Quantum Correlation NMR Research Articles

Extraction and characterisation of kudzu root residue lignin based on deep eutectic solvents.

Lignin has great potential as the most abundant renewable phenolic polymer. Studies have shown that lignin structure varies depending on different sources and different extraction methods. However, there are few studies on lignin in kudzu root residue. The aim of the study was to explore optimal extraction conditions of Pueraria lobata residue lignin (PLL) with deep eutectic solvents (DESs) and characterise the structure and morphology of PLL. Firstly, the chemical composition of kudzu root residue was determined by the Van-soest method. Then, betaine was used as hydrogen bond acceptor (HBA), nine kinds of common acids and alcohol were selected as hydrogen bond donor (HBD) to synthesise a DES to extract lignin from kudzu root residue. The influence of conditions on the extraction of PLL was explored by a betaine-based DES according to a single-factor experiment, and then the best process of PLL extraction was determined by an orthogonal experiment. Finally, the morphology and structure of PLL were analysed by scanning electron microscope (SEM), thermogravimetric analysis (TGA), gel permeation chromatography (GPC), Fourier transform infrared spectroscopy (FTIR), and NMR. Cellulose, hemicellulose, lignin, and ash content in kudzu root residue were 41.13%, 16.39%, 25.03%, and 0.41%, respectively. When the DES consisted of betaine and formic acid, the solid-liquid ratio was 1:45, the extraction time was 5.5 h at 160°C, the extraction yield of lignin was 89.29%, and the purity was 83.01%. PLL was composed of interconnected spherical particles with good thermal stability and narrow polydispersity index (PDI) distribution. FTIR and 2D-heteronuclear singular quantum correlation (HSQC) NMR illustrated that PLL was a typical G-type and S-type lignin. This study would fill the gap of research on lignin in kudzu root residue and provide a theoretical reference for the utilisation of lignin in kudzu roots as well as a new thinking for the recycling of kudzu root resources.

Expanding the Limits of Structural Characterization of Marine Dissolved Organic Matter Using Nonuniform Sampling Frequency-Reversed Edited HSQC NMR.

The multiplicity-edited heteronuclear single quantum correlation (ME-HSQC) NMR method is widely used for the structural characterization of marine dissolved organic matter (DOM), which is a complex molecular mixture comprising millions of individual compounds. However, the standard ME-HSQC suffers from significant signal cancellation and subsequent loss of crucial structural information due to the overlap between CH3/CH (positive) and CH2 (negative) cross-peaks in overcrowded regions. This study introduces nonuniform sampling in frequency-reversed ME-HSQC (NUS FR-ME-HSQC), highlighting its remarkable potential for the comprehensive structural characterization of marine DOM. By reversing the frequency of CH2 cross-peaks into an empty region, the FR-ME-HSQC method effectively simplifies the spectra and eliminates signal cancellation. We demonstrate that nonuniform sampling enables the acquisition of comparable spectra in half the time or significantly enhances the sensitivity in time-equivalent spectra. Comparative analysis also identifies vulnerable CH2 cross-peaks in the standard ME-HSQC that coincide with CH3 and CH cross-peaks, resulting in the loss of critical structural details. In contrast, the NUS FR-ME-HSQC retains these missing correlations, enabling in-depth characterization of marine DOM. These findings highlight the potential of NUS FR-ME-HSQC as an advanced NMR technique that effectively addresses challenges such as signal overcrowding and prolonged experimental times, enabling the thorough investigation of complex mixtures with implications in several fields, including chemistry, metabolomics, and environmental sciences. The advantages of NUS FR-ME-HSQC are experimentally demonstrated on two solid-phase-extracted DOM (SPE-DOM) samples from the surface and deep ocean. With this new technology, differences in the composition of DOM from various aquatic environments can be assigned to individual molecules.

Quantification of Phosphonate Drugs by 1H-31P HSQC Shows That Rats Are Better Models of Primate Drug Exposure than Mice.

The phosphonate group is a key pharmacophore in many antiviral, antimicrobial, and antineoplastic drugs. Due to its high polarity and short retention time, detecting and quantifying such phosphonate-containing drugs with LC/MS-based methods are challenging and require derivatization with hazardous reagents. Given the emerging importance of phosphonate-containing drugs, developing a practical, accessible, and safe method for their quantitation in pharmacokinetics (PK) studies is desirable. NMR-based methods are often employed in drug discovery but are seldom used for compound quantitation in PK studies. Here, we show that proton-phosphorous (1H-31P) heteronuclear single quantum correlation (HSQC) NMR allows for the quantitation of the phosphonate-containing enolase inhibitor HEX in plasma and tissues at micromolar concentrations. Although mice were shown to rapidly clear HEX from circulation (over 95% in <1 h), the plasma half-life of HEX was more than 1 h in rats and nonhuman primates. This slower clearance rate affords a significantly higher exposure of HEX in rat models compared to that in mouse models while maintaining a favorable safety profile. Similar results were observed for the phosphonate-containing antibiotic, fosfomycin. Our study demonstrates the applicability of the 1H-31P HSQC method to quantify phosphonate-containing drugs in complex biological samples and illustrates an important limitation of mice as preclinical model species for phosphonate-containing drugs.

Gelation of konjac glucomannan by acetylmannan esterases from Aspergillus oryzae

Konjac glucomannan (KGM) is a principal component of the gelatinous food Konjac. Konjac production through alkali treatment releases an undesirable amine-odor. Two acetylesterases (AME1 and AME2) active against konjac glucomannan (polymer or oligomer) were purified from the supernatant of Aspergillus oryzae RIB40 culture. We cloned the genes encoding AME1 and AME2 based on the genomic information of A. oryzae, constructed their expression systems in A. oryzae, and obtained the recombinant enzymes (rAME1 and rAME2). rAME1 did not act on the KGM polymer but only on the KGM oligomer, releasing approximately 60% of the acetic acid in the substrate. However, rAME2 was active against both KGM substrates, releasing approximately 80% and 100% of acetic acid from the polymer and oligomer, respectively. Both enzymes were active against xylan and exhibited a trace activity on ethyl ferulate. The acetyl group position specificities of both enzymes were analyzed via heteronuclear single quantum correlation NMR using oligosaccharides of glucomannan prepared from Aloe vera (AGM), which has a higher acetyl group content than KGM. rAME1 acted specifically on single-substituted acetyl groups and not on double-substituted ones. In contrast, rAME2 appeared to act on all the acetyl groups in AGM. Treatment of 3% KGM with rAME2 followed by heating to 90 °C resulted in gel formation under weakly acidic conditions. This is the first study to induce gelation of KGM under these conditions. A comparison of the breaking and brittleness properties of gels formed by alkaline and enzymatic treatments revealed similar texture of the two gels. Furthermore, scanning electron microscopy of the surface structure of both gels revealed that both formed a fine mesh structure. Our findings on enzymatic gelation of KGM should lead to the development of new applications in food manufacturing industry.

The calcium-free form of atorvastatin inhibits amyloid-β(1–42) aggregation in vitro

Alzheimer's disease is characterized by the presence of extraneuronal amyloid plaques composed of amyloid-beta (Aβ) fibrillar aggregates in the brains of patients. In mouse models, it has previously been shown that atorvastatin (Ator), a cholesterol-lowering drug, has some reducing effect on the production of cerebral Aβ. A meta-analysis on humans showed moderate effects in the short term but no improvement in the Alzheimer's Disease Assessment Scale—Cognitive Subscale behavioral test. Here, we explore a potential direct effect of Ator on Aβ42 aggregation. Using NMR-based monomer consumption assays and CD spectroscopy, we observed a promoting effect of Ator in its original form (Ator-calcium) on Aβ42 aggregation, as expected because of the presence of calcium ions. The effect was reversed when applying a CaCO3-based calcium ion scavenging method, which was validated by the aforementioned methods as well as thioflavin-T fluorescence assays and transmission electron microscopy. We found that the aggregation was inhibited significantly when the concentration of calcium-free Ator exceeded that of Aβ by at least a factor of 2. The 1H–15N heteronuclear single quantum correlation and saturation-transfer difference NMR data suggest that calcium-free Ator exerts its effect through interaction with the 16KLVF19 binding site on the Aβ peptide via its aromatic rings as well as hydroxyl and methyl groups. On the other hand, molecular dynamics simulations confirmed that the increasing concentration of Ator is necessary for the inhibition of the conformational transition of Aβ from an α-helix-dominant to a β-sheet-dominant structure.

Uncovering selective and active Ga surface sites in gallia-alumina mixed-oxide propane dehydrogenation catalysts by dynamic nuclear polarization surface enhanced NMR spectroscopy.

Gallia–alumina (Ga,Al)2O3(x : y) spinel-type solid solution nanoparticle catalysts for propane dehydrogenation (PDH) were prepared with four nominal Ga : Al atomic ratios (1 : 6, 1 : 3, 3 : 1, 1 : 0) using a colloidal synthesis approach. The structure, coordination environment and distribution of Ga and Al sites in these materials were investigated by X-ray diffraction, X-ray absorption spectroscopy (Ga K-edge) as well as 27Al and 71Ga solid state nuclear magnetic resonance. The surface acidity (Lewis or Brønsted) was probed using infrared spectroscopy with pyridine and 2,6-dimethylpyridine probe molecules, complemented by element-specific insights (Ga or Al) from dynamic nuclear polarization surface enhanced cross-polarization magic angle spinning 15N{27Al} and 15N{71Ga} J coupling mediated heteronuclear multiple quantum correlation NMR experiments using 15N-labelled pyridine as a probe molecule. The latter approach provides unique insights into the nature and relative strength of the surface acid sites as it allows to distinguish contributions from Al and Ga sites to the overall surface acidity of mixed (Ga,Al)2O3 oxides. Notably, we demonstrate that (Ga,Al)2O3 catalysts with a high Al content show a greater relative abundance of four-coordinated Ga sites and a greater relative fraction of weak/medium Ga-based surface Lewis acid sites, which correlates with superior propene selectivity, Ga-based activity, and stability in PDH (due to lower coking). In contrast, (Ga,Al)2O3 catalysts with a lower Al content feature a higher fraction of six-coordinated Ga sites, as well as more abundant Ga-based strong surface Lewis acid sites, which deactivate through coking. Overall, the results show that the relative abundance and strength of Ga-based surface Lewis acid sites can be tuned by optimizing the bulk Ga : Al atomic ratio, thus providing an effective measure for a rational control of the catalyst performance.

Lignin-carbohydrate complexes from coconut (Cocos nucifera) coir: Fractionation, structural elucidation, and potential applications

Coconut coir, a major type of tropical lignocellulosic waste, has been restricted from higher-value applications due to its chemical complexity and inherent variability. To better disclose the chemical relationships between polysaccharides and lignin in coir and further exploit its high value-added bio-based materials, two lignin-carbohydrate complexes (LCCs) designated as glucan-lignin (GL) and xylan-lignin (XL) were successively isolated from coir via a simplified and quantitative fractionation method. The characterization of chemical composition, molecular weight distribution, and constituent substructures of the isolated LCC fractions were examined, and the lignin-carbohydrate (LC) chemical linkages were revealed as γ-esters by a 2D heteronuclear singular quantum correlation (HSQC) NMR technique. Furthermore, XL was demonstrated as a more competitive candidate than GL for 2,2-diphenyl-1-picryl-hydrazyl (DPPH) removal in comparison with 2,6-di-tert-butyl-4-methylphenol (BHT, a commercial antioxidant), and was also featured as a more promising reinforcing agent for elevating the adsorption property of polyacrylamide-based hydrogels via the synergistic effect of physical and hydrogen bonding adsorptions.

Enhanced Enzymatic Hydrolysis and Lignin Extraction of Wheat Straw by Triethylbenzyl Ammonium Chloride/Lactic Acid-Based Deep Eutectic Solvent Pretreatment.

Efficient and feasible pretreatment of lignocellulosic biomass waste is an important prerequisite step to promote subsequent enzymatic hydrolysis and enhance the economics of biofuels production. This study focuses on the pretreatment of wheat straw (WS) with triethylbenzyl ammonium chloride/lactic acid (TEBAC/LA)-based deep eutectic solvents to enhance biomass fractionation and lignin extraction. Effects of pretreatment time, temperature, and TEBAC/LA molar ratio on pretreatment were evaluated systematically. Results suggested that 89.06 ± 1.05% of cellulose and 71.00 ± 1.03% of xylan were hydrolyzed with enzyme loadings of 35 FPU cellulase and 82 CBU β-glucosidase (per gram of dry biomass) after pretreatment by TEBAC/LA (1:9) at 373 K for 10 h. A total monosaccharide yield of 0.550 g/g WS (91.27% of the theoretical yield) was achieved with 79.73 ± 0.93% of lignin removal. Furthermore, the 1H–13C two-dimensional heteronuclear single quantum correlation (2D-HSQC) NMR spectroscopy showed that the regenerated lignin (75.69 ± 1.32% purity) was mainly composed of the syringyl units and the guaiacyl units. Overall, the results in this study provide an effective and facile pretreatment method for lignocellulosic biomass waste to enhance enzymatic hydrolysis saccharification.

Unveiling TMP Process Water Potential As an Industrial Sourcing of Valuable Lignin–Carbohydrate Complexes toward Zero-Waste Biorefineries

The development of zero-waste biorefineries is a promising yet challenging pathway toward a sustainable bioeconomy. Deep insights on the composition of biorefineries side streams along with the development of green technologies are mandatory to achieve such a goal. In this Article, a sustainable pilot-scale membrane process was used to recover valuable organic matter dissolved in the main side stream of a thermomechanical pulping (TMP) plant. A great number of analytical tools were combined to get an extensive characterization of the generated fraction, including inductively coupled plasma atomic emission spectroscopy (ICP-AES), high-performance ion chromatography coupled with pulse amperometric detection (HPIC-PAD), Fourier transform infrared (FTIR), size-exclusion chromatography (SEC), and C-13 and 2D heteronuclear single quantum correlation (HSQC) NMR. The results revealed the presence of a considerable amount of native lignin-carbohydrate complexes (LCCs) in the fraction representing 40% of the dry weight. The LCCs are composed of partially acetylated galactoglucomannans (GGMs) covalently linked to low condensed and ) beta-O-4-rich lignin through phenyl glycosidic bonds (25.5 bonds/100 aromatic rings (ar)) and characterized by a relatively low molecular weight around 7 kDa. Such complexes present an interesting potential for valorization with unique properties resulting from the combination of their aromatic and sugar moieties. Different applications are discussed in regards to the generated data in this work as an industrial sourcing of LCC is reported.

Structural Elucidation of Cisplatin and Hydrated cis-Diammineplatinum(II) Complex Conjugated with Cyanocobalamin by Liquid Chromatography with Electrospray Ionization-Mass Spectrometry and Multistage Mass Spectrometry.

Pt(II)-based derivatives bearing a cyanocobalamin (CNCbl) unit were synthesized in aqueous solutions, and the reaction mixtures were examined by reversed-phase liquid chromatography with electrospray ionization and linear ion trap mass spectrometry (MS). Isotopic pattern analysis, multistage mass-spectra (MS/MS and MS3) interpretation, and differential isotopic labeling were used to establish the chemical composition and to suggest the chemical structures of reaction products. When cisplatin (cis-[PtCl2(NH3)2]) was used as a Pt(II) drug derivative, a coordination bond between diamminemonochloroplatinum(II) and the cyano group of CNCbl, in turn linked covalently to the vitamin Co(III) ion, occurred. The resulting conjugate with a CoIII–CN–PtII bridge was MS detected as a doubly positive charged ion with the prevailing isotopologue at m/z 810.26 (empirical formula [C63H95ClCoIIIN16O14PPt]2+). Likewise, a peak signal centered at m/z 811.26 was observed when 15N-labeled cisplatin cis-[PtCl2(15NH3)2] was used as Pt(II) complex, thus confirming the presence of both the cisplatin amino groups in the conjugate. A bifunctional conjugate was obtained between CNCbl and the cis-diamminediaquaplatinum(II), that is, cis-[Pt(NH3)2(H2O)2]2+; in this case, the planar coordination complex of Pt(II) was also involved in a covalent bond with the oxygen atom of one of the CNCbl amide moieties. The peak signal detected at m/z 792.26 (empirical formula [C63H94CoIIIN16O14PPt]2+) changed to m/z 793.26 when the labeled cis-[Pt(15NH3)2(H2O)2]2+ complex was adopted for conjugation. Comparison between MS/MS spectra allowed an extended structural characterization of both conjugates, as such or 15N-labeled. Two-dimensional heteronuclear (1H–15N) single quantum correlation NMR spectroscopy, applied to 15N-labeled conjugates, supported the hypotheses made on the Pt(II) coordination in both cases.

Water Soluble Antioxidant Dextran-Quercetin Conjugate with Potential Anticancer Properties.

Quercetin, a naturally occurring potent antioxidant, is limited in therapeutic use, owing to its poor water solubility and stability. Herein, a method of conjugating quercetin to an aldehyde functionalized dextran via an HCl catalyzed condensation reaction to yield a water soluble quercetin functionalized polymer is reported. The prepared conjugate is characterized by 1 H and 1 H-13 C heteronuclear single quantum correlation (HSQC) NMR, which demonstrate that conjugation occurs via both the A- and B-rings of quercetin. The degree of quercetin functionalization can be tuned by varying the reaction temperature and/or the concentration of the HCl catalyst. However, as temperatures and HCl concentrations are increased above 40 °C and 2 m, respectively, the increase in functionalization is accompanied by an increase in the oxidation of the conjugated quercetin and a decrease in polymer yield. The prepared conjugate is shown to have improved stability compared with native quercetin while maintaining substantial free-radical scavenging activity. Anticancer activity is evaluated in vitro in a neuroblastoma cell line. The dextran-aldehyde-quercetin conjugate prepared at 40 °C and 2 m HCl is shown to be cytotoxic to neuroblastoma cells (SH-SY5Y-IC50 = 123 µg mL-1 and BE(2)-C-IC50 = 380 µg mL-1 ) but shows no activity against nonmalignant MRC-5 cells at concentrations up to 400 µg mL-1 .

Membrane separation and characterisation of lignin and its derived products obtained by a mild ethanol organosolv treatment of rice straw

Abstract An organosolv process using ethanol-water was optimized in order to recover high quality lignin from rice-straw previously pre-treated by autohydrolysis at 210 °C. The results showed a selective and appreciable removal of lignin under very mild conditions and the highest delignification yield occurred at 30 °C. The lignin extracts were characterised using capillary zone electrophoresis (CZE), size exclusion chromatography (SEC), Fourier transform infrared spectroscopy (FT-IR) and 31P-NMR, and two-dimensional heteronuclear single quantum correlation NMR spectroscopy (2D-HSQC NMR), which enabled the identification of low molecular weight lignins with a syringyl/guaiacyl ratio of about 0.74 containing phenolic compounds with potential bioactive properties. In order to separate the target compounds, membrane technology has been used and an enriched extract containing value-added phenolic compounds such as tricin, vanillin, ferulic acid and p-coumaric acid was obtained. High membrane efficiency (around 80%) was obtained for target compounds.

Thermal stability of low and high Mw fractions of bio-oil derived from lignin conversion in subcritical water

The thermal stability of bio-oil influences its application in industry and is, therefore, a very important factor that must be taken into consideration. In this study, the stability of low and high molecular weight (Mw) fractions of bio-oil obtained from the hydrothermal liquefaction (HTL) of lignin in subcritical water was studied at an elevated temperature (80 °C) for a period of 1 h, 1 day and 1 week. The changes in molecular weight (gel permeation chromatography (GPC)) and chemical composition (gas chromatography–mass spectrometry (GC–MS) and 2D heteronuclear single quantum correlation (HSQC) NMR (18.8 T, DMSO-d6)) of low and high Mw fractions of the HTL bio-oil (i.e. light oil (LO) and heavy oil (HO)) were evaluated before and after ageing. It was found that only a slight formation of high Mw insoluble structures was obtained during ageing at elevated temperature for 1 week: 0.5% for the LO and 3.1% for the HO. These higher Mw moieties might be formed from different polymerisation/condensation reactions of the reactive compounds (i.e. anisoles, guaiacols, phenols, methylene (–CH2–) groups in phenolic dimers and xanthene). The high Mw insolubles in both the LO and the HO were analysed for structural composition using 2D HSQC NMR to obtain a better understanding of the changes in the composition of bio-oil fractions during the accelerated ageing process. In addition, a chemical shift database in DMSO-d6 was analysed for a subset of phenolic model compounds to simplify the interpretation of the 2D HSQC NMR spectra.

Centrifugal partition chromatography: A preparative tool for isolation and purification of xylindein from Chlorociboria aeruginosa

A centrifugal partition chromatography (CPC) method was developed for the preparative-scale isolation and purification of xylindein from the wood-staining fungi, Chlorociboria aeruginosa. Xylindein, a blue–green pigment naturally secreted from the hyphae and fruiting bodies of the fungus, has great value in the decorative wood industry and textile coloration. Xylindein has great potential for use as a fluorescent labeling agent as well as in organic semiconductor applications. However, a primary limitation of xylindein is its poor solubility in most common HPLC solvents. Consequently, it is arduous to purify using preparative liquid chromatography or solid-phase extraction (SPE). Support-free, liquid–liquid chromatographic methods, including CPC, where solutes are separated based on their different distribution coefficients (KD) between two immiscible solvent systems, are promising alternatives for the purification of the compound on a preparative scale. In this work, a new biphasic solvent system suitable for CPC separation of xylindein was developed. Various groups of solvents were assessed for their suitability as xylindein extractants. A new solvent system suitable for CPC separation of xylindein, composed of heptane/THF/MEK/acetonitrile/acetic acid/water, was developed. This solvent system yielded a KD value for xylindein of 1.54±0.04, as determined by HPLC (n=3). The compositions of the upper phase and lower phase of the solvent system were determined by Heteronuclear Single Quantum Correlation (HSQC) NMR and proton NMR. A CPC system, equipped with a fraction collector, was used for the isolation of xylindein from crude extracts. The xylindein fractions isolated by the CPC were then analyzed using HPLC and presented as a fractogram. Based on the CPC fractogram, the purified xylindein fractions were achieved after 30min CPC separation time, yielding 71% extraction efficiency. The developed CPC method allowed for isolation of this naturally sourced xylindein in amounts suitable for further study.

Multifunctional quinoline-triazole derivatives as potential modulators of amyloid-β peptide aggregation

Metal ion dyshomeostasis is hypothesized to play a role in the toxicity and aggregation of the amyloid beta (Aβ) peptide, contributing to Alzheimer's disease (AD) pathology. We report on the synthesis and metal complexation ability of three bidentate quinoline-triazole derivatives 3-(4-(quinolin-2-yl)-1H-1,2,3-triazol-1-yl)propan-1-ol (QOH), 4-(2-(4-(quinolin-2-yl)-1H-1,2,3-triazol-1-yl)ethyl)morpholine (QMorph), and 4-(2-(4-(quinolin-2-yl)-1H-1,2,3-triazol-1-yl)ethyl)thiomorpholine (QTMorph). We further study the utility of these ligands to modulate Aβ peptide aggregation processes in the presence and absence of Cu2+ ions. Ligand-peptide interactions were first investigated using both 2-D 1H–15N band-selective optimized flip angle short transient heteronuclear multiple quantum correlation (SOFAST-HMQC) NMR spectroscopy and molecular modeling techniques, indicating interactions with glutamic acid (E3) and several residues in the hydrophobic region of Aβ. Native gel electrophoresis with western blotting along with transmission electron microscopy provided information on the ability of each ligand to modulate Aβ aggregation. While the ligands alone did not modify Aβ peptide aggregation at the 24h timepoint, signifying relatively weak ligand-peptide interactions, the ligands did modify the aggregation profile of the peptide in the presence of stoichiometric and suprastoichiometric Cu. Interestingly, the thioether derivative QTMorph exhibited the most pronounced effect on peptide aggregation in the presence of Cu. Overall, the quinoline-triazole ligand series were shown to interact with the hydrophobic region of the Aβ peptide, and modulate the Cu-Aβ aggregation process.

Mechanism and Kinetics of Oligosilsesquioxane Growth in the In Situ Water Production Sol–Gel Route: Dependence on Water Availability

AbstractThiol‐functionalized nanobuilding blocks (NBBs) were synthesized from 3‐mercaptopropyltrimethoxysilane by using the in situ water production (ISWP) process in which the water needed to hydrolyze the precursor was provided by means of an esterification reaction. In the present study the reaction between 1‐propanol and chloroacetic acid was used. Whereas the growth of the Si oligomers was followed at room temperature and 100 °C using 1D 29Si and 2D 1H–29Si heteronuclear single quantum correlation (HSQC) NMR spectroscopic experiments, the amount of water delivered along the process was followed by means of 1H NMR spectroscopy. The results show a good correlation between the evolution of the degree of condensation and the amount of water produced in situ. They also point to the preferential formation of cagelike structures and the narrowing of the species distribution over long reaction times. The average size of the growing oligomers was estimated from their diffusion coefficient, which was measured by means of 1H diffusion‐ordered NMR spectroscopy (DOSY NMR). Like gel permeation chromatography, DOSY NNR showed a plateau between 70 to 100 hours in the growth of the oligomers, a time at which, according to 29Si NMR spectroscopy, the well‐defined octakis(3‐mercaptopropylsilsesquioxane) is the major species.

Surface Properties of Squalene/Meibum Films and NMR Confirmation of Squalene in Tears.

Squalene (SQ) possesses a wide range of pharmacological activities (antioxidant, drug carrier, detoxifier, hydrating, emollient) that can be of benefit to the ocular surface. It can come in contact with human meibum (hMGS; the most abundant component of the tear film lipid layer) as an endogenous tear lipid or from exogenous sources as eyelid sebum or pharmaceuticals. The aims of this study were to determine (i) if SQ is in tear lipids and (ii) its influence on the surface properties of hMGS films. Heteronuclear single quantum correlation NMR confirmed 7 mol % SQ in Schirmer’s strips extracts. The properties of SQ/hMGS pseudo-binary films at the air/water interface were studied with Langmuir surface balance, stress-relaxation dilatational rheology and Brewster angle microscopy. SQ does not possess surfactant properties. When mixed with hMGS squalene (i) localized over the layers’ thinner regions and (ii) did not affect the film pressure at high compression. Therefore, tear SQ is unlikely to instigate dry eye, and SQ can be used as a safe and “inert” ingredient in formulations to protect against dry eye. The layering of SQ over the thinner film regions in addition to its pharmacological properties could contribute to the protection of the ocular surface.

Improving the resolution in proton-detected through-space heteronuclear multiple quantum correlation NMR spectroscopy

Connectivities and proximities between protons and low-gamma nuclei can be probed in solid-state NMR spectroscopy using two-dimensional (2D) proton-detected heteronuclear correlation, through Heteronuclear Multiple Quantum Correlation (HMQC) pulse sequence. The indirect detection via protons dramatically enhances the sensitivity. However, the spectra are often broadened along the indirect F1 dimension by the decay of heteronuclear multiple-quantum coherences under the strong 1H–1H dipolar couplings. This work presents a systematic comparison of the performances of various decoupling schemes during the indirect t1 evolution period of dipolar-mediated HMQC (D-HMQC) experiment. We demonstrate that 1H–1H dipolar decoupling sequences during t1, such as symmetry-based schemes, phase-modulated Lee–Goldburg (PMLG) and Decoupling Using Mind-Boggling Optimization (DUMBO), provide better resolution than continuous wave 1H irradiation. We also report that high resolution requires the preservation of 1H isotropic chemical shifts during the decoupling sequences. When observing indirectly broad spectra presenting numerous spinning sidebands, the D-HMQC sequence must be fully rotor-synchronized owing to the rotor-synchronized indirect sampling and dipolar recoupling sequence employed. In this case, we propose a solution to reduce artefact sidebands caused by the modulation of window delays before and after the decoupling application during the t1 period. Moreover, we show that 1H–1H dipolar decoupling sequence using Smooth Amplitude Modulation (SAM) minimizes the t1-noise. The performances of the various decoupling schemes are assessed via numerical simulations and compared to 2D 1H–{13C} D-HMQC experiments on [U–13C]–L-histidine⋅HCl⋅H2O at various magnetic fields and Magic Angle spinning (MAS) frequencies. Great resolution and sensitivity enhancements resulting from decoupling during t1 period enable the detection of heteronuclear correlation between aliphatic protons and ammonium 14N sites in L-histidine⋅HCl⋅H2O.

Selective hydrolysis of hen egg white lysozyme at Asp-X peptide bonds promoted by oxomolybdate

The activity of oxomolybdate(VI) towards hen egg white lysozyme (HEWL) was examined under physiological and slightly acidic pH conditions. Purely hydrolytic cleavage of HEWL in the presence of 10 to 100mM of oxomolybdate(VI) after incubation at pH5.0 and 60°C for 2 to 7days was observed in SDS-PAGE experiments. Four cleavage sites, which all occurred at Asp-X sequences and included the Asp18-Asn19, Asp48-Gly49, Asp52-Trp53 and Asp101-Gly102 peptide bonds, were identified with Edman degradation. The molecular interaction between [MoO4]2− and HEWL was studied by circular dichroism (CD) and 1H-15N heteronuclear single quantum correlation (HSQC) NMR spectroscopy. CD spectroscopy revealed a significant decrease in the α-helical content of HEWL upon addition of oxomolybdate, while 1H-15N HSQC NMR spectroscopy identified the residues which were most affected upon interaction with [MoO4]2−. 95Mo NMR measurements, performed on oxomolybdate solutions containing HEWL, identified the monomeric [MoO4]2− form as active species in the hydrolytic reaction. The hydrolysis of the Asp-Gly model peptide in the presence of oxomolybdate(VI) was studied by 1H NMR, further supporting a hydrolytic mechanism where polarisation of the carbonyl is followed by internal nucleophilic attack on the Asp residue.

Solution structure of lysine‐free (K0) ubiquitin

Lysine-free ubiquitin (K0-Ub) is commonly used to study the ubiquitin-signaling pathway, where it is assumed to have the same structure and function as wild-type ubiquitin (wt-Ub). However, the K0-Ub (15) N heteronuclear single quantum correlation NMR spectrum differs significantly from wt-Ub and the melting temperature is depressed by 19°C, raising the question of the structural integrity and equivalence to wt-Ub. The three-dimensional structure of K0-Ub was determined by solution NMR, using chemical shift and residual dipolar coupling data. K0-Ub adopts the same backbone structure as wt-Ub, and all significant chemical shifts can be related to interactions impacted by the K to R mutations.