OGs), well known for the ability to elicit defense responses and protect plants against pathogen infections. Arabidopsis thaliana plants treated with TFMF fractions displayed induction of defense responses and exhibited tolerance against microbial pathogens without adverse effects on growth and fitness. This study shows that pâté by-products can potentially be exploited in agriculture as sustainable plant phyto-protectant.

Comprehensive analysis of deflazacort oxidative degradation: Insights into novel degradation products and mechanisms

The oxidative degradation pathways of deflazacort (DFL) were investigated to address the gap in understanding its degradation products, focusing on reactions with oxidative stressors such as hydrogen peroxide and 4,4′-azobis (4-cyanovaleric acid) (ACVA). Using HPLC-PDA, high-resolution mass spectrometry (HRMS), NMR and IR spectroscopy, four novel degradation products were identified and structurally characterized. Two of these products were isolated using preparative HPLC before characterization. Hydrogen peroxide led to the formation of three novel products (DP-1, DP-2, and DP-3), while ACVA resulted in a single novel product (DP-4). Mechanistic and kinetic experiments supported the proposed degradation pathways under the various oxidative stress conditions studied, revealing distinct rates of formation for the degradation products during the time-course study. The identification and detailed structural elucidation of these degradation products provide critical insights into the chemical stability and potential reactivity of DFL under oxidative stress. These findings underscore the importance of comprehensive stability testing for ensuring drug safety and efficacy, and offer valuable data for future research on the toxicity and pharmacological impact of DFL degradation products.

Semisynthetic Studies Establish a Role for Conjugate Halide Exchange in the Formation of Chlorinated Pyrroloiminoquinones and Related Alkaloids.

Two novel pyrroloiminoquinone alkaloids, 6-chlorodamirone A and 6-bromodamirone A, have been identified for the first time from the marine sponge Latrunculia sp. (order: Poecilosclerida: family Latrunculiidae), sourced from Western Australia. Alongside these new compounds, seven previously known metabolites were also isolated. Despite being obtained in submilligram quantities, the structures of these natural products were successfully elucidated using high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy. To confirm the structures of these newly discovered alkaloids, a semisynthetic approach was employed starting from the more abundant metabolite, damirone A, additionally, single crystal X-ray crystallography was used to validate our structural proposals. The semisynthetic studies suggest that the chlorinated alkaloids are likely formed through a nonenzymatic conjugate halide substitution reaction rather than an enzymatic process. This reactivity parallels that observed in related metabolites, such as the caulibugulones B and C. Furthermore, a biomimetic cascade reaction was attempted to synthesize the spirodienone moiety characteristic of the discorhabdin alkaloids, inspired by the nucleophilic substitution observed in the tricyclic damirone A system. Albeit unsuccessful, these findings provide valuable insight into the reactivity of halogenated pyrroloiminoquinones under various conditions.

Extraction of In-Cell β-Amyloid Fibrillar Aggregates for Studying Molecular-Level Structural Propagations Using Solid-State NMR Spectroscopy.

Molecular-level structural polymorphisms of β-amyloid (Aβ) fibrils have recently been recognized as pathologically significant. High-resolution solid-state nuclear magnetic resonance (ssNMR) spectroscopy has been utilized to study these structural polymorphisms, particularly in ex-vivo fibrils seeded from amyloid extracts of post-mortem brain tissues of Alzheimer's disease (AD) patients. One unaddressed question in current ex-vivo seeding protocol is whether fibrillation from exogenous monomeric Aβ peptides, added to the extracted seeds, can be quantitatively suppressed. Addressing this issue is critical because uncontrolled fibrillation could introduce biased molecular structural polymorphisms in the resulting fibrils. Here, we present a workflow to optimize the key parameters of ex-vivo seeding protocols, focusing on the quantification of amyloid extraction and the selection of exogenous monomeric Aβ concentrations to minimize nonseeded fibrillation. We validate this workflow using three structurally different 40-residue Aβ (Aβ40) fibrillar seeds, demonstrating their ability to propagate their structural features to exogenous wild-type Aβ40.

Integrating HRMAS-NMR Data and Machine Learning-Assisted Profiling of Metabolite Fluxes to Classify Low- and High-Grade Gliomas.

Diagnosing and classifying central nervous system tumors such as gliomas or glioblastomas pose a significant challenge due to their aggressive and infiltrative nature. However, recent advancements in metabolomics and magnetic resonance spectroscopy (MRS) offer promising avenues for differentiating tumor grades both in vivo and ex vivo. This study aimed to explore tissue-based metabolic signatures to classify/distinguish between low- and high-grade gliomas. Forty-six histologically confirmed, intact solid tumor samples from glioma patients were analyzed using high-resolution magic angle spinning nuclear magnetic resonance (HRMAS-NMR) spectroscopy. By integrating machine learning (ML) algorithms, spectral regions with the most discriminative potential were identified. Validation was performed through univariate and multivariate statistical analyses, along with HRMAS-NMR analyses of 46 paired plasma samples. Amongst the various ML models applied, the logistics regression identified 46 spectral regions capable of sub-classifying gliomas with accuracy 87% (F1-measure 0.87, Precision 0.82, Recall 0.93), whereas the extra-tree classifier identified three spectral regions with predictive accuracy of 91% (F1-measure 0.91, Precision 0.85, Recall 0.97). Wilcoxon test presented 51 spectral regions significantly differentiating low- and high-grade glioma groups (p < 0.05). Based on sensitivity and area under the curve values, 40 spectral regions corresponding to 18 metabolites were considered as potential biomarkers for tissue-based glioma classification and amongst these N-acetyl aspartate, glutamate, and glutamine emerged as the most important markers. These markers were validated in paired plasma samples, and their absolute concentrations were computed. Our results demonstrate that the metabolic markers identified through the HRMAS-NMR-ML analysis framework, and their associated metabolic networks, hold promise for targeted treatment planning and clinical interventions in the future.

Geographical origin identification of cinnamon using HPLC-DAD fingerprints and chemometrics

The commercial value of cinnamon depends largely on its geographical origin. This study proposed high-performance liquid chromatography-diode array detection (HPLC-DAD) fingerprints combined with chemometrics to identify geographical origins of cinnamons. Alternating trilinear decomposition (ATLD) algorithm was applied to extract meaningful chemical information from the HPLC fingerprint data. Then, non-targeted HPLC fingerprints and chemical component information were used as chemical descriptors to distinguish geographical origins using principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA)/N-way partial least squares-discriminant analysis (NPLS-DA). Satisfactory classification results were acquired for each discriminant model and the correct classification rates (CCRs) of training and test sets were 100%. Moreover, four characteristic variables were screened by variable importance in projection (VIP) method, three of which were reasonably identified as cinnamic acid, cinnamaldehyde and 2-methoxycinnamaldehyde, which can be regarded as potential markers to distinguish cinnamon from different geographical origins. The results indicated that HPLC-DAD fingerprints combined with chemometrics can be a promising means to identify the geographical origins and screen potential markers of cinnamon samples, especially in the absence of high-cost and sophisticated analytical instrumentals (e.g., high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy). This work can help to prevent cinnamon fraud practices and maintain the normal order of the cinnamon market.

Utilizing cutting-edge analytical techniques for the Identification, Isolation, and structural characterization of donepezil HCl forced degradation products

The current study elucidates the stress degradation behavior of donepezil HCl, a medication used to treat Alzheimer-type dementia. Using hydrolytic, oxidative, thermal, and photolytic conditions, the forced degradation investigation was conducted in accordance with ICH recommendations. In neutral hydrolysis, oxidative, thermal, and photolytic conditions, donepezil is comparatively stable; nevertheless, significant degradation was noted in acid and base hydrolysis. Using sophisticated analytical methods such as LCMS, Preparative-HPLC, NMR, and HRMS, the degradation products were identified, isolated, and their structural validity was confirmed. Degradation products 1, 2, and 3 are the three degradation products that were found and isolated during acid degradation. Conversely, products 1 and 3 generated during base hydrolysis degradation. Degradation product 1 and 3 were known products, while degradation product 2 is new compound that have not yet been documented. Using an advanced analytical approach, this is the first recorded comprehensive structural characterization of all degradation products. In the current work, high-resolution mass spectroscopy and nuclear magnetic resonance spectroscopy were employed to get specific confirmation of degradation product structures. In the future, degradation products with lower runtime will also be identified using the present LCMS technique.

The role of NMR spectroscopy in lignocellulosic biomass characterisation: A mini review

Lignocellulosic biomass (LB) is promising feedstock for the production of various bio-based products. However, due to its heterogenous character, complex chemical structure and recalcitrance, it is necessary to know its structural composition in order to optimize pretreatment process and further (bio)conversion into bio-based products. Nuclear Magnetic Resonance (NMR) spectroscopy is a fast and reliable method that can provide advanced data on the molecular architecture and composition of lignocellulosic biomass. In this brief overview, characteristic examples of the use of high-resolution NMR spectroscopy for the investigation of various types of LB and their structural units are given and the main drawbacks and future perspectives are outlined.

High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy of Paired Clinical Liver Tissue Samples from Hepatocellular Cancer and Surrounding Region.

The global burden of liver cancer is increasing. Timely diagnosis is important for optimising the limited available treatment options. Understanding the metabolic consequences of hepatocellular carcinoma (HCC) may lead to more effective treatment options. We aimed to document metabolite differences between HCC and matched surrounding tissues of varying aetiology, obtained at the time of liver resection, and to interpret metabolite changes with clinical findings. High-resolution magic angle spinning nuclear magnetic resonance (HRMAS-NMR) spectroscopy analyses of N = 10 paired HCC and surrounding non-tumour liver tissue samples were undertaken. There were marked HRMAS-NMR differences in lipid levels in HCC tissue compared to matched surrounding tissue and more subtle changes in low-molecular-weight metabolites, particularly when adjusting for patient-specific variability. Differences in lipid-CH3, lipid-CH2, formate, and acetate levels were of particular interest. The obvious differences in lipid content highlight the intricate interplay between metabolic adaptations and cancer cell survival in the complex microenvironment of liver cancer. Differences in formate and acetate might relate to bacterial metabolites. Therefore, documentation of metabolites in HCC tissue according to histology findings in patients is of interest for personalised medicine approaches and for tailoring targeted treatment strategies.

Probing and gauging of D-Penicillamine xenobiotics in hepatic Wilson disease patients

D-penicillamine (PA) is the primary chelator of choice to treat Wilson disease (WD). There are limitations in obtaining comprehensive data on PA metabolites in biological specimens by conventional approaches. Hence, the aim of the present was to identify the major hepatic PA metabolites and draw clear conclusions of the drug's xenobiotic in WD. Urine samples were collected from children with hepatic WD (n = 63, aged 14.8 ± 4 years) 5 h after PA administration (16.3 ± 3.8 mg/kg/day) and age-matched healthy volunteers comprised as controls (n = 30). High-resolution 800 MHz nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry was applied to reveal unambiguous appraisals of different excretory by-products of PA metabolism. Four new products comprising penicillamine disulphide (PD), penicillamine cysteine disulphide (PCD), S-methyl penicillamine (SMP), and N-acetyl penicillamine (NAP) of PA xenobiotic metabolites were identified using high-resolution NMR spectroscopy. Quantitative levels of PCD and SMP were approximately three-fold higher than those of PD and NAP, respectively. High-resolution NMR identifies the major PA metabolites with certainty. Reduction, sulfation, and methylation are the predominant pathways of PA metabolism. There is a potential application for assessing therapeutic monitoring of chelation in hepatic WD.

Understanding the Solid-State Structure of Riboflavin through a Multitechnique Approach.

Crystalline riboflavin (vitamin B2) performs an important biological role as an optically functional material in the tapetum lucidum of certain animals, notably lemurs and cats. The tapetum lucidum is a reflecting layer behind the retina, which serves to enhance photon capture and vision in low-light settings. Motivated by the aim of rationalizing its biological role, and given that the structure of biogenic solid-state riboflavin remains unknown, we have used a range of experimental and computational techniques to determine the solid-state structure of synthetic riboflavin. Our multitechnique approach included microcrystal XRD, powder XRD, three-dimensional electron diffraction (3D-ED), high-resolution solid-state 13C NMR spectroscopy, and dispersion-augmented density functional theory (DFT-D) calculations. Although an independent report of the crystal structure of riboflavin was published recently, our structural investigations reported herein provide a different interpretation of the intermolecular hydrogen-bonding arrangement in this material, supported by all the experimental and computational approaches utilized in our study. We also discuss, more generally, potential pitfalls that may arise in applying DFT-D geometry optimization as a bridging step between structure solution and Rietveld refinement in the structure determination of hydrogen-bonded materials from powder XRD data. Finally, we report experimental and computational values for the refractive index of riboflavin, with implications for its optical function.

Discovery of Unprecedented Human Stercobilin Conjugates.

Two unique metabolites (M18 and M19) were detected in feces of human volunteers dosed orally with [14C]inavolisib with a molecular ion of parent plus 304 Da. They were generated in vitro by incubation with fecal homogenates and we have evidence that they are formed chemically and possibly enzymatically. Structural elucidation by high resolution mass spectrometry and nuclear magnetic resonance spectroscopy showed that the imidazole ring of inavolisib was covalently bound to partial structures derived from stercobilin, an end-product of heme catabolism produced by the gut microbiome. The structural difference between the two metabolites was the position of methyl and ethyl groups on the pyrrolidin-2-one moieties. We propose a mechanism of M18 and M19 generation from inavolisib and stercobilin whereby nucleophilic attack from the imidazole ring of inavolisib occurs to the bridging carbon of a stercobilin molecule. The proposed mechanism was supported by computational calculations of molecular orbitals and transition geometry. SIGNIFICANCE STATEMENT: We report the characterization of two previously undescribed conjugates of the phosphoinositide 3-kinase inhibitor inavolisib, generated by reaction with stercobilin, an end-product of heme catabolism produced by the gut microbiome. These conjugates were confirmed by generating them using in vitro fecal homogenate incubation via nonenzymatic and possibly enzymatic reactions. Given the unique nature of the conjugate, it is plausible that it may have been overlooked with other small molecule drugs in prior studies.