N-(2-Carboxyethyl)chitosans: regioselective synthesis,characterisation and protolytic equilibria

N-(2-Carboxyethyl)chitosans were obtained by reaction of low molecular weight chitosan with a low degree of acetylation and 3-halopropionic acids under mild alkaline media (pH 8-9, NaHCO3) at 60 degrees C. The chemical structure of the derivatives obtained was determined by 1H and 13C NMR spectroscopies. It was found that alkylation of chitosan by 3-halopropionic acids proceeds exclusively at the amino groups. The products obtained are described in terms of their degrees of carboxyethylation and ratio of mono-, di-substitution and free amine content. The protonation constants of amino and carboxylate groups of a series of N-(2-carboxyethyl)chitosans were determined by pH-titration at ionic strength 0.1 M KNO3 and 25 degrees C.     

Physicochemical characterization of chitin and chitosan from crab shells

Crab chitosan was prepared by alkaline N-deacetylation of crab chitin for 60, 90 and 120 min and the yields were 30.0-32.2% with that of chitosan C120 being the highest. The degree of N-deacetylation of chitosans (83.3–93.3%) increased but the average molecular weight (483–526 kDa) decreased with the prolonged reaction time. Crab chitosans showed lower lightness and WI values than purified chitin, chitosans CC and CS but higher than crude chitin. With the prolonged reaction time, the nitrogen (8.9–9.5%), carbon (42.2–45.2%) and hydrogen contents (7.9–8.6%) in chitosans prepared consistently increased whereas N/C ratios remained the same (0.21). Crab chitosans prepared showed a melting endothermic peak at 152.3–159.2 °C. Three chitosans showed similar microfibrillar crystalline structure and two crystalline reflections at 2θ = 8.8–9.0° and 18.9–19.1°. Overall, the characteristics of three crab chitosans were unique and differed from those of chitosan CC and CS as evidenced by the element analysis, differential scanning calorimetry, scanning electron microscopy and X-ray diffraction patterns.     

Extraction of chitin and chitosan from housefly,Musca domestica,pupa shells

Chitins and chitosans are some of the most abundant natural polysaccharide materials, and are used to increase innate immune response and disease resistance in humans and animals. In this work, chitin and chitosan from housefly, Musca domestica, pupa shells were obtained by treatment with HCl and NaOH. For chitin extraction, 2 N HCl and 1.25 N NaOH solutions were used to achieve decalcification and deproteinization, respectively. For chitosan extraction, 50% NaOH solution was used to achieve deacetylation. The yields of chitin and chitosan from pupa shells of M. domestica were 8.02% and 5.87%, respectively. The deacetylations of chitosan (from chitin C1 and C2) were 89.76% and 92.39%, respectively, after the first alkali treatment with 50% NaOH (w/w) solution at 105 °C for 3 h and 5 h, respectively. The viscosities of the chitosans (from chitin C1 and C2) were 33.6 and 19.2 cP, respectively.     

TEMPO-mediated oxidation of chitin, regenerated chitin and N-acetylated chitosan

A commercial chitin, regenerated chitin prepared from chitin solutions in 6.8% NaOH and N-acetylated chitosans with degrees of N-acetylation (DNAc) of 77–93% were subjected to oxidization in water with NaClO and catalytic amounts of 2,2,6,6-tetramethylpiperidinyloxy radical (TEMPO) and NaBr. When regenerated chitin with DNAc of 87% and N-acetylated chitosan with DNAc of 93% were used as starting materials, water-soluble β-1,4-linked poly-N-acetylglucosaminuronic acid (chitouronic acid) Na salts with degrees of polymerization of ca. 300 were obtained quantitatively within 70 min. On the other hand, the original chitin and N-acetylated chitosan with DNAc of 77% did not give water-soluble products, owing to incomplete oxidation. The high crystallinity of the original chitin brought about low reactivity, and the high C2-amino group content of the N-acetylated chitosan with DNAc of 77% led to degradations rather than the selective oxidation at the C6 hydroxyls. The obtained chitouronic acid had low viscosities in water, and clear biodegradability by soil microorganisms.     

Physicochemical behavior of homogeneous series of acetylated chitosans in aqueous solution: role of various structural parameters

Physicochemical properties of four different homogeneous series of chitosans with degrees of acetylation (DA) and weight-average degrees of polymerization (DP(w)) ranging from 0 to 70% and 650 to 2600, respectively, were characterized in an ammonium acetate buffer (pH 4.5). Then, the intrinsic viscosity ([eta](0)), the root-mean-square z-average of the gyration radius (R(G,z)), and the second virial coefficient (A(2)) were studied by viscometry and static light scattering. The conformation of chitosan, according to DA and DP(w), was highlighted through the variations of alpha and nu parameters, deduced from the scale laws [eta](0) = K(w)and R(G,z) = K', respectively, and the total persistence length (L(p,tot)). In relation with the different behaviors of chitosan in solution, the conformation varied according to two distinct domains versus DA with a transition range in between. Then, (i) for DA < 25%, chitosan exhibited a flexible conformation; (ii) a transition domain for 25 < DA < 50%, where the chitosan conformation became slightly stiffer and, (iii) for DA > 50%, on increasing DP(w) and DA, the participation of the excluded volume effect became preponderant and counterbalanced the depletion of the chains by steric effects and long-distance interactions. It was also highlighted that below and beyond a critical DP(w,c) (ranging from 1 300 to 1 800 for DAs from 70 to 0%, respectively) the flexibility of chitosan chains markedly increased then decreased (for DA > 50%) or became more or less constant (DA < 50%). All the conformations of chitosan with regards to DA and DP(w) were described in terms of short-distance interactions and excluded volume effect.     

The impact of dilute sulfuric acid on the selectivity of xylooligomer depolymerization to monomers

The disappearance of xylose and xylooligosaccharides with degrees of polymerization (DP) ranging from 2 to 5 was followed at 160 degrees C with sulfuric acid added to adjust the pH from near neutral to 1.45, and the impact on the yields of lower DP xylooligomers and xylose monomer was determined. In addition, the experimental data for the disappearance of these xylooligomers was kinetically modeled assuming first-order reaction kinetics for xylose degradation and xylooligomer hydrolysis to evaluate how the pH affected the selectivity of monomer formation from xylooligomers and direct oligomer degradation to unknown products. The yield of xylose from xylooligomers increased appreciably with increasing acid concentration but decreased with increasing xylooligomer DP at a given acid concentration, resulting in more acid being required to realize the same xylose yields for higher DP species. For example, the maximum xylose yields were 49.6%, 28.0%, 13.2% and 3.2% for DP values of 2, 3, 4, and 5, respectively, at pH 4.75. Kinetic modeling revealed that all the xylooligomers disappeared at a higher rate compared to xylose monomer and the disappearance rate constant increased with DP at all pH. The kinetics for lower DP oligomers of 2 and 3 showed that these species directly degrade to unknown compounds in the absence of acid. On the other hand, higher oligomers of DP 4 and 5 exhibited negligible losses to degradation products at all pH. Therefore, only xylooligomers of DP 2 and 3 were found to directly degrade to undesired products in the absence of acid, but more work is needed to determine how higher DP species behave. This study also revealed that the source of water and the material used for the construction of the reactor impacted xylose degradation kinetics.     

Precise derivatization of structurally distinct chitosans with rhodamine B isothiocyanate

Work to date shows that structurally distinct chitosans have reacted inefficiently and unpredictably with fluorescein isothiocyanate (FITC) in an acid–methanol solvent that maintains both chitosan and fluorophore solubility. Since isothiocyanate preferentially reacts with neutral amine groups, and chitosan solubility typically depends upon a minimal degree of protonation, we tested the hypothesis that precise derivatization of chitosan with rhodamine isothiocyanate (RITC) can be achieved by controlling the reaction time and the degree of protonation. Addition of 50% v/v methanol reduced the chitosan degree of protonation in acetic acid but not HCl solutions. At various degrees of protonation, chitosan reacted inefficiently with RITC as previously observed with FITC. Nevertheless, precise derivatization was achieved by allowing the reaction to proceed overnight at a given degree of protonation (p < 0.0001, n = 18) and fixed initial fluorophore concentration. A reproducible 2% to 4% fraction of neutral amines reacted with RITC in proportion to the initial fluorophore concentration (p < 0.005). Using our optimized protocol, chitosans with different degree of deacetylation and molecular weight were derivatized to either 1% or 0.5% mol/mol RITC/chitosan-monomer with a precision of 0.1% mol/mol. The average molecular weight of fluorescent RITC-chitosan was similar to the unlabeled parent chitosan. Precise molar derivatization of structurally distinct chitosans with RITC can be achieved by controlling chitosan degree of protonation, initial fluorophore concentration, and reaction time.     

Structural analysis of chitosan mediated DNA condensation by AFM: influence of chitosan molecular parameters

Chitosan is a nontoxic and biodegradable polysaccharide that has recently emerged as a promising candidate for gene delivery. Here the ability of various chitosans, differing in the fractional content of acetylated units (F(A)) and the degree of polymerization (DP), to compact DNA was studied. Polyplexes made from mixing plasmid DNA with chitosan yielded a blend of toroids and rods, as observed by AFM. The ratios between the fractions of toroids and rods were observed to decrease with increasing F(A) of the chitosan, indicating that the charge density of chitosan, proportional to (1 - F(A)), is important in determining the shape of the compacted DNA. The amount of chitosan required to fully compact DNA into well-defined toroidal and rodlike structures were found to be strongly dependent on the chitosan molecular weight, and thus its total charge. A higher charge ratio (+/-) was needed for the shorter chitosans, showing that an increased concentration of the low DP chitosan could compensate for the reduced interaction strength of the individual ligands with DNA. Employing chitosans with different molecular parameters offers the possibility of designing DNA-chitosan polyplexes with various geometries, reflecting various chitosan-DNA interaction strengths, which is necessary for the evaluation of efficient gene delivery vehicles.     

Characterization of Some Fungal Chitosans

Chitosan-like materials were extracted from five different fungal cells with NaOH and acetic acid, with the yields varying from 1.2 to 10.4% of the dry fungal cell weight. The degree of N-acetylation of the extracts measured by the colloidal titration method varied considerably depending on the individual species. By IR measurements and the Elson-Morgan method, four kinds of the extracts were characterized as chitosan while another one was not.

The degree of N-acetylation and the Cu²⁺ adsorption capacity of the fungal chitosans were measured and compared with those of authentic samples with various degrees of N-acetylation, which were prepared by chemical treatment of authentic chitin and chitosan derived from Crustacea. The Cu²⁺ adsorption capacity of the fungal chitosans was higher than that of the authentic chitosan samples with similar degrees of N-acetylation and independent of the molecular weight of the chitosans from the various sources.     

Glycosaminoglycan destabilization of DNA-chitosan polyplexes for gene delivery depends on chitosan chain length and GAG properties

Chitosan-based gene delivery systems are promising candidates for non-viral gene therapy. A wide range of chitosans has been studied to optimize the properties of the DNA-chitosan complexes to yield high transfection efficiencies. An important parameter to control is the polyplex stability to allow transport towards the cells, subsequent internalization and release of DNA intracellularly. The stability of the DNA-chitosan complexes was here studied after exposure to heparin and hyaluronic acid (HA) using atomic force microscopy (AFM) and ethidium bromide (EtBr) fluorescence assay. To study the effect of polycation chain length on the polyplex stability, chitosans with a degree of polymerization (DP) varying from approximately 10 to approximately 1000 were employed for DNA compaction. Whereas HA was unable to dissociate the complexes, the degree of dissociation caused by heparin depended on both the chitosan chain length and the amount of chitosan used for complexation. When increasing the chitosan concentration, larger heparin concentrations were required for polyplex dissociation. Furthermore, increasing the chitosan chain length yielded more stable complexes. Varying the chitosan chain length thus provides a tool for controlling the ability of the polyplex to deliver therapeutic gene vectors to cells.     

Glycosaminoglycan destabilization of DNA–chitosan polyplexes for gene delivery depends on chitosan chain length and GAG properties

Chitosan-based gene delivery systems are promising candidates for non-viral gene therapy. A wide range of chitosans has been studied to optimize the properties of the DNA–chitosan complexes to yield high transfection efficiencies. An important parameter to control is the polyplex stability to allow transport towards the cells, subsequent internalization and release of DNA intracellularly. The stability of the DNA–chitosan complexes was here studied after exposure to heparin and hyaluronic acid (HA) using atomic force microscopy (AFM) and ethidium bromide (EtBr) fluorescence assay. To study the effect of polycation chain length on the polyplex stability, chitosans with a degree of polymerization (DP) varying from ∼10 to ∼1000 were employed for DNA compaction. Whereas HA was unable to dissociate the complexes, the degree of dissociation caused by heparin depended on both the chitosan chain length and the amount of chitosan used for complexation. When increasing the chitosan concentration, larger heparin concentrations were required for polyplex dissociation. Furthermore, increasing the chitosan chain length yielded more stable complexes. Varying the chitosan chain length thus provides a tool for controlling the ability of the polyplex to deliver therapeutic gene vectors to cells.     

Production of inulo-oligosaccharides using endo-inulinase from a pseudomonas sp.

Inulo-oligosaccharides were produced from inulin by using high activities of an endo-acting inulinase. The total yields of oligosaccharide were slightly decreased as the concentration of inulin increased from 50 to 200g/l. Under the optimal reaction conditions, the products consist of inulo-oligosaccharides ranging from DP (degrees of polymerization)2 to DP7, where the major oligosaccharides are 29.8% DP2, 21.4% DP3, and 8.1% DP4 oligomer, respectively. The maximum yield was 75.6% when 50g inulin/l and 15 units/g substrate were used.     

Chitosan as an enabling excipient for drug delivery systems. I. Molecular modifications

Chitosan was physicochemically modified for its potential use as a matrix for an implantable antibiotic delivery system that could sustain bactericidal concentrations in the vicinity of an implant or prosthesis. Deacetylation and depolymerization of chitosan were implemented in order to increase the number or accessibility of the reactive amino groups on the polymer backbone for better polymer-drug interaction. The deacetylation process involved reaction of particulate chitosan/depolymerized chitosan with alkali. The rate of deacetylation of chitosan was directly proportional to the reaction temperature up to 80 degrees C; beyond 80 degrees C, rapid degradation of the polymer occurred. The depolymerization of chitosan involved acid digestion of the polymer followed by application of mechanical agitation. This depolymerized product, although water insoluble, possessed a molecular weight that was one to two orders of magnitude lower than that of commercially available chitosans. These products not only exhibited improved reactivity, but also showed increased crystallinity when compared with the parent chitosan. The reactivity was found to be inversely proportional to chitosan's molecular weight. The depolymerization and deacetylation treatments afforded formation of chitosan having a greater number of amino groups available for interactions with the anionic actives.     

Exclusive and complete introduction of amino groups and their N-sulfo and N-carboxymethyl groups into the 6-position of cellulose without the use of protecting groups

A new regioselective synthesis of 6-amino-6-deoxycellulose with a DS 1.0 (degree of substitution) at C-6, and its 6-N-sulfonated and its 6-N-carboxymethylated derivatives, without using protecting groups is described in this paper. The reaction conditions were optimized for preparing cellulose tosylate with full tosylation at C-6 and partial tosylation at C-2 and C-3. The nucleophilic substitution (S(N)) reaction of the tosyl group by NaN(3) at low temperature of 50 degrees C in Me(2)SO was achieved completely at C-6, whereas the tosyl groups at C-2 and C-3 were not displaced. In contrast to this, at 100 degrees C the tosyl groups at C-6, and also those at C-2 and C-3, were replaced by azido groups. This regioselective reaction that depends on temperature makes it possible to reach a selective and quantitative S(N) reaction at C-6 at low temperatures. In the subsequent reduction step with LiAlH(4), the azido group at C-6 was reduced to the amino group, and the tosyl groups at C-2 and C-3 were simultaneously completely removed. Also reported is a temperature-dependent, regioselective and complete iodination by nucleophilic substitution of the tosyl group at C-6 at 60 degrees C. At higher temperatures from 75 to 130 degrees C, substitution is also observed to occur at C-2. The selective iodination at 60 degrees C was employed to confirm the complete tosylation at C-6 of cellulose. The reaction products were identified by four different independent quantitative methods, namely 13C NMR, elemental analysis, ESCA, and fluorescence spectroscopy. 6-N-Sulfonated and 6-N-carboxymethylated cellulose derivatives were also synthesized. The new derivatives are potent candidates for structure-function studies, e.g., studies in relation to regioselectively 2-N-sulfonated and 2-N-carboxymethylated chitosan derivatives.     

Preparation of low-molecular-weight and high-sulfate-content chitosans under microwave radiation and their potential antioxidant activity in vitro

In the present paper microwave radiation has been used to introduce N-sulfo and O-sulfo groups into chitosan with a high degree of substitution and low-molecular weight. The sulfation of chitosan was performed in microwave ovens. It was found that microwave heating is a convenient way to obtain a wide range of products of different degrees of substitution and molecular weight only by changing reaction time or/and radiation power. Moreover, microwave radiation accelerated the degradation of sulfated chitosan, and the molecular weight of sulfated chitosan was considerably lower than that obtained by traditional heating. There are no differences in the chemical structure of sulfated chitosan obtained by microwave and by conventional technology. FTIR and 13C NMR spectral analyses demonstrated that a significantly shorter time is required to obtain a satisfactory degree of substitution and molecular weight by microwave radiation than by conventional technology. In this present paper, we also determined antioxidant activity of low-molecular-weight and high-sulfate-content chitosans (LCTS). The results showed LCTS could scavenge superoxide and hydroxyl radical. Its IC50 is 0.025 and 1.32 mg/mL, respectively. It is a potential antioxidant in vitro.     

Non-specific depolymerization of chitosan by pronase and characterization of the resultant products.

Pronase (type XXV serine protease from Streptomyces griseus) efficiently depolymerizes chitosan, a linear beta-->1,4-linked polysaccharide of 2-amino-deoxyglucose and 2-amino-2-N-acetylamino-D-glucose, to low-molecular weight chitosans (LMWC), chito-oligomers (degree of polymerization, 2-6) and monomer. The maximum depolymerization occurred at pH 3.5 and 37 degrees C, and the reaction obeyed Michaelis-Menten kinetics with a Km of 5.21 mg.mL(-1) and Vmax of 138.55 nmoles.min(-1).mg(-1). The molecular mass of the major product, LMWC, varied between 9.0 +/- 0.5 kDa depending on the reaction time. Scanning electron microscopy of LMWC showed an approximately eightfold decrease in particle size and characterization by infrared spectroscopy, circular dichroism, X-ray diffractometry and 13C-NMR revealed them to possess a lower degree of acetylation, hydration and crystallinity compared to chitosan. Chitosanolysis by pronase is an alternative and inexpensive method to produce a variety of chitosan degradation products that have wide and varied biofunctionalities.     

Synthesis of chitosan-based polymeric surfactants and their adsorption properties for heavy metals and fatty acids

Chitosan-based polymeric surfactants (CBPSs) were prepared by N-acylation of chitosans (chitosan 10 and 500) with several acid anhydrides such as hexanoic (C6), lauric (C12), and palmitic (C16) anhydrides. Among the CBPS samples, CBPSs having a good solubility at pH 4.0 were selected and observed for viscosity, surface tension, and adsorption of heavy metals (Cd2+, Co2+, Cr2O7(2-), and Pb2+) as well as the fatty acid (n-octanoic acid). The 1H NMR spectrum of chitosan 10 modified with C16 at the substitution ratio of 0.4 (CBPS10-C16,0.4) showed 85% of acylation in 1% DCl/D2O solutions. CBPS10 with the substitution ratio less than 0.4 showed a good solubility because of shorter repeating units and lesser amounts of hydrophobic substituents. The intrinsic viscosity of CBPS10 was slightly increased, while that of CBPS500 was decreased. As the substitution ratio and length of the carbon chain increased, the surface tension of CBPS10 tended to decrease. CBPS10-C16,0.2 had high adsorption ability for cationic metal ions such as Cd2+, Co2+, and Pb2+ comparable to chitosan. Interestingly, CBPS(10)-C(16,0.2) showed a unique pH optimum for the anionic metal ion such as Cr2O7(2-). In addition, CBPS10-C16,0.2 exhibited the highest adsorption ability for n-octanoic acid among the tested CBPS10 with different carbon chains.     

Chitosan-elicited synthesis of callose and of coumarin derivatives in parsley cell suspension cultures

In suspension cultured cells of parsley (Petroselinum crispum), chitosan elicited a rapid deposition of the 1,3-ß-glucan callose on the cell wall and a slower formation of coumarins. With cells remaining in conditioned growth medium, fully N-deacetylated chitosans and partially N-acetylated chitosans were about equally active, the potency increased with the degree of polymerization up to several thousand and addition of reduced glutathione increased the sensitivity of the cells. These results indicate common initial events in the induction of callose and coumarin synthesis although two fully independent metabolic pathways are involved. When the cells were suspended in fresh growth medium, less chitosan was required, and fully N-deacetylated chitosan became the best callose elicitor.Abbreviations DP average degree of polymerization - GSH reduced glutathione - PE pachyman equivalents - Pmg Phytophthora megasperma f. sp.glycinea     

Specific interactions in modified chitosan systems

This paper concerns the bulk and interfacial properties of a series of alkylated chitosans having different alkyl chain lengths grafted randomly along the main chitosan chain. Chitosan has a low degree of acetylation (5%); on chitosan derivatives, the role of the degree of grafting and of length of the alkyl chains are examined. The optimum alkyl chain length is C12 and the degree of grafting 5% to get physical gelation based on the formation of hydrophobic domains. The cross-linking is essentially controlled by the salt concentration: it is shown that 0.025 M AcONa is needed to screen electrostatic interchain repulsions. Hydrophobic interactions produce highly non-Newtonian behavior with large thinning behavior; this behavior is suppressed in the presence of cyclodextrins able to cap the hydrophobic alkyl chains.The interfacial properties of the chitosan derivatives were tested for the air/aqueous solution interfaces. Specifically, the role of their structure on the kinetic of film formation was examined showing that excess of external salt favors the stabilization of the interfacial film. The derivatives with a higher degree of substitution and longer alkyl chains are more efficient and give a higher elastic modulus compared to the model surfactant as a result of the chain properties.     

Proteinase inhibitor synthesis in tomato leaves : induction by chitosan oligomers and chemically modified chitosan and chitin

Soluble chemical derivatives of chitin and chitosan including ethylene glycol chitin, nitrous acid-modified chitosan, glycol chitosan, and chitosan oligomers, produced from chitosan by limited hydrolysis with HCl, were found to possess proteinase inhibitor inducing activities when supplied to young excised tomato (Lycopersicon esculentum var Bonnie Best) plants. Nitrous acid-modified chitosans and ethylene glycol chitin exhibited about 2 to 3 times the activity of acid hydrolyzed chitosan and 15 times more activity than glycol chitosan. The parent chitin and chitosans are insoluble in water or neutral buffers and cannot be assayed. Glucosamine and its oligomers from degree of polymerization = 2 through degree of polymerization = 6 were purified from acid-fragmented chitosan and assayed. The monomer was inactive and dimer and trimer exhibited weak activities. Tetramer possessed higher activity and the larger pentamer and hexamer oligomers were nearly as active as the total hydrolyzed mixture. None of the fragments exhibited more than 2% acetylation (the limits of detection). The contents of the acid-fragmented mixture of oligomers was chemically N-acetylated to levels of 13% and 20% and assayed. The N-acetylation neither inhibited nor enhanced the proteinase inhibitor inducing activity of the mixture. These results, along with recent findings by others that chitinases and chitosanases are present in plants, provide further evidence for a possible role of soluble chitosan fragments as signals to activate plant defense responses.