Diffusion of a highly charged supramolecular assembly: direct observation of ion association in water.

The diffusion coefficient of the self-assembled supramolecular cluster [Ga4L6]12- depends on the cationic counterions in solution. Diffusion coefficients were determined using the pulsed-gradient spin-echo 1H NMR method and fit using nonlinear least-squares refinement. Saturation studies revealed a small number of ion-association sites on the exterior of the assembly and the direct observation of ion association in water.

Synthesis of mono- and dideoxygenated alpha,alpha-trehalose analogs.

In this work, we describe the synthesis and NMR characterization of four mono- and four dideoxygenated analogs of alpha,alpha-D-trehalose. The symmetrical (2,2'-, 3,3'-, 4,4'- and 6,6'-) dideoxy analogs were obtained via selective protection and subsequent radical deoxygenation of the desired hydroxyl group set. The unsymmetrical (2'-, 3'-, 4'- and 6'-) monodeoxy analogs were synthesized by desymmetrization of alpha,alpha-trehalose and subsequent deoxygenation under radical conditions.

Supramolecular catalysis of unimolecular rearrangements: substrate scope and mechanistic insights.

A cavity-containing metal-ligand assembly is employed as a catalytic host for the 3-aza Cope rearrangement of allyl enammonium cations. Upon binding, the rates of rearrangement are accelerated for all substrates studied, up to 850-fold. Activation parameters were measured for three enammonium cations in order to understand the origins of acceleration.

Mechanistic investigation of the staudinger ligation.

The Staudinger ligation of azides and phosphines has found widespread use in the field of chemical biology, but the mechanism of the transformation has not been characterized in detail. In this work, we undertook a mechanistic study of the Staudinger ligation with a focus on factors that affect reaction kinetics and on the identification of intermediates. The Staudinger ligation with alkyl azides was second-order overall and proceeded more rapidly in polar, protic solvents.

Characterization of the acid stability of glycosidically linked neuraminic acid: use in detecting de-N-acetyl-gangliosides in human melanoma.

The glycosidic linkage of sialic acids is much more sensitive to acid hydrolysis than those of other monosaccharides in vertebrates. The commonest sialic acids in nature are neuraminic acid (Neu)-based and are typically N-acylated at the C5 position. Unsubstituted Neu is thought to occur on native gangliosides of certain tumors and cell lines, and synthetic de-N-acetyl-gangliosides have potent biological properties in vitro.

Exploring the glycan repertoire of genetically modified mice by isolation and profiling of the major glycan classes and nano-NMR analysis of glycan mixtures.

The production of mice with genetic alterations in glycosyltransferases has highlighted the need to isolate and study complex mixtures of the major classes of oligosaccharides (glycans) from intact tissues. We have found that nano-NMR spectroscopy of whole mixtures of N- and O-glycans can complement HPLC profiling methods for elucidating structural details. Working toward obtaining such glycan mixtures from mouse tissues, we decided to develop an approach to isolate not only N- and O-glycans, but also to separate out glycosphingolipids, glycosaminoglycans and glycosylphosphatidylinositol anchors.

Trans-sialidase from Trypanosoma cruzi catalyzes sialoside hydrolysis with retention of configuration.

The trans -sialidase from Trypanosoma cruzi is a member of the sialidase superfamily that functions as a sialidase in the absence of a carbohydrate acceptor. We have used(1)H nuclear magnetic resonance (NMR) spectroscopy to investigate the stereospecificity of the hydrolysis of two substrates, namely, 4-methyl-umbelliferyl- N -acetylneur-aminic acid and alpha(2-3)-sialyllactose, catalyzed by a recombinant T.cruzi trans -sialidase.

Molecular characterization and immunohistochemical localization of IV(4)GalNAcGgOse(4)Cer: a naturally occurring novel neutral glycosphingolipid in bovine brain.

A pair of novel neutral glycosphingolipids (Ngsls) has been identified in bovine brain. Their mobilities on thin layer chromatography were slightly different from a standard pentaglycosylceramide (nLcOse(5)Cer from bovine erythrocytes). The compounds were purified to homogeneity by column chromatography.

Accurate and precise measurement of heteronuclear long-range couplings by a gradient-enhanced two-dimensional multiple-bond correlation experiment.

We propose a phase-sensitive gradient-enhanced two-dimensional heteronuclear multiple-bond correlation (psge-2D HMBC) experiment for speedy, accurate, and precise measurement of 2JCH and 3JCH. The experiment does not suppress one-bond correlations. Rather, the value of a desired long-range JCH is obtained from the pertinent cross-peak pattern in the HMBC spectrum, using the corresponding 1JCH correlation pattern as reference.

A 1H NMR spectroscopic approach to the unambiguous determination of glycosyl linkage positions in oligosaccharides.

A sensitive (< 1 mg) and nondestructive method was devised for the unambiguous determination of the glycosyl linkage positions in oligosaccharides using 1H NMR spectroscopy. The technique is based on the absence of a hydroxyl group on the carbon atom that participates in the glycosidic linkage. The "missing" hydroxyl group is identified by recording 1H NMR spectra of the oligosaccharide in H2O and D2O.

Structure of the capsular polysaccharide of Clostridium perfringens Hobbs 5 as determined by NMR spectroscopy.

The complete primary structure of the capsular polysaccharide of Clostridium perfringens Hobbs 5, an anaerobic bacterium implicated in food poisoning, was determined. The polysaccharide was isolated from C. perfringens Hobbs 5 cells, after deproteination, by ethanol precipitation and by ion-exchange chromatography.

Molecular motions of a glycopeptide from human serum transferrin studied by 13C nuclear magnetic resonance.

The molecular motions of a 21-amino-acid glycopeptide (Gp21) containing multiple glycoforms of an N-linked diantennary oligosaccharide were studied by two-dimensional 1H-detected 13C relaxation measurements at natural abundance. Gp21 was derived from human serum transferrin, its amino acid sequence is QQQHLFGSNVTDCSGNFCLFR, and its N-glycan structure is [Formula: see text] The measured longitudinal and transverse relaxation rate constants and the nuclear Overhauser enhancements for the methine carbons of Gp21 were analyzed by using the model-free approach to obtain information about the internal motions in the molecule. The calculated order parameters S2 of the alpha carbons in both NH2- and COOH-terminal segments of the peptide are smaller than those in the interior segment of the Gp21 peptide moiety, implying that the internal motions in the terminal segments are less restricted than in the interior segment.

Complete 1H and 13C resonance assignments of a 21-amino acid glycopeptide prepared from human serum transferrin.

A 21-amino acid glycopeptide (Gp21) was isolated and purified in multi-milligram yields from commercially available human serum transferrin (HSTF) by a combination of tryptic digestion, Con A affinity chromatography, and reverse phase HPLC. The peptide chain of Gp21 contains a single N-glycosylation site to which a diantennary oligosaccharide is attached. The amino acid sequence and the glycan primary structure of Gp21 have been verified by peptide sequencing, electrospray mass spectrometry, and one-dimensional 1H NMR spectroscopy.

Molecular cloning of a developmentally regulated N-acetylgalactosamine alpha2,6-sialyltransferase specific for sialylated glycoconjugates.

A cDNA encoding a novel sialyltransferase has been isolated employing the polymerase chain reaction using degenerate primers to conserved regions of the sialylmotif that is present in all eukaryotic members of the sialyltransferase gene family examined to date. The cDNA sequence revealed an open reading frame coding for 305 amino acids, making it the shortest sialyltransferase cloned to date. This open reading frame predicts all the characteristic structural features of other sialyltransferases including a type II membrane protein topology and both sialylmotifs, one centrally located and the second in the carboxyl-terminal portion of the cDNA.

Chemical and immunological characterization of galactosyl-beta 1-3-globoside in bovine, human, and rat brain.

Our studies of bovine brain neutral glycosphingolipids (Ngsls) have revealed the presence of several short-chain (containing -CHO 1-4) and previously uncharacterized long-chain (-CHO > 4-5) Ngsls. We reported the structural characterization of brain GgOse4Cer (GA1) and have now purified another brain Ngsl to homogeneity. The purified Ngsl migrated close to standard GgOse4Cer and nLcOse5Cer on a TLC plate employing two different solvent systems.

Evidence for a transient interresidue hydrogen bond in sucrose in aqueous solution obtained by rotating-frame exchange NMR spectroscopy under supercooled conditions.

We have used the hydroxyl protons of sucrose dissolved in supercooled water as NMR probes for the detection of intramolecular hydrogen bonds. Neither the measured OH temperature shift coefficients, 3JHCOH scalar couplings, isotope-induced 13C chemical shifts (delta delta COH/COD), nor OH exchange rates allowed us to single out any hydroxyl group in sucrose with characteristics indicative of involvement in strong hydrogen bonding. However, two-dimensional rotating-frame exchange (ROESY) spectroscopy revealed a direct exchange between the glucosyl OH2 and fructosyl OH1 protons.

Molecular mass and carbohydrate structure of prostate specific antigen: studies for establishment of an international PSA standard.

Despite the widely accepted use of prostate specific antigen (PSA) as a marker of prostate cancer, this molecule has not yet been completely characterized. Past studies have well established, however, using both amino acid and cDNA sequencing techniques, that PSA contains 237 amino acids, with a molecular mass of 26,079 Da for the peptide moiety of the molecule. The present study reports analysis of this protein by ion spray mass spectrometry (ISMS) and analysis of its carbohydrate moiety by NMR spectroscopy.

Isolation and structural characterization of adhesin polysaccharide receptors.

The procedure for the purification of the adhesin polysaccharide receptor and its hexasaccharide repeating unit from whole S. oralis ATCC 55229 by chemical, enzymatic, and chromatographic techniques has been described. Chemical, chromatographic, and mass spectrometric procedures allow preliminary structural characterization of the hexasaccharide repeating unit and polysaccharide.

A revised structure for the disialosyl globo-series gangliosides of human erythrocytes and chicken skeletal muscle.

Disialosyl globo-series gangliosides have previously been isolated from chicken skeletal muscle (E. L. Hogan, R.

Sulfate detection in glycoprotein-derived oligosaccharides by artificial neural network analysis of Fourier-transform infrared spectra.

We report the use of an artificial neural network to analyze the fingerprint region of Fourier-transform infrared (ir) spectra of oligosaccharides for the presence of sulfate groups. This assay can rapidly and nondestructively detect the presence of sulfate in as little as 1 nmol (approximately 2 micrograms) of a glycoprotein-derived monosulfated decasaccharide. The neural network was trained to recognize the presence of sulfate groups by presenting it with 45 ir spectra of sulfated and nonsulfated mono- and oligosaccharides.

Branched monosialo gangliosides of the lacto-series isolated from bovine erythrocytes: characterization of a novel ganglioside, NeuGc-isooctaosylceramide.

We report the isolation, purification, and structural characterization of three monosialo gangliosides (G-1, G-2, and G-3) from bovine erythrocytes. Each of the purified compounds migrates as a single band on thin-layer chromatography in three solvent systems. All three gangliosides contain ceramide (Cer) as the lipid protein, with d18:1 sphingosine as the predominant long-chain base and with C18:0, C18:1, C20:0, C20:1, and C22:1 fatty acids, as determined by gas chromatography.

Carbohydrate dynamics at a micellar surface: GD1a headgroup transformations revealed by NMR spectroscopy.

The conformational dynamics of the carbohydrate headgroup of ganglioside GD1a, NeuAc alpha 2-->3Gal beta 1-->3GalNAc beta 1-->4[NeuAc alpha 2-->3]Gal beta 1-->4Glc beta 1-->1Cer, anchored in a perdeuterated dodecylphosphocholine micelle in aqueous solution, were probed by high resolution NMR spectroscopy. The observed 1H/1H NOE interactions revealed conformational averaging of the terminal NeuAc alpha 2-->3Gal and Gal beta 1-->3GalNAc glycosidic linkages. The pronounced flexibility of this trisaccharide moiety was substantiated further by two-dimensional proton-detected 13C T1, T1 rho and 1H/13C NOE measurements.

Structure determination of the intact major sialylated oligosaccharide chains of recombinant human erythropoietin expressed in Chinese hamster ovary cells.

Recombinant human erythropoietin (rHuEPO) is used abundantly in the clinic to stimulate red blood cell growth in anaemic patients. The efficacy of the drug depends strongly on the extent of sialylation of its carbohydrate moiety. Prompted by conflicting literature reports on the issue, we reinvestigated the structures of the intact sialylated carbohydrate chains of rHuEPO expressed in Chinese hamster ovary (CHO) cells.