Proton NMR Analysis Research Articles

NMR and Texture Analyses in Relation to Swelling Kinetics of 2‐Hydroxyethyl Methacrylate/N‐Vinylpyrrolidinone Hydrogels

AbstractThis study investigates the relationship between swelling kinetics and the relative stress relaxation (SR) values from texture analysis, as well as the relaxation times from proton NMR analysis of photopolymerized 2‐hydroxyethyl methacrylate (HEMA)/N‐vinyl pyrrolidinone (NVP) hydrogels with varying HEMA and NVP contents. Swelling tests in Milli‐Q water at 37 °C showed that hydrogels of higher NVP content displayed non‐Fickian diffusion kinetics. Texture analysis indicated the existence of a trend between SR values and diffusion kinetics with the hydrogels that displayed non‐Fickian diffusion having significantly lower SR values. Proton NMR analysis showed that the hydrogel with higher NVP content (non‐Fickian diffusion kinetics) displayed substantially longer relaxation times, which indicated slower exchange of water molecules between different sites within the meshes of the polymer matrix, attributable probably to hydrogen bonding and hydrophilic attraction between polymer and the water molecules. A relationship appeared to exist for the hydrogels understudy with hydrogels exhibiting non‐Fickian diffusion kinetics displaying lower SR values and longer relaxation times.

Development of tissue-targeted metabonomics. Part 1. Analytical considerations

Tissue-targeted metabonomics provides tissue specific metabolic information while still retaining the profiling approach of traditional metabonomics. Microdialysis sampling is used to generate site-specific samples of endogenous metabolites. The dialysate samples are subjected to proton NMR analysis with data analysis by principal components analysis and partial least squares regression. In this study, sample and data pretreatment methods were examined for their impact on the quality of the data analysis. Specifically, the effects of speedvacuuming, sample solubility, sample pH stability, and sample storage stability were examined. Data pretreatment methods examined included the effects of standardization and normalization to internal standards. In addition, the ability of tissue-targeted metabonomics to generate time trend data was explored and more fully characterized using principal components analysis and partial least squares regression.

Substantially monodispersed poly(ɛ-l-lysine)s frequently occurred in newly isolated strains of Streptomyces sp.

The presence of poly(epsilon-L-lysine) (epsilon-PL) was found quite frequently by screening various strains of Streptomyces sp. Most of the ten newly obtained epsilon-PLs, when they were produced from glucose, showed a polydispersity index of Mw/Mn = 1.01 using ion-pair chromatography analysis. The polymers were classified into five groups according to their chain lengths. The average numbers of residues in the five groups were 32, 28, 25, 19, and 16, respectively. The use of glycerol instead of glucose resulted in decreases of 10 to 20% in the Mn and slight increases in the Mw/Mn. These observations indicated the chain length and polydispersity of epsilon-PL were primarily determined by each producer strain. Proton and 13C NMR analysis revealed the signals of glycerol-derived ester at the C terminus of the polymer from several producers including the first discovered S. albulus strain, although the percentages of the ester were low under our culture conditions. These results, coupled with the previous observation that SO4(2-) was essential for the polymer production, led to discussion on the mechanistic aspects of monomer activation, elongation, and termination in the biosynthesis of epsilon-PL.

Identification of organic molecules from a structure database using proton and carbon NMR analysis results

A compound is identified by matching its proton and/or carbon NMR spectra to NIH PubChem molecular structures. The matching process involves analyzing 1D proton, 1D carbon, DEPT, and/or HSQC spectra, and comparing the number of NMR resonances, detected proton and carbon shifts, likely number of methyl- and methoxy-groups, and an optionally specified molecular formula to predicted proton and carbon shifts of PubChem structures. A structure verification module rates the consistency between experimental spectral analysis results and a proposed structure (not limited to PubChem structures) and assigns observed shifts to the proposed structure. The spectral analysis, structure identification, and structure verification are largely automated in a software package and can be performed in minutes.

Chemical modification of starch with hexamethylene diisocyanate derivatives

Chemical modification of potato starch in a two-step process has been presented. At the first stage the starch modifiers, i.e. isocyanate derivatives were synthesised by the equimolar reaction between hexamethylene diisocyanate (HMDI) and monoalcohol or monoamine. Proton NMR analysis has revealed that the prepared modifiers contain monoisocyanate compounds with respectively urethane or urea bonds, as the main products. Synthesis of HMDI-derived modifiers was followed using DSC method and the reaction products were characterized by the FTIR spectroscopy. At the second step starch was modified with the synthesised HMDI derivatives, in N-methylpyrrolidone slurry. Some properties of the obtained starch polymers were investigated and compared, i.e. efficiency of substitution, IR spectra, hydrophobic/hydrophilic features, DSC thermograms (melting temperatures) as well as moldability (hot press melt flow).

Glycosylated polyacrylate nanoparticles by emulsion polymerization

A selection of glycosylated polyacrylate nanoparticles has been prepared by radical-initiated emulsion polymerization in aqueous media. Using ethyl acrylate as a co-monomer, carbohydrate acrylates were incorporated into the poly(ethyl acrylate) framework to give stable emulsions of glyconanoparticles with an average particle size of around 40 nm. Using this technique a variety of glyconanoparticles were prepared from 3- O-acryloyl-1,2:5,6-di- O-isopropylidene-α- d-glucofuranose, 1- O-acryloyl-2,3:5,6-di- O-isopropylidene-α- d- mannofuranose, 6- O-acryloyl-1,2:3,4-di- O-isopropylidene-α- d-galactopyranose, 2- N-acryloyl-1,3,4,6-tetra- O-acetyl-β- d-glucosamine, 5- O-acryloyl-2,3-isopropylidene-1-methoxy-β- d-ribofuranose and 4- N-acetyl-5′- O-acryloyl-2′,3′- O-isopropylidene cytidine. Scanning electron microscopy, dynamic light scattering and proton NMR analysis of the emulsions indicated essentially 100% incorporation of the carbohydrate acrylate monomer into the polymer with the exception of O-benzyl- and O-benzoyl-protected carbohydrate acrylates, which gave incomplete incorporation. Formation of larger glyconanoparticles of ∼80 nm with (unprotected) 3- O-acryloyl- d-glucose and 5- O-acryloyl-1-methoxy-β- d-ribofuranose revealed the influence of free hydroxyl groups in the monomer on the particle size during polymerization, a feature which is also apparently dependent on the amount of carbohydrate in the matrix. This methodology allows for a new, simple route to the synthesis of polymeric glyconanoparticles with potential applications in targeted drug delivery and materials development.

Design and synthesis of pyrazolo[3,4- d]pyrimidine and triazolo[4,5- d]pyrimidine based dissymmetrical ‘Leonard linker’ compounds: 1H NMR and crystallographic evidence for folded conformation due to arene interactions

Proton NMR analyses of several newly synthesized dissymmetrical ‘Leonard/trimethylene linker’ compounds 1/2/3-[(4,6-dimethylsulfanyl-1 H-pyrazolo[3,4- d]pyrimidin-1-yl)propyl]-5,7-dimethyl/ethyl-sulfanyl-1 H/2 H/3 H-triazolo[4,5- d]pyrimidines ( 6 and 8) show intramolecularly stacked conformation in solution. X-ray crystallography of one dissymmetrical ‘Leonard/trimethylene’ linker compound ( 6b) based on two different heterocycles namely pyrazolo[3,4- d]pyrimidine core and triazolo[4,5- d]pyrimidine, for the first time, shows unusual U-motif formed due to intramolecular π–π interactions, which is similar to earlier related pyrazolo[3,4- d]pyrimidine core based ten symmetrical ( 1 and 2) and one dissymmetrical compound ( 3). Supramolecular structures of the new compound ( 6b) show unusual chain motif due to weak intermolecular CH…N and CH…S interactions. More importantly, the new compound ( 6b) shows S…arene interaction not shown by any earlier compounds ( 1, 2 and 3).

A Comparative Study of the Properties of Selected Melon Seed Oils as Potential Candidates for Development into Commercial Edible Vegetable Oils

AbstractA comprehensive compositional and characterization study was carried out on five seed oils from varieties of the melons Citrullus lanatus and C. colocynth in order to evaluate their suitability for large‐scale exploitation as edible vegetable oils. The oils were extracted by Soxhlet with a 3:1 mixture of n‐hexane/2‐propanol with yields that ranged from 24.8 to 30.0% (wt/wt). The refractive indices and relative densities of the oils fell within the narrow ranges of 1.465–1.469 and 0.874–0.954 g/cm3, respectively. Saponification values ranged between 182.1 and 193.8 mg KOH/g, whilst iodine values (IV) ranged from 95.8 to 124.0 (Wijs). The ranges of the values for free fatty acid (AV), 1.2–4.0 mg KOH/g, peroxide (PV), 1.1–10.9 meq/kg and p‐anisidine (p‐AV), 0.2–9.0, indicated that secondary oxidation products were barely present. GC analysis gave total unsaturation contents of 67.93–82.36%, with linoleic acid (18:2) being the dominant fatty acid (55.21–66.85%). The GC results agreed closely with those from proton NMR analysis of the fatty acid classes. The physicochemical and compositional properties determined in this study show that the qualities of the test Cucurbitacea seed oils are highly comparable to those of soybean, sunflower and groundnut seed oils. Therefore, the test melon seed oils could be developed into commercial products to serve as alternate vegetable oils in Southern and West Africa, the regions where these melons grow.

Quantification and Identification of Components in Solution Mixtures from 1D Proton NMR Spectra Using Singular Value Decomposition

One-dimensional proton NMR spectra of complex solutions provide rich molecular information, but limited chemical shift dispersion creates peak overlap that often leads to difficulty in peak identification and analyte quantification. Modern high-field NMR spectrometers provide high digital resolution with improved peak dispersion. We took advantage of these spectral qualities and developed a quantification method based on linear least-squares fitting using singular value decomposition (SVD). The linear least-squares fitting of a mixture spectrum was performed on the basis of reference spectra from individual small-molecule analytes. Each spectrum contained an internal quantitative reference (e.g., DSS-d6 or other suitable small molecules) by which the intensity of the spectrum was scaled. Normalization of the spectrum facilitated quantification based on peak intensity using linear least-squares fitting analysis. This methodology provided quantification of individual analytes as well as chemical identification. The analysis of small-molecule analytes over a wide concentration range indicated the accuracy and reproducibility of the SVD-based quantification. To account for the contribution from residual protein, lipid or polysaccharide in solution, a reference spectrum showing the macromolecules or aggregates was obtained using a diffusion-edited 1D proton NMR analysis. We demonstrated this approach with a mixture of small-molecule analytes in the presence of macromolecules (e.g., protein). The results suggested that this approach should be applicable to the quantification and identification of small-molecule analytes in complex biological samples.

Erratum: Proton NMR analysis of plasma is a weak predictor of coronary artery disease

Nat. Med. 12, 705–710 (2006); published online 28 May 2006; corrected after print 19 June 2006 In the version of this article initially published, the layout of the data in Tables 1 and 2 is incorrect. The error has been corrected in the HTML and PDF versions of the article.

Proton NMR analysis of plasma is a weak predictor of coronary artery disease

Multivariate analysis of 1H-NMR spectra of blood sera was reported previously to predict angiographically defined advanced coronary artery disease (CAD) with >90% accuracy and specificity. The analysis depended mainly on the major lipid regions of the spectra, but many variables, including gender and drug treatment, affect lipid composition and are potential confounders. We have determined the predictive power of the same methodology for angiographically defined CAD using plasma samples from groups of male patients, classified by statin treatment, who had normal coronary arteries (NCAs) or CAD. Predictions for NCA and CAD groups were only 80.3% correct for patients not treated with statins and 61.3% for treated patients, compared with random correct predictions of 50%. A confidence limit of >99% was achieved for 36.2% of predictions for untreated groups and 6.2% for treated groups. Detection of CAD by 1H-NMR with >99% confidence was therefore very weak compared with angiography.

Design and synthesis of pyrazolo[3,4- d]pyrimidine core based dissymmetrical ‘Leonard linker’ compounds: 1H NMR and crystallographic evidence for folded conformation due to arene interactions

Proton NMR analysis of two newly synthesized ‘Leonard/trimethylene linker’ dissymmetrical compounds 1-(4,6-dimethylsulfanyl-1 H-pyrazolo[3,4- d]pyrimidin-1-yl)-3-(5-methyl-6-methylsulfanyl-4-oxo-1,5-dihydropyrazolo[3,4- d]pyrimidin-1-yl)propane (8) and 1-(4-methoxy-6-methylsulfanyl-1 H-pyrazolo[3,4- d]pyrimidin-1-yl)-3-(6-methoxy-5-methyl-4-oxo-1,5-dihydropyrazolo[3,4- d]pyrimidin-1-yl)propane (10) show intramolecularly stacked conformation in solution. X-Ray crystallography of dissymmetrical ‘Leonard/trimethylene linker’ compound (8) based on pyrazolo[3,4- d]pyrimidine core, for the first time, shows unusual U-motif formed due to intramolecular π–π stacking interactions, which is similar to earlier related symmetrical compounds 1 & 2. Supramolecular structures of new compound (8) show additional CH…O, CH…N, CH…S and S…S interactions.

Products of laccase catalyzed reaction of 1-hydroxypyrene

The formation of residues in soils and sediments which are not readily extractable is an important fate for hydrophobic organic contaminants. Reaction of 1-hydroxypyrene, a known fungal and terrestrial invertebrate metabolite and photo-oxidation product of pyrene, with a commercially prepared laccase (an oxidoreductive enzyme) in solution provides a test system to study the fate of residues in nature. The reaction of dissolved 1-hydroxypyrene with laccase produced a distinct color change and caused a precipitate to form. At least six products were shown by HPLC analysis with photodiode array detection. The most prominent peak was composed of 1,6- and 1,8-pyrene-dione as confirmed by proton NMR analysis. LC/MS data on remaining peaks indicate that radical coupling produces acetonitrile soluble dimers and at least one trimer. Proposed structures consistent with MS and photodiode array data indicate that pyrene subunits are linked predominantly by ether bonds. Additional products, insoluble in acetonitrile, were also formed. The formation of complex products in vitro, indicates that the same mechanisms could produce bound residues in soil.

Fine tuning of folded conformation by change of substituents: 1H NMR and crystallographic evidence for folded conformation due to arene interactions in pyrazolo[3,4-d]pyrimidine core based ‘propylene linker’ compounds

Proton NMR and X-ray crystallographic analysis of two newly synthesized ‘trimethylene linker,’ (Leonard linker) compounds 1,3-bis(4-ethoxy-6-methylsulfanyl-lH-pyrazolo[3,4-d]pyrimidin-1-yl)propane (4c) and 1,3-bis(4-isopropoxy-6-methylsulfanyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propane (4d) show intramolecularly stacked conformations both in solution and solid state. Robustness of the unusual U-motif formed due to intramolecular stacking in earlier related compounds (1, n=3 and 3, n=3) is not only confirmed but additional fine tuning is also achieved in new compounds (4) formed by replacement of remote 4-alkylsulfanyl group in 1a by 4-alkoxy substituents.

Synthesis of Short-/Medium-Chain-Length Poly(hydroxyalkanoate) Blends by Mixed Culture Fermentation of Glycerol

Glycerol was used as a substrate in the bio-production of poly(hydroxyalkanoates) (PHAs) in an effort to establish an alternative outlet for glycerol and produce value-added products. Pseudomonas oleovorans NRRL B-14682 and Pseudomonas corrugata 388 grew and synthesized poly(3-hydroxybutyrate) (P3HB) and medium-chain-length PHA (mcl-PHA) consisting primarily of 3-hydroxydecanoic acid (C(10:0); 44 +/- 2 mol %) and 3-hydroxydodecenoic acid (C(12:1); 31 +/- 2 mol %), respectively, from glycerol at concentrations up to 5% (v/v). Cellular productivity maximized at 40% for P. oleovorans in 5% (v/v) glycerol and 20% for P. corrugata in 2% (v/v) glycerol after 72 h. Increasing the glycerol media concentration from 1% to 5% (v/v) caused a 61% and 72% reduction in the molar mass (M(n)) of the P3HB and mcl-PHA polymers, respectively. Proton-NMR analysis of the glycerol-derived P3HB revealed that the M(n) decrease was the result of esterification of glycerol onto the polymer in a chain terminating position. However, no evidence of glycerol-based chain termination was present in the mcl-PHA. The growth patterns of P. oleovorans and P. corrugata on glycerol permitted their use as mixed cultures to produce natural blends of P3HB and mcl-PHA. By incorporating a staggered inoculation pattern and varying the duration of the fermentations, P3HB/mcl-PHA ratios were achieved that varied from 34:66 to 96:4.

Block Copolymers of γ-Methacryloxypropyltrimethoxysilane and Methyl Methacrylate by RAFT Polymerization. A New Class of Polymeric Precursors for the Sol−Gel Process

Reversible addition−fragmentation chain transfer (RAFT) polymerization of the reactive monomer γ-methacryloxypropyltrimethoxysilane (γMPS) mediated by 2-cyanoprop-2-yl dithiobenzoate (CPDB) has been studied in dioxane using 2,2‘-azobis(isobutyronitrile) (AIBN) as initiator. Conditions were optimized for a polymerization temperature of 80 °C, and controlled PγMPS chains (up to Mn = 40 000 g mol-1) exhibiting low polydispersity indexes (PDI < 1.3) were synthesized. Their characterization by matrix-assisted laser desorption ionization time-of-flight (MALDI-ToF) mass spectrometry showed that the expected structure was obtained although degradation of the dithiobenzoate chain end occurred. Syntheses of block copolymers based on γMPS and methyl methacrylate (MMA) were performed starting from either the PγMPS (Mn = 32 100 g mol-1, PDI = 1.16, PMMA calibration) or the PMMA block (Mn = 21 000 g mol-1, PDI = 1.14). The success of the block copolymerization was showed by the shift toward higher molar mass of the size exclusion chromatography (SEC) chromatograms recorded before and after block copolymerization. Proton NMR analyses of P(MMA-b-γMPS) allowed to calculate the molar mass of the PγMPS blocks (17 400 g mol-1) which agreed with the targeted one (18 150 g mol-1).

Preparation and fractionation of conjugated trienes from α‐linolenic acid and their growth‐inhibitory effects on human tumor cells and fibroblasts

Conjugated alpha-linolenic acid (CLnA) was prepared from alpha-linolenic acid (9,12,15-18:3n-3, LnA) by alkaline treatment; we fractionated CLnA into three peaks by reversed-phase column-HPLC as evidenced by monitoring absorbance at 205, 235, and 268 nm. Peak I was a conjugated dienoic FA derived from LnA, whereas Peaks II and III were conjugated trienoic LnA. Proton NMR analysis showed that Peak III consisted of the all-trans isomer. The methylated Peak III was further divided into five peaks (Peaks IV-VIII) by silver ion column-HPLC. Peak V, a major constituent in the Peak III fraction, was identified as conjugated 10t,12t,14t-LnA by GC-EIMS and 1H NMR analysis. Peaks III and V, which consisted of conjugated all-trans trienoic LnA, had stronger growth-inhibitory effects on human tumor cell lines than the other collected peaks and strongly induced lipid peroxidation as compared with Peaks I, II, and LnA. We propose that conjugated all-trans trienoic FA have the strongest growth-inhibitory effect among the conjugated trienoic acids and conjugated dienoic acids produced by alkaline treatment of alpha-LnA, and that this effect is mediated by lipid peroxidation.

Atropisomeric property of 1‐(2,6‐difluorobenzyl)‐3‐[(2R)‐amino‐2‐phenethyl]‐5‐(2‐fluoro‐3‐methoxyphenyl)‐6‐methyluracil

1-(2,6-Difluorobenzyl)-3-[(2R)-amino-2-phenethyl]-5-(2-fluoro-3-methoxyphenyl)-6-methyluracil (6), a potent and orally active antagonist of the human gonadotropin-releasing hormone receptor, exists as a pair of atropisomers in solution, which was detected by NMR spectroscopy, and separable by HPLC. In addition to a (R)-configured benzylamine, there is a second stereogenic element due to the presence of a chiral axis between the substituted 5-phenyl group and the uracil core. The rate constant of the interconversion (k = 5.07 x 10(-5) s(-1)) of these two atropisomers was determined by proton NMR analysis of a diastereoisomer-enriched sample in aqueous solution at 25 degrees C, and the corresponding Gibbs free energy DeltaG(#) of rotation barrier (97.4 kJ mol(-1)) was calculated using the Eyring equation. The diastereoisomer half-life at physiological temperature (37 degrees C) in aqueous media was estimated to be about 46 min.

Tumor-targeted bioconjugate based delivery of camptothecin: design, synthesis and in vitro evaluation

Camptothecin (CPT) presents numerous challenges associated with optimal transport and delivery including variability in clinically observed effects, low target tissue concentrations and severe and unpredictable toxicity. The objective of the present study was to optimize the delivery of CPT by targeting it to cancer cells using an endogenous receptor system. A novel CPT bioconjugate was synthesized using carbodiimide chemistry with a linear poly(ethylene glycol) (PEG) and amino acid glycine as the spacer and linker respectively. Folic acid was used as the targeting ligand to take advantage of folate receptor mediated endocytosis. The bioconjugate was extensively characterized using MALDI, proton NMR, FT-IR and amino acid analysis. Furthermore, the bioconjugate was evaluated in vitro for specific targeting to folate receptor-expressing KB cells, a human nasopharyngeal carcinoma. Finally, the delivery system was evaluated for cytotoxicity using a MTT based assay. The results indicate significantly higher efficacy of the bioconjugate in comparison to CPT. A control conjugate without PEG demonstrated no improvement in efficacy over untargeted CPT emphasizing the importance of spacer between the anticancer compounds and targeting moiety. This bioconjugate represents the ‘first-in-series’ of targeted bioconjugates and serves as prototype for improving tumor cell concentration and efficacy.

Characterization and compositional studies of the oils from some legume cultivars, Phaseolus vulgaris, grown in Southern Africa

AbstractSeed oils from six legume cultivars of Phaseolus vulgaris, grown in the Kingdom of Lesotho, were extracted and their physicochemical properties and FA compositions were determined in order to compare their dietary lipids with those in P. vulgaris cultivars grown in other parts of the world. The oil content of the beans was very low, ranging from 1.5 to 2.0% (w/w). The acid values ranged from 11.0 to 19.2 mg KOH/g, whereas a combination of the PV and the p‐anisidine values in Holm's equation gave oxidation values that ranged from 11.0 to 15.0. Thus, considerable enzymatic hydrolysis and oxidation had taken place in the beans during storage. Iodine values ranged from 80.5 to 92.3 (Wijs method), indicating moderate unsaturation in the oils. However, capillary GC analysis, supported by proton NMR analysis of the FAME, gave a total unsaturation range from 79.67 to 84.24%. The dominant FA were α‐linolenic acid (36.47–48.81%) and linoleic acid (20.96–36.10%), with appreciable amounts of palmitic acid (14.33–18.23%). This FA composition pattern is quite similar to the FA distribution reported for low oil‐bearing legume seeds. Thus, notwithstanding the different climatic and soil conditions, the general properties of lipids in the southern African legume cultivars were quite similar to those of lipids in P. vulgaris cultivars grown in other parts of the world. The high content of α‐linolenic acid in the cultivars of P. vulgaris could very likely play a beneficial role in reducing the risk of coronary heart disease among the large populations consuming them in the southern African region.