NMR Characterization Research Articles

Hypericum triquetrifolium and H. neurocalycinum as Sources of Antioxidants and Multi-Target Bioactive Compounds: A Comprehensive Characterization Combining In Vitro Bioassays and Integrated NMR and LC-MS Characterization by Using a Multivariate Approach.

Hypericum triquetrifolium and H. neurocalycinum were evaluated for their phytochemical content and in vitro bioactivity. NMR analyses were performed on the methanol extract of the aerial parts of H. triquetrifolium to establish the main classes of phytoconstituents. Then, LC-DAD-MSn analyses were performed in order to compare the composition of aerial parts and roots extracts of both Hypericum species, obtained using either methanol or water as solvents. Results, processed using multivariate data analysis, showed a significantly higher phenolic content of methanol extracts compared to water extracts, while minor qualitative differences were observed between the two. Distinctive flavonoid and PAC patterns were observed for H. triquetrifolium and H. neurocalycinum, and specific compounds were exclusively detected in one or the other species. Specifically, the phloroglucinols 7-epiclusianone, hyperfirin and hyperforin were present only in H. neurocalycinum, while hyperforin was detected only in H. triquetrifolium. Extracts were assayed using different in vitro tests to evaluate their antioxidant properties and their inhibitory activity against several enzymes, showing significant antioxidant and metal chelating activities. Furthermore, inhibitory properties against acetylcholinesterase, butyrylcholinesterase and tyrosinase were observed. Multivariate approaches were used to correlate biological data with the phytochemical composition of the different extracts. The results, showing positive correlations between specific chemical constituents and the measured bioactivities, represent preliminary data that could guide future studies aimed at isolating bioactive constituents from H. neurocalycinum and H. triquetrifolium for further pharmacological evaluations.

A Biorefinery Approach to the Biomass of the Seaweed Undaria pinnatifida (Harvey Suringar, 1873): Obtaining Phlorotannins-Enriched Extracts for Wound Healing.

Brown seaweeds are recognized sources of compounds with a wide range of properties and applications. Within these compounds, phlorotannins are known to possess several bioactivities (e.g., antioxidant, anti-inflammatory, and antimicrobial) with potential to improve wound healing. To obtain phlorotannins enriched extracts from Undaria pinnatifida, a biorefinery was set using low-cost industry-friendly methodologies, such as sequential solid–liquid extraction and liquid–liquid extraction. The obtained extracts were screened for their antioxidant and antimicrobial activity against five common wound pathogens and for their anti-inflammatory potential. The ethanolic wash fraction (wE100) had the highest antioxidant activity (114.61 ± 10.04 mmol·mg−1 extract by Diphenyl-1-picrylhydrazyl (DPPH) and 6.56 ± 1.13 mM eq. Fe II·mg−1 extract by and Ferric Reducing Antioxidant Power (FRAP)), acting efficiently against Gram-negative (Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) bacteria, and showing a nitric oxide production inhibition over 47% when used at 0.01 µg·mL−1. NMR and FTIR chemical characterization suggested that phlorotannins are present. Obtained fraction wE100 proved to be a promising candidate for further inclusion as wound healing agents, while the remaining fractions analyzed are potential sources for other biotechnological applications, giving emphasis to a biorefinery and circular economy framework to add value to this seaweed and the industry.

NMR Characterization of the Interactions Between Glycosaminoglycans and Proteins.

Glycosaminoglycans (GAGs) constitute a considerable fraction of the glycoconjugates found on cellular membranes and in the extracellular matrix of virtually all mammalian tissues. The essential role of GAG-protein interactions in the regulation of physiological processes has been recognized for decades. However, the underlying molecular basis of these interactions has only emerged since 1990s. The binding specificity of GAGs is encoded in their primary structures, but ultimately depends on how their functional groups are presented to a protein in the three-dimensional space. This review focuses on the application of NMR spectroscopy on the characterization of the GAG-protein interactions. Examples of interpretation of the complex mechanism and characterization of structural motifs involved in the GAG-protein interactions are given. Selected families of GAG-binding proteins investigated using NMR are also described.

Synthesis, FT-IR and NMR Characterization, Antibacterial and Antioxidant Activities, and DNA Docking Analysis of a New Vanillin-Derived imine Compound

A new vanillin-derived imine compound, [5-((1E,15E)-16-(3-methoxy-4-hydroxyphenyl)hexadeca-1′,15′-di-imine)-2-methoxyphenol], was synthesized and characterized by DFT calculations, experimental and theoretical vibrational spectroscopy and NMR techniques. The most stable molecular structure of the title compound has been calculated by optimizing the molecular structures using DFT/B3LYP/6-311+G(d,p) level of theory. The fundamental vibrational wavenumbers, IR and Raman intensities for the optimized geometry of the compound under investigation were determined and compared to the experimental vibrational spectra. The vibration assignment of the molecule was made in accord with the potential energy distribution (PED) of the vibration modes and using the group frequencies. The molecular electrostatic potential (MEP), HOMO and LUMO orbitals were also calculated. The antibacterial activities of the new vanillin-derived imine compound against gram-positive and gram-negative bacteria was determined. The antioxidant activity of the title compound was also examined. Moreover, the molecular docking studies have been performed to understand the nature of binding of the compound with DNA. The results indicated that the investigated compound has a good binding affinity with DNA and interacted with the DG4 and DT7 residues via the intermolecular hydrogen bonds.

Quality properties of chicken meatballs prepared with varying proportions of woody breast meat.

The objective of this study was to explore the effect of woody breast (WB) on quality characteristics of chicken meatballs paired with the feasibility of its inclusion. Cook loss (CL), color (CIE L*, a*, b*), texture (hardness, springiness, chewiness and resilience), low-field NMR (bound water, immobilized water, and free water), microstructure, and sensory characteristics of chicken meatballs with different WB inclusion levels (0%, 25%, 50%, 75%, 100%) were analyzed. The results showed that the impairment of product quality traits such as CL, color, texture (hardness, chewiness), free water, microstructure, and sensory scores (appearance, organization, total score) increased as the percentage of WB meat increased in the product formulation, particularly when the WB incorporation level exceeded 25%. Indeed, cook loss, L*, a*, b* parameters, bound water, and immobilized water increased when the WB inclusion level was higher than 25% (P ≤ 0.05). However, free water, sensory characteristics, hardness, and chewiness parameters decreased (P ≤ 0.05). The microstructure of chicken meatballs also changed as the proportion of WB meat increased. Even though data suggest that the inclusion of WB meat up to 30% could be feasible to produce acceptable chicken meatballs, the optimal maximum incorporation rate of WB meat into chicken meatball recipes was 25% based on economic feasibility and final overall quality.

THF co-solvent pretreatment prevents lignin redeposition from interfering with enzymes yielding prolonged cellulase activity

BackgroundConventional aqueous dilute sulfuric acid (DSA) pretreatment of lignocellulosic biomass facilitates hemicellulose solubilization and can improve subsequent enzymatic digestibility of cellulose to fermentable glucose. However, much of the lignin after DSA pretreatment either remains intact within the cell wall or readily redeposits back onto the biomass surface. This redeposited lignin has been shown to reduce enzyme activity and contribute to rapid enzyme deactivation, thus, necessitating significantly higher enzyme loadings than deemed economical for biofuel production from biomass.ResultsIn this study, we demonstrate how detrimental lignin redeposition on biomass surface after pretreatment can be prevented by employing Co-solvent Enhanced Lignocellulosic Fractionation (CELF) pretreatment that uses THF–water co-solvents with dilute sulfuric acid to solubilize lignin and overcome limitations of DSA pretreatment. We first find that enzymatic hydrolysis of CELF-pretreated switchgrass can sustain a high enzyme activity over incubation periods as long as 5 weeks with enzyme doses as low as 2 mg protein/g glucan to achieve 90% yield to glucose. A modified Ninhydrin-based protein assay revealed that the free-enzyme concentration in the hydrolysate liquor, related to enzyme activity, remained unchanged over long hydrolysis times. DSA-pretreated switchgrass, by contrast, had a 40% drop in free enzymes in solution during incubation, providing evidence of enzyme deactivation. Furthermore, measurements of enzyme adsorption per gram of lignin suggested that CELF prevented lignin redeposition onto the biomass surface, and the little lignin left in the solids was mostly integral to the original lignin–carbohydrate complex (LCC). Scanning electron micrographs and NMR characterization of lignin supported this observation.ConclusionsEnzymatic hydrolysis of solids from CELF pretreatment of switchgrass at low enzyme loadings was sustained for considerably longer times and reached higher conversions than for DSA solids. Analysis of solids following pretreatment and enzymatic hydrolysis showed that prolonged cellulase activity could be attributed to the limited lignin redeposition on the biomass surface making more enzymes available for hydrolysis of more accessible glucan.

XRD, NMR, FT-IR and DFT structural characterization of a novel organic-inorganic hybrid perovskite-type hexabromotellurate material

The synthesis of a new hexabromotellurate of triazole ring-containing amino acid is described. XRD, NMR and DFT analysis of the structure of protonated [(5-amino-4-phenyl-4H-1,2,4-triazol-3-yl)sulfanyl]acetic acid testifies the protonation of the first nitrogen atom of the triazole ring. Hirshfeld surface analysis identifies numerous intermolecular interactions in the newly synthesized organic-inorganic hybrid hexabromotellurate. The DFT based comprehensive analysis of the structures of possible conformers also testifies the higher thermodynamic stability of the 1-protonated triazole cation, whereas the starting [(5-amino-4-phenyl-4H-1,2,4-triazol-3-yl)sulfanyl]acetic acid exists in solution in neutral form with the intermolecular hydrogen bond between the carboxylic acid OH group and the second nitrogen of the triazole ring. The experimentally observed NMR upfield shift of the signal of the exocyclic amino group was studied via GIAO computations and was explained through the analysis of the HOMOs of the neutral and protonated forms of the triazole compound.

Understanding the dynamic pore wetting by 1H LF NMR characterization. Part 2: Effect of liquid surface tension

The first paper (Part 1) of this study fully described the effect of the dynamic viscosity of liquids on dynamic pore wetting of porous materials. The surface tension, as an important physical properties of liquids may also play a vital role in the pore wetting process. However, the quantitative characterization of dynamic pore wetting is difficult. Thus, this paper (Part 2) investigated the effect of liquid surface tension (mixture of 1-propanol and pure water in different proportions) on dynamic wetting of pores of different sizes in hydrophilic and hydrophobic active carbon by a 1H low-field nuclear magnetic resonance (1H LF NMR), and the quantitative model between surface tension and pore wetting percentage was built. The Crispation number of liquids in the pores of different sizes was calculated. For the hydrophilic samples, the wetting processes of pores of different sizes (except for mesopores) were not changed with the surface tension. For the hydrophobic sample, the liquid with lower surface tension achieved a higher pore wetting percentage and the order of pore wetting rate was: micropores > transition pores > mesoproes. The 60-min wetting percentages in pores of different sizes showed a linear decrease relationship with the surface tension. As the Crispation number increased, the 60-min wetting percentages in different pore sizes had a first-order linear increase trend. This paper not only provides a novel method for characterizing the dynamic pore wetting of porous materials, but also have a significance for regulating pore wetting process.

Boron‐Catalyzed Polymerization of Dienyltriphenylarsonium Ylides: On the Way to Pure C5 Polymerization

AbstractThe first C5 polymerization is reported, where the main‐chain is growing by five carbon atoms of the monomer at a time. Three dienyltriphenylarsonium ylide monomers were synthesized and polymerized with triethylborane as an initiator, leading to random terpolymers (C1, C3, C5) with mainly C5 repeating units (up to 84.1 %). It has been found that the methyl group (electron‐donating substituent) on the conjugated double bond of the ylides facilitates the formation of C5 segments. A mechanism was proposed based on NMR characterization and DFT calculations. The high C5 content ensures that things are on the right track for pure C5 homopolymerization.

Boron-Catalyzed Polymerization of Dienyltriphenylarsonium Ylides: On the Way to Pure C5 Polymerization.

The first C5 polymerization is reported, where the main‐chain is growing by five carbon atoms of the monomer at a time. Three dienyltriphenylarsonium ylide monomers were synthesized and polymerized with triethylborane as an initiator, leading to random terpolymers (C1, C3, C5) with mainly C5 repeating units (up to 84.1 %). It has been found that the methyl group (electron‐donating substituent) on the conjugated double bond of the ylides facilitates the formation of C5 segments. A mechanism was proposed based on NMR characterization and DFT calculations. The high C5 content ensures that things are on the right track for pure C5 homopolymerization.

Fast & fluorinated – Development and validation of a rapid benchtop NMR approach and other routine screening methods for the detection and quantification of synthesized fluorofentanyl derivatives

Fluorinated fentanyl regioisomers have become increasingly common in forensic casework. This study reports the synthesis of eight fluorinated fentanyl derivatives as pure reference materials and their complete NMR, infrared and mass spectral characterisation. Presumptive colour tests and TLC results are presented as an initial screening method for these compounds. The fully validated GC–MS method (employing SIM mode) allows the quantification of the target analytes (LOD = 9–20 ng/mL, LOQ = 31–67 ng/mL), fentanyl, heroin, acetaminophen and caffeine, within 13 min. In most cases, the regioisomeric fluorofentanyls were resolved from each other except for the 3′- and 4′-fluorinated derivatives (5b and 5c). To achieve full separation, an orthogonal benchtop 19F NMR method allows the identification and quantification of target analogues (LOD = 74–400 μg/mL, LOQ = 290–1340 μg/mL). The 19F NMR method allows the detection of fluorinated fentanyl analogues at a low concentration (2.4%w/w) in heroin.

Spectroscopic and Theoretical Explorations on 6-Hydromethyl-4-Methoxy-4- Methoxytetrahydro-Pyran-2, 3, 5-Triol-A Biomolecule

The chemical compound 6-hydromethyl-4-methoxy-4- methoxytetrahydro-pyran-2, 3, 5-triol (C7H14O6), was derived from the ethanol extract of Senna auriculata flower via GC-MS. FT-IR and NMR characterization of the compound support covalent bonding information and degree of degeneracy, respectively. 3D molecular structure was optimized to enhance the accuracy of computations. MEP, NBO, HOMO-LUMO, and Mulliken charge distribution studies provide details like reactive sites, electron density dislocation, and molecular interactions, charge transfer within the molecule and frontier orbital energies, electron density on the individual atom, and these were computed using hybrid B3-LYP quantum level with 6-311++G(d, p) basis set. Thermodynamic properties such as heat capacity, entropy, enthalpy, and their relation with temperature changes for the title molecule were also analyzed using THERMO.PL software. Non-linear optical properties namely polarizability, first-order hyperpolarizability, and electronic dipole moment have also been computed at B3-LYP level with 6-311++G (d, p) basis set. Docking simulation was initiated on the title molecule using Autodock 4.2 software and found to be an excellent inhibitor of ALR2, an enzyme.

NMR Characterization of Ten Apple Cultivars from the Piedmont Region.

The metabolite profile of ten traditional apple cultivars grown in the Piedmont region (Italy) was studied by means of nuclear magnetic resonance spectroscopy, identifying an overall number of 36 compounds. A more complete assignment of the proton nuclear magnetic resonance (1H NMR) resonances from hydroalcoholic and organic apple extracts with respect to literature data was reported, identifying fructose tautomeric forms, galacturonic acid, γ-aminobutyric acid (GABA), p-coumaroyl moiety, phosphatidylcholine, and digalactosyldiacylglycerol. The chemical profile of each apple cultivar was defined by thorough quantitative NMR analysis of four sugars (fructose, glucose, sucrose, and xylose), nine organic acids (acetic, citric, formic, citramalic, lactic, malic, quinic, and galacturonic acids), six amino acids (alanine, asparagine, aspartate, GABA, isoleucine, and valine), rhamnitol, p-coumaroyl derivative, phloretin/phloridzin and choline, as well as β-sitosterol, fatty acid chains, phosphatidylcholine, and digalactosyldiacylglycerol. Finally, the application of PCA analysis allowed us to highlight possible differences/similarities. The Magnana cultivar showed the highest content of sugars, GABA, valine, isoleucine, and alanine. The Runsé cultivar was characterized by high amounts of organic acids, whereas the Gamba Fina cultivar showed a high content of chlorogenic acid. A significant amount of quinic acid was detected in the Carla cultivar. The knowledge of apple chemical profiles can be useful for industries interested in specific compounds for obtaining ingredients of food supplements and functional foods and for promoting apple valorization and preservation.

NMR Characterization of Angiogenin Variants and tRNAAla Products Impacting Aberrant Protein Oligomerization.

Protein oligomerization is key to countless physiological processes, but also to abnormal amyloid conformations implicated in over 25 mortal human diseases. Human Angiogenin (h-ANG), a ribonuclease A family member, produces RNA fragments that regulate ribosome formation, the creation of new blood vessels and stress granule function. Too little h-ANG activity leads to abnormal protein oligomerization, resulting in Amyotrophic Lateral Sclerosis (ALS) or Parkinson’s disease. While a score of disease linked h-ANG mutants has been studied by X-ray diffraction, some elude crystallization. There is also a debate regarding the structure that RNA fragments adopt after cleavage by h-ANG. Here, to better understand the beginning of the process that leads to aberrant protein oligomerization, the solution secondary structure and residue-level dynamics of WT h-ANG and two mutants i.e., H13A and R121C, are characterized by multidimensional heteronuclear NMR spectroscopy under near-physiological conditions. All three variants are found to adopt well folded and highly rigid structures in the solution, although the elements of secondary structure are somewhat shorter than those observed in crystallography studies. R121C alters the environment of nearby residues only. By contrast, the mutation H13A affects local residues as well as nearby active site residues K40 and H114. The conformation characterization by CD and 1D 1H NMR spectroscopies of tRNAAla before and after h-ANG cleavage reveals a retention of the duplex structure and little or no G-quadruplex formation.

Expression, purification and characterization of the second DUSP domain of deubiquitinase USP20/VDU2

Deubiquitinase USP20/VDU2 (VHL-interacting Deubiquitinating Enzyme 2) has been proved to play vital roles in multiple cellular processes by controlling the life-span of substrate proteins including hypoxia-inducible factor HIF1α, β2-adrenergic receptor, and type 2 iodothyronine deiodinase etc. USP20 contains four distinct structural domains, which include the N-terminal zinc-finger ubiquitin binding domain (ZnF-UBP), the catalytic domain (USP domain), and two tandem DUSP domains (DUSP1 and DUSP2). Here in this study, we report the setting up of the production approach for USP20 DUSP2, and the NMR characterization of the produced target protein. With the assistance of GB1 tag and glycerol, both the solubility and stability of USP20 DUSP2 are significantly enhanced. And by using the optimized protein production procedure, monomeric and stable 15N, 13C-labeled USP20 DUSP2 sample for NMR data acquisition was obtained. The secondary structural elements of USP20 DUSP2 were then revealed by the analysis of recorded NMR spectra, and USP20 DUSP2 forms an AB3 fold in solution. The production protocol and NMR characterization results reported in this manuscript could be utilized in the extended structural and functional studies of USP20 DUSP2.

Backbone and ILVM methyl resonance assignments of human thymidylate synthase in apo and substrate bound forms.

Human thymidylate synthase (hTS) is a 72kDa homodimeric enzyme responsible for the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP), making it the sole source of de novo dTMP in human cells. As a result, hTS is an attractive anti-cancer therapeutic target. Additionally, hTS is known to possess a number of interesting biophysical features, including adoption of active and inactive conformations, positively cooperative substrate binding, half-the-sites activity, and interacting with its own mRNA. The physical mechanisms underlying these properties, and how they may be leveraged to guide therapeutic development, are yet to be fully explored. Here, as a preface to detailed NMR characterization, we present backbone amide and ILVM methyl resonance assignments for hTS in apo and dUMP bound forms. In addition, we present backbone amide resonance assignments for hTS bound to a substrate analog and the native cofactor.

ATP biphasically modulates LLPS of SARS-CoV-2 nucleocapsid protein and specifically binds its RNA-binding domain

SARS-CoV-2 is a highly contagious coronavirus causing the ongoing pandemic. Very recently its genomic RNA of ∼30 kb was decoded to be packaged with nucleocapsid (N) protein into phase separated condensates. Interestingly, viruses have no ability to generate ATP but host cells have very high ATP concentrations of 2–12 mM. A key question thus arises whether ATP modulates liquid-liquid phase separation (LLPS) of the N protein. Here we discovered that ATP not only biphasically modulates LLPS of the viral N protein as we previously found on human FUS and TDP-43, but also dissolves the droplets induced by oligonucleic acid. Residue-specific NMR characterization showed ATP specifically binds the RNA-binding domain (RBD) of the N protein with the average Kd of 3.3 ± 0.4 mM. The ATP-RBD complex structure was constructed by NMR-derived constraints, in which ATP occupies a pocket within the positive-charged surface utilized for binding nucleic acids. Our study suggests that ATP appears to be exploited by SARS-CoV-2 to promote its life cycle by facilitating the uncoating, localizing and packing of its genomic RNA. Therefore the interactions of ATP with the viral RNA and N protein might represent promising targets for design of drugs and vaccines to terminate the pandemic.

Tethering-induced destabilization and ATP-binding for tandem RRM domains of ALS-causing TDP-43 and hnRNPA1

TDP-43 and hnRNPA1 contain tandemly-tethered RNA-recognition-motif (RRM) domains, which not only functionally bind an array of nucleic acids, but also participate in aggregation/fibrillation, a pathological hallmark of various human diseases including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), alzheimer's disease (AD) and Multisystem proteinopathy (MSP). Here, by DSF, NMR and MD simulations we systematically characterized stability, ATP-binding and conformational dynamics of TDP-43 and hnRNPA1 RRM domains in both tethered and isolated forms. The results reveal three key findings: (1) upon tethering TDP-43 RRM domains become dramatically coupled and destabilized with Tm reduced to only 49 °C. (2) ATP specifically binds TDP-43 and hnRNPA1 RRM domains, in which ATP occupies the similar pockets within the conserved nucleic-acid-binding surfaces, with the affinity slightly higher to the tethered than isolated forms. (3) MD simulations indicate that the tethered RRM domains of TDP-43 and hnRNPA1 have higher conformational dynamics than the isolated forms. Two RRM domains become coupled as shown by NMR characterization and analysis of inter-domain correlation motions. The study explains the long-standing puzzle that the tethered TDP-43 RRM1–RRM2 is particularly prone to aggregation/fibrillation, and underscores the general role of ATP in inhibiting aggregation/fibrillation of RRM-containing proteins. The results also rationalize the observation that the risk of aggregation-causing diseases increases with aging.

Monocationic Bis-Alkyl and Bis-Allyl Yttrium Complexes: Synthesis, 89Y NMR Characterization, Ethylene or Isoprene Polymerization, and Modeling

Monocationic complexes of yttrium with various bis-alkyl and bis-allyl ligands Y(CH2SiMe2Ph)2(THF)4][B(C6F5)4], [Y(CH2C6H4NMe2)2(THF)2][B(C6F5)4], and [Y[1,3-(SiMe3)2C3H3]2(THF)2][B(C6F5)4] have be...

Copolymer chain formation of 2-oxazolines by in situ 1H-NMR spectroscopy: dependence of sequential composition on substituent structure and monomer ratios.

In situ 1H NMR characterization of copolymerization reactions of various 2-oxazoline monomers at different molar ratios offers detailed insight into the build-up and composition of the polymer chains. Various 2-oxazolines were copolymerized in one single solvent, butyronitrile, with 2-dec-9′-enyl-2-oxazoline, where the double bond allows for post-polymerization modification and can function as a crosslinking unit to form polymer networks. The types of the monomers and their molar ratios in the feed have a strong effect on the microstructure of the forming copolymer chains. Copolymers comprising 2-dec-9′-enyl-2-oxazoline and either 2-ethyl-, 2-isopropyl-, 2-butyl-, 2-heptyl, 2-nonyl- or 2-phenyl-2-oxazoline, show significant differences in sequential structure of copolymers ranging from block to gradient and random ordering of the monomer units. 1H NMR was found to be a powerful tool to uncover detailed oxazoline copolymerization kinetics and evolution of chain composition.