Bacterial lipopolysaccharides (LPSs) are the major component of the outer membrane of Gram-negative bacteria. They have a structural role since they contribute to the cellular rigidity by increasing the strength of cell wall and mediating contacts with the external environment that can induce structural changes to allow life in different conditions. Furthermore, the low permeability of the outer membrane acts as a barrier to protect bacteria from host-derived antimicrobial compounds. They also have a very important role in the elicitation of the animal and plant host innate immunity since they are microbe-associated molecular patterns, namely, they are glycoconjugates produced only by Gram-negative bacteria and are recognized as a molecular hallmark of invading microbes. LPSs are amphiphilic macromolecules generally comprising three defined regions distinguished by their genetics, structures, and function: the lipid A, the core oligosaccharide and a polysaccharide portion, the O-chain. In some Gram-negative bacteria, LPS can terminate with the core portion to form rough-type LPS (R-LPS, LOS). In this chapter, we will describe the isolation of both kinds of LPSs and their full chemical analysis, pivotal operations in the complete description of the primary structure of such important glycoconjugates.

lipopolysaccharides, isolation, gram negative bacteria, methods, MS, chemical analysis

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,4,3,3	bXParf	109.3	76.8	33.2	85.2	70.5	19.9
3,4,3	aLFucp	95.4	85.2	76.4	71.4	67.0	16.4
3,4	aDManp	102.9	67.8	76.0	70.4	70.4	62.2
3	aLFucp	102.6	69.4	71.3	80.7	68.3	17.7
2	Ac	171.2	22.2
	aDGalpN	99.0	49.5	77.9	70.1	73.1	62.4


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,4,3,3	bXParf	5.12	4.28	2.01	4.11	3.83	1.18
3,4,3	aLFucp	5.15	4.10	4.19	3.96	4.37	1.21
3,4	aDManp	5.26	4.32	3.94	3.82	3.88	3.79-3.90
3	aLFucp	4.96	3.75	4.01	3.97	4.20	1.27
2	Ac	-	2.03
	aDGalpN	5.29	4.40	3.88	4.02	4.20	3.73-3.78


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,4,3,3	bXParf	109.3/5.12	76.8/4.28	33.2/2.01	85.2/4.11	70.5/3.83	19.9/1.18
3,4,3	aLFucp	95.4/5.15	85.2/4.10	76.4/4.19	71.4/3.96	67.0/4.37	16.4/1.21
3,4	aDManp	102.9/5.26	67.8/4.32	76.0/3.94	70.4/3.82	70.4/3.88	62.2/3.79-3.90
3	aLFucp	102.6/4.96	69.4/3.75	71.3/4.01	80.7/3.97	68.3/4.20	17.7/1.27
2	Ac		22.2/2.03
	aDGalpN	99.0/5.29	49.5/4.40	77.9/3.88	70.1/4.02	73.1/4.20	62.4/3.73-3.78

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

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

787.762 g/mol (C₃₂H₅₃R₂NO₂₁, R = next and previous repeats, not counted in mol weight)