FIGURE 47.5. NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F.

FIGURE 47.5

NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F. Mild base treatment of the polysaccharide specifically breaks the phosphodiester linkage between rhamnose (Rha) and arabinitol and removes O-acetylation to give an oligosaccharide with the structure shown in the box. p denotes a pyranose ring. (A) 1H-NMR spectrum of the intact polysaccharide (at 70°C). Prominent, well-resolved signals come from the anomeric protons (H-1), the H-6 of rhamnose, and the methyl protons of O-acetyl groups. The signal from rhamnose D H-2 is shifted downfield (to the left) because of an O-acetyl group at D C-2. Residue G is an alditol with no anomeric center; all of its signals are in the crowded central region and are hard to assign. (B) 1H-NMR spectrum of the oligosaccharide (at 30°C). The signals from the oligosaccharide are sharper: The O-acetyl methyl signal has disappeared and the D H-2 signal is no longer shifted. Residue A is usually phosphorylated at C-3; the good resolution of the oligosaccharide spectrum allows identification of a small signal (A′) from H-1 of the small proportion of A phosphorylated at C-2. (C) Part of the HSQC spectrum used to assign 13C signals of the glycan (rhamnose H-6/C-6 signals omitted). This two-dimensional spectrum has 1H and 13C chemical shifts as X and Y axes. The guidelines (for the anomeric signals) show how 13C shifts may be determined from this spectrum once the 1H spectrum is assigned. These and other NMR spectra, taken together, can provide information on the number and type of residues in the repeating unit and their anomeric configuration, ring size (pyranose [p] or furanose [f]), linkage positions, substitution positions, and sequence. (D) The MALDI spectrum showing a molecular ion at m/z 1473 [M + Na]+ corresponding to a permethylated glycan of the composition PdeoxyHex3Hex3pent-ol. (E) The ESI-MS/MS spectrum of m/z 748 [M + 2Na]2+ showing fragment ions that define the sequence shown in the cartoon in panel F.

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From: Chapter 47, Structural Analysis of Glycans

Cover of Essentials of Glycobiology
Essentials of Glycobiology. 2nd edition.
Varki A, Cummings RD, Esko JD, et al., editors.
Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2009.
Copyright © 2009, The Consortium of Glycobiology Editors, La Jolla, California.

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