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	97.22	78.02	74.28	68.09	77.46	61.80
3,4,2	Ac	174.04	21.32
3,4	bDManp	99.35	69.97	77.63	66.04	77.41	61.65
3	bDGlcpA	103.22	73.51	74.73	81.58	76.24	174.65
2	Ac	175.16	22.94
	aDGlcpN	99.40	53.77	81.21	68.91	71.90	60.81


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,4,3	bDManp	4.82	3.83	3.77	3.71	3.44	3.79-3.95
3,4,2	Ac	-	2.22
3,4	bDManp	4.81	5.53	4.04	3.54	3.47	3.77-3.94
3	bDGlcpA	4.57	3.40	3.61	3.78	3.79	-
2	Ac	-	2.03
	aDGlcpN	5.11	4.03	4.03	3.66	4.04	3.73-3.91


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,4,3	bDManp	97.22/4.82	78.02/3.83	74.28/3.77	68.09/3.71	77.46/3.44	61.80/3.79-3.95
3,4,2	Ac		21.32/2.22
3,4	bDManp	99.35/4.81	69.97/5.53	77.63/4.04	66.04/3.54	77.41/3.47	61.65/3.77-3.94
3	bDGlcpA	103.22/4.57	73.51/3.40	74.73/3.61	81.58/3.78	76.24/3.79	
2	Ac		22.94/2.03
	aDGlcpN	99.40/5.11	53.77/4.03	81.21/4.03	68.91/3.66	71.90/4.04	60.81/3.73-3.91

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](CO)[C@H]2O)O[C@H](CO)[C@@H](O)[C@@H]1O

745.637 g/mol (C₂₈H₄₃R₂NO₂₂, R = next and previous repeats, not counted in mol weight)