MHz 1H NMR Spectroscopy Research Articles

600 MHz 1H-NMR spectroscopy of human cerebrospinal fluid: Effects of sample manipulation and assignment of resonances

Extensive assignments of resonances in the 600 MHz 1H-NMR spectra of cerebrospinal fluid are reported. These have been achieved by the measurement of a combination of two-dimensional experiments comprising homonuclear J-resolved, COSY45, and double-quantum filtered COSY (DQCOSY) spectra. By these means the previous total of 18 endogenous metabolites, of which in general only selected resonances have been assigned, has been augmented to 46 molecules including all of the resonances of both alpha- and beta-anomers of glucose. With only a few exceptions all resonances have been assigned for all of the metabolites. In addition, the effect of freeze-drying on the 600 MHz 1H-NMR spectrum of human cerebrospinal fluid (CSF) is presented using both lyophilization with reconstitution into either H2O or D2O. Freeze-drying and reconstitution into H2O causes a significant sharpening of many small molecule resonances, including notably those of glutamate and glutamine as well as other amino acids and in addition causes the loss of volatile components, principally acetone. Further exchange of the H2O solvent by D2O causes no additional changes in the spectra.

1H-NMR studies on thymopoietin-type oligopeptides — assignment of the proton resonances and investigation of conformational preferences

The thymopoietin-type tripeptides TP3 (HArg-Lys-AspOH), TP( d-Asp)3(HArg-Lys- d-AspOH) and tetrapeptide TP4 (HArg-Lys-Asp-ValOH) were studied by one- and two-dimensional, 500 MHz 1H-NMR spectroscopy in H 2O and D 2O solutions at four different pH values. All proton resonances of the three oligopeptides were assigned by two-dimensional phase-sensitive TOCSY experiments at pH 12.2, 9.1, 5.9 and 3.6. At these pH-values well-defined stages of protonation and concomitant molecular charges exist, allowing different possibilities for intra-molecular and inter-residual orientations. Conformation-sensitive rotating frame nuclear Overhauser enhancement (ROESY) two-dimensional experiments were also performed at the above pH values. These experiments indicated no definite solution conformation of any of the molecules at any pH. Standard one-dimensional experiments were also carried out and three-bond coupling constants were measured for the NHCH and the Asp CHCH moieties. The coupling constants provided evidence that non-statistical orientations of the functional groups exist which are changed upon protonation of the basic sites.

ChemInform Abstract: Synthesis of Diastereomerically Pure 4′‐Alkoxy‐α(L)‐ and ‐β( D)‐nucleosides and Their Conformational Analysis by 500 MHz 1H NMR Spectroscopy.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Conformational characteristics of kessane studied by 600 MHz 1H NMR and molecular mechanics

AbstractThe conformational characteristics of kessane were investigated by means of 600 MHz 1H NMR spectroscopy and molecular mechanics (MM2) calculations. Accurate vicinal 1H1H coupling constants were obtained by means of extensive computer simulations. These experimental values were compared with coupling constants calculated by the combined use of MM2 calculations and the generalized Karplus equation. It is shown that kessane adopts a single conformation in solution which is only slightly different from the conformation predicted by the MM2 calculations. The cyclopentane ring of kessane is characterized by a pure 1E conformation. The cycloheptane ring adopts a conformation which is situated precisely between the twist‐chair TC9 and the chair C5 conformation on the pseudo‐rotational pathway of the seven‐membered ring.

Structures of asparagine linked oligosaccharides of immunoglobulins (IgY) isolated from egg-yolk of Japanese quail

Structures of the Asn linked oligosaccharides of quail egg-yolk immunoglobulin (IgY) were determined in this study. Asn linked oligosaccharides were cleaved from IgY by hydrazinolysis and labelled with p-aminobenzoic acid ethyl ester (ABEE) after N-acetylation. The ABEE labelled oligosaccharides were then fractionated by a combination of Concanavalin A-agarose column chromatography and anion exchange, normal phase and reversed phase HPLC before their structures were determined by sequential exoglycosidase digestion, methylation analysis, HPLC, and 500 MHz 1H-NMR spectroscopy. Quail IgY contained only neutral oligosaccharides of the following categories: the glucosylated oligomannose type (0.6% Glc alpha 1-3Glc alpha 1-3Man9GlcNAc2; 35.6%, Glc alpha 1-3Man7-9GlcNAc2). oligomannose type (15.0%, with the structure Man5-9GlcNAc2) and biantennary complex type with core structures of -Man alpha 1-3(-Man alpha 1-6)Man beta 1-4GlcNAc beta 1-4GlcNAc (9.9%), -Man alpha 1-3 (GlcNAc beta 1-4)(-Man alpha 1-6)Man beta 1-4GlcNAc beta 1-4GlcNAc (25.1%) and -Man alpha 1-3(GlcNAc beta 1-4)(-Man alpha 1-6)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-6)GlcNAc (11.4%). Although never found in mammalian proteins, glucosylated oligosaccharides (Glc1Man7-9GlcNAc2) have been located previously in hen IgY.

Conformation of dehydropeptides

Six model dipeptide methylamides containing dehydroaminobutyric acid (delta Abu) of the type Boc-X-delta z Abu-NHCH3 and Box-X-delta E Abu-NHCH3, X = Ala, Val, Phe (Boc = tert-butoxycarbonyl), have been synthesized and their solution conformations explored using 300 MHz 1H NMR and IR spectroscopy. Studies based on delineation of intramolecularly hydrogen bonded NH groups in CDCl3 and (CD3)2SO revealed that none of the NH groups is appreciably solvent shielded. Difference NOE (Nuclear Overhauser Effect) studies have also failed to detect the presence of any discernible turn structure in these peptides. These studies indicate that the conformational preferences of peptides containing, alpha, beta-dehydroaminobutyric acid are different from those of delta ZPhe and delta ZLeu. It appears that steric interactions due to the beta-substituent in the dehydroamino acid moiety play an important role. Unlike delta ZPhe and delta ZLeu, which have relatively large beta-substituents, phenyl and isopropyl, respectively, and stabilize a beta-turn, the beta-methyl group of delta ZAbu or delta EAbu is readily accommodated in extended conformation. Clearly, the size of beta-substituent in dehydroamino acid crucially influences the conformational preferences. Thus, it may be possible to use different dehydroamino acids to introduce variable but definite constraints in synthetic peptides.

750 MHz 1H-NMR spectroscopy of human blood plasma

The application of high-resolution 750 MHz 1H-NMR spectroscopy to a biological fluid is demonstrated for the first time and its advantages over 600 MHz 1H observation shown by reference to studies on human blood plasma. Improvements in signal dispersion were observed which facilitated improved signal assignments. Differences in lipid/lipoprotein signal line-widths between 600 and 750 MHz were noted indicating that ultrahigh field measurements may help to give insight into dynamic motional phenomena of lipids in whole plasma. The two-dimensional J-resolved (JRES) technique and spin-echo spectra measured at 750 MHz have enabled new signal assignments to be made in control plasma. The application of 750 MHz JRES to the clinical chemical problem of the detection of abnormal metabolites associated with chronic renal failure is also demonstrated.

Enzymatic characterization of CMP-NeuAc:Galβ1-4GlcNAc-R α(2-3)-sialyltransferase from human placenta

In this report we present the enzymatic characterization of CMP-NeuAc:Gal beta 1-4GlcNAc-R alpha(2-3)-sialyltransferase from human placenta using placenta membranes as an enzyme preparation. This sialyltransferase is highly sensitive to detergents and prefers type 2 chain (Gal beta 1-4GlcNAc) over type 1 chain (Gal beta 1-3GlcNAc) acceptors. Oligosaccharides and glycopeptides were better acceptor substrates than glycoproteins. Of the branched oligosaccharides, those with a bisected N-acetylglucosamine (GlcNAc) structure appeared to be poorer substrates, while triantennary structures containing a Gal beta 1-4GlcNAc beta 1-4Man alpha 1-3Man branch were preferred. Product characterization, using 400 MHz 1H-NMR spectroscopy, confirmed that sialic acid was introduced into the Gal beta 1-4GlcNAc-R units of the acceptor substrates in an alpha (2-3) linkage, and revealed that this sialyltransferase does not prefer either of the two branches of a complex type di-antennary glycopeptide acceptor for sialic acid attachment. These properties distinguish this enzyme from all other sialyltransferases characterized to date.

Transformation of a kinetically controlled nematic phase of a linear polymer into one which is thermodynamically controlled via cyclization [1

Abstract The synthesis and characterization of cyclic main chain liquid-crystalline oligomers based on 1-(4-hydroxy-4′-biphenyl)-2-(4-hydroxyphenyl)butane (TPB) with 1,7-dibromoheptane (TPB-7(c)] are described. These oligomers were synthesized by the phase transfer catalysed polyetherification of TPB with 1,7-dibromoheptane under high dilution conditions and separated by column chromatography. Their cyclic structure was confirmed by 200 MHz 1H NMR spectroscopy. The mesomorphic behaviour of TPB-7(c) was characterized by differential scanning calorimetry and polarized optical microscopy. The cyclic dimer is only crystalline, while the cyclic trimer, tetramer and pentamer exhibit an enantiotropic nematic mesophase. The high molecular weight linear homologue TPB-7(1) exhibits a nematic mesophase which has an isotropization transition temperature located in the very close proximity of its glass transition temperature. Therefore, this nematic phase is kinetically controlled. Due to the higher rigidity of cyclic...

Synthesis of Diastereomerically Pure 4'-Alkoxy-alpha-(L)- and -beta(D)-nucleosides and their Conformational Analysis by 500 MHz 1H NMR Spectroscopy.

Synthesis of Diastereomerically Pure 4'-Alkoxy-alpha-(L)- and -beta(D)-nucleosides and their Conformational Analysis by 500 MHz 1H NMR Spectroscopy.

Investigation of the Structural Heterogeneity in the Carbohydrate Portion of a Mouse Monoclonal Immunoglobulin A Antibody

A mouse immunoglobulin A monoclonal antibody was isolated from hybridoma culture fluid by affinity chromatography. Chemical analysis of the intact antibody showed a monosaccharide composition, which besides mannose also contained monosaccharides commonly found in N-linked complex type of carbohydrate structures. No N-acetylgalactosamine was found showing the absence of O-linked oligosaccharides. The carbohydrate chains were released from the polypeptide and after fractionation on immobilized concanavalin A and high-performance ion-exchange chromatography structural analysis was performed. The structures were determined by chemical analyses, periodate oxidation in combination with fast atom bombardment mass spectrometry, and 500 MHz 1H NMR spectroscopy. The data revealed a great structural heterogeneity, including partially sialylated bi- and triantennary type of structures. Both types contained in addition species with branches terminated by Galα1-3Gal sequences.

Macrocycles with clearing temperatures higher than their linear high-molecular-weight homologues

The synthesis and characterization of macrocycles based on the conformationally flexible 1-(4-hydroxybiphenyl-4′-yl)-2-(4-hydroxyphenyl)butane (TPB) with 1,11-dibromoundecane [TPB–(c)11(z)], and TPB with 1,12-dibromododecane [TPB–(c)12(z)](where c = cyclic and z defines the degree of oligomerization of the macrocycle, i.e. z= 2 is a dimer; z= 3 is a trimer etc.) are described. These cyclic oligomers were synthesized by the phasetransfer catalyzed polyetherification of TPB with 1,11-dibromoundecane or 1,12-dibromododecane under high dilution conditions and were separated by column chromatography. Their cyclic structure was confirmed by 200 MHz 1H NMR spectroscopy. Most of the chemical shifts of the protons of the TPB unit are highly dependent on ring size owing to the conformational isomerism of the TPB mesogenic unit. The mesomorphic behaviour of TPB–(c)11(z) and TPB–(c)12(z) was characterized by differential scanning calorimetry (DSC) and optical polarized microscopy. TPB–(c)11(z) with z= 3,4 or 5 and TPB–(c)12(z) with z= 3 or 4 display equal or higher isotropization temperatures of their nematic mesophases than those of their linear high-molecular-weight homologues. Therefore, the mesophases of these macrocycles are stable over a broader range of temperature than those of their linear homologues. This was explained by the higher rigidity of the cyclic structure than that of the linear polymers, the absence of chain ends in the cyclic structure, and a suitable correlation between the TPB and the flexible spacer lengths.

Regioselectivity and stereoelectronic factors in the reactions of aryloxide nucleophiles with 4-nitrobenzofuroxan

The reactivity of 4-nitrobenzofuroxan (NBF, 1) with several aryloxide nucleophiles has been monitored by 400 MHz 1H NMR spectroscopy in acetonitrile–glyme (CD3CN–[2H10] glyme. 1 : 1, v/v) at reduced temperatures and in dimethyl sulfoxide ([2H6] DMSO) at ambient temperature. With the ambident (O- and C-) nucleophile phenoxide ion, formation of a C-7 O-adduct (3b) is observed in CD3CN–[2H10]glyme at –40 °C. As the temperature is raised this adduct gives way to 7-hydroxy-4-nitrobenzofurazan (as the anion, 5) and a 7-phenoxynitronate derivative (4b) that arises from transfer of the sp3-bound proton (7-H) of the C-7 phenoxide O-adduct to the N-oxide function. These decomposition products are the sole species observed when the reaction is carried out in [2H6] DMSO. Thus in contrast to the reaction of 1 with methoxide ion, where the C-5 adduct (2a) is kinetically preferred while the C-7 adduct (3a) is thermodynamically more stable, no C-5 O-adduct of phenoxide is observed in the present system. In addition, C-adducts that could potentially arise from the ambident nature of phenoxide were not formed. A similar reaction sequence was found for the reaction of 1 with 3,5-di-tert-butylphenoxide ion and 2,4,6-trimethylphenoxide ion. However, C-attachment was observed in the reaction of 1 with 2,6-di-tert-butylphenoxide ion where O-attack is sterically blocked and, again, preference for C-7 attachment was found. Comparison is drawn between the present results concerning the reactivity of 1 with aryloxide nucleophiles and the reactivity displayed by methoxide ion. Stereoelectronic factors in the C-7 aryloxide O-adducts are also discussed.

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.

Structural analysis of a low-sulfated chondroitin sulfate chain in human urinary trypsin inhibitor.

The low-sulfated chondroitin 4-sulfate(LSC) chain from human urinary trypsin inhibitor was purified and the structure was characterized. After hyaluronidase SD digestion of LSC, an oligosaccharide which contains the linkage region could be obtained. The structure of oligosaccharide was analyzed by HPLC and 500 MHz 1H-NMR spectroscopy. The analytical results revealed that 4-O-sulfo GalNAc residues were located in the neighborhood of the linkage region.

Sea anemone Radianthus macrodactylus — A new source of palytoxin

A very potent non-protein toxin was isolated from the sea anemone Radianthus macrodactylus with the use of chromatography on polytetrafluoroethylene, CM-Sephadex C-25 and by cation and anion exchange HPLC. The toxin was identified as palytoxin by u.v.-, i.r.- and 500 MHz 1H NMR spectroscopy. Its ld50 was 0.74±0.29 μg/kg by i.v. injection into mice. So far, palytoxin has been associated with zoanthids only. The toxin caused the loss of haemoglobin from erythrocytes but only in about 2 hr after the beginning of incubation, which is characteristic for palytoxin from zoanthids. Sea anemone palytoxin was divided into major and minor components by HPLC. The latter proved to be a product of degradation of palytoxin.

Novel structure of the N-acetylgalactosamine containing N-glycosidic carbohydrate chain of batroxobin, a thrombin-like snake venom enzyme.

The structure of the Asn-linked carbohydrate chain of batroxobin, a thrombin-like enzyme from Bothrops atrox moojeni snake venom, has been determined. The sugar chain was isolated from batroxobin by hydrazinolysis followed by pyridylamination (PA). The PA-oligosaccharide chain was purified by HPLC on an anion exchange or reverse phase columns, and its structure was examined by sequential exoglycosidase digestion, 600 MHZ 1H NMR spectroscopy and methylation analysis. The results indicate that the oligosaccharide chain has the following structure involving a novel linkage, NeuAc alpha 2----3GalNAc.

Ganglio- N-tetraosylceramide (GA1) of bovine and human brain Molecular characterization and presence in myelin

During our studies of bovine brain neutral glycosphingolipids (Ngsl's), we have purified a compound that co-migrates on thin-layer chromatogram with standard GA1 (purified by acid hydrolysis of GM1) and close to penta- (nLc 5Cer) glycosylceramide from bovine erythrocytes. The structure of the purified Ngsl from brain has been established by permethylation and by stepwise exoglycosidase hydrolysis. 600 MHz 1H NMR spectroscopy of the oligosaccharide obtained from the Ngsl after endoglycoceramidase hydrolysis confirms the structure as ganglio- N-tetraosylceramide (GgOse 4Cer or GA1) as Ga1β1→3Ga1NAcβ1-4Galβ1→4G1cβ1→1Cer. We have identified GA1 in bovine, rat and human brain and myelin by TLC-immunostaining with monospecific anti-GA1 antiserum.

Alpha 1-6(alpha 1-3)-difucosylation of the asparagine-bound N-acetylglucosamine in honeybee venom phospholipase A2.

Chymotryptic glycopeptides were prepared from a honeybee (Apis mellifica) venom phospholipase A2 (E.C. 3.1.1.4) fraction, with high affinity towards lentil (Lens culinaris) lectin. Treatment of the glycopeptide mixture with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase A, followed by HPLC fractionation, yielded two oligosaccharides, which were analysed by 500 MHz 1H-NMR spectroscopy to give the following structures [formula: see text] This is the first report on a naturally occurring glycoprotein N-glycan with two fucose residues linked to the asparagine-bound N-acetylglucosamine.

ChemInform Abstract: Synthesis of Furo(2,3‐c)pyran‐β‐D‐nucleosides by Radical Cyclization and Their Conformational Analysis by 500 MHz 1H NMR Spectroscopy.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.