The effect of the length and chemical composition of the side-chains in comb-like branched polysaccharides on gelation with trivalent metal ions has been studied using xanthan and xylinan (acetan) with intact and truncated side-chains. Partial or complete removal of the terminal β-d-mannose, or removal of up to 22% of the trisaccharide side-chains of xanthan using partial acid hydrolysis, has only small effects on the Cr3+-induced gelation. In contrast, replacement of β-d-mannose by the trisaccharide [structure] to yield the polysaccharide xylinan totally inhibits the gelation with Cr3+ ions. Removal of the trisaccharide by partial acid hydrolysis, which leads to a series of polymers with structures converging towards the partially hydrolysed xanthans, restores the gelling ability with Cr3+ ions. These observations seem to support the gelation model where Cr3+-glucuronic acid interactions are involved in the cross-linking of chains. It is further suggested that this interaction can be suppressed due to steric hindrance caused by the bulky side-chains in xylinan.

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

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

936.772 g/mol (C₃₅H₅₂R₂O₂₉, R = next and previous repeats, not counted in mol weight)

Implementation of conjugate vaccine technology revolutionized the ability to effectively elicit long-lasting immune responses to bacterial capsular polysaccharides. Although expansion of conjugate vaccine serotype coverage is designed to target residual disease burden to pneumococcal serotypes not contained in earlier vaccine versions, details of polysaccharide Ag structure, heterogeneity, and epitope structure components contributing to vaccine-mediated immunity are not always clear. Analysis of Streptococcus pneumoniae serotype 12F polysaccharide by two-dimensional nuclear magnetic resonance spectroscopy and mass spectrometry revealed a partial substitution of N-acetyl-galactosamine by the keto sugar 2-acetamido-2,6-dideoxy-xylo-hexos-4-ulose (Sug) in up to 25% of the repeat units. This substitution was not described in previous published structures for 12F. Screening a series of contemporary 12F strains isolated from humans (n = 17) identified Sug incorporation at varying levels in all strains examined. Thus, partial Sug substitution in S. pneumoniae serotype 12F may have always been present but is now detectable by state-of-the-art analytical techniques. During the steps of conjugation, the serotype 12F Sug epitope is modified by reduction, and both polysaccharide PPSV23 and conjugate PCV20 vaccines contain 12F Ags with little to no Sug epitope. Both PCV20 and PPSV23 vaccines were evaluated for protection against circulating 12F strains with varying amounts of Sug in their repeat unit based on an opsonophagocytic killing assay involving HL-60 cells and rabbit complement. Both vaccines elicited human-derived neutralizing Abs against serotype 12F, independent of Sug level between ∼2 and 25 mol%. These findings suggest that the newly identified serotype 12F Sug epitope is likely not an essential epitope for vaccine-elicited protection.

Streptococcus pneumoniae, capsular polysaccharide, vaccine, serotype 12F

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
4,3,2	Ac	175.37	23.46
4,3,3	aDGalp	101.90	69.55	70.56	70.84	72.52	62.90
4,3	aLFucpN	100.44	49.57	74.55	80.34	68.9	16.98
4,2	Ac	175.24	23.38
4	bDGalpN	99.04	52.76	78.00	69.02	76.26	61.70
2	Ac	175.86	23.27
3,2	aDGlcp	96.23	72.57	73.88	70.83	73.00	62.00
3	aDGlcp	96.86	75.55	72.33	70.78	73.70	61.77
	bDManpNA	100.71	53.33	75.54	73.99	79.23	175.62


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
4,3,2	Ac	-	2.004
4,3,3	aDGalp	5.002	3.678	3.785	4.002	4.128	3.718-3.718
4,3	aLFucpN	4.905	4.345	4.045	4.074	4.106	1.225
4,2	Ac	-	2.002
4	bDGalpN	4.663	4.085	3.713	3.907	3.566	3.760-3.832
2	Ac	-	2.120
3,2	aDGlcp	5.281	3.711	3.834	3.445	3.899	3.774-3.858
3	aDGlcp	5.416	3.659	3.841	3.448	3.897	3.762-3.837
	bDManpNA	4.795	4.686	4.082	4.314	3.674	-


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
4,3,2	Ac		23.46/2.004
4,3,3	aDGalp	101.90/5.002	69.55/3.678	70.56/3.785	70.84/4.002	72.52/4.128	62.90/3.718-3.718
4,3	aLFucpN	100.44/4.905	49.57/4.345	74.55/4.045	80.34/4.074	68.9/4.106	16.98/1.225
4,2	Ac		23.38/2.002
4	bDGalpN	99.04/4.663	52.76/4.085	78.00/3.713	69.02/3.907	76.26/3.566	61.70/3.760-3.832
2	Ac		23.27/2.120
3,2	aDGlcp	96.23/5.281	72.57/3.711	73.88/3.834	70.83/3.445	73.00/3.899	62.00/3.774-3.858
3	aDGlcp	96.86/5.416	75.55/3.659	72.33/3.841	70.78/3.448	73.70/3.897	61.77/3.762-3.837
	bDManpNA	100.71/4.795	53.33/4.686	75.54/4.082	73.99/4.314	79.23/3.674	

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

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

1093.989 g/mol (C₄₂H₆₇R₂N₃O₃₀, R = next and previous repeats, not counted in mol weight)

Implementation of conjugate vaccine technology revolutionized the ability to effectively elicit long-lasting immune responses to bacterial capsular polysaccharides. Although expansion of conjugate vaccine serotype coverage is designed to target residual disease burden to pneumococcal serotypes not contained in earlier vaccine versions, details of polysaccharide Ag structure, heterogeneity, and epitope structure components contributing to vaccine-mediated immunity are not always clear. Analysis of Streptococcus pneumoniae serotype 12F polysaccharide by two-dimensional nuclear magnetic resonance spectroscopy and mass spectrometry revealed a partial substitution of N-acetyl-galactosamine by the keto sugar 2-acetamido-2,6-dideoxy-xylo-hexos-4-ulose (Sug) in up to 25% of the repeat units. This substitution was not described in previous published structures for 12F. Screening a series of contemporary 12F strains isolated from humans (n = 17) identified Sug incorporation at varying levels in all strains examined. Thus, partial Sug substitution in S. pneumoniae serotype 12F may have always been present but is now detectable by state-of-the-art analytical techniques. During the steps of conjugation, the serotype 12F Sug epitope is modified by reduction, and both polysaccharide PPSV23 and conjugate PCV20 vaccines contain 12F Ags with little to no Sug epitope. Both PCV20 and PPSV23 vaccines were evaluated for protection against circulating 12F strains with varying amounts of Sug in their repeat unit based on an opsonophagocytic killing assay involving HL-60 cells and rabbit complement. Both vaccines elicited human-derived neutralizing Abs against serotype 12F, independent of Sug level between ∼2 and 25 mol%. These findings suggest that the newly identified serotype 12F Sug epitope is likely not an essential epitope for vaccine-elicited protection.

Streptococcus pneumoniae, capsular polysaccharide, vaccine, serotype 12F

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
4,3,2	Ac
4,3,3	aDGalp	102.00	?	?	?	?	?
4,3	aLFucpN	99.93	49.49	74.88	80.50	69.09	16.77
4,2	Ac	175.01	23.25
4	bD6dxylHexp-4-ulo	98.29	55.49	80.42	94.04	74.90	12.36
2	Ac
3,2	aDGlcp	95.91	?	?	?	?	?
3	aDGlcp	96.91	?	?	?	?	?
	bDManpNA	100.81	?	75.64	73.92	?	175.53


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
4,3,2	Ac
4,3,3	aDGalp	5.009	3.683	?	?	?	?
4,3	aLFucpN	4.910	4.382	4.068	4.092	4.443	1.175
4,2	Ac	-	1.974
4	bD6dxylHexp-4-ulo	4.691	3.980	3.594	-	3.449	1.302
2	Ac
3,2	aDGlcp	5.276	3.764	3.822	?	?	?
3	aDGlcp	5.380	3.645	3.814	?	?	?
	bDManpNA	?	4.680	4.020	4.275	3.654	-


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
4,3,2	Ac
4,3,3	aDGalp	102.00/5.009	?/3.683	?/?	?/?	?/?	?/?
4,3	aLFucpN	99.93/4.910	49.49/4.382	74.88/4.068	80.50/4.092	69.09/4.443	16.77/1.175
4,2	Ac		23.25/1.974
4	bD6dxylHexp-4-ulo	98.29/4.691	55.49/3.980	80.42/3.594		74.90/3.449	12.36/1.302
2	Ac
3,2	aDGlcp	95.91/5.276	?/3.764	?/3.822	?/?	?/?	?/?
3	aDGlcp	96.91/5.380	?/3.645	?/3.814	?/?	?/?	?/?
	bDManpNA	100.81/?	?/4.680	75.64/4.020	73.92/4.275	?/3.654	

The spectrum also has 17 signals at unknown positions (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

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

1076.958 g/mol (C₄₂H₆₄R₂N₂O₃₀, R = next and previous repeats, not counted in mol weight)

The effect of the length and chemical composition of the side-chains in comb-like branched polysaccharides on gelation with trivalent metal ions has been studied using xanthan and xylinan (acetan) with intact and truncated side-chains. Partial or complete removal of the terminal β-d-mannose, or removal of up to 22% of the trisaccharide side-chains of xanthan using partial acid hydrolysis, has only small effects on the Cr3+-induced gelation. In contrast, replacement of β-d-mannose by the trisaccharide [structure] to yield the polysaccharide xylinan totally inhibits the gelation with Cr3+ ions. Removal of the trisaccharide by partial acid hydrolysis, which leads to a series of polymers with structures converging towards the partially hydrolysed xanthans, restores the gelling ability with Cr3+ ions. These observations seem to support the gelation model where Cr3+-glucuronic acid interactions are involved in the cross-linking of chains. It is further suggested that this interaction can be suppressed due to steric hindrance caused by the bulky side-chains in xylinan.

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*O[C@@H]1[C@@H](CO)O[C@@H](O[C@@H]2[C@@H](CO)O[C@@H](*)[C@H](O)[C@H]2O[C@H]2O[C@H](COC(C)=O)[C@@H](O)[C@H](O)[C@@H]2O[C@@H]2O[C@H](C(=O)O)[C@@H](O[C@H]3O[C@H](CO[C@@H]4O[C@H](CO[C@@H]5O[C@@H](C)[C@H](O)[C@@H](O)[C@H]5O)[C@@H](O)[C@H](O)[C@H]4O)[C@@H](O)[C@H](O)[C@H]3O)[C@H](O)[C@H]2O)[C@H](O)[C@H]1O

1175.008 g/mol (C₄₄H₇₀R₂O₃₆, R = next and previous repeats, not counted in mol weight)