The structure of the O-antigen polysaccharides (PS) from the enteroaggregative Escherichia coli strain 94/D4 and the international type strain E. coli O82 have been determined. Component analysis and (1)H, (13)C, and (31)P NMR spectroscopy experiments were employed to elucidate the structure. Inter-residue correlations were determined by (1)H, (13)C-heteronuclear multiple-bond correlation, and (1)H, (1)H-NOESY experiments. D-GroA as a substituent is linked via its O-2 in a phosphodiester-linkage to O-6 of the α-D-Glcp residue. The PS is composed of tetrasaccharide repeating units with the following structure: →4)-α-D-Glcp6-(P-2-D-GroA)-(1→4)-β-D-Galp-(1→4)-β-D-Glcp-(1→3)-β-D-GlcpNAc-(1→ Cross-peaks of low intensity from an α-D-Glcp residue were present in the NMR spectra and spectral analysis indicates that they originate from the terminal residue of the polysaccharide. Consequently, the biological repeating unit has a 3-substituted N-acetyl-D-glucosamine residue at its reducing end. Enzyme immunoassay using specific anti-E. coli O82 rabbit sera showed identical reactivity to the LPS of the two strains, in agreement with the structural analysis of their O-antigen polysaccharides.

Lipopolysaccharide, NMR, Escherichia coli, biological repeating unit, glyceric acid

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,4,4,6,0	lDGroA	175.48	77.10	63.41
3,4,4,6	P
3,4,4	aDGlcp	100.23	72.34	72.15	79.50	70.00	64.40
3,4	bDGalp	104.12	71.62	72.92	77.52	76.35	61.03
3	bDGlcp	103.83	73.58	75.02	79.62	75.60	60.93
2	Ac	175.45	23.16
	bDGlcpN	101.91	55.22	83.79	69.44	76.26	61.50


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,4,4,6,0	lDGroA	-	4.615	3.94
3,4,4,6	P
3,4,4	aDGlcp	4.86	3.58	3.84	3.67	4.30	4.06
3,4	bDGalp	4.49	3.58	3.73	4.01	3.78	3.84-3.91
3	bDGlcp	4.54	3.34	3.66	3.67	3.64	3.84-3.98
2	Ac	-	2.08
	bDGlcpN	4.65	3.94	3.80	3.58	3.57	3.80-3.95


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,4,4,6,0	lDGroA		77.10/4.615	63.41/3.94
3,4,4,6	P
3,4,4	aDGlcp	100.23/4.86	72.34/3.58	72.15/3.84	79.50/3.67	70.00/4.30	64.40/4.06
3,4	bDGalp	104.12/4.49	71.62/3.58	72.92/3.73	77.52/4.01	76.35/3.78	61.03/3.84-3.91
3	bDGlcp	103.83/4.54	73.58/3.34	75.02/3.66	79.62/3.67	75.60/3.64	60.93/3.84-3.98
2	Ac		23.16/2.08
	bDGlcpN	101.91/4.65	55.22/3.94	83.79/3.80	69.44/3.58	76.26/3.57	61.50/3.80-3.95

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*O[C@@H]1[C@@H](COP(=O)(O)O[C@H](CO)C(=O)O)O[C@H](O[C@H]2[C@@H](CO)O[C@@H](O[C@@H]3[C@@H](CO)O[C@@H](O[C@@H]4[C@@H](NC(C)=O)[C@H](*)O[C@H](CO)[C@H]4O)[C@H](O)[C@H]3O)[C@H](O)[C@H]2O)[C@H](O)[C@H]1O

857.658 g/mol (C₂₉H₄₈R₂NO₂₆P, R = next and previous repeats, not counted in mol weight)