Stéphane Trombotto

Chemical Preparation and Structural Characterization of a Homogeneous Series of Chitin/Chitosan Oligomers

The preparation of a homogeneous series of chitin/chitosan oligomers (chito-oligomers) with the same distribution of degrees of polymerization (DP) ranging from 2 to 12, but with various average degrees of N-acetylation (DA) from 0 to 90% is described. This DA-series was obtained according to a two-step chemical process involving (i) the production of a well-defined mixture of glucosamine (GlcN) oligomers obtained by acid hydrolysis of a fully N-deacetylated chitosan and after selective precipitations of the hydrolysis products, and (ii) the partial N-acetylation of the GlcN units of these oligomers from a hydro-alcoholic solution of acetic anhydride in a controlled manner. The characterization of this series of samples with different DAs by proton nuclear magnetic resonance (1H NMR) spectroscopy and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) allowed us to determine their average DA and identify the main oligomer structures constituting each mixture. Furthermore, MALDI-TOF MS was particularly helpful to study the distribution evolution of the diverse oligomers as a function of DA for the main DPs from 3 to 7. The modeling of these distributions by means of a binomial law displayed that the chemical N-acetylation of low DP GlcN oligomers, produced in a homogeneous medium, occurs randomly along the oligosaccharide chains in accordance with a statistical (Bernoullian) arrangement. In this case, the relative proportion of each chito-oligomer present in the mixture can be estimated precisely as a function of DA considering oligomers of same DP.

Polysaccharide gels based on chitosan and modified starch: structural characterization and linear viscoelastic behavior.

We investigated the properties of polymeric systems formed by cross-linking chitosan with modified starch (oxidized maltodextrin). Such a macromolecular cross-linker proved to be efficient to react with chitosan with potentially minimal toxicity. The structural characterization of modified starch alone and of the two-polysaccharide reactive systems was performed using (1)H NMR and FTIR. The rheological behaviors of all systems, from solutions to gels, were also characterized. Depending on experimental parameters, such as chitosan concentration, cross-linking pH, degree of oxidation of starch, and molar ratio of reactive groups, different kinds of systems ranging from pure viscoelastic solutions to stiff hydrogels were formed. These versatile systems could be used in biomedical applications because of the good biocompatibility of their constituents.

Structural Characterization of Chitin and Chitosan Obtained by Biological and Chemical Methods

Chitin production was biologically achieved by lactic acid fermentation (LAF) of shrimp waste (Litopenaeus vannameii) in a packed bed column reactor with maximal percentages of demineralization (D(MIN)) and deproteinization (D(PROT)) after 96 h of 92 and 94%, respectively. This procedure also afforded high free astaxanthin recovery with up to 2400 μg per gram of silage. Chitin product was also obtained from the shrimp waste by a chemical method using acid and alkali for comparison. The biologically obtained chitin (BIO-C) showed higher M(w) (1200 kDa) and crystallinity index (I(CR)) (86%) than the chemically extracted chitin (CH-C). A multistep freeze-pump-thaw (FPT) methodology was applied to obtain medium M(w) chitosan (400 kDa) with degree of acetylation (DA) ca. 10% from BIO-C, which was higher than that from CH-C. Additionally, I(CR) values showed the preservation of crystalline chitin structure in BIO-C derivatives at low DA (40-25%). Moreover, the FPT deacetylation of the attained BIO-C produced chitosans with bloc copolymer structure inherited from a coarse chitin crystalline morphology. Therefore, our LAF method combined with FPT proved to be an affective biological method to avoid excessive depolymerization and loss of crystallinity during chitosan production, which offers new perspective applications for this material.

Polysaccharide-Based Adhesive for Biomedical Applications: Correlation between Rheological Behavior and Adhesion

Adhesion to biological tissues is a challenge especially when the adhesive is in contact with physiological fluids. Abdominal hernia is a disease that often requires the implantation of a mesh within the abdominal wall. To minimize pain and postsurgical complications, gluing the mesh appears to be a convenient method. For this purpose, a bioadhesive system based on solutions of chitosan and modified starch (oxidized maltodextrin) has been developed. Mixtures of these polysaccharides form either viscoelastic solutions or hydrogels, depending on various experimental parameters (chitosan concentration, starch degree of oxidation, molar ratio between amine and aldehyde functions, pH, etc.). An adhesion test was developed to assess the adherence of such systems under conditions similar to the intended use. The rheological behavior of each formulation was correlated to its adherence, and it was found that optimum adhesion is obtained for systems exhibiting an intermediate behavior between the viscoelastic solution and the gel.

Kinetics Study of the Solid-State Acid Hydrolysis of Chitosan: Evolution of the Crystallinity and Macromolecular Structure

The heterogeneous hydrolysis of fully deacetylated chitosan solid samples was carried out with concentrated HCl. The hydrolysis kinetics was studied at different temperatures and HCl concentrations. From 5 to 50 degrees C in the hydrolysis time range up to 50 h, a monomodal distribution of molecular weights was observed connected to the only degradation of amorphous domains. Between 70 and 90 degrees C and for the hydrolysis longest times, a multimodal distribution appeared with the additional hydrolysis of the crystalline phase. The crystallinity index increased from 57 to 73% with the elimination and partial recrystallization of amorphous regions. X-ray diffraction patterns revealed the presence of the anhydrous polymorph, absent in the starting materials only containing the hydrated polymorph. The apparent crystallite width (from the Scherrer equation) of both the anhydrous and hydrated allomorphs did not vary significantly with time despite the increase in the fraction of anhydrous allomorph. Therefore, the hydrolysis in the solid state was complex, revealing several regimes. The activation energy parameters were deduced, and the mechanisms were discussed.

Partially Acetylated Chitosan Oligo-and Polymers Induce an Oxidative Burst in Suspension Cultured Cells of the Gymnosperm Araucaria angustifolia

Suspension-cultured cells were used to analyze the activation of defense responses in the conifer A. angustifolia , using as an elicitor purified chitosan polymers of different degrees of acetylation (DA 1-69%), chitin oligomers of different degrees of polymerization (DP 3-6), and chitosan oligomer of different DA (0-91%). Suspension cultured cells elicited with chitosan polymers reacted with a rapid and transient generation of H2O2, with chitosans of high DA (60 and 69%) being the most active ones. Chitosan oligomers of high DA (78 and 91%) induced substantial levels of H2O2, but fully acetylated chitin oligomers did not. When cultivated for 24-72 h in the presence of 1-10 microg mL(-1) chitosan (DA 69%), cell cultures did not show alterations in the levels of enzymes related to defense responses, suggesting that, in A. angustifolia , the induction of an oxidative burst is not directly coupled to the induction of other defense reactions.

Synthesis of new glucosylated porphyrins bearing an α-D-linkage

This paper presents the synthesis of two new glucosyl tritolylporphyrins in which the carbohydrate moiety is connected through a carboxymethyl glycosidic α‐D‐linkage. These compounds have been obtained by reaction between porphyrins bearing an amino function with a lactone prepared from the available disaccharide isomaltulose. The photocytotoxicity of these compounds against K562 human chronic myelogenous leukemia cells has been evaluated in comparison to Photofrin II.

Extensively deacetylated high molecular weight chitosan from the multistep ultrasound-assisted deacetylation of beta-chitin

High intensity ultrasound irradiation was used to convert beta-chitin (BCHt) into chitosan (CHs). Typically, beta-chitin was suspended in 40% (w/w) aqueous sodium hydroxide at a ratio 1/10 (gmL(-1)) and then submitted to ultrasound-assisted deacetylation (USAD) during 50min at 60°C and a fixed irradiation surface intensity (52.6Wcm(-2)). Hydrogen nuclear magnetic resonance spectroscopy and capillary viscometry were used to determine the average degree of acetylation (DA‾) and viscosity average degree of polymerization (DPv‾), respectively, of the parent beta-chitin (DA‾=80.7%; DPv‾=6865) and USAD chitosans. A first USAD reaction resulted in chitosan CHs1 (DA‾=36.7%; DPv‾=5838). Chitosans CHs2 (DA‾=15.0%; DPv‾=5128) and CHs3 (DA‾=4.3%; DPv‾=4889) resulted after repeating the USAD procedure to CHs1 consecutively once and twice, respectively. Size-exclusion chromatography analyzes allowed the determination of the weight average molecular weight (Mw‾) and dispersity (Ð) of CHs1 (Mw‾=1,260,000gmol(-1); Ð=1.4), CHs2 (Mw‾=1,137,000gmol(-1); Ð=1.4) and CHs3 (Mw‾=912,000gmol(-1); Ð=1.3). Such results revealed that, thanks to the action of high intensity ultrasound irradiation, the USAD process allowed the preparation of unusually high molecular weight, randomly deacetylated chitosan, an important breakthrough to the development of new high grade chitosan-based materials displaying superior mechanical properties.

Enzymatic hydrolysis of chitin pretreated by rapid depressurization from supercritical 1,1,1,2-tetrafluoroethane toward highly acetylated oligosaccharides.

The hydrolysis of chitin treated under supercritical conditions was successfully carried out using chitinases obtained by an optimized fermentation of the fungus Lecanicillium lecanii. The biopolymer was subjected to a pretreatment based on suspension in supercritical 1,1,1,2-tetrafluoroethane (scR134a), which possesses a critical temperature and pressure of 101°C and 40bar, respectively, followed by rapid depressurization to atmospheric pressure and further fibrillation. This methodology was compared to control untreated chitins and chitin subjected to steam explosion showing improved production of reducing sugars (0.18mg/mL), enzymatic hydrolysis and high acetylation (FA of 0.45) in products with degrees of polymerization between 2 and 5.

Access to tetra-N-acetyl-chitopentaose by chemical N-acetylation of glucosamine pentamer

Nowadays, the easy access of tetra-N-acetyl-chitopentaose and its counterparts is highly interesting since such chemical compounds are precursors of biological signal molecules with a strong agro-economic impact. The chemical synthesis of tetra-N-acetyl-chitopentaose by controlled N-acetylation of the glucosamine pentamer hydrochloride under mild conditions is described herein. A systematic study on the influence of the different parameters involved in this reaction, such as the solvent, the acetylating agent, and the base used for the deprotonation of ammonium groups of the starting material was carried out. The characterization of final reaction products by HPLC and MALDI-TOF mass spectrometry showed that each of these parameters affects differently the acetylation reaction. Whereas the solvent plays an important role in the N- or O-acetylation selectivity, the acetylating agent and the base were found to influence both the degree of N-acetylation and the distribution of the partially N-acetylated derivatives in the product mixtures. Based on these results, optimized reaction conditions have been established allowing tetra-N-acetyl-chitopentaose to be synthesized in a one-pot deprotonation/N-acetylation of the glucosamine pentamer hydrochloride in a moderate yield (ca 30%).