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Solutions of compound 1 exhibit dramatic, characteristic color changes in response to sugar analytes. Structurally related saccharides including glucose phosphates and amino and carboxylic acid sugars can be readily distinguished by visual inspection. These findings should promote the design of unique color sensory materials based on readily available, functional macrocyclic hosts.
[structure: see text] Heating aqueous DMSO solutions of five saccharides in the presence of 1-3 reveals that each receptor promotes solution colors characteristic for each sugar. New compound 3 allows the direct correlation of sugar concentration with visible region absorbance and/or fluorescence intensities.
In this work we have evaluated the potential to use wheat germ agglutinin (WGA) for weak affinity chromatography (WAC) of N-acetyl derivatives of mono-, di-, tri- and tetrasaccharides. WGA was used as a ligand in a high performance liquid affinity chromatography (HPLAC) system. Isocratic affinity chromatography was conducted where similar N-acetyl saccharides were separated according to their binding strength to WGA. Affinities are weak and lie typically in the mM range. For example, for 3'sialyllactose, the dissociation constant (Kd) was found to be 2.4 mM at 8 degrees C. It was interesting to note that the WGA-HPLC column can distinguish between the anomeric forms of N-acetylglucosamine. Weak affinity chromatography with immobilised WGA was used in an enzyme assay to detect the activity of GlcNAc-transferases.
Secreted epithelial mucins are large macromolecules which exhibit extreme polydispersity, the molecular basis of which is not fully understood. We have obtained partial sequences of two genes (BSM1 and BSM2) coding for two distinct molecules. This is the first time that such closely-related genes have been identified for any mucin from an animal. We propose that a combination of multiple homologous genes, alternative splicing, differential glycosylation, and additional post-translational processing all contribute to the extreme polydispersity of mucins. The multiple domain structure and non-identical tandem repeats are also very important for the generation of the saccharide diversities of mucins.
This review provides a chronological survey of over fifty fluorescent chemosensors for carbohydrates from the period between 1992 to the present. The survey contains only those sensors that are synthetic or chemosensory, utilize boronic acids and display a fluorescence response in the form of intensity changes or shifts in wavelength. With each compound listed, a description of the saccharide probe is given with regard to concentration, excitation and emission wavelengths, pH and solvent mixture proportions. In addition, the selectivity of each chemosensor is provided as well as the trends in binding constants. Where possible, a description of the fluorescence signaling mechanism is given as well as commentary on the probe's unique features within this class of sensors.
Galectins are beta-galactoside-binding proteins that occur intra- and extracellularly in many animal tissues. They have been proposed to form networks of glycoconjugates on the cell surface, where they may modulate various cell response pathways such as growth, activation and adhesion. The high resolution X-ray crystallographic analyses of three crystal forms of bovine galectin-1 in complex with biantennary saccharides of N-acetyllactosamine type reveal infinite chains of lectin dimers cross-linked through N-acetyllactosamine units located at the end of the oligosaccharide antenna. The oligosaccharide adopts a different low energy conformation in each of the three crystal forms.
A modular electrochemical saccharide sensor using ferrocene has been prepared which contains two boronic acid receptor groups and hexamethylene linker.
Glycopeptides that bind to MHC molecules on antigen presenting cells may elicit carbohydrate selective T cells. In order to investigate how the cellular immune response depends on the size of the carbohydrate moiety, a trigalactosylated derivative of an immunogenic peptide from hen egg-white lysozyme (HEL52-61) was prepared. Synthesis was accomplished by assembly of an alpha-1,4-linked trigalactose peracetate which was coupled to Fmoc serine. After activation as a pentafluorophenyl ester the resulting building block was used in solid-phase synthesis In contrast to the corresponding mono- and digalactosylated derivatives of HEL52-61, the trigalactosylated HEL52-61 was not immunogenic. Somewhat surprisingly, this was found to be because the trigalactosyl derivative bound approximately two orders of magnitude weaker to I-Ak MHC molecules than the mono- and digalactosyl peptides. Our observation suggests an explanation for previous findings, which show that glycopeptides isolated from MHC molecules in nature usually carry small saccharides.
Highly efficient boron removal from water was achieved by using mesoporous silica materials functionalised with saccharides.
Solid-state polycondensation of a natural saccharide was found to take place in the presence of H3PO4(5 mol%) at 110 degrees C under a N2 flow, giving a highly branched polysaccharide (conv. 11-84%, Mw = 1400-19000, Mn = 1200-3700); the reaction mixture was powdery throughout the polymerization. Interestingly, alpha- and beta-anomers showed different polymerization behaviour; the former was polymerized more slowly, however, they gave comparable molecular weight polymers. The polysaccharide product was per-O-methylated and subjected to structure analyses. The acid-hydrolysis products, the partially O-methylated monosaccharides, suggested that the polysaccharide products have highly branched structures. MALDI-TOF mass analysis revealed that intramolecular glycosylation and acetal exchange reactions are involved in the polymerization mechanism.
Pseudorotaxane formed by reacting beta-cyclodextrin bearing a phenylboronic acid residue with 1-heptyl-4-(4'-dimethylaminostyryl)pyridinium functioned as a novel fluorescent saccharide receptor having unique responses.
The minimal signalling unit for tyrosine kinase receptors is two protomers dimerized by one or more ligands. However, it is clear that maximal signalling requires the formation of larger complexes of many receptors at discrete foci on the cell surface. The biological interactions that lead to this are likely to be diverse and have system specific components. In the present study, we demonstrate that, in the FGF (fibroblast growth factor)-FGFR (FGF receptor) system, multimers of the minimal complex composed of two FGF1 and two FGFR2 protomers can form on a single chain of the co-receptor heparin. Using size-exclusion chromatography, we show that two complexes can form on heparin chains as small as 16 saccharide units. We also show by MS that discrete complexes containing exactly two copies of the minimal signalling unit are formed. However, the doublet of complexes appears to be less co-operative than the formation of the 2:2:1 FGF1:FGFR2:heparin complex, suggesting that this mechanism is one of a number of weaker interactions that might be involved in the formation of a focal complex on the cell surface.
Particulate membrane fractions from pig brain catalyse the synthesis of lipid-linked sugar derivatives of the dolichyl phosphate pathway. Flavomycin, a phosphoglycolipid antibiotic produced by various species of streptomycetes, interferes with the formation of these glycolipids to a different extent. The formation of dolichyl phosphate glucose was shown to be most susceptible to the antibiotic, being blocked by about 50% in the presence of 0.2mm-flavomycin, whereas the synthesis of dolichyl diphosphate N-acetylglucosamine, dolichyl diphosphate chitobiose and dolichyl diphosphate chitobiosyl mannose required higher concentrations to achieve a comparable inhibition. Although the formation of dolichyl phosphate mannose was hardly affected, the accumulation of oligosaccharides with five to seven sugar units was observed, when dolichyl diphosphate oligosaccharides were synthesized with GDP-[(14)C]mannose in the presence of 1mm-flavomycin. This indicates that the inhibition of the synthesis of larger-sized oligosaccharides, known to be mediated by lipid-bound mannose, was not caused by an actual deficiency in dolichyl phosphate mannose. At flavomycin concentrations that inhibited the formation of dolichyl phosphate glucose by 50%, the transfer of lipid-linked saccharides to either the hexapeptide Tyr-Asn-Gly-Thr-Ser-Val or endogenous protein acceptors was hardly influenced. The mode of action of flavomycin is still obscure, but seems not to be of a competitive nature, since the inhibition was unaffected by increasing concentrations of dolichyl phosphate. Some evidence indicates that, besides a direct interaction of the antibiotic with some transferases, a non-specific incorporation into the membrane and alteration of its properties might be responsible for those inhibitory effects on all enzymes which were observed at high concentrations of flavomycin.
The conformation of the L-iduronate residue in non-sulphated di-, tetra- and hexa-saccharides and their alditol derivatives derived from rooster comb dermatan sulphate was investigated by 400 MHz 1H-n.m.r. spectroscopy. The ratio of conformational isomers is obtained by the average spin-spin coupling constants of a mixture of nearly isoenergetic conformers (1C4, 4C1 and 2S0). The non-reducing terminal L-iduronate residue in the tetrasaccharides (I-H-I-H and I-H-G-H) and their alditols (I-H-I-H-ol and I-H-G-H-ol) is in equilibrium with three conformers (1C4, 30%; 4C1, 40%; 2S0, 30%) of nearly equal population. Whereas the internal L-iduronate residue in the tetrasaccharides (I-H-I-H and G-H-I-H) exists as an equilibrium mixture of 1C4 (54%) and 2S0 (42-44%) conformers, that of their alditols (I-H-I-H-ol and G-H-I-H-ol) is in equilibrium between 2S0 conformer (66%) and 1C4 conformer (28%). The conformational population for the internal L-iduronate residue 2I in the hexasaccharide (3I-H-2I-H-1I-H) is also calculated and compared with that for the L-iduronate residue in native dermatan sulphate, which was calculated on the basis of the spin-spin coupling constants reported by Gatti, Casu, Torri & Vercellotti [(1979) Carbohydr. Res. 68, c3-c7].
A simple and efficient synthetic glycosylation method suitable for use in solid-phase enzymic oligosaccharide synthesis and site-specific glycosylation of recombinant proteins to produce defined glycoforms is described. This strategy utilizes N-(beta-saccharide) haloacetamides for attaching oligosaccharides specifically to cysteine residues of proteins in solution to form neoglycoproteins. The alkylation reaction was tested using N-(beta-chitotriose) bromoacetamide and an unprotected synthetic hexapeptide containing a single cysteine residue. The glycosylated product was confirmed by amino acid and hexosamine analyses as well as laser desorption mass spectrometry. Similarly N-(beta-chitotriose) iodoacetamide was covalently linked to non-reduced BSA to produce a defined glycoform of this protein. The specific attachment of chitotriose at the single cysteine residue in non-reduced serum albumin was suggested by Ellman's assay for free thiols. This was verified by amino acid sequencing of tryptic glycopeptide derived from this neoglycoprotein. Multiple sugar attachment was accomplished using fully reduced serum albumin as demonstrated by the formation of two neoglycoproteins using iodoacetamide derivatives of galactose beta 1-3-N-acetylgalactosamine (Gal beta 1-3GalNAc) and the major xylose/fucose-class plant-type oligosaccharide of horseradish peroxidase. These two neoglycoproteins with an average of 18-21 sugar residues attached were assayed positively for binding to peanut agglutinin and a sugar-specific anti-(horseradish peroxidase) monoclonal antibody YZ1/2.23 respectively. Sialylation of the neoglycoprotein containing Gal beta 1-3GalNAc was accomplished using alpha-2,3-sialyltransferase and radiolabelled CMP-N-acetylneuraminic acid. Significantly, glycan attachment using this conjugation method is reversible as demonstrated by the release of oligosaccharides from these two neoglycoproteins using hydrazinolysis. Therefore this method could provide invaluable reagents for many glycobiological studies.
The presence of sulphate groups on various saccharide residues of N-linked carbohydrate units has now been observed in a number of glycoproteins. To explore the cell specificity of this post-translational modification, we evaluated sulphate incorporation into virus envelope glycoproteins by a variety of cells, since it is believed that assembly of their N-linked oligosaccharides is to a large extent dependent on the enzymic machinery of the host. Employing the vesicular stomatitis virus (VSV) envelope glycoprotein (G protein) as a model, we noted that the addition of [35S]sulphate substituents into its complex carbohydrate units occurred in Madin-Darby canine kidney (MDCK), Madin-Darby bovine kidney, LLC-PK1 and BHK-21 cell lines but was not detectable in BRL 3A, BW5147.3, Chinese hamster ovary, HepG2, NRK-49F, IEC-18, PtK1 or 3T3 cells. The sulphate groups were exclusively located on C-3 of galactose [Gal(3-SO4)] and/or C-6 of N-acetylglucosamine [GlcNAc(6-SO4)] residues in the N-acetyllactosamine sequence of the branch chains. Moreover, we observed that the pronounced host-cell-dependence of the terminal galactose sulphation was reflected by the 3'-phosphoadenosine 5'-phosphosulphate:Gal-3-O-sulphotransferase activity assayed in vitro. Comparative studies carried out on the haemagglutinin of the influenza virus envelope formed by MDCK and LLC-PK1 cells indicated that sulphate in this glycoprotein was confined to its complex N-linked oligosaccharides where it occurred as Gal(3-SO4) and GlcNAc(6-SO4) on peripheral chains as well as on the mannose-substituted N-acetylglucosamine of the core. Since sulphation in both internal and peripheral locations of the virus glycoproteins was found to be arrested by the alpha1-->2 mannosidase inhibitor, kifunensine, as well as by the intracellular migration block imposed by brefeldin A, it was concluded that this modification is a late biosynthetic event which most likely takes place in the trans-Golgi network.
The positions of the ordered hydrogen isotopes of a protein and its bound solvent can be determined by using neutron crystallography. Furthermore, by collecting neutron data at cryo temperatures, the dynamic disorder within a protein crystal is reduced, which may lead to improved definition of the nuclear density. It has proved possible to cryo-cool very large Con A protein crystals (>1.5 mm3) suitable for high-resolution neutron and x-ray structure analysis. We can thereby report the neutron crystal structure of the saccharide-free form of Con A and its bound water, including 167 intact D2O molecules and 60 oxygen atoms at 15 K to 2.5-A resolution, along with the 1.65-A x-ray structure of an identical crystal at 100 K. Comparison with the 293-K neutron structure shows that the bound water molecules are better ordered and have lower average B factors than those at room temperature. Overall, twice as many bound waters (as D2O) are identified at 15 K than at 293 K. We note that alteration of bound water orientations occurs between 293 and 15 K; such changes, as illustrated here with this example, could be important more generally in protein crystal structure analysis and ligand design. Methodologically, this successful neutron cryo protein structure refinement opens up categories of neutron protein crystallography, including freeze-trapped structures and cryo to room temperature comparisons.
Three allelic variants of P-pilus-associated G-adhesins (lectins) with different cell-binding properties were recently described. Here we have analyzed Escherichia coli HB101 strains expressing the recombinant G-adhesin variants for their ability to agglutinate erythrocytes from various species as this relates to the glycosphingolipid (GSL) composition in the erythrocyte membranes. All three variants exhibit similar specificities for the globo-series GSLs affixed to artificial surfaces. However, only the PapGJ96 adhesin induces agglutination of erythrocytes having globotriaosylceramide (GbO3) [Gal(alpha 1-4)LacCer] as the major GSL. Furthermore, only PapGAD110 induces strong agglutination of erythrocytes having globotetraosylceramide (GbO4) [GalNAc(beta 1-3)Gal(alpha 1-4)LacCer] as the major GSL, while PrsGJ96 alone agglutinates those containing globopentaosylceramide (GbO5) [GalNAc(alpha 1-3)GalNAc(beta 1-3)Gal(alpha 1-4)LacCer]. Molecular modeling of these globo-GSLs demonstrates different saccharide orientations to the membrane surface for these isoreceptors. We suggest that the differential binding of the three G-adhesin variants results from differences in epitope presentation at the membrane among these globo-GSLs.