Mild acid hydrolysis of the lipopolysaccharide of Escherichia coli O54 afforded an O-polysaccharide, which was studied by sugar analysis, solvolysis with anhydrous trifluoroacetic acid, and 1H and 13C NMR spectroscopy. Solvolysis cleaved predominantly the linkage of β-d-Ribf and, to a lesser extent, that of β-d-GlcpNAc, whereas the other linkages, including the linkage of α-l-Rhap, were stable under selected conditions (40 °C, 5 h). The following structure of the O-polysaccharide was established: →4)-α-d-GalpA-(1 → 2)-α-l-Rhap-(1 → 2)-β-d-Ribf-(1 → 4)-β-d-Galp-(1 → 3)-β-d-GlcpNAc-(1→ The O-antigen gene cluster of E. coli O54 was analyzed and found to be consistent in general with the O-polysaccharide structure established but there were two exceptions: i) in the cluster, there were genes for phosphoserine phosphatase and serine transferase, which have no apparent role in the O-polysaccharide synthesis, and ii) no ribofuranosyltransferase gene was present in the cluster. Both uncommon features are shared by some other enteric bacteria.

O-antigen, Escherichia coli, O-polysaccharide, bacterial polysaccharide structure, O-antigen gene cluster

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,4,2,2	aDGalpA	99.3	69.4	70.4	80.0	72.1	174.5
3,4,2	aLRhap	99.7	77.7	70.6	73.2	70.7	17.8
3,4	bDRibf	108.1	82.2	71.3	83.9	63.8
3	bDGalp	104.7	72.1	74.1	76.8	76.0	62.5
2	Ac	176.1-176.2	23.7
	bDGlcpN	103.1	56.1	83.6	69.9	76.3	62.3


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,4,2,2	aDGalpA	5.05	3.72	4.05	4.42	4.88	-
3,4,2	aLRhap	5.07	4.07	3.89	3.47	3.77	1.30
3,4	bDRibf	5.44	4.22	4.24	4.01	3.66-3.83
3	bDGalp	4.44	3.50	3.74	3.99	3.71	3.73-3.73
2	Ac	-	2.03-2.05
	bDGlcpN	4.70	3.80	3.78	3.52	3.38	3.73-3.86


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,4,2,2	aDGalpA	99.3/5.05	69.4/3.72	70.4/4.05	80.0/4.42	72.1/4.88	
3,4,2	aLRhap	99.7/5.07	77.7/4.07	70.6/3.89	73.2/3.47	70.7/3.77	17.8/1.30
3,4	bDRibf	108.1/5.44	82.2/4.22	71.3/4.24	83.9/4.01	63.8/3.66-3.83
3	bDGalp	104.7/4.44	72.1/3.50	74.1/3.74	76.8/3.99	76.0/3.71	62.5/3.73-3.73
2	Ac		23.7/2.03-2.05
	bDGlcpN	103.1/4.70	56.1/3.80	83.6/3.78	69.9/3.52	76.3/3.38	62.3/3.73-3.86

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*O[C@H]1[C@@H](C(=O)O)O[C@H](O[C@H]2[C@H](O[C@H]3[C@H](O[C@H]4[C@@H](CO)O[C@@H](O[C@@H]5[C@@H](NC(C)=O)[C@H](*)O[C@H](CO)[C@H]5O)[C@H](O)[C@H]4O)O[C@H](CO)[C@H]3O)O[C@@H](C)[C@H](O)[C@H]2O)[C@H](O)[C@H]1O

819.716 g/mol (C₃₁H₄₉R₂NO₂₄, R = next and previous repeats, not counted in mol weight)