Plesiomonas shigelloides is a Gram-negative opportunistic pathogen associated with gastrointestinal and extraintestinal infections, which especially invades immunocompromised patients and neonates. The lipopolysaccharides are one of the major virulence determinants in Gram-negative bacteria and are structurally composed of three different domains: the lipid A, the core oligosaccharide and the O-antigen polysaccharide. In the last few years we elucidated the structures of the O-chain and the core oligosaccharide from the P. shigelloides strain 302-73. In this paper we now report the characterization of the linkage between the core and the O-chain. The LPS obtained after PCP extraction contained a small number of O-chain repeating units. The product obtained by hydrazinolysis was analysed by FTICR-ESIMS and suggested the presence of an additional Kdo in the core oligosaccharide. Furthermore, the LPS was hydrolysed under mild acid conditions and a fraction that contained one O-chain repeating unit linked to a Kdo residue was isolated and characterized by FTICR-ESIMS and NMR spectroscopy. Moreover, after an alkaline reductive hydrolysis, a disaccharide α-Kdo-(2→6)-GlcNol was isolated and characterized. The data obtained proved the presence of an α-Kdo in the outer core and allowed the identification of the O-antigen biological repeating unit as well as its linkage with the core oligosaccharide.

NMR spectroscopy, structure elucidation, Plesiomonas, outer-core Kdo, reductive hydrolysis

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6	C7	C8
5,3,4,4,4,4	%Ac
5,3,4,4,4	aL6dTalpN	102.5	52.7	65.1	72.2	68.8	16.6
5,3,4,4,2	Ac
5,3,4,4	aLFucpN	99.5	51.23	68.1	80.8	69.1	16.4
5,3,4,2	Ac
5,3,4	aLFucpN	99.5	51.23	67.8	81.7	69.1	16.7
5,3,2	Ac
5,3	aLFucpN	98.4	50.3	67.9	80.5	69.0	16.8
5,2	Ac
5,4	l?3HOBut	?	46.5	66.0	23.7
5	bDQuipN4N
	?XKdo?	?	97.6	34.7	67.8	75.8	71.7	70.7	63.6


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6	H7	H8
5,3,4,4,4,4	%Ac
5,3,4,4,4	aL6dTalpN	4.84	4.28	4.22	3.82	4.42	1.18
5,3,4,4,2	Ac
5,3,4,4	aLFucpN	4.95	4.20	4.15	3.87	4.45	1.11
5,3,4,2	Ac
5,3,4	aLFucpN	4.95	4.20	4.15	3.85	4.51	1.21
5,3,2	Ac
5,3	aLFucpN	5.13	4.14	3.84	3.78	3.89	1.22
5,2	Ac
5,4	l?3HOBut	-	2.36	4.19	1.23
5	bDQuipN4N	4.69	3.94	3.79	3.64	3.87	?
	?XKdo?	-	-	1.84	4.16	4.06	3.79	3.95	3.65-3.80


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6	C7/H7	C8/H8
5,3,4,4,4,4	%Ac
5,3,4,4,4	aL6dTalpN	102.5/4.84	52.7/4.28	65.1/4.22	72.2/3.82	68.8/4.42	16.6/1.18
5,3,4,4,2	Ac
5,3,4,4	aLFucpN	99.5/4.95	51.23/4.20	68.1/4.15	80.8/3.87	69.1/4.45	16.4/1.11
5,3,4,2	Ac
5,3,4	aLFucpN	99.5/4.95	51.23/4.20	67.8/4.15	81.7/3.85	69.1/4.51	16.7/1.21
5,3,2	Ac
5,3	aLFucpN	98.4/5.13	50.3/4.14	67.9/3.84	80.5/3.78	69.0/3.89	16.8/1.22
5,2	Ac
5,4	l?3HOBut		46.5/2.36	66.0/4.19	23.7/1.23
5	bDQuipN4N	NMR TSV error 2: unequal length of 13C and 1H datasets
	?XKdo?			34.7/1.84	67.8/4.16	75.8/4.06	71.7/3.79	70.7/3.95	63.6/3.65-3.80

The spectrum also has 2 signals at unknown positions (not plotted).

There are 2 chemically distinct structures. Please, select:
%Ac(1-4)aL6dTalpN(1-4)[Ac(1-2)]aLFucpN(1-4)[Ac(1-2)]aLFucpN(1-4)[Ac(1-2)]aLFucpN(1-3)[l?3HOBut?(1-4),Ac(1-2)]bDQuipN4N(1-5)?XKdop %Ac(1-4)aL6dTalpN(1-4)[Ac(1-2)]aLFucpN(1-4)[Ac(1-2)]aLFucpN(1-4)[Ac(1-2)]aLFucpN(1-3)[l?3HOBut?(1-4),Ac(1-2)]bDQuipN4N(1-5)xXKdoa

SVGMWSMILES3D molIonize

 

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

1259.273 g/mol (C₅₂H₈₆N₆O₂₉)