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
3,4,0,6,2	Ac	174.55	22.89
3,4,0,6,3,2	Ac	174.61	23.19
3,4,0,6,3	aLQuipN	99.20	54.28	71.91	76.07	69.80	17.38
3,4,0,6	aDGalpN	98.47	50.06	74.75	72.11	72.06	61.75
3,4,0	aDGlcp	96.06	72.31	73.79	70.02	72.57	66.53
3,4	P
3,2	Ac	174.79	23.03
3	aLQuipN	98.04	54.28	70.96	81.24	67.80	17.34
2	Ac	174.90	23.53
	bDGlcpN	99.28	57.73	79.14	69.87	76.87	61.80


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,4,0,6,2	Ac	-	1.980
3,4,0,6,3,2	Ac	-	2.076
3,4,0,6,3	aLQuipN	4.945	3.968	3.626	3.306	3.683	1.317
3,4,0,6	aDGalpN	4.861	4.427	4.085	4.334	4.044	3.720-3.768
3,4,0	aDGlcp	5.562	3.584	3.727	3.584	3.995	3.708-4.009
3,4	P
3,2	Ac	-	2.085
3	aLQuipN	4.970	3.988	3.865	3.863	4.218	1.286
2	Ac	-	2.118
	bDGlcpN	5.165	3.567	3.972	3.525	3.472	3.762-3.916


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,4,0,6,2	Ac		22.89/1.980
3,4,0,6,3,2	Ac		23.19/2.076
3,4,0,6,3	aLQuipN	99.20/4.945	54.28/3.968	71.91/3.626	76.07/3.306	69.80/3.683	17.38/1.317
3,4,0,6	aDGalpN	98.47/4.861	50.06/4.427	74.75/4.085	72.11/4.334	72.06/4.044	61.75/3.720-3.768
3,4,0	aDGlcp	96.06/5.562	72.31/3.584	73.79/3.727	70.02/3.584	72.57/3.995	66.53/3.708-4.009
3,4	P
3,2	Ac		23.03/2.085
3	aLQuipN	98.04/4.970	54.28/3.988	70.96/3.865	81.24/3.863	67.80/4.218	17.34/1.286
2	Ac		23.53/2.118
	bDGlcpN	99.28/5.165	57.73/3.567	79.14/3.972	69.87/3.525	76.87/3.472	61.80/3.762-3.916

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

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

1022.898 g/mol (C₃₈H₆₃R₂N₄O₂₆P, R = next and previous repeats, not counted in mol weight)