Fine structure of the O-polysaccharide chain of the lipopolysaccharide (O-antigen) defines the serospecificity of bacterial cells, which is the basis for O-serotyping of medically and agriculturally important gram-negative bacteria including Escherichia coli. In order to obtain the O-polysaccharide for structural analysis, the lipopolysaccharide was isolated from cells of E. coli O84a by phenol/water extraction and degraded with mild acid. However, the O-polysaccharide was cleaved at a highly acid-labile β-l-fucopyranosyl phosphate (β-l-Fucp-1-P) linkage to give mainly a pentasaccharide that corresponded to the O-polysaccharide repeat. Therefore, the lipopolysaccharide and the pentasaccharide as well as their O-deacylated derivatives were studied using sugar analysis, NMR spectroscopy, and (for oligosaccharides) ESI HR MS, and the O84-polysaccharide structure was established. The O-polysaccharide is distinguished by the presence of β-l-Fucp-1-P and randomly di-O-acetylated 6-deoxy-d-talose, which are found for the first time in natural carbohydrates. The gene cluster for the O84-antigen biosynthesis was analysed and its content was found to be consistent with the O-polysaccharide structure.

Escherichia coli, O-specific polysaccharide, Glycosyl phosphate, bacterial polysaccharide structure, O-antigen gene cluster, 6-deoxy-D-talose

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
2,3,3,4	P
2,3,3	aDGlcp	101.3	72.8	73.5	75.0	72.5	61.7
2,3	aLFucp	101.0	68.5	78.7	73.0	68.1	16.3
2,2	Ac	175.7-175.8	23.5
2	bDGlcpN	102.5	56.9	81.3	70.1	77.2	62.8
3	aD6dTalp	103.5	70.7	66.7	73.4	68.9	16.8
	bLFucp	97.4	79.7	78.5	72.4	71.8	16.6


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
2,3,3,4	P
2,3,3	aDGlcp	5.20	3.60	3.94	3.91	3.87	3.80
2,3	aLFucp	5.01	3.89	3.91	3.94	4.34	1.14
2,2	Ac	-	2.01
2	bDGlcpN	4.71	3.85	3.66	3.47	3.46	3.73-3.97
3	aD6dTalp	5.26	4.05	3.95	3.74	4.08	1.24
	bLFucp	4.57	3.67	3.78	3.76	3.77	1.21


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
2,3,3,4	P
2,3,3	aDGlcp	101.3/5.20	72.8/3.60	73.5/3.94	75.0/3.91	72.5/3.87	61.7/3.80
2,3	aLFucp	101.0/5.01	68.5/3.89	78.7/3.91	73.0/3.94	68.1/4.34	16.3/1.14
2,2	Ac		23.5/2.01
2	bDGlcpN	102.5/4.71	56.9/3.85	81.3/3.66	70.1/3.47	77.2/3.46	62.8/3.73-3.97
3	aD6dTalp	103.5/5.26	70.7/4.05	66.7/3.95	73.4/3.74	68.9/4.08	16.8/1.24
	bLFucp	97.4/4.57	79.7/3.67	78.5/3.78	72.4/3.76	71.8/3.77	16.6/1.21

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CC(=O)N[C@H]1[C@H](O[C@@H]2[C@@H](O)O[C@@H](C)[C@@H](O)[C@H]2O[C@H]2O[C@H](C)[C@H](O)[C@H](O)[C@@H]2O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@@H]1O[C@@H](C)[C@@H](O)[C@@H](O[C@H]2O[C@H](CO)[C@@H](OP(=O)(O)O)[C@H](O)[C@H]2O)[C@@H]1O

901.755 g/mol (C₃₂H₅₆NO₂₆P)