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 1H and 13C 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 13C 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)[a-l-QuipNAc-(1-3)]-a-d-GalpNAc6Ac-(1-6)-a-d-Glcp-(1-P-4)-a-l-QuipNAc-(1-3)-b-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.

O-antigen, Escherichia coli, NMR spectroscopy, structure elucidation, polysaccharides

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
6,3,2	Ac	174.65	23.14
6,3	aLQuipN	99.07	54.31	71.73	76.01	69.81	17.37
6,2	Ac	174.65	22.80
6,4,3,2	Ac	174.95	23.02
6,4,3	aLQuipN	98.36	54.71	71.51	76.22	68.85	17.15
6,4,2	Ac	174.95	23.42
6,4	bDGlcpN	99.53	57.45	79.08	69.70	76.89	61.66
6	aDGalpN	98.24	50.02	74.62	72.14	72.01	61.74
	aDGlcp	93.03	72.38	73.78	70.29	71.10	66.88


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
6,3,2	Ac	-	2.073
6,3	aLQuipN	4.947	3.962	3.614	3.313	3.682	1.314
6,2	Ac	-	1.975
6,4,3,2	Ac	-	2.088
6,4,3	aLQuipN	4.969	3.949	3.689	3.240	4.139	1.244
6,4,2	Ac	-	2.120
6,4	bDGlcpN	5.113	3.627	3.915	3.536	3.472	3.764-3.921
6	aDGalpN	4.859	4.420	4.104	4.333	4.048	3.719-3.773
	aDGlcp	5.239	3.533	3.717	3.501	3.698	3.701-3.953


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
6,3,2	Ac		23.14/2.073
6,3	aLQuipN	99.07/4.947	54.31/3.962	71.73/3.614	76.01/3.313	69.81/3.682	17.37/1.314
6,2	Ac		22.80/1.975
6,4,3,2	Ac		23.02/2.088
6,4,3	aLQuipN	98.36/4.969	54.71/3.949	71.51/3.689	76.22/3.240	68.85/4.139	17.15/1.244
6,4,2	Ac		23.42/2.120
6,4	bDGlcpN	99.53/5.113	57.45/3.627	79.08/3.915	69.70/3.536	76.89/3.472	61.66/3.764-3.921
6	aDGalpN	98.24/4.859	50.02/4.420	74.62/4.104	72.14/4.333	72.01/4.048	61.74/3.719-3.773
	aDGlcp	93.03/5.239	72.38/3.533	73.78/3.717	70.29/3.501	71.10/3.698	66.88/3.701-3.953

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CC(=O)N[C@@H]1[C@H](O[C@H]2[C@H](O)[C@@H](CO)O[C@@H](O[C@H]3[C@@H](CO)O[C@H](OC[C@H]4O[C@H](O)[C@H](O)[C@@H](O)[C@@H]4O)[C@H](NC(C)=O)[C@H]3O[C@@H]3O[C@@H](C)[C@H](O)[C@@H](O)[C@@H]3NC(C)=O)[C@@H]2NC(C)=O)O[C@@H](C)[C@H](O)[C@H]1O

960.934 g/mol (C₃₈H₆₄N₄O₂₄)