Opportunistic human pathogens of the genus Providencia from the family Enterobacteriaceae are serotyped by their O-antigens, which represent the O-polysaccharide chains of the lipopolysaccharides (LPSs) on the cell surface. In this work, the O-polysaccharide of Providencia alcalifaciens O9 was obtained by mild acid degradation of a long-chain S-form LPS. The structure of the hexasaccharide repeat (O-unit) of the O-polysaccharide containing one D-Gal, two D-Glc, and three D-GalNAc residues was established by sugar and methylation analyses along with one- and two-dimensional (1)H and (13)C NMR spectroscopy. Another degradation product was derived from a short-chain SR-form LPS and found to consist of a core oligosaccharide bearing one O-unit. Its studies by NMR spectroscopy and electrospray ionization mass spectrometry enabled identification of one of the GalNAc residues as the first monosaccharide of the O-unit, whose glycosidic linkage links the O-units to each other and the first O-unit to the core. The core is distinguished by the occurrence of two glycoforms differing in the nature of a lateral monosaccharide, which is either D-Glc or D-GlcNAc. Although composed of common monosaccharides, the O-polysaccharide of P. alcalifaciens O9 has a unique structure among bacterial polysaccharides, whereas the oligosaccharide region belongs to one of several core types recognized in the LPSs of Providencia.

core oligosaccharide, O-polysaccharide, Providencia, lipopolysaccharide structure, biological repeating unit

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,4,6,6	bDGlcp	104.3	77.9	75.8	71.2	77.0	62.1
3,4,6	aDGalp	101.6	69.8	70.5	70.9	68.6	71.8
3,4,2	Ac	175.	23.2
3,4	aDGalpN	95.3	51.4	70.9	68.7	69.4	66.1
3,2	Ac	175.6	23.4
3,3	bDGlcp	106.2	74.3	76.6	71.2	77.2	62.3
3	aDGalpN	94.3	49.9	77.8	75.7	73.6	61.5
2	Ac	176.0	23.9
	aDGalpN	97.7	48.8	73.0	65.5	71.6	62.0


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,4,6,6	bDGlcp	4.65	3.51	3.58	3.45	3.49	3.74-3.94
3,4,6	aDGalp	4.88	3.80	3.87	3.96	4.08	3.72-4.13
3,4,2	Ac	-	2.05
3,4	aDGalpN	4.92	4.24	4.08	4.15	4.59	3.67-3.70
3,2	Ac	-	2.09
3,3	bDGlcp	4.45	3.32	3.53	3.48	3.47	3.74-3.91
3	aDGalpN	5.15	4.56	3.84	4.29	3.93	3.68-3.73
2	Ac	-	2.11
	aDGalpN	5.49	4.48	4.04	4.23	4.28	3.78-3.78


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,4,6,6	bDGlcp	104.3/4.65	77.9/3.51	75.8/3.58	71.2/3.45	77.0/3.49	62.1/3.74-3.94
3,4,6	aDGalp	101.6/4.88	69.8/3.80	70.5/3.87	70.9/3.96	68.6/4.08	71.8/3.72-4.13
3,4,2	Ac		23.2/2.05
3,4	aDGalpN	95.3/4.92	51.4/4.24	70.9/4.08	68.7/4.15	69.4/4.59	66.1/3.67-3.70
3,2	Ac		23.4/2.09
3,3	bDGlcp	106.2/4.45	74.3/3.32	76.6/3.53	71.2/3.48	77.2/3.47	62.3/3.74-3.91
3	aDGalpN	94.3/5.15	49.9/4.56	77.8/3.84	75.7/4.29	73.6/3.93	61.5/3.68-3.73
2	Ac		23.9/2.11
	aDGalpN	97.7/5.49	48.8/4.48	73.0/4.04	65.5/4.23	71.6/4.28	62.0/3.78-3.78

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

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

1096.005 g/mol (C₄₂H₆₉R₂N₃O₃₀, R = next and previous repeats, not counted in mol weight)