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The molecular structural dynamics of PEG in aqueous solution has been addressed with a series of experimental data correlating macroscopic solution properties of PEG with structural and hydration properties at molecular level, by using thermodynamic, UV Brillouin and Raman spectroscopy data. Water activity measured in PEG 600 solutions by a novel dynamical calorimetry approach shows that data reveal some non-equilibrium process for dilute solutions. Brillouin scattering data on aqueous solutions in the UV range made possible the measurement of the viscoelastic relaxation of the system with a characteristic temperature, TM, as precursor of the glass transition process at lower T, while at constant temperature, the addition of water to liquid PEG 600 first lead to a slight decrease and then to an increase in the solution viscoelastic relaxation consistent with the slowest polymer dynamics observed in the concentrated solution. Specific Raman bands corresponding to trans and gauche conformations of the C–C and C–O bonds and their sequences in the PEG chain have been identified and their relative intensities as a function of concentration evidence non-monotonous variations with a rather unusual concentration dependence for the frequency of C–O bonds in the range of physiological temperatures. The heuristic result is that the extended time-space domains approach suggests an overall quasi-regular solution behavior of semidilute PEG, with opposite concentration dependence at low and at high water content, similar to discontinuities observed in excess thermodynamic properties and eutectic phase diagram. The cross-over of these distinct behaviors occurs at the concentration close to that usually employed to mimic the cellular crowding in biomolecular systems, highlighting the interplay of water molecules in solute-solute interactions.

CE applications to charged polysaccharides are briefly reported. A simple procedure is presented to determine the esterification degree of a hyaluronan derivative. In this case the degree of substitution was as low as 14 %.The molecular weight distribution of mannuronic oligosaccharides mixture produced by hydrolysis of native polymannuronic is readily calculated from peak area of the species resolved by CE on the basis of a specific degree of polymerization.The influence of the applied electric field strength on the free solution mobility of hyaluronan samples is briefly addressed for molar masses of the order of 10(5) and 10(6) g/mol. The data are compared with the results obtained for a 50 % galactose substituted HA.Mobility data obtained as a function of buffer pH for a native HA sample as well as for two galactose-amide HA derivatives, having slightly different degrees of substitution, are presented and discussed in terms of the polymer charge density parameters ξ.In most cases, more questions than answers arise from the application of CE to charged polysaccharides. However, perspectives are disclosed for a further understanding of the reliability of CE applied for the structural elucidation of such macromolecules.

The objective of this paper is to emphasize the fact that while consistent interest has been paid to the industrial use of chitosan, minor attention has been devoted to spread the knowledge of a good characterization of its physico-chemical properties. Therefore, the paper attempts to critically comment on the conflicting experimental results, highlighting the facts, the myths and the controversies. The goal is to indicate how to take advantage of chitosan versatility, to learn how to manage its variability and show how to properly tackle some unexpected undesirable features. In the sections of the paper various issues that relate chitosan properties to some basic features and to advanced solutions and applications are presented. The introduction outlines some historical pioneering works, where the chemistry of chitosan was originally explored. Thereafter, particular reference is made to analytical purity, characterization and chain modifications. The macromolecular characterization is mostly related to molecular weight and to degree of acetylation, but also refers to the conformational and rheological properties and solution stability. Then, the antimicrobial activity of chitosan in relation with its solubility is reviewed. A section is dedicated to the formulation of chitosan biomaterials, from gel to nanobeads, exploring their innovative application as active carrier nanoparticles. Finally, the toxicity issue of chitosan as a polymer and as a constructed nanomaterial is briefly commented in the conclusions.

Capillary electrophoresis (CE) applications to charged polysaccharides are briefly reported. A simple procedure is presented to determine the esterification degree of a hyaluronan derivative. In this case, the degree of substitution was as low as 14%. The molecular weight distribution of mannuronic oligosaccharides mixture produced by hydrolysis of native polymannuronic is readily calculated from peak area of the species resolved by CE on the basis of a specific degree of polymerization. The influence of the applied electric field strength on the free solution mobility of hyaluronan samples is briefly addressed for molar masses of the order of 10(5) and 10(6) g/mol. The data are compared with the results obtained for a 50% galactose-substituted hyaluronic acid (HA). Mobility data obtained as a function of buffer pH for a native HA sample as well as for two galactose-amide HA derivatives, having slightly different degrees of substitution, are presented and discussed in terms of the polymer charge density parameters xi. In most cases, more questions than answers arise from the application of CE to charged polysaccharides. However, perspectives are disclosed for a further understanding of the reliability of CE applied for the structural elucidation of such macromolecules.

Evidences for the involvement of the Galectin-1 in the interaction of pig chondrocytes with a lactose-modified chitosan, namely Chitlac, are reported. The Chitlac glycopolymer has been shown to promote pig chondrocyte aggregation and to induce extracellular matrix production. Highly pure Galectin-1 was obtained from pig spleen by affinity chromatography and its identity was determined by ion spray mass spectrometry analysis of tryptic peptide fragments obtained after in-gel digestion. The complete sequence of pig Galectin-1 CDS was obtained by screening a pig EST database using human Galectin-1 sequence as template. The Galectin-1 cDNA was cloned into a pGEX-4T-1 expression vector and the recombinant protein was purified, characterized and used to produce a rabbit anti-serum. Recombinant Galectin-1 interacts in a dose-dependent manner with Chitlac as determined with ELISA assay. Expression level of galectin-1 gene, quantified by real-time PCR, was significantly higher in chondrocytes cultivated on Chitlac. In the same way, the presence of Chitlac stimulates secretion of Galectin-1 in culture medium that, by immunohistochemical analysis, revealed to be clustered on the surface of Chitlac-induced aggregates. These data indicate the role of Galectin-1 as a bridging agent between Chitlac and chondrocyte aggregates.

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.

Aim: To test the efficacy and toxicity of exosomal doxorubicin (exoDOX) compared with free doxorubicin. Materials & methods: The cytotoxic effects of exoDOX were tested in vitro and in nude mice by measuring the tumor volume. The toxic effects were evaluated by measuring the bodyweight and through histopathologic analyses. The biodistribution of DOX was assessed by MS. Results: In vitro and in vivo studies showed that exosomes did not decrease the efficacy of DOX. Surprisingly, exoDOX showed no cardiotoxicity as observed in DOX-treated mice and MS studies confirmed that the accumulation of exoDOX in the heart was reduced by approximately 40%. Conclusion: We demonstrated that exoDOX was less toxic than DOX through its altered biodistribution.

The present work reports a thorough conformational analysis of iodinated contrast media: iomeprol, iopamidol (the world's most utilized contrast agent), and iopromide. Its main aim is the understanding of the complex structural features of these atropisomeric molecules, characterized by the presence of many conformers with hindered rotations, and of the role of atropisomerism in the physicochemical properties of their aqueous solutions. The problem was tackled by using an extensive analysis of (13)C NMR data on the solutions of whole molecules and of simple precursors in addition to FT-IR investigation and molecular simulations. This analysis demonstrated that out of the many possible atropisomers, only a few are significantly populated, and their relative population is provided. The conformational analysis also indicated that the presence of a sterically hindered amidic bond, allowing a significant population of cis forms (E in iopromide and exo in iomeprol), may be the basis for an increased thermodynamic solubility of concentrated solutions of iomeprol.

Structural properties of several cross-linked hyaluronan derivatives, obtained by scanning electron microscopy, monodimensional NMR microscopy and small angle X-ray scattering of synchrotron radiation, are presented and compared with those observed for non-modified hyaluronic acid, used as a reference material. The experimental results, obtained in different media, showed a consistent picture of the synthesized matrices. In particular, the presence of zones of denser polymeric material observed by electron microscopy resulted in a higher transversal relaxation rate of the bulk water protons as well as in a decrease of the diffusion coefficient obtained by NMR microscopy. Moreover, the presence of polymer junction zones gave rise to the appearance of a well-defined correlation peak in the pattern of intensity of the scattered X-radiation.

The synthesis of some glycosylamines (1-amino-1-deoxy D-glucose, 1-amino-1-deoxy-D-galactose and 1-amino-1-deoxylactose) was carried out by treatment of the corresponding reducing sugars with ammonium hydrogencarbonate in concentrated ammonia. The reaction mixture was first analyzed by capillary electrophoresis with indirect absorbance detection and high performance anion-exchange chromatography with pulsed amperometric detection. Beside glycosylcarbamate, a known reaction by-product, fructose and lactulose were detected during the synthesis of 1-amino-1-deoxyglucose and 1-amino-1-deoxylactose, respectively. Quantification of glycosylamines was carried out by micellar electrokinetic chromatography with UV detection of their 9-fluorenylmethyloxycarbonyl (Fmoc) derivatives; lactulosylamine was thus detected in the synthesis mixture of 1-amino-1-deoxylactose. The Fmocglycosylamines were easily purified from the other components of the crude synthesis mixtures.

Exosomes are nanovesicles produced by cells embedded of cellular information and represent a formidable natural cargo for communication. The name “FedExosomes” denotes the general idea that exosomes could deliver cargo that conveniently manipulated could be of help for patient therapy. Up to now, a few papers demonstrated that modified exosomes could deliver drugs specifically to cancer cells and many laboratories are now studying their properties and compositions. Differently from previously published papers that focused on the efficacy of the doxorubicin encapsulated in exosomes (exoDOX), for the first time, we demonstrated that unmodified exosomes loaded with DOX are less toxic than free DOX by altering the biodistribution of the drug. The heart tissue of mice treated with a toxic dosage of DOX exhibited scattered cytoplasmic paranuclear vacuoles and focal intense eosinophilic cytoplasm, devoid of their transversal striations with moderate disarrangement of cardiomyocytes. The heart tissue of exoDOX treated mice at the same dosage appeared normal. Mass spectrometry studies confirmed that although the quantity of exoDOX in the blood was the same compared to DOX, accumulation in the heart was reduced of about forty percent. Maximum tolerated dose of exoDOX treated mice is double than free DOX. Mice treated at double concentration of exoDOX vs DOX have reduced tumor burden compared to free DOX without observable toxicity. We demonstrated that exoDOX increased the therapeutic index of DOX by reducing toxic side effects and envisage a similar mechanism for others chemotherapeutic drugs. Since the exosomes could be prepared directly from the patients, our preclinical approach could have a possible immediately application in the clinic. Citation Format: Flavio Rizzolio, Mohamad Hadla, Giuseppe Corona, Isabella Caligiuri, Stefano Palazzolo, Sabrina Semeraro, Amelia Gamini, Vincenzo Canzonieri, Giuseppe Toffoli. Exosomal encapsulation of doxorubicin reduces the cardiac toxicity of mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2205.

A survey of the more recent applications and developments of capillary electrophoresis (CE) to study reactions involving saccharide-bearing molecules is presented. Selected studies are cited to highlight the potentialities as well as the versatility of capillary electrophoresis. CE strategies that are applied to monitoring the synthesis of carbohydrate-based molecules are discussed. One strategy is the monitoring of mono- and oligosaccharides synthesis; another is the preparation of glycoconjugates, including those bearing nonsaccharidic backbone. The biological implications are also discussed, such as the action of enzymes on carbohydrate-based species, particularly on polysaccharides, in terms of kinetic parameters, enzyme activity, and substrate specificity evaluation, as well as active site identification. Studies showing how capillary electrophoretic strategies are applied to investigate naturally occurring processes, pursuing the ultimate goal of monitoring single cell reactions, are also presented.

The physicochemical solution properties of the galactoglucan excreted by Rhizobium meliloti strain YE-2(S1) have been investigated by capillary viscometry, potentiometric titration, isothermal mixing microcalorimetry, and circular dichroism. Potentiometric and chiro-optical data, as a function of the degree of ionisation, indicate the absence of a co-operative conformational transition. Solution properties, as a function of ionic strength and temperature, suggest that the galactoglucan adopts a disordered conformation characterised by moderate flexibility. Polyelectrolyte theory is used to fit the enthalpy of dilution data with a suitable linear charge-density parameter. Conformational calculations and chain modelling, using molecular mechanics, give an unperturbed characteristic ratio, (C infinity) of 20, which was smaller than that estimated from intrinsic-viscosity and molecular-weight data for an expanded-coil chain model.

Literature data and unpublished results have been used to correlate the primary and secondary structures of gellan, welan, rhamsan, succinoglycan and galactoglucan in solution with their rheological properties. In dilute solution the chain conformation ranges from that of the random coil of galactoglucan to that of the stiffer double helix of gellan, and possibly a similar conformation exists for welan and rhamsan. In semidilute solution the rheological behaviour of the above microbial polysaccharides is mainly determined by their conformation in solution. Ordered regions along the polysaccharide chains seem to be the prerequisite to establish intermolecular interactions which lead to non-transient polymer networks. The lack of ordered secondary structure leads to entangled networks in which the topological constraints govern the rheological behaviour. Experimental evidence is reported to support the hypothesis that the chain conformation and rigidity can be qualitatively predicted on the basis of rheological measurements.

N-linked or O-linked glycans derived from glycoprotein processing carry, an N-acetylglucosamine or an N-acetylgalactosamine respectively, at their reducing termini. The presence of the N-acetylamino group on C-2 of reducing sugar residues has been reported to hamper the derivatization reaction with a chromophore at the anomeric centre. In this paper N-acetyllactosamine, N-acetylglucosamine, N-acetylgalactosamine and several other neutral monosaccharides are coupled to three different dyes (4-aminobenzonitrile, 2-aminopyridine, 2-aminobenzoic acid (2-AA)) by reductive amination and analysed by CE with UV detection. The 2-AA derivatives showed the lowest concentration detection limits, varying approximately in the 2-3 muM range for the saccharides tested including the N-acetamido ones. The possibility to separate and detect with the same sensitivity ten 2-AA-labelled monosaccharides mainly found in mammalian or plant glycoproteins in a single CE run is highlighted. The analysis has been carried out in less than 25 min using the borate-complexation method in CZE mode. The influence of the strength of the acid used as catalyst in the chemical modification of the sugars with 2-AA is also shortly addressed.

Capillary electrophoresis (CE) applications to charged polysaccharides are briefly reported. A simple procedure is presented to determine the esterification degree of a hyaluronan derivative. In this case, the degree of substitution was as low as 14%. The molecular weight distribution of mannuronic oligosaccharides mixture produced by hydrolysis of native polymannuronic is readily calculated from peak area of the species resolved by CE on the basis of a specific degree of polymerization. The influence of the applied electric field strength on the free solution mobility of hyaluronan samples is briefly addressed for molar masses of the order of 10(5) and 10(6) g/mol. The data are compared with the results obtained for a 50% galactose-substituted hyaluronic acid (HA). Mobility data obtained as a function of buffer pH for a native HA sample as well as for two galactose-amide HA derivatives, having slightly different degrees of substitution, are presented and discussed in terms of the polymer charge density parameters xi. In most cases, more questions than answers arise from the application of CE to charged polysaccharides. However, perspectives are disclosed for a further understanding of the reliability of CE applied for the structural elucidation of such macromolecules.

Experimental results obtained by membrane equilibria, osmotic pressure, viscosity and circular dichroism measurements on alginate and pectate solutions in the presence of Ca2+ ions are presented. From equilibrium dialysis data both electrostatic and cooperative interactions seem to describe the binding process of Ca2+ ions onto polymer chains. An increase of the number-average molecular weight Mn for both poly-saccharides with calcium ion concentration is observed. An increase of polymer dimensions can well account for the observed increase of the intrinsic viscosity [η] with bound Ca2+ ion concentration at several ionic strengths.

Among disaccharides, trehalose is demonstrated to be the most efficient bioprotecting agent against dehydration and freezing and it has been identified, at high concentration, in several natural organisms with important applications in the preservation of mammalian cells. However, despite its relevance in biopreservation, the physical mechanism underlying the effectiveness of trehalose has not yet been definitely clarified. One reason could rely on the experimental difficulty in measuring the dynamics of the trehalose–water system at high concentration, as its behaviour is often derived as an extrapolation from low-concentration data. In this report we present the results of a feasibility experiment in which the dynamic structure factor S(Q, ω) of a trehalose–water solution at high trehalose concentration (79% w) has been measured for the first time over a rather large temperature range (235 K

Rapid and reliable methods for the analysis of O- and C-allyl galactopyranosides and glucopyranosides are presented, based on capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MEKC). In MEKC, the formation of chromophoric and charged complexes between the saccharides and borate as well as the hydrophobic interactions with micelles jointly contributed to the selective separation and sensitive detection of all the investigated anomeric couples. Some non-purified synthesis mixtures of C-allyl glycosides were successfully characterised without pre-treatment. MEKC buffer conditions for which glycosides separation was successfully achieved were then exported and applied to reverse-phase liquid chromatography (RP-HPLC), for the quantitative isolation of each allyl glycoside anomer. Identification of the obtained anomeric products was performed by electrospray mass spectrometry and (13)C NMR spectroscopy. Glycoside-solvent interactions driving the selective anomeric separation were shortly addressed and discussed on the basis of sugar derivatives structural differences.

A reductive amination reaction (N-alkylation) obtained exploiting the aldheyde group of lactose and the amino group of the glucosamine residues of chitosan (d.a. 89%) afforded a highly soluble engineered polysaccharide (chitlac) for a potential application in the repair of the articular cartilage. Chitosan derivatives with 9% and 64% of side chain groups introduced have been prepared and characterized by means of potentiometric titration, 1H-NMR and intrinsic viscosity. Both polymers, with respect to the unmodified chitosan, induce cell aggregation when in contact with a primary culture of pig chondrocytes, leading to the formation of nodules of considerable dimensions (up to 0.5–1 mm in diameter). The nodules obtained from chondrocytes treated with chitlac with the higher degree of substitution have been studied by means of optical and electron microscopy (SEM, TEM) and the production of glycosaminoglycans (GAGs) and collagen has been measured by means of colorimetric assays. The chondro-specificity of GAG and collagen was determined by RT-PCR. The results show that the lactose-modified chitosan is non-toxic and stimulates the production of aggrecan and type II collagen.

Galactose moieties have been introduced on the uronic groups of alginates from different sources via an N-glycosidic bond, thus affecting the net charge on the polymer chain. The modified polymers have been analyzed by means of viscosity and of high-performance size-exclusion chromatography combined with refractive index multiple angle laser light scattering (HPSEC-RI-MALLS) measurements. The latter technique enabled us to determine the molecular weight of the modified polymers, proving that the synthetic procedure did not affect the chemical integrity of the chain. The intrinsic viscosity and the radius of gyration data showed that the hydrodynamic properties of the polymer chain varied with the degree and the pattern of substitution. In the presence of a relatively low galactose content (up to 19%), a decrease of the hydrodynamic dimensions of the coil was experienced, while on increasing the degree of substitution (especially on GG diads) a re-extension of the chain was discovered. Measurements of intrinsic viscosity at different values of the degree of dissociation have demonstrated that this effect cannot be solely explained by the reduction of the charge density of the polymer. Rather, it implies the occurrence of conformational changes of the chain that are specific to the chemical nature of the site of substitution. These data have been supported by the values of the persistence length of the natural and modified polymers obtained with the Doty-Benoit equation. The chiro-optical properties of the modified polymers studied by means of circular dichroism (CD) spectroscopy confirmed that conformational variations occurred to the polymeric chain upon introduction of galactose residues.

Coupling of alginate with 1-amino-1-deoxygalactose in the presence of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide results in a substituted polymer containing galactose side linked via an amide bond. To clarify the degree and pattern of substitution, a (1)H NMR study on the anomeric region of modified alginate, polymannuronate, alginate enriched in guluronic acid (G-enriched alginate), and polyalternating MG, was carried out (G, alpha-l-guluronic acid; M, beta-d-mannuronic acid). From the resonance of the proton at position 1 of galactosylamine, it was possible to determine the amount of galactose linked to mannuronic and to guluronic residues, respectively. Furthermore, (1)H NMR spectroscopy revealed a higher reactivity of guluronic residues for low degrees of conversion. Modified alginates with 7% and 19% of substitution are both able to form stable beads in the presence of calcium ions. The effect of galactose substitution on the dimensions, swelling, and stability of the beads has been studied and the cytotoxicity of the modified polymer evaluated in preliminary biological tests.

A trisaccharide (Glca1–4Glca1–6Glc) and a tetrasaccharide (Glca1–4Glca1–4Glca1–6Glc) the structures of which are related to that of repeating unit of pullulan have been obtained, exploiting the transglycolytic activity of Aspergillus niger cyclodextrin glucanotransferase. Bothproducts were obtained in one-pot reaction using as a donor th e a-cyclodextrin and as an acceptor the disaccharide isomaltose. The regioselectivity of the reaction was 85% for the tetrasaccharide and 80% for the trisaccharide. The yield of reaction resulted to be 42% for the synthesis of trisaccharide and 25% for that of tetrasaccharide. Purification of products was performed by size exclusion chromatography and by semipreparative reverse phase HPLC after reversible derivatization with 2-aminopyridine. Structural characterization was performed by capillary electrophoresis, ion-spray mass spectrometry, and by 13 C-NMR spectroscopy. A comparison of these results with those obtained by using a-D-glucosidase, which had been effective for the synthesis of the disaccharide isomaltose, is reported. 2002 Elsevier Science (USA). All rights reserved.

Three derivatives of the biocompatible polymer poly(styrene-co-maleic anhydride) (SMA) were obtained with 1-amino-1-deoxy-beta-D-galactose, 1-amino-1-deoxy-beta-D-glucose, and 1-amino-1-deoxy-beta-D-lactose, respectively. The amino sugars were chemically conjugated via formation of an amide bond between the anomeric amino group of the sugar residue and the anhydride of the copolymer, giving the corresponding glycoconjugate derivatives. Colorimetric assay of the unreacted amino groups and elemental analysis were used to determine the degree of substitution. About 56%, 54%, and 94% of the available anhydride groups reacted to give galactosyl-amide (SMA-Gal), glucosyl-amide (SMA-Gluc), and lactosyl-amide (SMA-Lac) branched polymers, respectively. The synthesized glycopolymers were characterized by Fourier transform infrared spectroscopy, gel permeation chromatography, circular dichroism, and UV and fluorescence spectroscopy. The release of glucosylamine from the glucosyl-amide branched polymer, by basic hydrolysis, was monitored by high-performance anion-exchange chromatography and by capillary electrophoresis, providing for an additional check of the degree of substitution of this specific polymer derivative. Biological activity tests showed that both SMA-Gal and SMA-Lac allow adhesion of HepG2 hepatic cells about five times larger than that of hydrolyzed, underivatized SMA.

An efficient and rapid synthesis of the derivative of the biocompatible polymer poly(styrene co-maleic acid) with Linear B disaccharide (Galili antigen) was achieved. The oligosaccharide portion was obtained by a transglycosylation reaction catalyzed by coffee bean alpha-D-galactosidase using p-nitrophenyl-alpha-D-galactopyranoside both as donor and as acceptor. The reaction was carried out in aqueous buffer without any organic cosolvent. The molar yield (30%) and the regioselectivity (82%) were significantly improved with respect to the data so far reported in the literature. The selective reduction of the p-nitrophenyl group afforded the p-aminophenyl derivative of Linear B disaccharide. Linkage of this derivative via an amidic bond to the poly(styrene co-maleic acid) was obtained by using N'-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide. The products were chemically characterized by ionspray mass spectrometry, infrared, (13)C- and (1)H-nuclear magnetic resonance. The glycopolymer specifically reacts with human serum containing antibodies and with a mixture of partially purified human IgG and IgM anti-Linear B. It efficiently protects pig kidney PK15 cells from cytotoxic effects of human serum.

Structural properties of several cross-linked hyaluronan derivatives, obtained by electron microscopy, monodimensional NMR microscopy and low angle X-ray scattering by synchrotron radiation, are presented and compared with those observed for non-modified hyaluronic acid used as reference material. The experimental results, obtained in different media by the different techniques, showed a consistent picture of the synthesized matrices. In particular, the presence of zones of more dense polymeric material observed by electron microscopy resulted in a higher transversal relaxation rate of the bulk water protons as well as in a decrease of the diffusion coefficient obtained by NMR microscopy. Moreover the presence of polymer junction zones gave rise to the appearance of an additional correlation peak in the pattern of the intensity of the scattered X-radiation.

A modification of the Counterion Condensation (CC) theory of linear polyelectrolytes has been developed to take into account the presence of specific interactions between the polymer and the counterions. The particular case in which the polymer is the Ionic Polysaccharide (IPS) K-carrageenan and the ions are monovalent alkaline ions with high atomic number (e.g. K + , Cs + ) has been considered. K-carrageenan in the presence of such ions undergoes a conformational transition eventually leading to the formation of side-by-side ordered pairs of chains, acting as junctions in a 3-D gel network. The presence of the conformational equilibrium leading to the fundamental ordered single chain (1h) and of the following one leading to chain pairing (1h.1h) complicates the description of the overall process. Nevertheless, they have been successfully accommodated together with the specific ion-polymer interactions to give a self-consistent set of thermodynamic equations. The application of the model to the experimental calorimetric data of mixing K-carrageenan with Cs + ions has been successful. The thermodynamic parameters for the main chain conformational changes, as well as those for the specific interactions, have been calculated. In addition, the approach allowed for the evaluation of the number of polymer repeating units in the cooperative units of both 1h and of 1h.1h. They are as large as 50 and 35, respectively.

The enzymatic synthesis of 6-O-alpha-glucopyranosyl-glucopyranose (isomaltose) was achieved. The regiospecific transglycosylation reaction was catalyzed by a crude preparation of alpha-D-glucosidase from Aspergillus niger, using p-nitrophenyl alpha-D-glucopyranose as the donor and glucopyranose as the acceptor. The yield of the reaction was 59% on a molar basis with respect to the donor. The structural identity of the product was fully determined by HPLC, HPAEC-PAD, ionspray mass spectrometry and (13)C NMR.

The conformation of agarose in dimethyl sulfoxide (Me 2 SO) or in aqueous solutions has been studied by 1 H and 13 C NMR spectroscopy. Partial assignment of the 1 H NMR spectrum of agarose in Me 2 SO- d 6 has been obtained using 1D and 2D techniques. Coupling constant, chemical shift temperature dependence and exchange rate data for the hydroxyl protons have been used to establish the presence of intramolecular hydrogen bonds. The reported data show that the protons of HO-2 in the 3,6- anhydro-α- l -Gal p residue (A) and HO-4 in the β- d -Gal p residue (G) have a less rotational freedom and a higher thermal stability than the other hydroxyl protons. On the basis of the exchange rate data, obtained from 1D NOESY experiments with selective excitation of the residual water signal, an inter-residue hydrogen bond is proposed only for G HO-4 in Me 2 SO, while the involvement of A HO-2 in the agarose-Me 2 SO complex, recently reported, is suggested. The latter hypothesis is supported by the 13 C NMR data obtained in Me 2 SO- d 6 , in a Me 2 SO- d 6 -D 2 O mixture and in D 2 O, which also show that a relatively small conformational rearrangement of the chain segments occurs in aqueous solution. Deuterium isotope effect data indicate the persistence in water of the inter-residue G HO-4 mediated hydrogen bond with the additional involvement of G HO-6. In addition, the negligible isotope effect on the C-2 atom of A would indicate that the corresponding OH group, although free from the agarose-Me 2 SO complex, is not available for further strong interactions. The hydrogen bonding and conformation of agarose in methyl sulfoxide or in aqueous solutions were studied by NMR spectroscopy. Based on 1 H NMR data, an inter-residue H-bond is proposed for HO-4 of β- d -Gal p in Me 2 SO, while the involvement of HO-2 of 3,6- anhydro -α- l -Gal p in an agarose-Me 2 SO complex is suggested. These hypotheses are supported by the 13 C NMR data.

The secondary structure of xanthan in solutions of relatively low salt concentration and at room temperature has been investigated using static light scattering experiments. Additional evidence has been found for a dimeric structure at 25 degrees C in 0.01 M NaCl. From the experimental z-average mean square (ms) radius of gyration, a value for the persistence length p has been estimated, taking explicitly into account the polydispersity of the three samples used, which has been established by gel permeation chromatography (GPC) measurements. The experimental particle scattering functions of the three samples are consistent with theoretical estimates for polydisperse systems with the same value of p = 65 +/- 10 nm and the molar mass per unit length for a dimeric structure. This secondary structure remains unaffected by the ionic strength in the 0.005-0.01 M range. Partial aggregation seems to occur at higher NaCl concentrations. Light scattering and GPC data show that heating the xanthan 0.01 M NaCl solutions to about 70 degrees C considerably reduces the Mw of the low molar mass sample (2.3 x 10(5) g.mol-1), contrary to what is observed for the high molar mass sample (1.8 x 10(6) g.mol-1). These experimental findings can be accounted for by a partial temperature-induced dissociation of the xanthan dimers according to an all-or-none mechanism.

O -Carboxymethylagar was produced from a commercial, low methoxylated agar sample. The degree of substitution of the sample was 1·2 carboxymethyl groups per disaccharide repeating unit. The procedure used for the synthesis lowered the polymer molecular weight slightly. Carboxymethylagar exhibited different binding behaviour with different divalent cations. Interaction with ions was monitored by polarography, circular dichroism, and isothermal microcalorimetry. The polycarboxylate interacted strongly with lead ions, and showed lower affinity for copper ions. Little interaction was detected with respect to other divalent cations.

The interaction of ions with certain carbohydrate ligands in aqueous solution is examined. Simple systems are analysed to understand the role of conformational transitions and the possible specificity often advanced as an explanation for the complexity of the binding. Evidence of an extrinsic chirality of bound ionic chromophores is presented for nearly enantiomeric complexes of alginate and pectins with Pb2+ ions. Data on the thermodynamics of interaction between divalent cations and typical ionic polysaccharides are presented with a view to offering an interpretation based upon current theoretical models of polyelectrolyte solutions. In particular, reference is made to the linear correlation between thermodynamic state functions.