Carbohydrate-Derived Polytriazole Nanoparticles Enhance the Anti-Inflammatory Activity of Cilostazol.

Poly(amide-triazole) and poly(ester-triazole) synthesized from d-galactose as a renewable resource were applied for the synthesis of nanoparticles (NPs) by the emulsification/solvent evaporation method. The NPs were characterized as stable, spherical particles, and none of their components, including the stabilizer poly(vinyl alcohol), were cytotoxic for normal rat kidney cells. These NPs proved to be useful for the efficient encapsulation of cilostazol (CLZ), an antiplatelet and vasodilator drug currently used for the treatment of intermittent claudication, which is associated with undesired side-effects. In this context, the nanoencapsulation of CLZ was expected to improve its therapeutic administration. The carbohydrate-derived polymeric NPs were designed taking into account that the triazole rings of the polymer backbone could have attractive interactions with the tetrazole ring of CLZ. The activity of the nanoencapsulated CLZ was measured using a matrix metalloproteinase model in a lipopolysaccharide-induced inflammation system. Interestingly, the encapsulated drug exhibited enhanced anti-inflammatory activity in comparison with the free drug. The results are very promising since the stable, noncytotoxic NP systems efficiently reduced the inflammation response at low CLZ doses. In summary, the NPs were obtained through an innovative methodology that combines a carbohydrate-derived synthetic polymer, designed to interact with the drug, ease of preparation, adequate biological performance, and environmentally friendly production.

Synthesis, characterization and chemical degradation of poly(ester-triazole)s derived from d-galactose.

α-Azide-ω-alkynyl ester monomers were designed and synthesized in order to obtain hydrolytically degradable polymers. The monomers were prepared from d-galactose, as a renewable resource. Environmentally benign azido-alkyne cycloaddition polymerizations were conducted to afford poly(ester-triazole)s, with complete atom economy. Although polymer formation prevailed under optimized polymerization conditions, variable proportions of cyclic oligomer byproducts were detected. The Cu-catalyzed click polymerization led regioselectively to 1,4-disubstituted triazole linkages, while the thermal, metal-free polymerization produced a random distribution of 1,4- and 1,5-disubstituted triazoles in the polymer backbone. The poly(ester-triazole)s exhibited high molecular weights (M w in the range 35-85 kDa). They were soluble in organic solvents but highly insoluble in water, thus removal of the Cu(i) catalyst was simplified. The polymers were stable up to 300 °C, and had T g values in the range 90-100 °C. The materials were hydrolysed under either basic or strong acid conditions, and the degradation products have been characterized.

Synthesis and characterization of poly-O-methyl-[n]-polyurethane from a d-glucamine-based monomer.

Aminoalditol 1-amino-1-deoxy-D-sorbitol (1) was readily converted into 2,3,4,5-tetra-O-methyl derivative 5, a key precursor of a sugar-based [n]-polyurethane. For the polymerization, the free amino or primary hydroxyl groups of 5 were selectively activated and employed as starting monomers in two alternative procedures. Thus, the amino function of 5 was converted into the isocyanate derivative by treatment with di-tert-butyltricarbonate, and polymerized in situ in the presence of Zr(IV) acetylacetonate. The resulting poly(1-amino-1-deoxy-2,3,4,5-tetra-O-methyl-D-sorbitol)urethane (8) had a moderate molecular weight and showed the presence of urea units. The alternative synthesis of 8 involved the activation of the free hydroxyl group of 5 as the corresponding phenylcarbonate. The polymerization of this α-amino-ω-phenylcarbonate alditol monomer does not require a metal catalyst. The resulting material exhibited an improved molecular weight and higher purity than that obtained via the isocyanate. [n]-polyurethane 8 was highly soluble in water as well as in common organic solvents (chloroform, acetone, ethyl acetate, etc) and was obtained as an amorphous material which was characterized thermally and spectroscopically.

Structural characterization of native high-methoxylated pectin using nuclear magnetic resonance spectroscopy and ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Comparative use of 2,5-dihydroxybenzoic acid and nor-harmane as UV-MALDI matrices.

The successful analysis by ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (UV-MALDI-TOF MS) of native and hydrolyzed high-methoxylated pectin samples is described. In order to find the optimal conditions for UV-MALDI-TOF MS analysis several experimental variables were studied such as: different UV-MALDI matrices (nor-harmane, 2,5-dihydroxybenzoic acid), sample preparation methods (mixture, sandwich), inorganic salt addition (doping salts, NaCl, KCl, NH(4)Cl), ion mode (positive, negative), linear and reflectron mode, etc. nor-Harmane has never been used as a UV-MALDI matrix for the analysis of pectins but its use avoids pre-treatment of the sample, such as an enzymatic digestion or an acid hydrolysis, and there is no need to add salts, making the analysis easier and faster. This study suggested an alternative way of analyzing native high-methoxylated pectins, with UV-MALDI-TOF MS, by using nor-harmane as the matrix in negative ion mode. The analysis by (1)H and (13)C nuclear magnetic resonance (NMR) spectroscopy of the native and hydrolyzed pectin is also briefly described.

Synthesis of polyhydroxy amino acids based on D- and L-alanine from D-glycero-D-gulo-heptono-1,4-lactone.

2-Amino-2,3-dideoxy-D-manno-heptonic acid (7) has been synthesized from 2,5,6,7-tetra-O-acetyl-3-deoxy-D-gluco-heptono-1,4-lactone (1), which was readily prepared from D-glycero-D-gulo-heptono-1,4-lactone. O-Deacetylation of 1 followed by treatment with 13:1 (v/v) 2,2-dimethoxypropane/acetone in the presence of p-toluenesulfonic acid gave methyl 3-deoxy-4,5:6,7-di-O-isopropylidene-D-gluco-heptonate (3) as a crystalline product (80% yield). The free hydroxyl group (OH-2) of 3 was mesylated and substituted by azide to give the corresponding azide derivative 5. Hydrogenolysis and further hydrolysis of the ester function of 5 afforded alpha-amino acid 7 (43% overall yield from 1). Compound 7 is an analog of L-alanine having a polyhydroxy chain attached to C-3. The diastereoisomer of 7 at C-2, 2-amino-2,3-dideoxy-D-gluco-heptonic acid (12) was also prepared from 3, by a route that involved 2,3-dideoxy-2-iodo derivative 8 as a key intermediate.

Structural analysis and antiviral activity of a sulfated galactan from the red seaweed Schizymenia binderi (Gigartinales, Rhodophyta).

Aqueous extraction of gametophytic Schizymenia binderi afforded a polysaccharide composed of galactose and sulfate groups in a molar ratio of 1.0:0.89 together with uronic acids (6.8 wt%) and minor amounts of other neutral sugars. Alkali-treatment of the polysaccharide afforded a polysaccharide devoid of 3,6-anhydrogalactose. 13C NMR spectroscopy of the desulfated alkali-treated polysaccharide showed a backbone structure of alternating 3-linked beta-D-galactopyranosyl and 4-linked alpha-galactopyranosyl units that are predominantly of the D-configuration and partly of the L-configuration. Methylation, ethylation and NMR spectroscopic studies of the alkali-treated polysaccharide indicated that the sulfate groups are located mainly at positions O-2 of 3-linked beta-D-galactopyranosyl residue and at position O-3 of 4-linked-alpha-galactopyranosyl residues, the latter is partially glycosylated at position O-2. The sulfated galactan from S. binderi exhibited highly selective antiviral activity against Herpes simplex virus types 1 and 2, with selectivity indices (ratio cytotoxicity/antiviral activity) >1000 for all assayed virus strains. This compound was shown to interfere with the initial adsorption of viruses to cells.

Matrix-assisted ultraviolet laser desorption/ionization time-of-flight mass spectrometry of beta-(1 --> 3), beta-(1 --> 4)-xylans from Nothogenia fastigiata using nor-harmane as matrix.

Three xylan fractions isolated from the red seaweed Nothogenia fastigiata (Nemaliales) were analyzed by ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (UV-MALDI-TOFMS). UV-MALDI-TOFMS was carried out in the linear and reflectron modes, and as routine in the positive and negative ion modes. Of the several matrices tested, nor-harmane was the only effective one giving good spectra in the positive ion mode. The number-average molar masses of two of the fractions, calculated from the distribution profiles, were lower than those determined previously by (1)H NMR analysis, suggesting a decrease in the ionization efficiency with increasing molecular weight; weight-average molar mass and polydispersity index were also determined. As the xylans retained small but significant quantities of calcium salts, the influence of added Ca(2+) as CaCl(2) on UV-MALDI-MS was investigated. The simultaneous addition of sodium chloride and calcium chloride was also analyzed. Addition of sodium chloride did not change the distribution profile of the native sample showing that the inhibitory effect is due to Ca(2+) and not to Cl(-). Addition of calcium chloride with 1:1 analyte/salt molar ratio gave spectra with less efficient desorption/ionization of oligomers; the signals of these oligomers were completely suppressed when the addition of the salt became massive (1:100 analyte/salt molar ratio).

Desulfation of sulfated galactans with chlorotrimethylsilane. Characterization of beta-carrageenan by 1H NMR spectroscopy.

A desulfation method using chlorotrimethylsilane for treatment of pyridinium salts of sulfated galactans was developed. It proved to be appropriate for desulfation of polysaccharides of both agar and carrageenan families. In order to evaluate its efficiency in presence of the maximum content of 3,6-anhydrogalactose, it was applied to commercial kappa-carrageenan, leading to obtention of a product mainly composed by beta-carrageenan. Best experimental conditions for achieving desulfation of kappa-carrageenan--in terms of low sulfate content, high recovery and low degradation of the product--were found. In addition, the complete assignment of the 1H NMR spectrum of beta-carrageenan was achieved by means of 1D and 2D NMR techniques.

Sulfated polysaccharides from the red seaweed Georgiella confluens.

The water-soluble polysaccharides from Georgiella confluens, collected in Antarctica, were fractionated with cetrimide. The complexed material was subjected to fractional solubilization in solutions of increasing sodium chloride concentration. The initially extracted polysaccharide and the major fraction isolated, soluble in 0.5 M NaCl, were studied. These are sulfated xylogalactans naturally rich in methylated sugar residues, comprising of 3,6-anhydro-2-O-methyl-L-galactose, 2-O-methyl-L-galactose and 6-O-methyl-D-galactose. Structural analysis was carried out by methylation, ethylation, desulfation-ethylation, desulfation-methylation, Smith degradation, 13C NMR spectroscopy and determination of the absolute configuration of monosaccharides by gas chromatography of diastereomeric derivatives produced by reductive amination. The results indicated the presence of an agaran backbone with an unusual substitution pattern: sulfation mainly at the 3-position of the alpha-L-galactose units and the presence of xylose side chains at the 4-position of the beta-D-galactose residues.