A computerized method that uses predicted functions of glycosyltransferases (GTs) in conjunction with unassigned NMR data has been developed for the structural elucidation of bacterial polysaccharides. In this approach information about the action of glycosyltransferases (consisting of possible sugar residues used as donors and/or acceptors, as well as the anomeric configuration and/or substitution position in the respective glycosidic linkages) is extracted from the E. coli O-antigen database (ECODAB) and is submitted, together with the unassigned NMR data, to the CASPER program. This time saving methodology, that alleviates the need for chemical analysis, was successfully implemented in the structural elucidation of the O-antigen polysaccharide (PS) of E. coli O59. The repeating unit of the O-specific chain was determined using the O-deacylated PS and has a branched structure, namely, →6)[α-D-GalpA3Ac/4Ac-(1→3)]-α-D-Manp-(1→3)-α-D-Manp-(1→3)-β-D-Manp-(1→3)-α-D-GlcpNAc-(1→. The identification of the O-acetylation positions were efficiently performed by comparison of the 1H,13C-HSQC NMR spectra of the O-deacylated lipopolysaccharide (LPS-OH) and the lipid-free polysaccharide in conjunction with chemical shift predictions made by the CASPER program. The side-chain D-GalpA residue carries one equivalent of O-acetyl groups at the O-3 and O-4 positions distributed in the LPS in a 3:7 ratio, respectively. The presence of O-acetyl groups in the repeating unit of the E. coli O59 PS is consistent with the previously proposed acetyltransferase WclD in the O-antigen gene cluster.

Escherichia coli, glycosyltransferases, CASPER, O-antigen polysaccharide, ECODAB

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,3,3,3	aDGalpA	101.73	69.46	70.56	71.58	73.06	176.19
3,3,3	aDManp	103.47	70.62	80.72	65.69	72.53	65.74
3,3	aDManp	103.19	70.66	79.85	66.59	74.16	61.60
3	bDManp	101.18	71.44	81.61	66.78	76.94	61.76
2	Ac	174.97	22.90
	aDGlcpN	97.86	53.27	82.33	69.38	72.53	61.45


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,3,3,3	aDGalpA	5.264	3.870	3.987	4.327	4.460	-
3,3,3	aDManp	5.128	4.242	4.014	4.147	3.997	3.608-4.145
3,3	aDManp	5.092	4.156	3.975	3.839	3.826	3.848
3	bDManp	4.742	3.994	3.720	3.721	3.443	3.757-3.929
2	Ac	-	2.052
	aDGlcpN	4.885	4.129	3.943	3.628	3.765	3.839


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,3,3,3	aDGalpA	101.73/5.264	69.46/3.870	70.56/3.987	71.58/4.327	73.06/4.460	
3,3,3	aDManp	103.47/5.128	70.62/4.242	80.72/4.014	65.69/4.147	72.53/3.997	65.74/3.608-4.145
3,3	aDManp	103.19/5.092	70.66/4.156	79.85/3.975	66.59/3.839	74.16/3.826	61.60/3.848
3	bDManp	101.18/4.742	71.44/3.994	81.61/3.720	66.78/3.721	76.94/3.443	61.76/3.757-3.929
2	Ac		22.90/2.052
	aDGlcpN	97.86/4.885	53.27/4.129	82.33/3.943	69.38/3.628	72.53/3.765	61.45/3.839

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

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

865.741 g/mol (C₃₂H₅₁R₂NO₂₆, R = next and previous repeats, not counted in mol weight)