A glucuronomannan (GM) was derived by removal, through Smith degradation, of xylose from the native (3-O-acetylglucurono)xylomannan exopolysaccharide isolated from Tremella mesenterica. 13C-N.m.r. chemical shifts measured at various pD values were compared for p-nitrophenyl β-D-glucopyranosiduronic acid (1) and two GMs (2 and 3) differing in GlcA content (Man:GlcA; 2, 10:1; and 3, 5:1). Also measured and compared were pKa values for 1 and 2. One-dimensional and two-dimensional (COSY and HETCOR) n.m.r. data allowed unambiguous assignments of pD-sensitive chemical shifts due to 2-O-β-D-GlcpA substituents attached to a (1----3)-linked α-D-Manp backbone. The pKa and n.m.r. data indicated that the CO2H groups in either GM are independent of each other, and are similar in behavior to those of p-nitrophenyl β-D-glucopyranosiduronic acid molecules. The n.m.r. data confirmed the previous, chemically deduced, structural role of GlcpA in the native polysaccharide from T. mesenterica, and indicated that significant pD-induced changes occur in the stabilities of the glycosidic orientations in the GM. Previous 13C-n.m.r. assignments for 2-O-β-D-GlcpA in polysaccharides derived from Cryptococcus neoformans serotype A-variant were confirmed, except for the signal due to the anomeric carbon atom. This signal is now known to be pD-sensitive. In acidic solutions, it is coincident with the signal (104.5 p.p.m.) due to the anomeric carbon atoms of the unsubstituted α-D-Manp backbone residues. In basic solutions, the 2-O-β-D-GlcpA anomeric carbon resonance is shifted upfield by approximately 0.2 p.p.m., and is observed as a separate signal.

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,3,3,3,3,3,3,3,3	aDManp	104.56	72.32	80.85	68.86	76.18	63.72
3,3,3,3,3,3,3,3	aDManp	104.56	72.32	80.85	68.86	76.18	63.72
3,3,3,3,3,3,3	aDManp	104.56	72.32	80.85	68.86	76.18	63.72
3,3,3,3,3,3	aDManp	104.56	72.32	80.85	68.86	76.18	63.72
3,3,3,3,3	aDManp	105.19	72.54	81.07	69.28	76.30	63.65
3,3,3,3,2	bDGlcpA	104.32	75.32	78.13	74.35	79.47	178.03
3,3,3,3	aDManp	102.41	79.77	79.30	69.08	76.06	63.31
3,3,3	aDManp	104.72	72.54	81.12	69.28	76.30	63.65
3,3	aDManp	104.56	72.32	80.85	68.86	76.18	63.72
3	aDManp	104.56	72.32	80.85	68.86	76.18	63.72
	aDManp	104.56	72.32	80.85	68.86	76.18	63.72


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,3,3,3,3,3,3,3,3	aDManp	5.13	4.22	4.03	3.79	3.83	3.80
3,3,3,3,3,3,3,3	aDManp	5.13	4.22	4.03	3.79	3.83	3.80
3,3,3,3,3,3,3	aDManp	5.13	4.22	4.03	3.79	3.83	3.80
3,3,3,3,3,3	aDManp	5.13	4.22	4.03	3.79	3.83	3.80
3,3,3,3,3	aDManp	5.11	4.22	4.02	3.68	?	4.42
3,3,3,3,2	bDGlcpA	4.49	3.38	3.50	3.58	3.65	-
3,3,3,3	aDManp	5.25	4.28	4.09	3.84	3.98	3.84
3,3,3	aDManp	5.19	4.22	3.96	3.68	?	4.42
3,3	aDManp	5.13	4.22	4.03	3.79	3.83	3.80
3	aDManp	5.13	4.22	4.03	3.79	3.83	3.80
	aDManp	5.13	4.22	4.03	3.79	3.83	3.80


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,3,3,3,3,3,3,3,3	aDManp	104.56/5.13	72.32/4.22	80.85/4.03	68.86/3.79	76.18/3.83	63.72/3.80
3,3,3,3,3,3,3,3	aDManp	104.56/5.13	72.32/4.22	80.85/4.03	68.86/3.79	76.18/3.83	63.72/3.80
3,3,3,3,3,3,3	aDManp	104.56/5.13	72.32/4.22	80.85/4.03	68.86/3.79	76.18/3.83	63.72/3.80
3,3,3,3,3,3	aDManp	104.56/5.13	72.32/4.22	80.85/4.03	68.86/3.79	76.18/3.83	63.72/3.80
3,3,3,3,3	aDManp	105.19/5.11	72.54/4.22	81.07/4.02	69.28/3.68	76.30/?	63.65/4.42
3,3,3,3,2	bDGlcpA	104.32/4.49	75.32/3.38	78.13/3.50	74.35/3.58	79.47/3.65	
3,3,3,3	aDManp	102.41/5.25	79.77/4.28	79.30/4.09	69.08/3.84	76.06/3.98	63.31/3.84
3,3,3	aDManp	104.72/5.19	72.54/4.22	81.12/3.96	69.28/3.68	76.30/?	63.65/4.42
3,3	aDManp	104.56/5.13	72.32/4.22	80.85/4.03	68.86/3.79	76.18/3.83	63.72/3.80
3	aDManp	104.56/5.13	72.32/4.22	80.85/4.03	68.86/3.79	76.18/3.83	63.72/3.80
	aDManp	104.56/5.13	72.32/4.22	80.85/4.03	68.86/3.79	76.18/3.83	63.72/3.80

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

[*]O[C@H]1[C@H](O)[C@@H](CO)O[C@H](O[C@@H]2[C@H](O)[C@@H](O[C@@H]3[C@H](O)[C@@H](O[C@@H]4[C@H](O)[C@@H](O[C@@H]5[C@H](O)[C@@H](O[C@@H]6[C@H](O[C@@H]7O[C@H](C(=O)O)[C@@H](O)[C@H](O)[C@H]7O)[C@@H](O[C@@H]7[C@H](O)[C@@H](O[C@@H]8[C@H](O)[C@@H](O[C@@H]9[C@H](O)[C@@H](O[C@@H]%10[C@H](O)[C@@H]([*])O[C@H](CO)[C@H]%10O)O[C@H](CO)[C@H]9O)O[C@H](CO)[C@H]8O)O[C@H](CO)[C@H]7O)O[C@H](CO)[C@H]6O)O[C@H](CO)[C@H]5O)O[C@H](CO)[C@H]4O)O[C@H](CO)[C@H]3O)O[C@H](CO)[C@H]2O)[C@H]1O

1797.534 g/mol (C₆₆H₁₀₈R₂O₅₆, R = next and previous repeats, not counted in mol weight)

A glucuronomannan (GM) was derived by removal, through Smith degradation, of xylose from the native (3-O-acetylglucurono)xylomannan exopolysaccharide isolated from Tremella mesenterica. 13C-N.m.r. chemical shifts measured at various pD values were compared for p-nitrophenyl β-D-glucopyranosiduronic acid (1) and two GMs (2 and 3) differing in GlcA content (Man:GlcA; 2, 10:1; and 3, 5:1). Also measured and compared were pKa values for 1 and 2. One-dimensional and two-dimensional (COSY and HETCOR) n.m.r. data allowed unambiguous assignments of pD-sensitive chemical shifts due to 2-O-β-D-GlcpA substituents attached to a (1----3)-linked α-D-Manp backbone. The pKa and n.m.r. data indicated that the CO2H groups in either GM are independent of each other, and are similar in behavior to those of p-nitrophenyl β-D-glucopyranosiduronic acid molecules. The n.m.r. data confirmed the previous, chemically deduced, structural role of GlcpA in the native polysaccharide from T. mesenterica, and indicated that significant pD-induced changes occur in the stabilities of the glycosidic orientations in the GM. Previous 13C-n.m.r. assignments for 2-O-β-D-GlcpA in polysaccharides derived from Cryptococcus neoformans serotype A-variant were confirmed, except for the signal due to the anomeric carbon atom. This signal is now known to be pD-sensitive. In acidic solutions, it is coincident with the signal (104.5 p.p.m.) due to the anomeric carbon atoms of the unsubstituted α-D-Manp backbone residues. In basic solutions, the 2-O-β-D-GlcpA anomeric carbon resonance is shifted upfield by approximately 0.2 p.p.m., and is observed as a separate signal.

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,3,3,3	aDManp	104.56	72.32	80.85	68.86	76.18	63.72
3,3,3	aDManp	105.19	72.54	81.07	69.28	76.30	63.65
3,3,2	bDGlcpA	104.32	75.31	78.13	74.35	79.47	178.03
3,3	aDManp	102.41	79.77	79.30	69.08	76.06	63.31
3	aDManp	104.72	72.54	81.12	69.28	76.30	63.65
	aDManp	104.56	72.32	80.85	68.86	76.18	63.72


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,3,3,3	aDManp	5.13	4.22	4.03	3.79	3.83	3.80
3,3,3	aDManp	5.11	4.22	4.02	3.68	?	4.42
3,3,2	bDGlcpA	4.49	3.38	3.50	3.58	3.65	-
3,3	aDManp	5.25	4.28	4.09	3.84	3.98	3.84
3	aDManp	5.19	4.22	3.96	3.68	?	4.42
	aDManp	5.13	4.22	4.03	3.79	3.83	3.80


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,3,3,3	aDManp	104.56/5.13	72.32/4.22	80.85/4.03	68.86/3.79	76.18/3.83	63.72/3.80
3,3,3	aDManp	105.19/5.11	72.54/4.22	81.07/4.02	69.28/3.68	76.30/?	63.65/4.42
3,3,2	bDGlcpA	104.32/4.49	75.31/3.38	78.13/3.50	74.35/3.58	79.47/3.65	
3,3	aDManp	102.41/5.25	79.77/4.28	79.30/4.09	69.08/3.84	76.06/3.98	63.31/3.84
3	aDManp	104.72/5.19	72.54/4.22	81.12/3.96	69.28/3.68	76.30/?	63.65/4.42
	aDManp	104.56/5.13	72.32/4.22	80.85/4.03	68.86/3.79	76.18/3.83	63.72/3.80

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

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

986.829 g/mol (C₃₆H₅₈R₂O₃₁, R = next and previous repeats, not counted in mol weight)

Cryptococcus neoformans, the etiologic agent of cryptococcal meningoencephalitis, produces glucuronoxylomannan (GXM) as the major capsule component. Purified GXMs obtained from eight serotype A isolates of C. neoformans were treated by ultrasonic irradiation and then O-deacetylated prior to their comprehensive chemical analysis by GLC, GLC-MS, and 13C NMR spectroscopy. The average xylose: mannose: glucuronic acid molar ratio of the eight isolates is 1.96 +/- 0.25: 3.00: 0.58 +/- 0.10. Methylation analyses and 13C NMR spectroscopy show a general structure for GXM that is comprised of a linear (1----3)-α-D-mannopyranan substituted with β-D-GlcpA and with β-D-Xylp at O-2. Variable quantities of unsubstituted (1----3)-α-D-manp were observed between the eight isolates studied. In several isolates some of the (1----3)-α-D-manp residues are disubstituted with β-D-GlcpA at O-2 and with β-D-Xylp at O-4; this type of substitution was not previously thought to occur in serotype A isolates. Heterogeneity, between isolates, in the disposition of the substituents along the mannopyranan backbone was revealed by 13C NMR spectroscopy. The eight isolates, and three isolates previously studied, were each assigned to one of four distinct groups based on the 13C NMR chemical shifts of the anomeric carbons. Six of the eleven isolates gave identical spectra (Group I). The six major anomeric resonances from Group I were assigned to specific glycosidic linkages present in GXM. The remaining five isolates gave more complex spectra that are indicative of additional linkages and comprise the remaining three groups. Three of these five isolates contain substantial amounts of linkages previously thought to be distinctive of serotypes B and C, i.e., Manp residues that are 4-O-glycosylated with β-D-Xylp. Methylation analyses only predicted an average repeating unit, whereas 13C NMR spectroscopy demonstrated that GXM from each isolate may be categorized into four groups by the occurrence of distinct sequences of carbohydrate residues.

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

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

986.829 g/mol (C₃₆H₅₈R₂O₃₁, R = next and previous repeats, not counted in mol weight)