The structure of lipopolysaccharide (LPS) expressed by non-typeable Haemophilus influenzae (NTHi) strains 1008 and 1247 has been investigated by mass spectrometry and NMR analyses on O-deacylated LPS and core oligosaccharide material. Both strains express the conserved triheptosyl inner core, [L-α-D-Hepp-(1→2)-[PEtn→6]-L-α-D-Hepp-(1→3)-L-α-D-Hepp-(1→5)-[PPEtn→4]-α-Kdo-(2→6)-Lipid A] with PCho→6)-β-D-Glcp (GlcI) substituting the proximal heptose (HepI) at O-4. Strain 1247 expresses the common structural motifs of H. influenzae; globotetraose [β-D-GalpNAc-(1→3)-α-D-Galp-(1→4)-β-D-Galp-(1→4)-β-D-Glcp-(1→] and its truncated versions globoside [α-D-Galp-(1→4)-β-D-Galp-(1→4)-β-D-Glcp-(1→] and lactose [β-D-Galp-(1→4)-β-D-Glcp-(1→] linked to the terminal heptose of the inner core and GlcI. A genetically distinct NTHi strain, 1008, expresses identical structures to strain 1247 with the exception that it lacks GalNAc. A lpsA mutant of strain 1247 expressed LPS of reduced complexity that facilitated unambiguous structural determination of the oligosaccharide from HepI. By CE-ESI-MS/MS we identified disialylated glycoforms indicating disialyllactose [α-Neu5Ac-(2→8)-α-Neu5Ac-(2→3)-β-D-Gal-(1→4)-β-D-Glcp-(1→] as an extension from GlcI which is a novel finding for NTHi LPS.

Lipopolysaccharide, Haemophilus influenzae, otitis media, Digalactoside, disialyllactoside

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
5,3,2,2,4,4	aDGalp	100.9	68.9	69.7	70.1	?	?
5,3,2,2,4	bDGalp	103.6	71.3	72.3	77.8	75.7	?
5,3,2,2	bDGlcp
5,3,2	aXLDmanHepp
5,3,6,0	xXEtN
5,3,6	P
5,3	aXLDmanHepp
5,4,4,4,4	aDGalp
5,4,4,4	bDGalp
5,4,4	bDGlcp
5,4,6,0	xXCho
5,4,6	P
5,4	bDGlcp
5	aXLDmanHepp
4,0,0	xXEtN
4,0	%xXP?
4	P
	aXKdop


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
5,3,2,2,4,4	aDGalp	4.96	3.84	3.92	4.05	?	?
5,3,2,2,4	bDGalp	4.51	3.57	3.75	4.06	3.79	?
5,3,2,2	bDGlcp
5,3,2	aXLDmanHepp
5,3,6,0	xXEtN
5,3,6	P
5,3	aXLDmanHepp
5,4,4,4,4	aDGalp
5,4,4,4	bDGalp
5,4,4	bDGlcp
5,4,6,0	xXCho
5,4,6	P
5,4	bDGlcp
5	aXLDmanHepp
4,0,0	xXEtN
4,0	%xXP?
4	P
	aXKdop


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
5,3,2,2,4,4	aDGalp	100.9/4.96	68.9/3.84	69.7/3.92	70.1/4.05	?/?	?/?
5,3,2,2,4	bDGalp	103.6/4.51	71.3/3.57	72.3/3.75	77.8/4.06	75.7/3.79	?/?
5,3,2,2	bDGlcp
5,3,2	aXLDmanHepp
5,3,6,0	xXEtN
5,3,6	P
5,3	aXLDmanHepp
5,4,4,4,4	aDGalp
5,4,4,4	bDGalp
5,4,4	bDGlcp
5,4,6,0	xXCho
5,4,6	P
5,4	bDGlcp
5	aXLDmanHepp
4,0,0	xXEtN
4,0	%xXP?
4	P
	aXKdop

The spectrum also has 3 signals at unknown positions (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

C[N+](C)(C)CCOP(=O)(O)OC[C@H]1O[C@@H](O[C@@H]2[C@@H]([C@@H](O)CO)O[C@H](O[C@@H]3[C@H](OP(=O)(O)OP(=O)(O)OCCN)C[C@](O)(C(=O)O)O[C@@H]3[C@H](O)CO)[C@@H](O)[C@H]2O[C@H]2O[C@H]([C@H](CO)OP(=O)(O)OCCN)[C@@H](O)[C@H](O)[C@@H]2O[C@H]2O[C@H]([C@@H](O)CO)[C@@H](O)[C@H](O)[C@@H]2O[C@@H]2O[C@H](CO)[C@@H](O[C@@H]3O[C@H](CO)[C@H](O[C@H]4O[C@H](CO)[C@H](O)[C@H](O)[C@H]4O)[C@H](O)[C@H]3O)[C@H](O)[C@H]2O)[C@H](O)[C@@H](O)[C@@H]1O[C@@H]1O[C@H](CO)[C@@H](O[C@@H]2O[C@H](CO)[C@H](O[C@H]3O[C@H](CO)[C@H](O)[C@H](O)[C@H]3O)[C@H](O)[C@H]2O)[C@H](O)[C@H]1O

2441.892 g/mol (C₈₀H₁₄₆N₃O₇₃P₄⁺)