High Field 1H NMR Spectroscopy Research Articles

An NMR‐based metabolomic approach to the analysis of the effects of xenobiotics on endogenous metabolite levels in plants

High field 1H NMR spectroscopy has been employed to obtain, in conjunction with chemometric analysis, information regarding fluctuations in endogenous metabolic profiles for Crotalaria cobalticola plant cells following exposure to cobalt chloride.Such ‘metabolomic’ type data analysis is often confounded by experimental, environmental or genetic factors that are not correlated to the classifications of interest and serve only to complicate the extraction of meaningful answers from a dataset. This work demonstrates the application of data filtering to remove extraneous data that result from spectrometer variation rather than being correlated with the classes of interest. Samples were analysed from Crotalaria cobalticola suspension cell culture following exposure to cobalt chloride using 2 spectrometers. Removal of confounding data due to spectrometer variation resulted in clear separation between control and dosed classes. It was then possible to use the model to determine key changes in biochemical status caused as a result of exposure to cobalt. Branched chain amino acids, succinate and secondary metabolite precursors phenylalanine and tyrosine were all higher in the control samples, whilst choline, glutamate, alanine and lactate were higher in the dosed samples.

Structural determination of Torlon ® 4000T polyamide–imide by NMR spectroscopy

High field 1H and 13C NMR spectroscopy and published information in the scientific literature were combined in the determination of the chemical structure of the commercially available Torlon ® 4000T, a polyamide–imide which is widely used in applications where good mechanical, thermal and oxidative properties are required. It was found from qualitative and quantitative NMR analysis that the three monomers used to synthesize Torlon are as follows: 1.0 trimellitic anhydride chloride, 0.7 4,4′-oxydianiline, 0.3 m-phenylenediamine. It was also shown that the sequence of appearance of the polymer's amide and imide functional groups follows a certain distribution of Amide–Imide, Amide–Amide and Imide–Imide repeat units due to the dual reactive sites, acid chloride and anhydride, of the monomer trimellitic anhydride chloride.

Effects of chronic lithium and sodium valproate on concentrations of brain amino acids

The present study was designed to determine if the mood stabilizers, lithium and valproate, have common effects on concentrations of amino acid neurotransmitters which may be related to their mechanisms of action. Two separate groups of rats were administered therapeutic doses of lithium, sodium valproate, or saline for 2 weeks. Whole brain extracts were then examined using either high-field 1H NMR spectroscopy or HPLC. Both drugs decreased whole brain concentrations of aspartate, glutamate, and taurine while brain concentrations of γ-aminobutyric acid (GABA) and alanine decreased following chronic sodium valproate administration but not following chronic lithium administration. These findings indicate that lithium and sodium valproate share common effects on the concentrations of certain amino acid neurotransmitters in whole brain which may be related to their mechanisms of action in bipolar disorder.

Multi-component metabolic classification of commercial feverfew preparations via high-field 1H-NMR spectroscopy and chemometrics.

There is increasing interest in evaluating the clinical efficacy of herbal medicines. However, there are significant analytical problems associated with quality control and the measurement of the overall composition of such complex, multi-component mixtures as normally required in the pharmaceutical industry. Here we describe a novel NMR spectroscopic and pattern recognition analytical approach to investigate composition and variability of a commonly used herbal medicine. 600 MHz (1)H-NMR spectroscopy and principal components analysis (PCA) was used to discriminate between batches of 14 commercially available feverfew samples based on multi-component metabolite profiles. Two of the batches were significantly different from the other twelve. The twelve remaining classes could be classified into discrete groups by PCA on the basis of minor differences in overall chemical composition. NMR based pattern recognition (PR) analysis of extracts proved to be superior to PR analysis of HPLC traces of the same mixtures. This work indicates the potential value of NMR combined with PCA for the characterisation of complex natural product mixtures, and the discrimination of samples containing allegedly identical ingredients.

Stereochemistry in solution of spiro-2,2′-dioxybiphenyl-cyclotriphosphazenes: an NMR spectroscopic study combined with molecular dynamics simulations

The stereochemistry in solution of spirocyclophosphazenes 1–3 containing 2,2′-dioxybiphenyl groups, tris(2,2′-dioxybiphenyl)cyclotriphosphazene [N3P3(O2C12H8)3] (1) and the chloro derivatives [N3P3Cl2(O2C12H8)2] (2) and [N3P3Cl4(O2C12H8)] (3), was studied using high field 1H, 13C and 31P NMR spectroscopy (variable temperature experiments) combined with molecular dynamics (MD) simulations. The ambient temperature spectra, after full assignments, showed a unique set of signals (four and six for hydrogens and carbons, respectively) for the corresponding nuclei of all the biphenoxy systems. This isochrony was compatible with symmetry equivalence as well as with fast interconversions on the NMR time scale between the stereoisomers RRR, SSS, RRS, SSR (1), RR, SS, RS, SR (2) and R, S (3). Dynamic NMR studies evidenced that atropoisomers of 1–3 are populated at low temperature (≤ 183 K) bearing out fast racemization of the NP(O2C12H8) units in solution at ambient temperature. The MD trajectories, which were long enough (50 ns) to monitor time-averaged processes on the NMR scale, were selectively compatible with DNMR results, and provided insights into the motional properties of 1–3. They significantly complemented the experimental information on the exchange process of 3, due to the inherent limitation of the single use of the NMR technique when NMR active diastereotopic nuclei are absent in the investigated system.

Synthesis of the three isomeric mono-2-, 3-, or 6-hydroxy permethylated β-cyclodextrins and unambiguous high field NMR characterisation

The three isomeric mono-2-, 3- or 6-hydroxy permethylated β-cyclodextrins are good precursors for a wide variety of mono functionalised ‘permethyl’ β-cyclodextrins. In this work, we describe the selective access to mono-6-hydroxy (via the mono-6-tertbutyldimethylsilyl derivative), mono-2-hydroxy (via the mono-2-benzyl derivative) and mono-3-hydroxypermethylated-β-cyclodextrins (by under-methylation of heptakis (2,6-di- O-methyl)-β-cyclodextrin). These derivatives were characterised by high field 1H and 13C NMR spectroscopy. The position of the free hydroxyl group was confirmed unambiguously by 13C NMR after methylation with 13C-labelled methyl iodide.

Transesterification characteristics of poly(bisphenol A carbonate) with ethylene terephthalate-Caprolactone copolyester

The transesterification of poly(bisphenol A carbonate) (PC) with ethylene terephthalate (ET)–caprolactone (CL) copolyester (TCL) was investigated with high-field 1H NMR spectroscopy. Under an uncatalyzed condition, ET and CL segments in TCL reacted more easily with PC than the corresponding homopolymers poly(ethylene terephthalate) and poly(ϵ-caprolactone). In a comparison of the transferring rates, aromatic ET segments had higher reaction activity than aliphatic CL segments. Furthermore, the reaction activity appeared to be strongly dependent on the TCL compositions. High and low ET-segment contents were unfavorable to the exchange reaction for both ET and CL segments. These characteristics are discussed on the basis of the TCL structures. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 39: 232–238, 2001

The role of N-acetylcysteine in protecting synovial fluid biomolecules against radiolytically-mediated oxidative damage: A high field proton NMR study

High field proton (1H) NMR spectroscopy has been employed to evaluate the abilities of the antioxidant thiol drug N-acetylcysteine and exogenous cysteine to protect metabolites present in intact inflammatory synovial fluid samples against oxidative damage arising from gamma-radiolysis (5.00 kGy) in the presence of atmospheric O2. Although oxidation of urate to allantoin by radiolytically-generated *OH radical was readily circumventable by pre-treatment of synovial fluids with N-acetylcystine (1.00 or 3.00 × 10-3 mol · dm-3) or cysteine (1.00, 2.00 or 5.00 × 10-3 mol · dm-3), both thiols offered only a limited protective capacity with respect to hyaluronate depolymerisation and the production of formate from carbohydrates in general. Radiolytic products generated from the added thiols (predominantly their corresponding disulphides) were simultaneously detectable in 1H Hahn spin-echo spectra of gamma-irradiated synovial fluids, permitting a quantitative evaluation of the radioprotective capacity of these agents. It is concluded that the multicomponent analytical ability of high field 1H NMR spectroscopy provides much useful molecular information regarding mechanisms associated with the radioprotectant actions of thiols in intact biofluids.

A cycloartane triterpene 3β, 16β diglucoside from Astragalus trigonus and its non natural 6-hydroxy epimer

The detailed investigation of Astralagus trigonus DC roots, led to the isolation of a new cycloartan-3β, 16β diglucoside, whose structure was elucidated by extensive use of 1D- and 2D- high field (600 MHz) NMR techniques. The NaBH 4 reduction of the natural 6-oxo 3β, 16β diglucoside, previously isolated from the same plant, afforded the 6-epi-derivative of the new compound. The conformational preference of both these compounds has been investigated by high field 1H NMR spectroscopy and molecular modeling techniques.

Mono- and disubstituted-3,8-diazabicyclo[3.2.1]octane derivatives as analgesics structurally related to epibatidine: synthesis, activity, and modeling.

A series of 3,8-diazabicyclo[3.2.1]octanes substituted either at the 3 position (compounds 1) or at the 8 position (compounds 2) by a chlorinated heteroaryl ring were synthesized, as potential analogues of the potent natural analgesic epibatidine. When tested in the hot plate assay, the majority of the compounds showed significant effects, the most interesting being the 3-(6-chloro-3-pyridazinyl)-3,8-diazabicyclo[3.2.1]octane (1a). At a subcutaneous dose of 1 mg/kg, 1a induced a significant increase in the pain threshold, its action lasting for about 45 min. 1a also demonstrated good protection at a dose of 5 mg/kg in the mouse abdominal constriction test, while at 20 mg/kg it completely prevented the constrictions in the animals. Administration of naloxone (1 mg/kg i.p.) did not antagonize its antinociception while mecamylamine (2 mg/kg i.p.) did, thus suggesting the involvement of the nicotinic system in its action. Binding studies confirmed high affinity for the alpha 4 beta 2 nAChR subtype (Ki = 4.1 +/- 0.21 nM). nAChR functional activity studies on three different cell lines showed that 1a was devoid of any activity at the neuromuscular junction. Finally, due to the analogy in their pharmacological profile with that of epibatidine, compounds were compared from a structural and conformational point of view through theoretical calculations and high-field 1H NMR spectroscopy. Results indicate that all of them present one conformation similar to that of epibatidine.

A convenient route to carbon-substituted derivatives of nido-5,6-C 2B 8H 12

An alternative route to the parent nido-5,6-C 2B 8H 12 dicarbaborane is reported together with a convenient synthesis of its carbon-substituted derivatives. The method is based on reactions between 4-(Me 2S)- arachno-B 9H 13 and alkynes R 1R 2C 2 (where R 1R 2=H,H; Me, Me; Ph,H, and Ph,Ph) in toluene at reflux. The characteristic reaction mode is a dicarbon insertion into the 9-vertex arachno cluster to produce a series of the 5,6-R 1R 2- nido-5,6-C 2B 8H 10 species combined with concomitant elimination of one {BH} vertex. The products were characterised by high-field 1H and 11B NMR spectroscopy and mass spectrometry associated with [ 11B– 11B]-COSY and 1H{ 11B(selective)} measurements that permitted complete assignments of all resonances to individual cluster {BH} units.

Probing the Origins of Asymmetric Induction by 3-Aminopyrrolidine Lithium Amides Complexes: A 6Li/1H/13C NMR Study

The structure of two chiral N,N‘-disubstituted-3-aminopyrrolidine lithium amides 3 and 4 in solution in THF-d8 has been studied at low temperatures by high-field 1H, 13C, and 6Li NMR spectroscopy. Despite their structural analogy, these two compounds adopt very different conformations in solution: while the amide 3 pyrrolidinic ring undergoes only minor changes with respect to its amino precursor 1, amide 4 presents a norbornyl-like bridged structure around 6Li+. When excess Bu6Li is added to both amide solutions, 1:1 amide−Bu6Li complexes arise, and their structures appear, this time, very similar and organized around a parallepipedic N−Li2−C core, the two lithium cations bridging the amide and the alkyl chain. The 4−Bu6Li complex appears very tight, with two distinct signals corresponding to each of the diastereotopic α-protons of butyllithium, by contrast with the looser 3−Bu6Li complex. From all these data, we propose a model to interpret the results obtained using these chiral amides in the asymmetr...

Determination of the absolute configuration of monosaccharides by 1H NMR spectroscopy of their per- O-( S)-2-methylbutyrate derivatives

An empirical method was developed to determine the absolute configuration of monosaccharides, based on high-field 1H NMR spectroscopy of their per- O-( S)-2-methylbutyrate (SMB) derivatives. The SMB derivatives of the D and L forms of a given monosaccharide are diastereomers, allowing them to be distinguished on the basis of differences in their 1H NMR chemical shifts. The reproducibility of these chemical shift differences may allow the absolute configuration of a wide range of monosaccharides to be routinely determined by comparing the spectra of their SMB derivatives to standard spectra in a data base, making it unnecessary to prepare and analyze new standards for each analysis. The derivatization procedure uses inexpensive, easy-to-handle reagents and is virtually complete. Application of the method to three complex glycans provided an unambiguous determination of the absolute configurations of their constituent monosaccharides. The enantiomeric forms of several monosaccharides are distinguishable by high-field 1H NMR spectroscopy of their per- O-( S)-2-methylbutyrate derivatives.

Evidence for Distinct Behaviour of Phosphatidylcholine and Sphingomyelin at the Low Density Lipoprotein Surface

This study demonstrates that the use of high field1H NMR spectroscopy permits individual detection of phosphatidylcholine and sphingomyelin molecules at the surface of native low density lipoprotein (LDL) particles. Distinct behaviour was observed for the choline head group −N(CH3)3resonances of these different phospholipids revealing preferential immobilisation for phosphatidylcholine. This suggests the existence of reversible and irreversible phosphatidylcholine-apolipoprotein B interactions and is consistent with microdomain formation at the surface monolayer of LDL. The novel resonance assignment and results show that1H NMR can provide efficient and practical means for future studies on the structure and dynamics at the LDL surface.

The structure of the keratan sulphate chains attached to fibromodulin isolated from articular cartilage.

Fibromodulin has been isolated from bovine and equine articular cartilage and the attached keratan sulphate chains subjected to digestion by keratanase II. The oligosaccharides generated have been reduced and subsequently isolated by strong anion-exchange chromatography. Their structures have been determined by high-field 1H-NMR spectroscopy and high-pH anion-exchange chromatography. Both alpha(2-6)- and alpha(2-3)-linked N-acetylneuraminic acid have been found in the capping oligosaccharides, and, fucose which is alpha(1-3)-linked to N-acetylglucosamine has been found as a branch in both repeat region and capping oligosaccharides. These data demonstrate that there are fundamental differences between the structures present in the N-linked keratan sulphate chains attached to fibromodulin from articular cartilage and those from tracheal cartilage, which lack both alpha(2-6)-linked N-acetylneuraminic acid and alpha(1-3)-linked fucose. It has been confirmed that the keratan sulphate chains are short, being only eight or nine disaccharides in length. Very significant differences in the levels of galactose sulphation have been identified at the non-reducing end of the chain. The galactose residue adjacent to the non-reducing cap is sulphated in only 1-3% of chains, compared with a sulphation level of over 40% closer to the reducing end. This highlights the difference between the chain termini and the repeat region in terms of structure and points to the potential for functional importance. The repeat region and capping fragments of the N-linked keratan sulphates from bovine and equine articular cartilage fibromodulin have been found to have the following general structure: NeuAc-(alpha 2-3/6)Gal[6SO3-](beta 1-4)GlcNAc6SO3-(beta 1-3)Gal[6SO3-] (beta 1-4)¿[Fuc(alpha 1-3)]0-1GlcNAc6SO3-(beta 1-3)Gal-[6SO3-](beta 1-4)¿ 6-7GlcNAc6SO3-.

The structure of the keratan sulphate chains attached to fibromodulin isolated from bovine tracheal cartilage: oligosaccharides generated by keratanase II digestion

The repeat region and chain caps of the N-linked keratan sulphates attached to bovine tracheal cartilage fibromodulin were fragmented by digestion with keratanase II, and the oligosaccharides generated were isolated by strong anion-exchange chromatography. Each of these oligosaccharides has been examined by both HPAE chromatography and high field 1H-NMR spectroscopy. All of the capping oligosaccharides isolated terminated with alpha(2-6)-linked N-acetyl-neuraminic acid chain terminators, nor fucose alpha(1-3)-linked to N-acetylglucosamine were found. The keratan sulphate chains were short, with average lengths of five to seven disaccharides, and the level of galactose sulphation varied along the length of the chain. The repeat region and chain cap were confirmed as having the following general structure: [formula: see text]. This study has identified a novel structure in fibromodulin, namely a cap containing a sulphated galactose adjacent to a non-reducing terminal N-acetyl-neuraminic acid. We have also confirmed that the general structure of the repeat units and chain caps of N-linked keratan sulphate attached to fibromodulin isolated from bovine tracheal cartilage, is similar to that of O-linked keratan sulphate chains attached to aggrecan from non-articular cartilage. However, there are important differences in chain lengths and sulphation patterns.

Detection and investigation of the molecular nature of low-molecular-mass copper ions in isolated rheumatoid knee-joint synovial fluid

Low-molecular-mass copper(II) species have been detected and quantified in ultrafiltrates ( n = 7) of rheumatoid synovial fluid (SF) by a highly-sensitive HPLC-based assay system with the ability to determine Cu(II) concentrations of < 10 −7 mol·dm t-3 . High field 1H NMR spectroscopy demonstrated that addition of Cu(II) (aq.) to isolated samples of RA SF ultrafiltrates resulted in complexation by histidine > alanine > formate > threonine > lactate > tyrosine > phenylalanine, their effectiveness in this context being in the given order. CD spectra of Cu(II)-treated samples of intact SF exhibited absorption bands typical of copper(II)-albumin complexes, in addition to a band attributable to a low-molecular-mass histidinate complex ( λ min 610 nm). Since both albumin and histidine are potent radical scavengers, these results indicate that any .OH radical generated from bound copper ions will be ‘site-specifically’ scavenged. Hence, low-molecular-mass copper complexes with the ability to promote the generation of .OH radical which can then escape from the metal ion co-ordination sphere (and in turn, cause damage to critical biomolecules) appear to be absent from inflammatory SF.

The synthesis of homogeneous triglycerides of eicosapentaenoic acid and docosahexaenoic acid by lipase

A highly efficient synthesis of homogeneous triglycerides of either pure eicosapentaenoic acid, 1, or docosahexaenoic acid, 2, by an immobilized non-regiospecific yeast lipase from Candida antarctica is described. Two methods were used: Interesterification of tributyrin and direct esterification of glycerol with stoichiometric amount of 99 % EPA or DHA as ethyl esters and free fatty acids, respectively. Both processes were performed under vacuum at 65°C in the absence of any solvent. The volatile co-products were condensed into a cooled trap, thus shifting the equilibrium toward completion. Complete incorporation was reached in less than 72 h for all cases, but the direct esterification was found to proceed considerably faster than the transesterification as were both processes involving EPA as compared to DHA. High-field 1H NMR spectroscopy analysis offered detailed investigation of the intermediate glycerides during the direct esterification reaction. The purity of the resulting crude products which were afforded in excellent yields was very high.

Generation of lipid peroxidation products in culinary oils and fats during episodes of thermal stressing: A high field 1H NMR study

The oxidative deterioration of glycerol-bound polyunsaturated fatty acids (PUFAs) in culinary oils and fats during episodes of heating associated with normal usage (30–90 min at 180°C) has been monitored by high field 1H NMR spectroscopy. Thermal stressing of PUFA-rich culinary oils generated high levels of n-alkanals, trans-2-alkenals, alka-2,4-dienals and 4-hydroxy- trans-2-alkenals via decomposition of their conjugated hydroperoxydiene precursors, whereas only low concentrations of selected aldehydes were produced in oils with a low PUFA content, lard and dripping when subjected to the above heating episodes. Samples of repeatedly used, PUFA-rich culinary oils obtained from restaurants also contained high levels of each class of aldehyde. The dietary, physiological and toxicological ramifications of the results obtained are discussed.

Evaluation of liquid chromatography coupled with high-field 1H NMR spectroscopy for drug metabolite detection and characterization: the identification of paracetamol metabolites in urine and bile.

The applicability of coupled reversed-phase high performance liquid chromatography (HPLC)-NMR spectroscopy for the detection and identification of paracetamol (N-(4-hydroxyphenyl)acetamide) and its sulfate, glucuronide and N-acetylcysteinyl metabolites in the unprocessed biological fluids, human urine, rat urine and rat bile, is investigated. Analysis of these samples was performed by gradient HPLC elution and directly coupled 500 MHz 1H NMR spectroscopy detection using a combination of one- and two-dimensional NMR methods in stopped-flow mode. The stopped-flow approach is demonstrated to be an efficient technique for identification of drug metabolites which have, for example, a UV-chromophore. Stopped-flow HPLC analysis with NMR detection is a viable technique and halting the chromatographic process several times during a run has a negligible effect on the separation and NMR characterization. The post-acquisition data processing method of 'quantified maximum entropy' is shown to provide a means of improving the quality of spectra for minor components, thus aiding NMR resonance assignments.