The glycosaminoglycan hyaluronan is involved in a diverse range of physiological and diseases processes and comprises repeated disaccharide units of N-acetyl-d-glucosamine (GlcNAc) and d-glucuronic acid (GlcA). A molecular description of the solution conformation of HA is required to account for this biology, which is best attained using nuclear magnetic resonance (NMR). NMR studies of the polymer, however, are frustrated by resonance overlap arising from the highly degenerate structure. In contrast, end-effects in oligosaccharides can produce some chemical shift dispersion, giving the possibility that their conformational properties can be measured and extrapolated to models of the polymer. We report the complete resolution and assignment of (1)H, (13)C and (15)N nuclei in hyaluronan oligosaccharides with seven different naturally occurring terminal rings. At 900MHz, all (1)H nuclei in the hexasaccharide GlcA-β-(1→3)-GlcNAc-β-(1→4)-GlcA-β-(1→3)-GlcNAc-β-(1→4)-GlcA-β-(1→3)-GlcNAc-OH were uniquely resolved and the two central rings were found to be a good model for the polymer environment. These assignments now allow resolved, unambiguous structural restraints to be acquired on this oligosaccharide and extrapolated to models for the solution conformation of the polymer.

coupling constant, resolution, hyaluronan, strong coupling, end-effect

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,4,3,4,3	bDGlcpA	104.13	73.98	76.61	72.96	76.86	?
3,4,3,4,2	Ac	?	23.73
3,4,3,4	bDGlcpN	101.67	55.45	84.29	69.75	76.59	61.79
3,4,3	bDGlcpA	104.30	73.75	74.84	81.09	77.42	?
3,4,2	Ac	?	23.73
3,4	bDGlcpN	101.72	55.52	83.72	69.64	76.53	61.77
3	bDGlcpA	104.22	73.68	74.90	81.18	77.51	?
2	Ac	?	23.47
	?DGlcpN


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,4,3,4,3	bDGlcpA	4.455	3.319	3.495	3.496	3.722	-
3,4,3,4,2	Ac	-	2.027
3,4,3,4	bDGlcpN	4.554	3.843	3.705	3.539	3.477	3.776-3.918
3,4,3	bDGlcpA	4.457	3.344	3.577	3.736	3.704	-
3,4,2	Ac	-	2.021
3,4	bDGlcpN	4.548	3.839	3.707	3.519	3.476	3.764-3.910
3	bDGlcpA	4.463	3.363	3.578	3.741	3.699	-
2	Ac	-	2.012
	?DGlcpN


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,4,3,4,3	bDGlcpA	104.13/4.455	73.98/3.319	76.61/3.495	72.96/3.496	76.86/3.722	
3,4,3,4,2	Ac		23.73/2.027
3,4,3,4	bDGlcpN	101.67/4.554	55.45/3.843	84.29/3.705	69.75/3.539	76.59/3.477	61.79/3.776-3.918
3,4,3	bDGlcpA	104.30/4.457	73.75/3.344	74.84/3.577	81.09/3.736	77.42/3.704	
3,4,2	Ac		23.73/2.021
3,4	bDGlcpN	101.72/4.548	55.52/3.839	83.72/3.707	69.64/3.519	76.53/3.476	61.77/3.764-3.910
3	bDGlcpA	104.22/4.463	73.68/3.363	74.90/3.578	81.18/3.741	77.51/3.699	
2	Ac		23.47/2.012
	?DGlcpN

The spectrum also has 6 signals at unknown positions (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CC(=O)N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@@H]1O[C@H](C(=O)O)[C@@H](O[C@@H]2O[C@H](CO)[C@@H](O)[C@H](O[C@@H]3O[C@H](C(=O)O)[C@@H](O[C@@H]4O[C@H](CO)[C@@H](O)[C@H](O[C@@H]5O[C@H](C(=O)O)[C@@H](O)[C@H](O)[C@H]5O)[C@H]4NC(C)=O)[C@H](O)[C@H]3O)[C@H]2NC(C)=O)[C@H](O)[C@H]1O

1155.969 g/mol (C₄₂H₆₅N₃O₃₄)