High Field 1H NMR Spectroscopy Research Articles

High-field NMR spectroscopy of cystocarpic and tetrasporic carrageenans from Iridaea undulosa

High-field 1H and 13C NMR spectroscopy has been used to characterize the fine structure of the cystocarpic and tetrasporic carrageenans from Iridaea undulosa. The proportion of different carrageenans from the κ-family in cystocarpic samples were calculated. In the 13C NMR spectra of the tetrasporic samples, twelve signals of approximately equal intensity were observed, indicating a nearly “perfect” λ-structure.

Ultra high field NMR spectroscopic studies on human seminal fluid, seminal vesicle and prostatic secretions

Ultra high field 1H-NMR spectroscopic methods have been used to analyse the composition of seminal fluid and its component secretions, prostatic and seminal vesicle fluids from normal human subjects and those with vasal aplasia and non-obstructive infertility. The 1H-NMR spectrum of whole seminal fluid is extremely complex and many resonances are extensively overlapped in single pulse spectra even when measured at 600 or 750 MHz 1H resonance frequency. A combination of 2-D 1H-NMR methods (including J-Resolved and various 1H homonuclear correlation and 1H 13C heteronuclear correlation techniques) were applied at 600 or 750 MHz in order to extensively assign the signals from the organic components of seminal fluid. Prostatic fluid (PF) gives a much less complex metabolite profile than whole seminal fluid and can be completely analysed using 500 MHz 1H-NMR spectroscopy. The 1H-NMR spectra of prostatic fluid are dominated by signals from citrate, spermine and myo-inositol, whereas the spectra of seminal vesicle fluid (SVF) show extensively overlapped signals from complex peptide mixtures together with strong signals for glycerophosphocholine (GPC) and lactate. Whole seminal fluid is a combination of the PF and SVF constituents together with further substances that appear after mixing due to the operation of PF enzymes on SVF, e.g. peptidase activity causes rapid cleavage of peptides to amino acids and GPC is hydrolysed to choline, glycerol and inorganic phosphate. It is also shown that vasal aplasia leads to highly characteristic abnormal metabolite profiles in seminal fluid that can be readily observed in single-pulse 500 and 600 MHz 1H-NMR spectra. Measurement of the molar citrate to choline, or spermine to choline ratios in seminal fluid both show differences of 2 orders of magnitude between vasal aplasia (greater for both ratios) and non-obstructed infertile patients. This work gives an indication of the potential of high field 1H-NMR spectroscopy in the investigation and assessment of the secretory functions of the male genital tract and the evaluation of the infertile male subject.

Guaianolides and an ethylcyclohexane lactone from Andriala integrifolia

The reinvestigation of the aerial parts of Andriala integrifolia afforded three new lactones, a guaianolide, a guaianolide glucopyranoside and an ethylcyclohexane lactonic derivative with a quinol structure, together with five known compounds. Their structures were established by high field 1H NMR spectroscopy and chemical correlation.

Sesquiterpenes of Cladanthus arabicus

A methanol extract of Cladanthus arabicus gave two sesquiterpenes. One was a new compound, cladantholide, identified as 2-oxy- 1αH,5αH,6βH,7αH,11α H-guai-3,4-en-6,12-olide. The other compound was sintenin elucidated as 3β,9β-diacetoxy-6βH,7αH-germacra- 1(10),4(5)-diene-6,12-olide. The structures of these compounds were substantiated by high field 1H and 13C NMR spectroscopy. The structure of cladantholide was confirmed by single X-ray diffraction.

Sesquiterpene glycosides from Calendula arvensis.

The extract of the aerial parts of Calendula arvensis afforded four new sesquiterpene glycosides 1-4 in addition to three known compounds. The structures of the new compounds were established by high field 1H-nmr spectroscopy.

On the formation of 2,3-dihydroxyacetophenone from pentoses or hexuronic acids

The structure of a previously isolated intermediate in the title reaction has been revised to 3-acetyl-2,3,4-trihydroxycyclohexanone by high-field 1H NMR spectroscopy. The three hydroxyl groups are mutually cis-related.

NMR spectroscopy as a novel approach to the monitoring of renal transplant function

High field 1H NMR spectroscopy was used for the rapid multicomponent analysis of low molecular wt compounds in urine in order to investigate the patterns of metabolic changes associated with early renal allograft dysfunction. Urine samples were collected daily for 14 days from 33 patients who underwent primary renal allograft transplantation, and analyzed by 500 and/or 600 MHz 1H NMR spectroscopy. All patients received 20 mg prednisolone and 5 mg/kg b.d. oral cyclosporin A (CsA) solution. In this study no patient showed clinical or histopathological evidence of CsA nephrotoxicity. For each patient the NMR-generated metabolite data were correlated with the clinical observations, graft biopsy pathology, and data from conventional laboratory techniques for assessing renal function. The NMR spectra of urine from patients with immediate functioning grafts were similar with respect to their patterns of amino acids, organic acids and organic amines, whereas the patients with delayed or non-functioning grafts showed significantly different metabolite excretion patterns. In longitudinal studies on individual patients there were increased urinary levels of trimethylamine-N-oxide (TMAO), dimethylamine (DMA), lactate, acetate, succinate, glycine and alanine during episodes of graft dysfunction. However, only the urinary concentration of TMAO was statistically significantly higher (P < 0.025) in the urine collected from patients during episodes of graft dysfunction (410 +/- 102 microM TMAO/mM creatinine) than in patients with good graft function (91 +/- 18 microM TMAO/mM creatinine) or healthy control subjects (100 +/- 50 microM TMAO/mM creatinine). These findings suggest that graft dysfunction is associated with damage to the renal medulla which causes the release of TMAO into the urine from the damaged renal medullary cells. This provides a possible novel urinary marker for post-transplant graft dysfunction. This study shows that NMR spectroscopy of biofluids, when used in combination with conventional laboratory techniques, is a valuable aid to renal transplant monitoring.

Arylbutanoid and diarylheptanoid glycosides from inner bark of Betula pendula

From the inner bark of Betula pendula, four new disaccharide glycosides, two of (−)-rhododendrol and two of platyphyllone, were isolated along with a new trisaccharide glycoside of (−)-centrolobol and several known compounds. The structures were elucidated by high field 1H and 13C NMR spectroscopy. Assignments of 1H and 13C NMR spectra were made by H,H- and C,H-COSY.

Determination of the structures of cystocarpic carrageenans from Gigartina skottsbergii by methylation analysis and NMR spectroscopy

The combined use of methylation analysis and high-field 1H and 13C NMR spectroscopy allows the determination of the fine structure of the carrageenans produced by the cystocarpic stage of Gigartina skottsbergii.

Ent-labdanes, manoyloxide and helipterol derivatives from Chrysocephalum ambiguum

The aerial parts of Chrysocephalum ambiguum afforded, in addition to two known diterpenes, 28 new ones, 22 ent-labdanes, four ent-manoyloxide derivatives and two monocyclic diterpenes derived from helipterol. The structures were elucidated by high field 1H NMR spectroscopy.

Structures of α- and β-turmerone

Procedures are described for isolating turmerones from Curcuma longa rhizomes and for chromatographically separating α- from β-turmerone. Using a combination of spectroscopic techniques (especially high field 1H NMR spectroscopy and chiroptical methods) these compounds are shown to be (1′R, 6S)-2-methyl-6-(4-methylcyclohexa-2,4-dienyl)hept-2-en-4-one 2(α-turmerone) and (1′R, 6S)-2-methyl-6-(4-methylenecyclohex-2-enyl)hept-2-en-4-one 3(β-turmerone).

Studies of the interaction of a meta-hydroxy analogue of hoechst 33258 with DNA by melting temperature, footprinting and high-resolution 1H NMR spectroscopy

A new analogue of Hoechst 33258 1 with a small structural change, i.e., the phenolic OH group being located meta rather than para, has been shown by footprinting, DNA melting studies and high-field 1H NMR spectroscopy to bind to the AATT sequences of DNA in a manner grossly similar to Hoechst 33258, although analysis by 1H NMR spectroscopy indicates that the phenolic end of the ligand could be prised away from the minor groove relative to Hoechst 33258, and that the major groove structure of d(CGCGAATTCGCG)2 is significantly perturbed by the binding of this Hoechst 33258 analogue.

Disaccharide compositional analysis of heparin and heparan sulfate using capillary zone electrophoresis

Capillary zone electrophoresis (CZE) was used to separate eight commercial disaccharide standards of the structure ΔUA2X(1 → 4)- d-GlcNY6X (where ΔUA is 4-deoxy-α- l- threo-hex-4-enopyranosyluronic acid, GlcN is 2-deoxy-2-aminoglucopyranose, S is sulfate, Ac is acetate, X may be S, and Y is S or Ac). These eight disaccharides had been prepared from heparin, heparan sulfate, and derivatized heparins. A similar CZE method was recently reported for the analysis of eight chondroitin and dermatan sulfate disaccharides (A. Al-Hakim and R. J. Linhardt, Anal. Biochem. 195, 68–73, 1991). Two of the standard heparin/heparan sulfate disaccharides, having an identical charge of −2, ΔUA2S(1 → 4)- d-GlcNAc and ΔUA(1 → 4)- d-GlcNS, were not fully resolved using standard sodium borate/boric acid buffer. This buffer had proven effective in separating chondroitin/dermatan sulfate disaccharides of identical charge. Resolution of these two heparin/heparan sulfate disaccharides could be improved by extending the capillary length, preparing the buffer in 2H 2O, or eliminating boric acid. Baseline resolution was achieved in sodium dodecyl sulfate in the absence of buffer. The structure and purity of each of the eight new commercial heparin/heparan sulfate disaccharide standards were confirmed using fast-atom-bombardment mass spectrometry and high-field 1H-NMR spectroscopy. Heparin and heparan sulfate were then depolymerized using heparinase (EC 4.2.2.7), heparin lyase II (EC 4.2.2.-), heparinitase (EC 4.2.2.8), and a combination of all three enzymes. CZE analysis of the products formed provided a disaccharide composition of each glycosaminoglycan. As little as 50 fmol of disaccharide could be detected by ultraviolet absorbance.

Probing the relationship between .alpha.-helix formation and calcium affinity in troponin C: proton NMR studies of calcium binding to synthetic and variant site III helix-loop-helix peptides

Three 34-residue peptides corresponding to the high-affinity calcium-binding site III and two variant sequences from the muscle protein troponin C (TnC) were synthesized by solid-phase techniques. The two variant 34-residue peptides had amino acid modifications at either the coordinating positions or both the coordinating and noncoordinating positions, which corresponded to the residues found in the low-affinity calcium-binding site II of TnC. High-field 1H NMR spectroscopy was used to monitor calcium binding to each peptide to determine the effect these amino acid substitutions had on calcium affinity. The dissociation constant of the native site III peptide (SCIII) was 3 x 10(-6) M, smaller than that of the peptide incorporating the ligands from site II (LIIL), 8 x 10(-6) M, and that with the entire site II loop (LII), 3 x 10(-3) M, which bound calcium very weakly. These calcium dissociation constants demonstrate that very minor amino acid substitutions have a significant effect on the dissociation constant and give some insight into why the dissociation constants for site III and IV in TnC are 100-fold smaller than those for sites I and II. The results suggest that the differences in coordinating ligands between sites II and III have very little effect on Ca2+ affinity and that the noncoordinating residues in the site II loop are responsible for the low affinity of site II compared to the high affinity of site III in TnC.

β-hydroxybutyrate: a urinary marker of imipenem induced nephrotoxicity in the cynomolgus monkey detected by high field 1H NMR spectroscopy

The use of 1H NMR as a complement to conventional clinical chemistry and histopathology resulted in the detection of hitherto unsuspected changes in urine composition as a result of imipenem induced nephrotoxicity. Large quantities of beta-hydroxybutyrate, as well as other ketone bodies were detected, indicating a disruption of energy metabolism. beta-Hydroxybutyrate may provide a useful non-invasive marker for imipenem toxicity.

Guaianolides from Brachylaena species

Guaianolides were isolated from two Brachylaena species. The compounds were all derived from desacyl cynaropicrin. The structures were elucidated by high field 1H NMR spectroscopy and the chemotaxonomy of the genus is discussed briefly.

Electrochemical Property and Formation of the Cation Radical and Dication of Dinaphtho[1,8-b,c]-1,5-dithiocin

A new symmetrical dithio peri-bridged naphthalene, dinaphtho[l,8-b,c]-1,5-dithiocin (1) has been synthesized. The cyclic voltammogram of 1 showed one reversible oxidation peak. ESR spectrum of the coned H2SO4 solution of 1 showed the formation of the cation radical. The formation of the dication of 1 was observed in the reaction of the corresponding monosulfoxide with concd D2SO4 by both high-field 1H and 13C NMR spectroscopy.

Molecular Recognition and Binding of a GC Site-Avoiding Thiazole-Lexitropsin to the Decadeoxyribonucleotide d-[CGCAATTGCG]2:1H-NMR Evidence for Thiazole Intercalation

The structural and dynamic aspects of the interaction of the thiazole containing lexitropsin (1) with an oligodeoxyribonucleotide were studied by high field 1H-NMR spectroscopy. Complete assignment of the 1H-NMR resonances of lexitropsin 1 was accomplished by 2D-NMR techniques. The complexation-induced chemical shifts and NOE cross peaks in the NOESY map of the 1:1 complex of lexitropsin (1) and d-[CGCAATTGCG]2 reveal that the thiazole ring of the lexitropsin (1) intercalates between dA4.A5 bases and the rest of the ligand resides in the minor groove of the AT rich core of decamer, thus occupying the 5'-AATT sequence on the DNA. Intercalation of the thiazole moiety of the drug has been detected by the presence of intermolecular NOEs both in the major and the minor groove of the decamer helix. The absence of intranucleotide NOEs between base protons and H1'/H2' protons suggested local unwinding of the binding site on the DNA. From COSY and NOESY methods of 2D-NMR, it was established that the N-formyl (amino) terminus of the thiazole lexitropsin (1) is projecting into the major groove towards A5H8 while the amidinium terminus lies in the minor groove towards the T7G8 base pairs of the opposite strand. The expected intranucleotide NOEs confirmed that the decadeoxyribonucleotide in the 1:1 complex exists in a right handed B-conformation. The presence of exchange signals along the binding site 5'-AATT indicated an exchange of the bound drug process wherein the rate of exchange between the two equivalent sites was estimated to be congruent to 130 s-1 at 30 degrees C and with delta G degrees of 62.4 kJ mol-1. Force field and Pi calculations permitted a rationalization of the experimentally observed binding mode in terms of preferred conformation of the ligand and repeat length in lexitropsins compared with the DNA receptor.

Chemical Composition, Particle Size Range, and Biological Activity of some Low Molecular Weight Heparin Derivatives

Several low molecular weight (LMW) heparin sodium derivatives from different sources, as well as some related regular heparin sodium preparations, were examined for chemical composition by high field (300 MHz) 1H NMR spectroscopy, for particle size range by quasi‐elastic light scattering (QELS) methods, and for anti‐coagulation potency and anti‐factor Xa activity by the standard U.S. Pharmacopeial assays described for regular heparin. The NMR spectra provided insight into possible modes of depolymerization used to generate the LMW heparins, as well as into the presence of dermatan sulfate or other chemical contaminants. The QELS analysis permitted the heparin preparations to be characterized and compared by virtue of their distinctive particle size distributions.

Irregular oxygenated monoterpenes from Achillea fragrantissima

From the aerial parts of A. fragrantissima, a santoline aldehyde and two highly oxygenated santoline derivatives were isolated in addition to several known compounds. The structures were elucidated by high field 1H NMR spectroscopy.