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The present work aims at understanding the structural basis of the biological recognition of Tn antigen (GalNAc-α-O-L-Ser), a specific epitope expressed by tumor cells, and the role of its amino acidic moiety in the interaction with its receptor (the isolectin B4 extracted from Vicia villosa). An NMR structural characterization of the α and β anomers, based on J couplings and molecular modeling revealed a structure in very good agreement with data reported in literature for variants of the same molecules. In order to demonstrate the involvement of the amino acid in the ligand–receptor recognition, also GalNAc-α-O-D-Ser was studied; the change in the stereochemistry is in fact expected to impact on the interaction only in case the serine is part of the epitope. Relaxation properties in the presence of the receptor clearly indicated a selective recognition of the natural L form, probably due to the formation of a water-mediated hydrogen bond with Asn 129 of the protein.

Circular dichroism (CD) is presented as a reliable and sensitive method of determining the diadic frequency composition of alginate (F(GG), F(MM) and F(GM+MG)). The availability of samples, very largely or even completely conforming to the limiting structures of polymannuronate (MM)(n), polyguluronate (GG)(n) and polyalternating MG (MG)(n), respectively, allowed the limiting CD spectra for each alginate diad to be obtained. These showed very different CD behaviour, thus pointing out the crucial importance of the neighbouring residue in chiroptical properties. Using an iterative best-fit procedure, the diadic composition of commercial alginates could be obtained from their respective CD spectra by means of a linear combination of the spectra of the three limiting diads. The results were found in excellent agreement with the composition parameters obtained by 1H NMR spectroscopy.

The synthesis of the disaccharide 6-O-beta-D-xylopyranosyl-2-acetamido-2-deoxy-D-glucopyranose (N-acetylprimeverosamine), structurally related to the natural disaccharide 6-O-beta-D-xylopyranosyl-D-glycopyranose (primeverose), was obtained via a transglycosylation reaction catalyzed by a crude preparation of beta-D-xylosidase from Aspergillus niger, using p-nitrophenyl beta-D-xylopyranoside as the donor and 2-acetamido-2-deoxy-D-glucopyranose as the acceptor. The yield of the reaction was 36% on a molar basis with respect to the donor. The chemical identity of the product was assessed by HPLC, ionspray mass spectrometry and NMR spectroscopy.

The reaction of hyaluronic acid benzyl ester derivative (1) with trimethyl orthoacetate in the presence of an acid catalyst in N,N-dimethylformamide gave the expected 4,6-orthoester 2, along with its hydrolysed products the 6-O- (3) and the 4-O- (4) acetate hyaluronic acid benzyl esters as minor components. Acid hydrolysis of 2 followed by O-4→O-6 acetyl migration using t-butylamine afforded a mixture which contained the 6-acetate 3 as the major and the 4-acetate 4 as the minor compound. Synthesis of a mixture of 2′,3′,6- (6) and 2′,3′,4- (7) tri-acetates was achieved by peracetylation of 2 followed by treatment with aqueous acetic acid. Compound 1 on treatment with 2,2-dimethoxypropane in dimethyl sulfoxide in the presence of p-toluenesulfonic acid afforded the corresponding 4,6-O-isopropylidene derivative 8. The 2′,3′-di-O-acetate 10 was obtained from 8 by conventional acetylation followed by de-acetylation using trifluoroacetic acid. Acetylation of 1 with acetic anhydride in a mixture of N,N-dimethylformamide and pyridine gave the expected 2′,3′,4,6-tetraacetate derivative 11. In order to facilitate the NMR assignments of the acetate groups in 3–11, the 2′,3′-diacetate-4,6-di(trideuterioacetate) 12 and the 2′,3′-di(trideuterioacetate)-4,6-diacetate 13 were prepared. The effect of substituents on conformational changes around the glycosidic linkages has been investigated.

The 1 H- and 13 C-NMR spectra of the ethyl and benzyl esters and the tetrabutylammonium and tetraethylammonium salts of hyaluronic acid { 2)-β- d -Glc p A-(1 → 3)-β- d -Glc p NAc-(1 n } in Me 2 SO- d 6 have been assigned using 1D and 2D techniques. The chemical shifts of the resonance of GlcNAc C-3 suggest that the relative orientations of the monosaccharides at the (1 → 3) linkage in the esters and salts are different. Small differences in the chemical shifts of the resonance GlcA C-4 suggest only a slight conformational variation around the (1 → 4) linkage. The 13 C-NMR data also suggest similarities in conformation between the esters in Me 2 SO- d 6 and the salts in water. The chemical shifts of the 1 H resonances for NH and OH groups and their temperature dependence for the esters and salts in Me 2 SO reveal markedly stronger inter-residue hydrogen bonds between the carboxyl and NH groups and between HO-4 of GlcA and O-5 of GlcNAc for the salts. The 3 J 2,NH values indicate a slightly different orientation for the acetamido group. For solutions in Me 2 SO, the higher segmental flexibility of the esters is supported by the line widths, whereas the reduced viscosity for the tetrabutylammonium salt showed a sigmoidal concentration dependence and suggests association of chains which could contribute to the segmental rigidity. The linear concentration dependence for the benzyl ester suggests a higher overall flexibility without chain association.

The conformation of κ-carrageenan in solution was studied combining 1 H and 13 C NMR with molecular mechanics. The experimental conditions were chosen to characterize the disordered conformation of the polymer. Particular attention has been given to explore a wide range of experimental conditions as to the dependence on solvent (water and Me 2 SO), polymer concentration, temperature, pH, presence of a denaturing agent (guanidinium chloride), and of ions otherwise able to induce conformational order of the carrageenan chains, either in solution (I − ) or in the gel state (Rb + ). Two-dimensional NOE experiments were analyzed to obtain information on internuclear distances, and molecular mechanics provided the range of energetically accessible conformations. Two inter-residue topological constraints were clearly identified: their combination is rather restricting for the chain and suggests that the disordered conformation of κ-carrageenan is characterized by an intrinsic stiffness with high values of persistent length and characteristic ratio. They also rule out any postulated interchain hydrogen bonds. In contrast, experiments on the temperature dependence of the chemical shift in Me 2 SO reveal the existence of two inter-residue intramolecular H-bonds which might contribute positively to the rigidity of the polymer chain. The overall picture emerging from the present results is that of a locally elongated ‘loose single helix’.

A series of oligosaccharides was prepared from hyaluronate by depolymerisation with bovine testicular hyaluronidase. Complete assignment of the 1 H and 13 C NMR spectra was obtained for the disaccharide, the tetrasaccharide, and the NaBH 4 -treated tetrasaccharide, by using various 1D and 2D NMR methods. The 1 H assignments for the tetrasaccharide differ from the incomplete data reported recently (ref. 11). The 13 C NMR spectra of the aqueous di, tetra-, hexa-, and octa-saccharides of this series show that all resonances, apart from those subject to obvious end effects, have chemical shifts comparable to those of the corresponding resonances of hyaluronate in D 2 O. The observed 13 C chemical shifts suggests that cooperative intramolecular hydrogen bonds probably play a minor role in determining the conformation of hyaluronate in water.

By adding finely powdered sodium nitrite to solutions of amylose in 85% (w/w) orthophosphoric acid, a series of d -glucurono- d -glucans was prepared, having number-average molecular weights of ∼104 and GlcA/Glc ratios varying from 0.5 to 3.0. The products were soluble in water, both as sodium salts and free acids, formed complexes with iodine having λmax in the range of 508–574 nm, and exhibited strongly pH-dependent chiroptical properties.

Whereas acetylation does not severely affect the molecular weight and polydispersity index of alginate, it greatly enhances the swelling ability of calcium gels made from these polymers. The affinity of alginate for calcium ions is diminished by acetylation as shown by the effect on the c.d. spectra and by the decrease in gel strength. Viscosity data suggest that a modest degree of acetylation (d.a. up to ∼11%) causes an expansion of the molecular chain, whereas a higher d.a. generates more flexible polymers.