O-Polysaccharides (O-antigens) are highly diverse glycopolymers present on the cell surface of Gram-negative bacteria, including the human opportunistic pathogen Providencia alcalifaciens. They define serospecificity of strains and are used for the serotyping of bacteria. In this work, a phosphorylated O-polysaccharide was isolated from P. alcalifaciens O22 and analyzed by chemical methods, ESI-MS, and 1H, 13C, and 31P NMR spectroscopy. It was found to contain two unusual components, 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose (D-FucNAc4N) and D-glyceramide 2-phosphate (D-GroAN-2-P), the latter being identified for the first time in bacterial polysaccharides. The structure of the trisaccharide repeating unit of the O-polysaccharide was established to be →4)-(D-GroAN-2→P→3)-D-GalNAc-(1→4)-D-Gal-(1→3)-D-FucNAc4N-(1→.

carbohydrates, lipopolysaccharides, antigens, structure elucidation

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,4,2	Ac	175.7-176.6	23.6-23.8
3,4	bDGalpN	104.3	54.0	72.7	76.5	75.5	62.0
3	bDGalp	106.2	71.9	73.6	78.1	75.5	62.4
2	Ac	175.7-176.6	23.6-23.8
	bDFucpN4N	103.3	52.5	78.4	56.0	68.8	16.8


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,4,2	Ac	-	2.05-2.07
3,4	bDGalpN	4.64	3.84	3.84	4.09	3.68	3.76-3.82
3	bDGalp	4.48	3.43	3.74	4.08	3.69	3.81-3.81
2	Ac	-	2.05-2.07
	bDFucpN4N	4.75	3.95	4.17	3.76	3.98	1.34


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,4,2	Ac		23.6-23.8/2.05-2.07
3,4	bDGalpN	104.3/4.64	54.0/3.84	72.7/3.84	76.5/4.09	75.5/3.68	62.0/3.76-3.82
3	bDGalp	106.2/4.48	71.9/3.43	73.6/3.74	78.1/4.08	75.5/3.69	62.4/3.81-3.81
2	Ac		23.6-23.8/2.05-2.07
	bDFucpN4N	103.3/4.75	52.5/3.95	78.4/4.17	56.0/3.76	68.8/3.98	16.8/1.34

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*O[C@H]1[C@@H](CO)O[C@@H](O[C@H]2[C@@H](CO)O[C@@H](O[C@@H]3[C@@H](NC(C)=O)[C@H](*)O[C@H](C)[C@@H]3N)[C@H](O)[C@H]2O)[C@H](NC(C)=O)[C@H]1O

551.546 g/mol (C₂₂H₃₇R₂N₃O₁₃, R = next and previous repeats, not counted in mol weight)