Fusobacterium nucleatum is an anaerobic bacterium found in the human mouth where it causes periodontitis. Recently, it has been gaining attention as a potential causative agent for colorectal cancer and is strongly linked with pregnancy complications including pre-term and still births. Little is known about the virulence factors of this organism, and thus we have initiated studies to examine the bacterium's surface glycochemistry. We isolated lipopolysaccharide (LPS) from F. nucleatum strain 25586 and purified and performed structural analysis on the O-antigen polysaccharide. The polysaccharide contained two novel sugars, 2-acetamido-2,6-dideoxy-l-altrose (l-6dAltNAc) and a 5-acetimidoylamino-3,5,9-trideoxy-gluco-non-2-ulosonic acid (Non5Am), which was tentatively assigned the l-glycero-l-gluco configuration. The polysaccharide was found to have a trisaccharide repeating unit, which is phosphorylated with phosphocholine (PCho), and the following structure was established: -[-4-β-Nonp5Am-4-α-l-6dAltpNAc3PCho-3-β-d-QuipNAc-]- We propose the trivial name 'fusaminic acid' for the novel nonulosonic acid. It is the first occurrence of a 9-deoxynonulosonic acid with a hydroxyl group at C-7, which is occupied by an amino group in all monosaccharides of this class described so far.

Lipopolysaccharide, NMR, structure, Fusobacterium, Fusobacterium nucleatum, Fusaminic acid

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6	C7	C8	C9
3,4,5	Ac
3,4	bXFusp	?	?	32.3	74.0	47.7	71.2	72.2	67.6	18.2
3,2	Ac
3,3,0	xXCho	60.5	67.5	55.4
3,3	P
3	aL6dAltpN	100.6	52.9	74.9	72.8	63.6	17.4
2	Ac
	bDQuipN	99.9	57.0	81.4	75.2	73.2	18.0


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6	H7	H8	H9
3,4,5	Ac
3,4	bXFusp	-	-	2.02-2.60	4.08	4.21	3.95	3.48	3.95	1.19
3,2	Ac
3,3,0	xXCho	4.36	3.71	3.26
3,3	P
3	aL6dAltpN	4.73	4.13	4.63	3.74	4.37	1.10
2	Ac
	bDQuipN	4.62	3.76	3.49	3.17	3.50	1.30


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6	C7/H7	C8/H8	C9/H9
3,4,5	Ac
3,4	bXFusp			32.3/2.02-2.60	74.0/4.08	47.7/4.21	71.2/3.95	72.2/3.48	67.6/3.95	18.2/1.19
3,2	Ac
3,3,0	xXCho	60.5/4.36	67.5/3.71	55.4/3.26
3,3	P
3	aL6dAltpN	100.6/4.73	52.9/4.13	74.9/4.63	72.8/3.74	63.6/4.37	17.4/1.10
2	Ac
	bDQuipN	99.9/4.62	57.0/3.76	81.4/3.49	75.2/3.17	73.2/3.50	18.0/1.30

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

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

[*]O[C@@H]1C[C@](O[C@H]2[C@H](C)O[C@@H](O[C@@H]3[C@@H](NC(C)=O)[C@H]([*])O[C@H](C)[C@H]3O)[C@H](NC(C)=O)[C@H]2OP(=O)(O)OCC[N+](C)(C)C)(C(=O)O)O[C@@H]([C@@H](O)[C@H](C)O)[C@H]1NC(C)=O

815.784 g/mol (C₃₂H₅₆R₂N₄O₁₈P⁺, R = next and previous repeats, not counted in mol weight)

Fusobacterium nucleatum is an anaerobic bacterium found in the human mouth where it causes periodontitis. Recently, it has been gaining attention as a potential causative agent for colorectal cancer and is strongly linked with pregnancy complications including pre-term and still births. Little is known about the virulence factors of this organism, and thus we have initiated studies to examine the bacterium's surface glycochemistry. We isolated lipopolysaccharide (LPS) from F. nucleatum strain 25586 and purified and performed structural analysis on the O-antigen polysaccharide. The polysaccharide contained two novel sugars, 2-acetamido-2,6-dideoxy-l-altrose (l-6dAltNAc) and a 5-acetimidoylamino-3,5,9-trideoxy-gluco-non-2-ulosonic acid (Non5Am), which was tentatively assigned the l-glycero-l-gluco configuration. The polysaccharide was found to have a trisaccharide repeating unit, which is phosphorylated with phosphocholine (PCho), and the following structure was established: -[-4-β-Nonp5Am-4-α-l-6dAltpNAc3PCho-3-β-d-QuipNAc-]- We propose the trivial name 'fusaminic acid' for the novel nonulosonic acid. It is the first occurrence of a 9-deoxynonulosonic acid with a hydroxyl group at C-7, which is occupied by an amino group in all monosaccharides of this class described so far.

Lipopolysaccharide, NMR, structure, Fusobacterium, Fusobacterium nucleatum, Fusaminic acid

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6	C7	C8	C9
4,4,2	Ac	?	22.9-23.7
4,4	bDQuipN	99.6	57.1	75.2	76.6	73.4	17.9
4,5	Ac
4	bXFusp	173.0	101.3	31.8	73.6	47.6	71.4	72.4	67.7	18.1
2	Ac
	aL6dAltpN	93.2	54.0	69.9	75.2	65.3	18.1


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6	H7	H8	H9
4,4,2	Ac	-	2.04-2.12
4,4	bDQuipN	4.70	3.67	3.49	3.21	3.52	1.32
4,5	Ac
4	bXFusp	-	-	1.83-2.67	4.12	4.22	4.04	3.42	3.95	1.19
2	Ac
	aL6dAltpN	4.98	4.05	4.13	3.78	4.21	1.20


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6	C7/H7	C8/H8	C9/H9
4,4,2	Ac		22.9-23.7/2.04-2.12
4,4	bDQuipN	99.6/4.70	57.1/3.67	75.2/3.49	76.6/3.21	73.4/3.52	17.9/1.32
4,5	Ac
4	bXFusp			31.8/1.83-2.67	73.6/4.12	47.6/4.22	71.4/4.04	72.4/3.42	67.7/3.95	18.1/1.19
2	Ac
	aL6dAltpN	93.2/4.98	54.0/4.05	69.9/4.13	75.2/3.78	65.3/4.21	18.1/1.20

The spectrum also has 1 signal at unknown position (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CC(=O)N[C@H]1[C@H](O[C@@H]2O[C@H](C)[C@@H](O)[C@H](O)[C@H]2NC(C)=O)C[C@](O[C@H]2[C@H](C)O[C@@H](O)[C@H](NC(C)=O)[C@H]2O)(C(=O)O)O[C@H]1[C@@H](O)[C@H](C)O

667.662 g/mol (C₂₇H₄₅N₃O₁₆)

Fusobacterium nucleatum is an anaerobic bacterium found in the human mouth where it causes periodontitis. Recently, it has been gaining attention as a potential causative agent for colorectal cancer and is strongly linked with pregnancy complications including pre-term and still births. Little is known about the virulence factors of this organism, and thus we have initiated studies to examine the bacterium's surface glycochemistry. We isolated lipopolysaccharide (LPS) from F. nucleatum strain 25586 and purified and performed structural analysis on the O-antigen polysaccharide. The polysaccharide contained two novel sugars, 2-acetamido-2,6-dideoxy-l-altrose (l-6dAltNAc) and a 5-acetimidoylamino-3,5,9-trideoxy-gluco-non-2-ulosonic acid (Non5Am), which was tentatively assigned the l-glycero-l-gluco configuration. The polysaccharide was found to have a trisaccharide repeating unit, which is phosphorylated with phosphocholine (PCho), and the following structure was established: -[-4-β-Nonp5Am-4-α-l-6dAltpNAc3PCho-3-β-d-QuipNAc-]- We propose the trivial name 'fusaminic acid' for the novel nonulosonic acid. It is the first occurrence of a 9-deoxynonulosonic acid with a hydroxyl group at C-7, which is occupied by an amino group in all monosaccharides of this class described so far.

Lipopolysaccharide, NMR, structure, Fusobacterium, Fusobacterium nucleatum, Fusaminic acid

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6	C7	C8	C9
4,4,2	Ac	?	22.9-23.7
4,4	bDQuipN	99.6	57.1	75.2	76.6	73.4	17.9
4,5	Ac
4	bXFusp	173.0	101.3	31.8	73.6	47.6	71.4	72.4	67.7	18.1
2	Ac
	bL6dAltpN	91.3	55.1	70.7	75.1	70.1	18.0


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6	H7	H8	H9
4,4,2	Ac	-	2.04-2.12
4,4	bDQuipN	4.70	3.67	3.49	3.21	3.52	1.32
4,5	Ac
4	bXFusp	-	-	1.83-2.67	4.12	4.22	4.04	3.42	3.95	1.19
2	Ac
	bL6dAltpN	5.26	4.06	4.22	3.67	3.90	1.19


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6	C7/H7	C8/H8	C9/H9
4,4,2	Ac		22.9-23.7/2.04-2.12
4,4	bDQuipN	99.6/4.70	57.1/3.67	75.2/3.49	76.6/3.21	73.4/3.52	17.9/1.32
4,5	Ac
4	bXFusp			31.8/1.83-2.67	73.6/4.12	47.6/4.22	71.4/4.04	72.4/3.42	67.7/3.95	18.1/1.19
2	Ac
	bL6dAltpN	91.3/5.26	55.1/4.06	70.7/4.22	75.1/3.67	70.1/3.90	18.0/1.19

The spectrum also has 1 signal at unknown position (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CC(=O)N[C@H]1[C@H](O[C@@H]2O[C@H](C)[C@@H](O)[C@H](O)[C@H]2NC(C)=O)C[C@](O[C@H]2[C@H](C)O[C@H](O)[C@H](NC(C)=O)[C@H]2O)(C(=O)O)O[C@H]1[C@@H](O)[C@H](C)O

667.662 g/mol (C₂₇H₄₅N₃O₁₆)