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PubMed2003

Anionic adducts of oligosaccharides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The formation and decomposition (postsource decay, PSD) of anionic adducts in negative ion matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry have been studied. Chloride, a small inorganic anion, has been found to form stable anionic adducts with a variety of neutral oligosaccharides that can survive the MALDI process to give readily identifiable signals (with characteristic isotope patterns) allowing subpicomole detection in the best cases. The matrixes that can aid the formation of chloride adducts of oligosaccharides have gas-phase acidities lower than or close to that of HCl (1373 kJ/mol). In PSD experiments, precursor chloride adducts of oligosaccharides yield fragment ions that retain the charge on the sugar molecule rather than solely forming Cl-, and these fragments can provide structurally informative product ions. In negative ion MALDI, highly acidic oligosaccharides do not form adducts with chloride anions, but mildly acidic saccharides (e.g., containing a carboxylic acid group) form both deprotonated molecules and chloride adducts, and each may provide structural information concerning the oligosaccharide upon decomposition.

Acids↗
PubMed2003

Infrared multiphoton dissociation of alkali metal-coordinated oligosaccharides.

Infrared multiphoton dissociation (IRMPD) of alkali metal-coordinated oligosaccharides was obtained in a Fourier transform mass spectrometer. Fragmentation of the oligosaccharides was observed for Li+- and Na+-coordinated species. For larger alkali metal ions (K+, Rb+, and Cs+), the major products were the alkali metal ions. IRMPD experiments were performed on milk oligosaccharides, and the dissociation thresholds were determined. The threshold values were found to differ for the isomers. It is suggested that the threshold may be useful for differentiating isomeric compounds. Additionally, oligosaccharide alditols from biological samples were analyzed. Comparison of the collision-induced dissociation (CID) and IRMPD spectra of oligosaccharide alditols revealed that IRMPD could be used as a complementary method to obtain structural information.

Carbohydrate Sequence↗
PubMed2005

Heparin sequencing using enzymatic digestion and ESI-MSn with HOST: a heparin/HS oligosaccharide sequencing tool.

Mass spectrometry, and specifically sequential stages of mass spectrometry (MSn), is an established tool for the analysis of carbohydrates, proteins, and more recently glycosaminoglycans. As this trend continues, the development of algorithms for the rapid and automatic interpretation of mass spectra to identify glycan structure is also expected to grow as an active field of research. The methodology described herein utilizes a combination of enzymatic digestion, ESI-MS, and MSn for the sequencing of small heparin oligosaccharides. The heparin oligosaccharide sequencing tool (HOST) is a basic software application that was developed to aid in the integration and analysis of the data generated from these experiments, facilitating the process involved in arriving at sequence information. The sequences of several heparin oligosaccharides were determined using this method to illustrate proof of principle. Tandem MS is a very rapid and efficient tool for oligosaccharide analysis when limited amounts of material are available. Having a means, such as HOST, for automating the interpretation of the MSn data generated from glycosaminoglycans, provides a practical methodology for the future analysis of heparin/HS oligosaccharides of unknown structure.

Amino Acid Sequence↗
PubMed2006

Multistage mass spectrometric sequencing of keratan sulfate-related oligosaccharides.

To establish a universal protocol for sequencing keratan sulfate (KS) using mass spectrometry (MS), systematic electrospray ionization-MSn fragmentation experiments were carried out for 10 KS-related oligosaccharides of defined structure. Under the experimental conditions employed, fully charged molecular-related ions were observed as dominant peaks in all MS(1) spectra, which clearly reflected the number of sulfates and sialic acids in the oligosaccharide structures. In the subsequent MS2, almost all of the oligosaccharides gave fragment ions corresponding to their dehydrated molecular-related ions as well as (0,2)A(r) scission ions (according to the nomenclature developed by Domon and Costello, where "r" represents the reducing end in this study). Further fragmentation of the (0,2)A(r) ions in MS3 predominantly yielded the corresponding (2,4)A(r) ions. Finally, in MS(4), these (2,4)A(r) ions were subjected to extensive glycosidic cleavage. Hence, the MS4 data of KS oligosaccharides provided sufficient information for their sequence determination. In addition, some important features of MSn fragmentation became evident. These findings should lead to the establishment of consensus rules applied for KS oligosaccharides, including those previously unidentified, and also accelerate functional studies on KS, i.e., KS-related glycosaminoglycomics.

Carbohydrate Conformation↗
PubMed2006

Sequence determination of sulfated carrageenan-derived oligosaccharides by high-sensitivity negative-ion electrospray tandem mass spectrometry.

Negative-ion electrospray tandem mass spectrometry with collision-induced dissociation is assessed for sequence determination of multiply sulfated oligosaccharide fragments of carrageenan obtained from partial depolymerization of the polysaccharides by either enzymatic digestion or mild acid hydrolysis. Carrageenan oligosaccharides with homogeneous disaccharide compositions were used to establish their fragmentation pattern, which was then applied to sequence determination of unusual oligosaccharides with either "hybrid" biose compositions or odd-numbered residues. As sulfate groups are labile, sulfate loss during collision-induced association was prevented by sodium adduction. The product ion spectra of [M - Na]- (where M represents the sodium salt of oligosaccharides) feature an extensive series of B- and C-type glycosidic cleavages, whereas the Y-type cleavage occurs mainly at the sulfated residues. The assignment of reducing or nonreducing terminal fragments was assisted by oligosaccharide reduction and the product ion spectra of the derived alditols. Due to the anionic nature of the sulfate present, high-sensitivity detection (1-5 pmol, using a hexasaccharide as an example) was obtained.

Carbohydrate Sequence↗
PubMed1999

Catalog-library approach for the rapid and sensitive structural elucidation of oligosaccharides.

We obtained the nearly complete structural elucidation of oligosaccharide components, including sequence, linkage, and even stereochemistry in the picomolar levels. The "catalog-library" approach is used for elucidating the structures of minor components in a mixture of oligosaccharides. Oligosaccharides released from a family of glycoproteins are often composed of a small finite set of monosaccharides. In this regard, the numerous oligosaccharide species are analogous to the products found in syntheses involving combinatorial libraries. The great structural diversity in the library is the result of the nearly infinite combinations in which even a small number of monosaccharides can be arranged. Fortunately, structural similarities exist between different oligosaccharides, as specific substructural motifs are preserved among different compounds. We propose that a catalog of substructural motifs can be identified and characterized by collision-induced dissociation mass spectrometry. The catalog is constructed from a set of known compounds that have been fully structurally elucidated by, for example, nuclear magnetic resonance. The catalog consists of the characteristic fragmentation patterns belonging to a set of specific substructural motifs. Collision-induced dissociation is used to determine the presence of these motifs and reconstruct the structures of less abundant components.

Carbohydrate Conformation↗
PubMed1993

Modification of oligosaccharide antenna flexibility induced by exoglycosidase trimming.

We have investigated the solution conformation of a triantennary glycopeptide using resonance energy transfer [Rice et al. (1991) Biochemistry 30, 6646-6655]. Triantennary glycopeptide was derivatized with a donor fluorophore on the N-terminus and with an acceptor fluorophore attached individually to each terminal galactose residue, resulting in three isomeric donor-acceptor pairs. Time-resolved energy-transfer experiments revealed two distinct donor-acceptor distance populations for two of the three antennae of the oligosaccharide. An extended conformation and a folded conformation were detected for the two flexible antennae whereas the third antenna on the oligosaccharide was rigid, containing only an extended conformer. The ratios of the extended to folded conformers of the two flexible antennae were reversibly modulated by temperature, allowing the calculation of delta H and delta S for the conformational change [Wu et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 9355-9359]. In the present study, we have trimmed the fluorescent glycopeptides with exoglycosidases which specifically removed the unmodified antennae of the oligosaccharide. The resulting single-chain isomeric glycopeptides each contained identical core sugar residues and a terminally located donor and acceptor, but differed only in the linkage configuration of the sugar residues. Analysis of these glycopeptides by time-resolved energy transfer indicated that each antenna of the oligosaccharide is, by itself, maintained exclusively in the extended conformation. Temperature modulation failed to induce antenna folding as was previously observed for the complete triantennary structure. These data suggest that interantenna interactions modulate the conformation of individual antenna of complex oligosaccharides.

Carbohydrate Conformation↗
PubMed1982

Rabbit muscle phosphorylase derivatives with oligosaccharides covalently bound to the glycogen storage site.

Linear maltooligosaccharides, e.g., maltoheptaose or terminal 4-O-methylmaltoheptaose, activated by cyanogen bromide, react covalently with rabbit muscle phosphorylases b and a (EC 2.4.1.1). Site-specific modification prevents further binding to glycogen and shifts the phosphorylase a tetramer-dimer equilibrium in favor of the dimer. Use was made of these properties to separate by affinity chromatography and gel filtration phosphorylase a dimers with specifically bound oligosaccharide from unspecifically modified products. The phosphorylase a-maltoheptaose derivative carries one oligosaccharide residue per monomer and can be distinguished from the native enzyme by its electrophoretic mobility in polyacrylamide gels or by affinity electrophoresis. Phosphorylase a preparations with covalently bound maltooligosaccharides are enzymatically active in the presence of a primer and alpha-D-glucopyranose 1-phosphate (glucose-1-P). Methylation of the nonreducing chain terminus of the bound oligosaccharide has no effect on glycogen synthesis. These findings exclude the participation of bound oligosaccharides in chain elongation. Purified covalent phosphorylase a-maltoheptaose complexes are stable dimers. They are no longer activated by glycogen. The properties of covalently modified phosphorylase-oligosaccharides are consistent with and provide direct evidence for the existence of a glycogen storage site in rabbit muscle phosphorylases. Covalent occupation of the storage site renders the affinity of glucose-1-P to phosphorylase a independent of modulation by glycogen, supporting the assumption that the glycogen storage site is involved in interactions with the catalytic site.

Animals↗
PubMed1984

Glycosylation and processing of high-mannose oligosaccharides of thyroid-stimulating hormone subunits: comparison to nonsecretory cell glycoproteins.

Thyroid-stimulating hormone (TSH) subunit glycosylation was compared to that of total cell glycoproteins in mouse thyrotropic tumors. Lipid-linked oligosaccharides, total cell glycoproteins, and TSH subunits were labeled with either [3H]mannose, [3H]galactose, or [3H]glucose in pulse and pulse-chase experiments. The various oligosaccharides were isolated respectively by lipid extraction and mild acid hydrolysis, by selective immunoprecipitation, or by acid precipitation followed by trypsin and endoglycosidase H treatment. The nature of the oligosaccharides was assessed by their migration in paper chromatography, their relative incorporation of different precursors, and also their resistance to alpha-mannosidase. At 60 min, lipid-linked oligosaccharides were found to be composed of Glc3-2Man9GlcNAc2, Man9-8GlcNAc2, and Man5GlcNAc2. At 10 or 60 min of labeling, total cell proteins contained Glc3Man9GlcNAc2, Glc1Man9GlcNAc2, Man9GlcNAc2, Glc1Man8GlcNAc2, Man8GlcNAc2, and Man7GlcNAc2. The largest oligosaccharide, Glc3Man9GlcNAc2, had an unusually long half-life of about 2 h. In contrast, no Glc3Man9GlcNAc2 was found either on TSH + alpha subunits or on free beta subunits isolated either by immunoprecipitation or by sodium dodecyl sulfate gel electrophoresis. Instead, primarily Man9GlcNAc2 was found after a 10-min pulse both on TSH + alpha subunits and on beta subunits. When the pulse was followed by a chase up to 2 h, there was a progressive increase in Man8GlcNAc2 in higher amounts on TSH + alpha-subunit carbohydrate chains than on beta subunits.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Control of glycoprotein synthesis. Bovine milk UDPgalactose:N-acetylglucosamine beta-4-galactosyltransferase catalyzes the preferential transfer of galactose to the GlcNAc beta 1,2Man alpha 1,3- branch of both bisected and nonbisected complex biantennary asparagine-linked oligosaccharides.

Bovine milk UDPgalactose:N-acetylglucosamine beta-4-galactosyltransferase has been used to investigate the effect of a bisecting GlcNAc residue (linked beta 1,4 to the beta-linked mannose of the trimannosyl core of asparagine-linked complex oligosaccharides) on galactosylation of biantennary complex oligosaccharides. Columns of immobilized lectins (concanavalin A, erythroagglutinating phytohemagglutinin, and Ricinus communis agglutinin 120) were used to separate the various products of the reactions. Preferential galactosylation of the GlcNAc beta 1,2Man alpha 1,3 arm occurred both in the absence and in the presence of a bisecting GlcNAc residue; the ratio of the rates of galactosylation of the Man alpha 1,3 arm to the Man alpha 1,6 arm was 6.5 in the absence of a bisecting GlcNAc and 2.8 in its presence. The bisecting GlcNAc residue reduced galactosylation of the Man alpha 1,3 arm by about 78% probably due to steric hindrance of the GlcNAc beta 1,2Man alpha 1,3 beta 1,4 region of the substrate by the bisecting GlcNAc. This steric hindrance prevents the action of four other enzymes involved in assembly of complex asparagine-linked oligosaccharides and indicates the importance of the bisecting GlcNAc residue in the control of glycoprotein biosynthesis. The Man alpha 1,3 arm of biantennary oligosaccharides is believed to be freely accessible to enzyme action whereas the Man alpha 1,6 arm is believed to be folded back toward the core. This may explain the preferential action of Gal-transferase on the Man alpha 1,3 arm of both bisected and nonbisected oligosaccharides.

Acetylglucosamine↗
PubMed1986

Poly(N-acetyllactosaminyl) oligosaccharides in glycoproteins of PC12 pheochromocytoma cells and sympathetic neurons.

Endo-beta-galactosidase treatment of glycopeptides derived from the trypsinate and membranes of PC12 pheochromocytoma cells and cultured sympathetic neurons demonstrated the presence of poly(N-acetyllactosaminyl) units on tri- and tetraantennary oligosaccharides, some of which have a core fucose residue and a 2,6-substituted alpha-linked mannose residue. Nerve growth factor induced differentiation of the PC12 cells led to a small but significant decrease in the proportion of these oligosaccharides. Poly(N-acetyllactosaminyl) oligosaccharides were also identified in a major 230 000-Da cell-surface glycoprotein (the nerve growth factor inducible large external, or NILE, glycoprotein) of PC12 cells and appear to account for much or all of the difference in size between this glycoprotein as compared to the immunochemically cross-reactive 205 000-Da species present in postnatal brain. Glycoproteins containing poly(N-acetyllactosaminyl) oligosaccharides were selectively labeled by treatment of PC12 cells with endo-beta-galactosidase to expose N-acetylglucosamine residues, followed by incubation with galactosyltransferase and UDP-[14C]galactose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography revealed the presence of a number of distinct PC12 cell glycoproteins that contain these oligosaccharides and have apparent molecular weights in the range of 25 000-250 000. Treatment of PC12 cells with nerve growth factor (NGF) altered the relative labeling of several of the glycoprotein bands, with a time course similar to the effects of NGF on neurite outgrowth.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Gland Neoplasms↗
PubMed1989

Separation of the complex asparagine-linked oligosaccharides of the glycoprotein fetuin and elucidation of three triantennary structures having sialic acids linked only to galactose residues.

Asparagine-linked oligosaccharides of the glycoprotein fetuin were isolated as reducing oligosaccharides after hydrazinolysis/re-N-acetylation/mild acid treatment of the Pronase-digested glycoprotein. The sialylated oligosaccharides were separated by high-performance liquid chromatography in two different systems, which resulted in greater than 35 fractions, comprising di-, tri-, tetra-, and pentasialylated oligosaccharides. The major components were isomeric structures comprising the tri- and tetrasialylated fractions. In this and the accompanying paper (Cumming et al., 1989), the structures of 10 of the major components of the tri-, tetra-, and pentasialylated oligosaccharide fractions are described. Separation protocols and three isolated structures having sialic acid linked only to galactose are presented in this paper.

Asparagine↗
PubMed1990

Structure of the oligosaccharide of hen phosvitin as determined by two-dimensional 1H NMR of the intact glycoprotein.

The major form of the oligosaccharide of hen phosvitin was studied with two-dimensional 1H NMR of the intact glycoprotein. Its structure was determined from an analysis of the chemical shifts of the structural reporter groups, and it was further confirmed by comparison to several related model oligosaccharides. The oligosaccharide is N-linked and is present in a 1:1 stoichiometry to the protein. It has a complex type 1 triantennary structure with two NeuAc alpha 2,6Gal beta 1,4GlcNAc beta 1,2 arms linked to the Man-4 and Man-4' and a third Gal beta 1, 4GlcNAc beta 1,4 arm attached to the Man-4. The oligosaccharide contains the common core sequence which is present in all N-linked glycoproteins [Man alpha 1,3(Man alpha 1,6)-Man beta 1,4GlcNAc beta 1,4GlcNAc beta 1,N]. In the course of this study, we have found that unique spin systems for the GlcNAc and NeuAc are obtained for spectra recorded in 90% H2O. Their NH peaks were assigned at low pH, and these assignments proved useful for confirming the identity of cross-peaks in the anomeric region. In addition, the protons of GlcNAc-1 could be correlated to the NH of the asparagine link. The cross-peak patterns determined in phase-sensitive 2D experiments for the H1,H2 protons have a different appearance for each type of monosaccharide, and this information was also used for making first-order assignments. A comparison with model compounds suggests that the solution conformation of the oligosaccharide is not affected by its attachment to the protein.

Animals↗
PubMed2006

Complete lipopolysaccharide of Plesiomonas shigelloides O74:H5 (strain CNCTC 144/92). 1. Structural analysis of the highly hydrophobic lipopolysaccharide, including the O-antigen, its biological repeating unit, the core oligosaccharide, and the linkage between them.

The lipopolysaccharide of Plesiomonas shigelloides serotype O74:H5 (strain CNCTC 144/92) was obtained with the hot phenol/water method, but unlike most of the S-type enterobacterial lipopolysaccharides, the O-antigens were preferentially extracted into the phenol phase. The poly- and oligosaccharides released by mild acidic hydrolysis of the lipopolysaccharide from both phenol and water phases were separated and investigated by (1)H and (13)C NMR spectroscopy, MALDI-TOF mass spectrometry, and sugar and methylation analysis. The O-specific polysaccharide and oligosaccharides consisting of the core, the core with one repeating unit, and the core with two repeating units were isolated. It was concluded that the O-specific polysaccharide is composed of a trisaccharide repeating unit with the [-->2)-beta-d-Quip3NAcyl-(1-->3)-alpha-l-Rhap2OAc-(1-->3)-alpha-d-FucpNAc-(1-->] structure, in which d-Qui3NAcyl is 3-amino-3,6-dideoxy-d-glucose acylated with 3-hydroxy-2,3-dimethyl-5-oxopyrrolidine-2-carboxylic acid. The major oligosaccharide consisted of a single repeating unit and a core oligosaccharide. This undecasaccharide contains information about the biological repeating unit and the type and position of the linkage between the O-specific chain and core. The presence of a terminal beta-d-Quip3NAcyl-(1--> residue and the -->3)-beta-d-FucpNAc-(1-->4)-alpha-d-GalpA element showed the structure of the biological repeating unit of the O-antigen and the substitution position to the core. The -->3)-beta-d-FucpNAc-(1--> residue has the anomeric configuration inverted compared to the same residue in the repeating unit. The core oligosaccharide was composed of a nonphosphorylated octasaccharide, which represents a novel core type of P. shigelloides LPS characteristic of serotype O74. The similarity between the isolated O-specific polysaccharide and that found on intact bacterial cells and lipopolysaccharide was confirmed by HR-MAS NMR experiments.

Amides↗
PubMed1996

Influence of core fucosylation on the flexibility of a biantennary N-linked oligosaccharide.

Fluorescence energy transfer was used to study the conformation of each antenna of a complex biantennary oligosaccharide. A core fucosylated biantennary oligosaccharide was converted to a glycosylamine which allowed coupling of a naphthyl donor fluorophore directly to the reducing-end GlcNAc 1. After generating an aldehyde at C-6 of residue 6 or 6' using galactose oxidase, a dansyl ethylenediamine acceptor fluorophore was coupled to either antenna of the oligosaccharide resulting in two donor-acceptor pairs. [Formula: see text] The fluorescence properties of the naphthyl group allowed determination of the end-to-end donor-acceptor distance and antenna flexibility of each isomer by steady-state and time-resolved fluorescence energy transfer at temperatures ranging from 0 to 40 degrees C. Extended (20.6 A) and folded (11.4 A) donor-acceptor distance populations were identified for the isomer containing dansyl attached to Gal 6', whereas only a single extended population (19.7 A) was determined when dansyl was attached to Gal 6. The presence of Fuc 1' had a dramatic effect on the conformation of the 6' antenna. Temperature modulation failed to alter the ratio of extended/folded populations when fucose was present. However, following the removal of fucose, the ratio of the extended/folded populations for 6' exhibited a temperature dependent conformational equilibrium allowing calculation of the enthalpy and entropy of unfolding. These results established a unique conformational property for the 6' antenna of a biantennary oligosaccharide that is influenced by core fucosylation. Comparison of the results obtained for the 6 antenna of biantennary with previous fluorescence energy transfer studies on a triantennary glycopeptide also established conformational differences in this antenna which are dependent on oligosaccharide structure.

Carbohydrate Sequence↗
PubMed1996

Mutational analysis of the oligosaccharide recognition site at the active site of Escherichia coli maltodextrin phosphorylase.

A mutagenesis approach was applied to identify specific amino acid residues that are tentatively involved in binding of the oligosaccharide substrate at the active site of Escherichia coli maltodextrin phosphorylase. From ten residues located within a proposed channel connecting the enzyme surface with the active site, nine displayed significant effects on the reaction with oligosaccharide substrates when exchanged by mutagenesis. While several mutant enzymes (N258A/D259A/N260A, N307A, E350A, and Y578F) exhibited moderate decreases in apparent binding (about 4-17-fold), two mutations, H536L and E67A, weakened apparent binding of oligosaccharide substrates by 2 orders of magnitude. Two further mutant enzymes (T346G and H310A) displayed a 10-fold increase in the apparent Km of the oligosaccharide in the degradation reaction, while binding in the synthesis direction seemed less affected, indicating partially differential binding modes of oligosaccharides in synthesis and degradation. Quite uniquely, the H310A mutant enzyme exhibits a more than 100-fold-lowered Ki for gluconolactone, indicating the existence of an inhibitor binding site similar to that expected for a carbonium ion-like transition state. This is further confirmed by the finding that glucose, which does not inhibit wild-type enzyme, became an inhibitor of the H310A mutant enzyme (Ki = 20 mM). Since mutation of D308 did reduce kcat about 10-100-fold while Km values remained unchanged, a participation of this residue in transition state binding is probable. The insight into substrate recognition derived from this mutagenesis study corroborates a binding model where maltopentaose fits into the phosphorylase b structure in a distorted form.

Amino Acid Sequence↗
PubMed1996

Minor influence of sialic acid on conformation of a membrane-bound oligosaccharide recognition site.

Wideline 2H NMR spectroscopy was used to assess the conformational and orientational effects of N-acetylneuraminic acid (NeuAc) (sialic acid) as a component of a particular oligosaccharide chain at a bilayer membrane surface. For this purpose, three glycosphingolipids, sharing a neutral core tetrasaccharide and differing only in the number of sialic acid residues, were compared. The starting compound was GD1A, which has terminal sialic acid attached to the second and fourth sugars of its neutral tetrasaccharide core. GD1A was probe-labeled in a non-perturbing fashion on both of these sialic acid residues and on its single GalNAc residue by replacement of -COCH3 with -COCD3 giving [(d3NeuAc)2,d3-GalNAc]GA1a. This represents the most complex glycolipid to have been studied by 2H NMR spectroscopy at a bilayer membrane surface. The sialic acid residue on the fourth sugar from the membrane was subsequently removed to produce the glycolipid [d3NeuAc,d3GalNAc]GM1, deuterated at the two remaining amino sugars. The neutral glycolipid [d3GalNAc]asialo-GM1 was then generated by removal of the second sialic acid residue, leaving an uncharged species deuterated at one (internal) oligosaccharide chain site (GalNAc). The effect of sialic acid was futher examined by selective deuteration of GM1 and asialo-GM1 at C6 of the terminal Gal residue, giving [d2Gal]GM1 and [d2Gal]asialo-GM1. Spectra of the three glycosphingolipids were compared at 7.7 mol % in unsoncicated fluid bilayers of 1-palmitoyl-2-oleoylphosphatidylcholine containing 23 mol % cholesterol. For liposomes suspend in buffered salt solutions with 2 mM Ca2+, 2H NMR spectra demonstrated the presence of well defined average conformation for each oligosaccharide chain. This preferred average conformation persisted over a wide temperature range, consistent with there being a single major oligosaccharide conformer in each case. Spectral features arising from both deuterated amino sugar (GalNAc) of asialo-GM1 could be identified, little changed, in spectra of GM1 and GD1A. Similarly, deuterons in the terminal Gal residue of asialo-Gm1 produced the same spectrum seen for this residue in GM1. Our findings indicate that certain major conformational and orientational features of this complex oligosaccharide recognition site are preserved, within maximum angular deviation + or -5 degrees or less upon addition or removal of a sialic acid residue.

Carbohydrate Conformation↗
PubMed2001

First successful molecular design of an artificial Lewis oligosaccharide binding system utilizing positive homotropic allosterism.

We have designed phenylboronic acid group appended Ce(IV) bis(porphyrinate) double decker 1 and meso-meso linked porphyrin 2, useful for the allosteric binding of biologically important saccharides, Lewis oligosaccharides. Compound 1 binds Lewis oligosaccharides in aqueous media because of the boronic acid-diol interaction, but the complexation event can occur only above the critical concentrations because of the sigmoidal [oligosaccharide] versus [complex] isotherm. Compound 1 has a sufficiently high affinity with Lewis oligosaccharides (K = 10(5)-10(6) M(-2)) with Hill coefficients n of 1.8-2.0, and Lewis(X) series and Lewis(a) series give opposite, symmetrical CD spectra. This is the first example of efficient binding of Lewis oligosaccharides to the artificial receptor, which has become possible by positive homotropic allosterism.

Carbohydrate Conformation↗