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Structural Elucidation of the O-Antigen Polysaccharide from Escherichia coli O181.

Fontana C, Weintraub A, Widmalm G - ChemistryOpen (2014)

Bottom Line: Analysis of the high-resolution mass spectrum of the oligosaccharide (OS), obtained by dephosphorylation of the O-deacetylated PS with aqueous 48 % hydrofluoric acid, revealed a pentasaccharide composed of two QuiNAc, one GlcNAc, one GalNAc, and one Glc residue.The structure of the native PS was determined using NMR spectroscopy, and it consists of branched pentasaccharide repeating units joined by phosphodiester linkages: →4)[α-l-QuipNAc-(1→3)]-α-d-GalpNAc6Ac-(1→6)-α-d-Glcp-(1→P-4)-α-l-QuipNAc-(1→3)-β-d-GlcpNAc-(1→; the O-acetyl groups represent 0.4 equivalents per repeating unit.Both the OS and PSs exhibit rare conformational behavior since two of the five anomeric proton resonances could only be observed at an elevated temperature.

View Article: PubMed Central - PubMed

Affiliation: Arrhenius Laboratory, Department of Organic Chemistry, Stockholm University S-106 91 Stockholm (Sweden).

ABSTRACT
Shiga-toxin-producing Escherichia coli (STEC) is an important pathogen associated to food-borne infection in humans; strains of E. coli O181, isolated from human cases of diarrhea, have been classified as belonging to this pathotype. Herein, the structure of the O-antigen polysaccharide (PS) from E. coli O181 has been investigated. The sugar analysis showed quinovosamine (QuiN), glucosamine (GlcN), galactosamine (GalN), and glucose (Glc) as major components. Analysis of the high-resolution mass spectrum of the oligosaccharide (OS), obtained by dephosphorylation of the O-deacetylated PS with aqueous 48 % hydrofluoric acid, revealed a pentasaccharide composed of two QuiNAc, one GlcNAc, one GalNAc, and one Glc residue. The (1)H and (13)C NMR chemical shift assignments of the OS were carried out using 1 D and 2 D NMR experiments, and the OS was sequenced using a combination of tandem mass spectrometry (MS/MS) data and NMR (13)C NMR glycosylation shifts. The structure of the native PS was determined using NMR spectroscopy, and it consists of branched pentasaccharide repeating units joined by phosphodiester linkages: →4)[α-l-QuipNAc-(1→3)]-α-d-GalpNAc6Ac-(1→6)-α-d-Glcp-(1→P-4)-α-l-QuipNAc-(1→3)-β-d-GlcpNAc-(1→; the O-acetyl groups represent 0.4 equivalents per repeating unit. Both the OS and PSs exhibit rare conformational behavior since two of the five anomeric proton resonances could only be observed at an elevated temperature.

No MeSH data available.


Related in: MedlinePlus

The MS/MS spectra of the pentasaccharide compound obtained after cleavage of the phosphodiester groups of the O-deacetylated PS of E. coli O181: a) pseudomolecular [M+Na]+ ion m/z 983.4 recorded in positive mode; b) pseudomolecular [M−H]− ion m/z 959.4 recorded in negative mode. The detected ions are shown in the structure located on the top of each spectrum, and the fragmentation pathway[18] is indicated in parentheses. Double fragmentations are indicated with dashed lines. The monosaccharide residues are denoted A–E according to the nomenclature used in Table 1 and correspond to the respective primed or double primed characters.
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fig03: The MS/MS spectra of the pentasaccharide compound obtained after cleavage of the phosphodiester groups of the O-deacetylated PS of E. coli O181: a) pseudomolecular [M+Na]+ ion m/z 983.4 recorded in positive mode; b) pseudomolecular [M−H]− ion m/z 959.4 recorded in negative mode. The detected ions are shown in the structure located on the top of each spectrum, and the fragmentation pathway[18] is indicated in parentheses. Double fragmentations are indicated with dashed lines. The monosaccharide residues are denoted A–E according to the nomenclature used in Table 1 and correspond to the respective primed or double primed characters.

Mentions: Information about the monosaccharide sequence in the oligosaccharide was obtained by MS/MS in both the positive and negative modes from the precursor pseudomolecular ions m/z 983.4 (Figure 3 a) and 959.4 (Figure 3 b), respectively, which produced the corresponding daughter ions via A1-, B-, C- and E-type cleavages.[18] The daughter ion m/z 796.3 (Figure 3 a) is consistent with the loss of a terminal QuiNAc residue (residue C′/C′′ or D′/D′′ in Table 1). Furthermore, the daughter ion m/z 413.2 corresponds to a QuiNAc-HexNAc fragment obtained via an A1-type cleavage, whereas the daughter ion m/z 593.2 corresponds to the loss of a QuiNAc-HexNAc moiety (cf. Figure 3 a). According to these fragmentation patterns, the aforementioned HexNAc residue is monosubstituted and thus can be assigned to the GlcNAc residue (B′/B′′) of Table 1. From the NMR analysis it was revealed that a 6-substituted Glc residue (A′/A′′) is located at the reducing end of the oligosaccharide, which is consistent with the daughter ion m/z 839.3 observed in the spectrum of Figure 3 b. Furthermore, since both QuiNAc residues are located at terminal non-reducing ends (cf. Table 1) the fragment m/z 593.2 in Figure 3 a can be assigned to a QuiNAc-GalNAc-Glc moiety, where QuiNAc is either residue C′/C′′ or D′/D′′, GalNAc is residue E′/E′′, and Glc is residue A′/A′′. Due to the broadening of the resonances of residue B′/B′′, the inter-residue correlations involving these monosaccharides could not be observed in the 1H,13C-HMBC spectrum. However, unambiguous correlations were observed from the anomeric protons of residues D′/D′′ to C3 of residue E′/E′′, and from the anomeric proton of residues E′/E′′ to C6 of residues A′/A′′. Thus, the sequence in the oligosaccharide is as defined in Figure 3. Having considered this, the fragments m/z 592.2 and 202.2, observed in the MS/MS spectrum of Figure 3 b, are attributed to a β-elimination processes taking place at the C3 position of a 3-substituted HexNAc residue after a C-type cleavage of the HexNAc glycosidic bond.[22]


Structural Elucidation of the O-Antigen Polysaccharide from Escherichia coli O181.

Fontana C, Weintraub A, Widmalm G - ChemistryOpen (2014)

The MS/MS spectra of the pentasaccharide compound obtained after cleavage of the phosphodiester groups of the O-deacetylated PS of E. coli O181: a) pseudomolecular [M+Na]+ ion m/z 983.4 recorded in positive mode; b) pseudomolecular [M−H]− ion m/z 959.4 recorded in negative mode. The detected ions are shown in the structure located on the top of each spectrum, and the fragmentation pathway[18] is indicated in parentheses. Double fragmentations are indicated with dashed lines. The monosaccharide residues are denoted A–E according to the nomenclature used in Table 1 and correspond to the respective primed or double primed characters.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4380953&req=5

fig03: The MS/MS spectra of the pentasaccharide compound obtained after cleavage of the phosphodiester groups of the O-deacetylated PS of E. coli O181: a) pseudomolecular [M+Na]+ ion m/z 983.4 recorded in positive mode; b) pseudomolecular [M−H]− ion m/z 959.4 recorded in negative mode. The detected ions are shown in the structure located on the top of each spectrum, and the fragmentation pathway[18] is indicated in parentheses. Double fragmentations are indicated with dashed lines. The monosaccharide residues are denoted A–E according to the nomenclature used in Table 1 and correspond to the respective primed or double primed characters.
Mentions: Information about the monosaccharide sequence in the oligosaccharide was obtained by MS/MS in both the positive and negative modes from the precursor pseudomolecular ions m/z 983.4 (Figure 3 a) and 959.4 (Figure 3 b), respectively, which produced the corresponding daughter ions via A1-, B-, C- and E-type cleavages.[18] The daughter ion m/z 796.3 (Figure 3 a) is consistent with the loss of a terminal QuiNAc residue (residue C′/C′′ or D′/D′′ in Table 1). Furthermore, the daughter ion m/z 413.2 corresponds to a QuiNAc-HexNAc fragment obtained via an A1-type cleavage, whereas the daughter ion m/z 593.2 corresponds to the loss of a QuiNAc-HexNAc moiety (cf. Figure 3 a). According to these fragmentation patterns, the aforementioned HexNAc residue is monosubstituted and thus can be assigned to the GlcNAc residue (B′/B′′) of Table 1. From the NMR analysis it was revealed that a 6-substituted Glc residue (A′/A′′) is located at the reducing end of the oligosaccharide, which is consistent with the daughter ion m/z 839.3 observed in the spectrum of Figure 3 b. Furthermore, since both QuiNAc residues are located at terminal non-reducing ends (cf. Table 1) the fragment m/z 593.2 in Figure 3 a can be assigned to a QuiNAc-GalNAc-Glc moiety, where QuiNAc is either residue C′/C′′ or D′/D′′, GalNAc is residue E′/E′′, and Glc is residue A′/A′′. Due to the broadening of the resonances of residue B′/B′′, the inter-residue correlations involving these monosaccharides could not be observed in the 1H,13C-HMBC spectrum. However, unambiguous correlations were observed from the anomeric protons of residues D′/D′′ to C3 of residue E′/E′′, and from the anomeric proton of residues E′/E′′ to C6 of residues A′/A′′. Thus, the sequence in the oligosaccharide is as defined in Figure 3. Having considered this, the fragments m/z 592.2 and 202.2, observed in the MS/MS spectrum of Figure 3 b, are attributed to a β-elimination processes taking place at the C3 position of a 3-substituted HexNAc residue after a C-type cleavage of the HexNAc glycosidic bond.[22]

Bottom Line: Analysis of the high-resolution mass spectrum of the oligosaccharide (OS), obtained by dephosphorylation of the O-deacetylated PS with aqueous 48 % hydrofluoric acid, revealed a pentasaccharide composed of two QuiNAc, one GlcNAc, one GalNAc, and one Glc residue.The structure of the native PS was determined using NMR spectroscopy, and it consists of branched pentasaccharide repeating units joined by phosphodiester linkages: →4)[α-l-QuipNAc-(1→3)]-α-d-GalpNAc6Ac-(1→6)-α-d-Glcp-(1→P-4)-α-l-QuipNAc-(1→3)-β-d-GlcpNAc-(1→; the O-acetyl groups represent 0.4 equivalents per repeating unit.Both the OS and PSs exhibit rare conformational behavior since two of the five anomeric proton resonances could only be observed at an elevated temperature.

View Article: PubMed Central - PubMed

Affiliation: Arrhenius Laboratory, Department of Organic Chemistry, Stockholm University S-106 91 Stockholm (Sweden).

ABSTRACT
Shiga-toxin-producing Escherichia coli (STEC) is an important pathogen associated to food-borne infection in humans; strains of E. coli O181, isolated from human cases of diarrhea, have been classified as belonging to this pathotype. Herein, the structure of the O-antigen polysaccharide (PS) from E. coli O181 has been investigated. The sugar analysis showed quinovosamine (QuiN), glucosamine (GlcN), galactosamine (GalN), and glucose (Glc) as major components. Analysis of the high-resolution mass spectrum of the oligosaccharide (OS), obtained by dephosphorylation of the O-deacetylated PS with aqueous 48 % hydrofluoric acid, revealed a pentasaccharide composed of two QuiNAc, one GlcNAc, one GalNAc, and one Glc residue. The (1)H and (13)C NMR chemical shift assignments of the OS were carried out using 1 D and 2 D NMR experiments, and the OS was sequenced using a combination of tandem mass spectrometry (MS/MS) data and NMR (13)C NMR glycosylation shifts. The structure of the native PS was determined using NMR spectroscopy, and it consists of branched pentasaccharide repeating units joined by phosphodiester linkages: →4)[α-l-QuipNAc-(1→3)]-α-d-GalpNAc6Ac-(1→6)-α-d-Glcp-(1→P-4)-α-l-QuipNAc-(1→3)-β-d-GlcpNAc-(1→; the O-acetyl groups represent 0.4 equivalents per repeating unit. Both the OS and PSs exhibit rare conformational behavior since two of the five anomeric proton resonances could only be observed at an elevated temperature.

No MeSH data available.


Related in: MedlinePlus