The structure of the O-antigen from the international reference strain Escherichia coli O93:-:H16 has been determined. A nonrandom modal chain-length distribution was observed for the lipopolysaccharide, a pattern which is typical when long O-specific polysaccharides are expressed. By a combination of (i) bioinformatics information on the gene cluster related to O-antigen synthesis including putative function on glycosyl transferases, (ii) the magnitude of NMR coupling constants of anomeric protons and (iii) unassigned 2D 1H,13C-HSQC and 1H,1H-TOCSY NMR spectra it was possible to efficiently elucidate the structure of the carbohydrate polymer in an automated fashion using the computer program CASPER. The polysaccharide also carries O-acetyl groups and their locations were determined by 2D NMR experiments showing that ~½ of the population was 2,6-di-O-acetylated, ~¼ was 2-O-acetylated, whereas ~¼ did not carry O-acetyl group(s) in the 3-O-substituted mannosyl residue of the repeating unit. The structure of the tetrasaccharide repeating unit of the O-antigen is given by: →2)-β-d-Manp-(1→3)-β-d-Manp2Ac6Ac-(1→4)-β-d-GlcpA-(1→3)-α-d-GlcpNAc-(1→, which should also be the biological repeating unit and it shares structural elements with capsular polysaccharides from E. coli K84 and K50. The structure of the acidic O-specific polysaccharide from Cellulophaga baltica strain NN015840T differs to that of the O-antigen from E. coli O93 by lacking the O-acetyl group at O6 of the O-acetylated mannosyl residue.

Lipopolysaccharide, NMR spectroscopy, bioinformatics, CASPER, CarbBuilder

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,4,3	bDManp	97.17	78.41	74.18	68.09	77.41	61.89
3,4,2	Ac	173.95	21.32
3,4,6	Ac	174.67	20.99
3,4	bDManp	99.61	69.87	77.33	65.73	74.73	63.88
3	bDGlcpA	103.22	73.44	74.73	82.14	76.24	174.65
2	Ac	175.16	22.94
	aDGlcpN	99.66	53.77	81.30	68.80	71.86	60.74


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,4,3	bDManp	4.82	3.81	3.77	3.71	3.44	3.79-3.95
3,4,2	Ac	-	2.22
3,4,6	Ac	-	2.17
3,4	bDManp	4.82	5.55	4.07	3.61	3.67	4.37-4.46
3	bDGlcpA	4.57	3.40	3.61	3.75	3.79	-
2	Ac	-	2.03
	aDGlcpN	5.08	4.03	4.03	3.66	4.04	3.74-3.93


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,4,3	bDManp	97.17/4.82	78.41/3.81	74.18/3.77	68.09/3.71	77.41/3.44	61.89/3.79-3.95
3,4,2	Ac		21.32/2.22
3,4,6	Ac		20.99/2.17
3,4	bDManp	99.61/4.82	69.87/5.55	77.33/4.07	65.73/3.61	74.73/3.67	63.88/4.37-4.46
3	bDGlcpA	103.22/4.57	73.44/3.40	74.73/3.61	82.14/3.75	76.24/3.79	
2	Ac		22.94/2.03
	aDGlcpN	99.66/5.08	53.77/4.03	81.30/4.03	68.80/3.66	71.86/4.04	60.74/3.74-3.93

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

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

787.674 g/mol (C₃₀H₄₅R₂NO₂₃, R = next and previous repeats, not counted in mol weight)