Enteropathogenic Escherichia coli O125, the cause of infectious diarrheal disease, is comprised of two serogroups, viz., O125ab and O125ac, which display the aggregative adherence pattern with epithelial cells. Herein, the structure of the O-antigen polysaccharide from E. coli O125ac:H6 has been elucidated. Sugar analysis revealed the presence of fucose, mannose, galactose and N-acetyl-galactosamine as major components. Unassigned 1H and 13C NMR data from one- and two-dimensional NMR experiments of the O125ac O-antigen in conjunction with sugar components were used as input to the CASPER program, which can determine polysaccharide structure in a fully automated way, and resulted in the following branched pentasaccharide structure of the repeating unit: →4)[β-d-Galp-(1→3)]-β-d-GalpNAc-(1→2)-α-d-Manp-(1→3)-α-l-Fucp-(1→3)-α-d-GalpNAc-(1→, where the side chain is denoted by square brackets. The proposed O-antigen structure was confirmed by 1H and 13C NMR chemical shift assignments and determination of interresidue connectivities. Based on this structure, that of the O125ab O-antigen, which consists of hexasaccharide repeating units with an additional glucosyl group, was possible to establish in a semi-automated fashion by CASPER. The putative existence of gnu and gne in the gene clusters of the O125 serogroups is manifested by N-acetyl-d-galactosamine residues as the initial sugar residue of the biological repeating unit as well as within the repeating unit. The close similarity between O-antigen structures is consistent with the presence of two subgroups in the E. coli O125 serogroup.

Lipopolysaccharide, NMR spectroscopy, CASPER, CarbBuilder

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,3,2,2	Ac	?	23.36
3,3,2,3	bDGalp	106.00	71.55	73.48	69.64	75.90	61.97
3,3,2	bDGalpN	100.91	52.63	78.94	74.43	76.31	61.29
3,3	aDManp	100.08	77.71	70.37	68.39	74.33	62.58
3	aLFucp	101.79	68.49	77.98	72.42	67.88	16.05
2	Ac	?	23.01
	aDGalpN	98.09	49.85	76.38	69.27	70.90	60.82


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,3,2,2	Ac	-	2.05
3,3,2,3	bDGalp	4.45	3.54	3.63	3.93	3.66	3.77-3.80
3,3,2	bDGalpN	4.59	4.20	3.98	4.29	3.75	3.72-3.74
3,3	aDManp	5.16	4.21	3.95	3.58	3.74	3.66-3.90
3	aLFucp	5.11	3.89	4.02	3.97	4.18	1.22
2	Ac	-	2.07
	aDGalpN	4.99	4.44	4.09	4.14	4.50	3.80


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,3,2,2	Ac		23.36/2.05
3,3,2,3	bDGalp	106.00/4.45	71.55/3.54	73.48/3.63	69.64/3.93	75.90/3.66	61.97/3.77-3.80
3,3,2	bDGalpN	100.91/4.59	52.63/4.20	78.94/3.98	74.43/4.29	76.31/3.75	61.29/3.72-3.74
3,3	aDManp	100.08/5.16	77.71/4.21	70.37/3.95	68.39/3.58	74.33/3.74	62.58/3.66-3.90
3	aLFucp	101.79/5.11	68.49/3.89	77.98/4.02	72.42/3.97	67.88/4.18	16.05/1.22
2	Ac		23.01/2.07
	aDGalpN	98.09/4.99	49.85/4.44	76.38/4.09	69.27/4.14	70.90/4.50	60.82/3.80

The spectrum also has 2 signals at unknown positions (not plotted).

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

876.812 g/mol (C₃₄H₅₆R₂N₂O₂₄, R = next and previous repeats, not counted in mol weight)