Saccharide sequencing

The RIC for mjz 1470 is shown in Fignre 5.29, while the MS-MS spectra from each of their [M-H] ions is presented in Fignre 5.30. A detailed stndy of these spectra and a comparison with spectra from compounds with known saccharide sequences is reqnired before strnctnral assignments may be made. It is not considered appropriate to carry ont snch an exercise in a text of this nature, nor to be necessary for the reader to be able to appreciate the value of this example. For this reason, the following paragraph is taken directly from reference [23] and interested readers may, at their leisure, examine Figure 5.30 in conjunction with this ... 

The methodology employed for the study of glycoproteins is somewhat different in that it is not just the saccharide sequence that is required but the position at which it is joined to the peptide backbone. 

Low-molecular-weight fragments produced by chemical depolymerization and extraction of standard heparin consist of heterogeneous polysaccharide chains of molecular weight 2,000 to 9,000. The LMWH molecules contain the pentasaccharide sequence necessary for binding to antithrombin III but not the 18-saccharide sequence needed for binding to thrombin. Compared to standard heparin, LMWH has a 2- to 4-fold greater antifactor Xa activity than antithrombin activity. 

The structures of the compounds were elucidated by a combination of NMR techniques (lH-, 13C-, and 13C-DEPT NMR) and chemical transformation, enzymatic degradation, and as well as mass spectrometry, which gives information on the saccharide sequence. A more recent approach consists of an extensive use of high-resolution 2D NMR techniques, such as homonuclear and heteronuclear correlated spectroscopy (DQF-COSY, HOHAHA, HSQC, HMBC) and NOE spectroscopy (NOESY, ROESY), which now play the most important role in the structural elucidation of intact glycosides. These techniques are very sensitive and non destructive and allow easy recovery of the intact compounds for subsequent biological testing. 

The details of the synthetic approach we have adopted for the preparation of all possible di- and trisaccharide sequences of the Y polysaccharide repeating unit have been published (16-19), together with the syntheses of two of the four possible tetrasaccharides (19,20). The synthesis of a third tetra-saccharide sequence not previously reported is described below. 

Factor Xa inhibitors are structurally diverse ranging fi-om peptides to proteins to heparin saccharidic sequences (59,60). They can be either naturally derived, recombinant or synthetic in origin. Molecular size differs between the inhibitors, as does specificity and lilies of fector Xa inhibition. The targeted binding site on fector Xa can differ between the inhibitors they can be direct binding to fector Xa or indirect via a cofector such as... 

Laine, R A, A calculation of all possible oligosaccharide isomers both branched and hnear yields 1.05 X lO stmctures for a reducing hexasaccharide the Isomer Barrier to development of singlemethod saccharide sequencing or synthesis systems, Glycobiology, 4, 759-767, 1994. 

The interesting chemistry of sialic acid has been discussed in several reviews [213,214,215]. Sialic acid is an important component of complex oligosaccharides, when it is placed in the terminal position, and often masks the penultimate saccharide sequences. In this position, it interacts with numerous receptors including the influenza virus neuraminidase, and receptors mediating intercellular interactions such as the selectins (members of the C-t) e animal lectin family) and siglecs (or sialoadhesins, members of the immunoglobulin superfamily). The structure of influenza B neuraminidase in complex with sialic acid has been solved by X-ray crystallography at 2.2 A resolution (O Fig. 17) [216]. 

FAST ATOM BOMBARDMENT (FAB) mass spectra gave the molecular weight of the whole glycoside and information on the saccharide sequence showing the sequential loss of more external monosaccharide units ... 

Studies by Ishihara (1994) and by Guimond et al. (1993) indicate that the interaction of FGFs with low-affinity receptors exhibits some degree of specificity, as different members of the FGF family require distinct saccharide sequences for bind-... 

Determination of saccharide sequences and interresidue linkage positions ... 

Multiple nucleotide/peptide/saccharide synthesis refers to the simultaneous (parallel) synthesis of a multitude of nucleotide/peptide/saccharide sequences, irrespective of the chain... 

In those eukaryotes which have been studied in some detail (i.e. those which are multicellular organisms) the opposite pressures may act. If complex saccharides are involved in cell adhesion, recognition or some other social process, there may be strong pressures to conserve structures, or to vary them only gradually and within strict rules. Indeed, many types of saccharide sequence do seem to be stongly conserved, but there is insufficient information about primitive eukaryotes to test whether this also applies to them. 

Figure 1.6 Complex Saccharide Structures of Glycoproteins. A large number of complex-type structures are possible and those shown in this figure illustrate some typical features. Fucose is commonly, but not always, found linked to C-6 of the first residue of A/-acetyl-glucosamine. Rarely short hetero-saccharide sequences occur linked to C-3 of either or both of the A-acetylglucosaminyl residues of the di-A-acetylchitobiosyl sequence. The attachment of A-acetylglucosamine to C-4 of the P-mannosyl residue is another variable feature. The outer chains vary in number and may be linear or branched, complete or incomplete and of various lengths. Sequences with two linear outer chains are termed biantennate , those with three are triantennate etc. (Yoshima, Furthmayr and Kobata, 1980 Yoshima, Takasaki and Kobata, 1980 Kornfeld, 1978.)...

E. Construction of Saccharide Sequences by Mechanisms Independent of Dolichol... 

Figure 4.13 The Structures of Saccharides with Blood Group I and i Specificities. These are an example of the presence of antigenic sequences inside saccharide sequences, unlike A and B which are always terminal.

Their experiments have depended upon assembling saccharide sequences in a wholly determined system. Purified glycosyltransferases from three different sources have been used to modify natural and partially synthetic substrates, in an attempt to discover how accurate glycosyl transfers are and what effect one transfer may have on another. Because the system is artifically constructed of enzymes and subtrates from disparate sources any specificities in the sequence of the products are a reflection of the extent to which the enzymes alone are the determinants of this. 

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