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.     

Chitin fractionation and characterization in N,N-dimethylacetamide/lithium chloride solvent system

A detailed study of the interaction of chitin molecular species with the solvent system N,N-dimethylacetamide (DMAc)/lithium chloride (LiCl) allowed the development of a new method for chitin fractionation by coacervate extraction. The controlled increase of the extracting power of the solvent was carried out using slight modification of the solvent composition. Partial extractions of molecular species were done between coacervation and complete dissolution limits using different mixtures of DMAc/LiCl of increasing extracting power. Fractions were characterized in DMAc/LiCl 5% (w/w) by viscometry and size exclusion chromatography with refractive index and multi-angle laser light scattering detectors. Fractions obtained by coacervate extraction range from 80,000 to 710,000 g mol⁻¹ with polydispersity index between 1.28 and 1.44. The Mark–Houwink–Sakurada equation constants a and K for chitin in DMAc/LiCl 5% (w/w) were found to be 0.95 and 7.6×10⁻⁵ dl g⁻¹, respectively.     

Alkaline N-deacetylation of chitin enhanced by flash treatments. Reaction kinetics and structure modifications

A new method for N-deacetylation of chitin is proposed in which a polymer almost free of N-acetyl groups is obtained by flash treatment. The reaction is carried out in 40% NaOH solution for 30–270 s at 140–190°C, using saturated steam.

Flash treatment was found to proceed faster and with a higher activation energy for the deacetylation reaction (E_a = 36 kcal mol⁻¹) compared with the traditional treatment (E_a = 11 kcal mol⁻¹). X-Ray diffractometry, CP-MAS ¹³C-NMR and FTIR spectroscopy show that the flash treatment induces structure modifications; in particular, higher crystallinity indexes and specific area values are observed together with changes in the local and chain conformation.     

Preparation of biodegradable chitin/gelatin membranes with GlcNAc for tissue engineering applications

Chitin is a natural biopolymer have been used for several biomedical applications due to its biodegradability and biocompatibility. By using the calcium solvent system, chitin regenerated hydrogel (RG) was prepared by using -chitin. And also, the swelling hydrogel (SG) was prepared by using β-chitin with water. Then, both RG and SG were mixed with gelatin and N-acetyl-d-(+)-glucosamine (GlcNAc) at 120 °C for 2 h. The chitin/gelatin membranes with GlcNAc were also prepared by using RG and SG with GlcNAc. The prepared chitin/gelatin membranes with or without GlcNAc were characterized by mechanical, swelling, enzymatic degradation, thermal and growth of NIH/3T3 fibroblast cell studies. The stress and elongation of chitin/gelatin membrane with GlcNAc prepared from RG was showed higher than the chitin/gelatin membranes without GlcNAc. But, the chitin/gelatin membranes prepared from SG with GlcNAc was showed higher stress and elongation than the chitin/gelatin membranes without GlcNAc. It is due to the crosslinking effect of GlcNAc. The chitin/gelatin membranes prepared from SG showed higher swelling than the chitin/gelatin membranes prepared from RG. In contrast, the chitin/gelatin membranes prepared from RG showed higher degradation than the chitin/gelatin membranes prepared from SG. And also, these chitin/gelatin membranes are showing good growth of NIH/3T3 fibroblast cell. So these novel chitin/gelatin membranes are useful for tissue engineering applications.     

N-Deacetylation and depolymerization reactions of chitin/chitosan: Influence of the source of chitin

The deacetylation and depolymerization reactions of chitin/chitosan from three crustacean species (Paralomis granulosa, Lithodes antarcticus and Palinurus vulgaris) were evaluated under the same conditions. The average molecular weight and the mole fraction of N-acetylated units were the parameters studied in the resulting chitosans. During the N-deacetylation process P. granulosa, L. antarcticus and P. vulgaris follow a pseudo-first order kinetics and their apparent rate constants are very similar. However, the degradation rate of chitosan in the first 45 min of this process is higher for P. vulgaris. The depolymerization process follows a pseudo-first order kinetics for the three species, but in the first 9 min P. vulgaris shows a slightly lower depolymerization rate. Hence, depending on the ash contents, crystallinity and the physicochemical characteristics of chitin from these sources, the obtained chitosans show different qualities.     

Interaction of oxidized and reduced N-methylphenazonium methosulfate (PMS) with Photosystem II

In 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) poisoned chloroplasts, the restoration of the fluorescence induction is presumed to be due to a back reaction of the reduced primary acceptor (Q⁻) and the oxidized primary donor (Z⁺) of Photosystem II. Carbonylcyanide m-chlorophenylhydrazone (CCCP) is known to inhibit this back reaction. The influence of reduced N-methylphenazonium methosulfate (PMS) in the absence of CCCP and of oxidized PMS in the presence of CCCP on the back reaction was investigated and the following results were obtained:

1. (1) Reduced PMS at the concentration of 1 μM inhibits the back reaction as effectively as hydroxylamine, suggesting an electron donating function of reduced PMS for System II.

2. (2) The inhibition of the back reaction by CCCP is regenerated to a high degree by oxidized PMS which led to assume a cyclic System II electron flow catalysed by PMS.

3. (3) At concentrations of reduced PMS higher than 1 μM it is shown that both the fast initial emission and more significantly the variable emission are quenched.

Biohydroxylation of N,N-dialkylarylamines by the isolated topsoil bacterium Bacillus megaterium

Topsoil microorganisms were screened for their acceptability of the standard substrate N,N-dimethylaniline in bacterial ‘whole-cell’ incubations. One bacterium converted N,N-dimethylaniline and was identified as Bacillus megaterium by 16S rDNA analysis and DNA/DNA-hybridization. In contrast to the well-known C-hydroxylation by liver microsomes, leading to p-hydroxylation, B. megaterium formed o- and p-monohydroxylated products, i.e. N,N-dimethyl-2-aminophenol and N,N-dimethyl-4-aminophenol, both identified by gas chromatography–mass spectrometry (GC–MS) using synthesized reference compounds. The observed hydroxylation showed slight regioselectivity in favour of the o-hydroxylated product. Two further substrates, N,N-diethylaniline and N-ethyl-N-methylaniline, were also successfully biohydroxylated by B. megaterium with corresponding regioselectivity. Interestingly, aniline, known to be transformed easily by cytochrome P-450meg into p-aminophenol, was not accepted as substrate.     

Binding of metal cations by N-carboxymethyl chitosans in water

The binding of metal cations by N-carboxymethyl chitosan in dilute aqueous solution was studied as a function of charge density, temperature and pH. The techniques employed were absorption and circular dichroism measurements, potentiometric titration, dilatometry and isothermal microcalorimetry. The composition of the polymer significantly affected the binding of Ni(II), Co(II) and Pb(II), and had a small influence on the extent of binding of Cu(II). In any case, the density of glycine residues affected the binding constant with all the counterions studied, thus confirming that this residue is engaged in the complex formation. The whole of the optical, chirooptical and thermodynamic evidence showed that, at least in the case of Cu(II), the ions are bound by both carboxylic and amino groups. The temperature did not affect the extent of binding, at least in the case of Cu(II) and Pb(II) ions. Circular dichroism spectra, however, showed that these counterions are able to interact with ---NH---CO---groups at high temperatures (61°C), very likely owing to the enhanced mobility of the polymer chain. Lowering pH depressed the binding ability of N-carboxymethyl chitosan towards Cu(II) and Pb(II) ions, the effect being much more pronounced in the case of Pb(II). Moreover, a refined method for collecting absorption spectra of the complex between the polymer and Cu(II) or Pb(II) ions, without interference with absorbance by undesired species, was tested and applied. The charge-transfer band exhibited by the N-carboxymethyl chitosan-Cu(II) system, recorded following this method, showed interesting features, on the basis of which the site of the binding of Cu(II) on to N-carboxymethyl chitosan was confirmed.     

N-Ethylmaleimide inhibition of the catalytic activities of the Dunaliella salina coupling factor 1 (CF1) and the restoration of the inhibition of the CF1 ATPase activity by N-ethylmaleimide

The sensitivity of the catalytic activities of the D. salina chloroplast coupling factor 1 (CF₁) to chemical modification by N-ethylmaleimide has been investigated. (i) When D. salina thylakoid membranes are treated with N-ethylmaleimide, both photophosphorylation and the inducible CF₁ ATPase activity are partially (approx. 60%) inhibited. The inhibition of both activities does not require the presence of a proton-motive force, and the inhibition of photophosphorylation is directly related to the N-ethylmaleimide-covalent modification of CF₁ as shown by (a) the time-course for the inhibition and (b) the maximal extent of inhibition. (ii) Treatment of the purified, latent, D. salina CF₁ with low concentrations of N-ethylmaleimide also results in the partial (approx. 60%) inhibition of the inducible ATPase activity (I₅₀ ≈ 50 μM). The inhibition does not require the presence of the chemical modifier during the activation of the enzyme. (iii) N-ethylmaleimide-induced inhibition of the ATPase activity of either membrane-bound or solubilized CF₁ is partially reversed by either (a) prolonged incubation at low concentrations of N-ethylmaleimide or (b) short incubation times at high concentrations of N-ethylmaleimide. The results are interpreted as indicating multiple binding sites on the D. salina CF₁ that have different rates of reactivity with N-ethylmaleimide. Those sites (or site) that react rapidly with N-ethylmaleimide cause(s) an inhibition of both ATP synthase and ATPase activities, whereas those sites (or site) that react more slowly partially restore(s) the original-ATPase activity. The effects of N-ethylmaleimide on the catalytic activity of D. salina CF₁ are probably mediated by N-ethylmaleimide-induced conformational changes of the enzyme.     

Degradation of fully water-soluble, partially N-acetylated chitosans with lysozyme

Chitosans, prepared by homogeneous N-deacetylation of chitin, with degrees of N-acetylation ranging from 4 to 60% (F_A = 0·04 to 0·60) exhibiting full water solubility and known random distribution of acetyl groups, were degraded with lysozyme. Initial degradation rates (r) were determined from plots of the viscosity decrease (Δ1/[η]) against time of degradation. The time course of degradation of chitosans with lysozyme were non-linear, while the time course of degradation of chitosans with an oxidative-reductive depolymerization reaction (using H₂O₂) showed the expected linear relationship for a first-order, random depolymerization reaction, independent of the chemical composition of the chitosan.

The effect of lysozyme concentration and substrate concentration on the initial degradation rates were determined, showing that this lysozyme-chitosan system obeys Michaelis-Menten kinetics.

The initial degradation rates of chitosan with lysozyme increased strongly with increasing fraction of acetylated units (F_A). From a Michaelis-Menten analysis of the degradation data that assumes different catalytic activities of lysozyme for the different hexameric substrates in the polysaccharide chain, it is concluded that the hexameric substrates that contain three-four or more acetylated units contribute mostly to the initial degradation rate when lysozyme degrades partially N-acetylated chitosans.

A chitosan with a very low fraction of acetylated units (F_A = 0·010) was studied as an enzyme inhibitor. Initial degradation rates of chitosan (with different F_A values) decreased as the inhibitor concentration increased, while the relative rates stayed constant, indicating that the ratio between initial reaction rates for productive sites (hexamers containing three-four or more N-acetylated units) are unaffected by non-productive sites, as deduced from the theory of competing substrates.     

Activity studies of glucose oxidase immobilized onto poly(N-vinylimidazole) and metal ion-chelated poly(N-vinylimidazole) hydrogels

Poly(N-vinylimidazole), PVIm, gels were prepared by γ-irradiation polymerization of N-vinylimidazole in aqueous solutions. These affinity gels with a water swelling ratio of 1800% for plain polymeric gel and between 30 and 80% for Cu(II) and Co(II)-chelated gels at pH 6.0 in phosphate buffer were used in glucose oxidase (GOx) adsorption–desorption studies. Different amounts of Cu(II) and Co(II) ions (maximum 3.64 mmol/g dry gel for Cu(II) and 1.72 mmol/g dry gel for Co(II)) were loaded onto the gels by changing the initial concentration of Cu(II) and Co(II) ions, and pH. GOx adsorption on these gels from aqueous solutions containing different amount of GOx at different pH was investigated in batch reactors. Immobilized glucose oxidase activity onto the poly(N-vinylimidazole), and Cu(II) and Co(II)-chelated poly(N-vinylimidazole) were investigated with changing pH and the initial glucose oxidase concentration. Maximum activity of immobilized glucose oxidase onto the PVIm, Cu(II) and Co(II)-chelated PVIm gels was investigated and pH dependence was observed to be at pH 6.5 for free enzyme, pH 7.0 for PVIm, pH 7.5 for Cu(II) and Co(II)-chelated PVIm gels, respectively. The stability of the immobilized enzyme is very high for all gels and the residual activity was higher than 93% in the first 10 days.     

Glycosylation with N-Troc-protected glycosyl donors

N-Troc-protected (Troc = 2,2,2-trichloroethoxycarbonyl) glucosamine and galactosamine glycosyl donors (1-O-acetyl sugar, bromo sugar, and thioglycoside) were compared with the corresponding N-Phth-protected derivatives in glycosylations of 2-(trimethylsilyl)ethanol, 2-bromoethanol, methyl 3-mercaptopropionate, N-Fmoc-protected serine, and 2-(trimethylsilyl)ethyl

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. The N-Troc-protected donors gave pure β-glycosides in somewhat higher yields than the N-Phth-protected counterparts. The N-Troc protecting group can be removed by reduction with zinc, which allows selective N-deprotection in oligosaccharides containing both N-Troc and N-Phth groups.     

H NMR study of the interaction of N,N′,N″-triacetyl chitotriose with Ac-AMP2, a sugar binding antimicrobial protein isolated from Amaranthus caudatus

The interaction between Ac-AMP2, a lectin-like small protein with antimicrobial and antifungal activity isolated from Amaranthus caudatus, and N,N′,N″-triacetyl chitotriose was studied using ¹H NMR spectroscopy. Changes in chemical shift and line width upon increasing concentration of N,N′,N″-triacetyl chitotriose to Ac-AMP2 solutions at pH 6.9 and 2.4 were used to determine the interaction site and the association constant K_a. The most pronounced shifts occur mainly in the C-terminal half of the sequence. They involve the aromatic residues Phe¹⁸, Tyr²⁰ and Tyr²⁷ together with their surrounding residues, as well as the N-terminal Val-Gly-Glu segment. Several NOEs between Ac-AMP2 and the N,N′,N″-triacetyl chitotriose resonances are reported.     

Characteristic properties of N-Carboxybutyl chitosan

N-Carboxybutyl chitosans obtained from levulinic acid (4-oxo-pentanoic acid) and five crustacean chitosans (heteropolymers of N-acetylglucosamine and glucosamine) have been instrumentally characterized and found to have degree of N-carboxyalkylation 0·27. They dissolve in water and in water-ethanol mixtures without the need of any acid and give more viscous solutions than the corresponding chitosans. Their compatibility with other polymers and with salts has been surveyed, and the soluble chelates of Cr(III) and Pb(II) have been studied. The bacteriostatic activity of the N-carboxybutyl chitosans, together with other favorable properties, such as the viscosifying action, the enhanced film-forming ability, the moisturizing effect and the stabilization of emulsions, make these novel modified chitosans most suitable as functional cosmetic ingredients.     

Regenerated chitin from phosphoric acid solutions

Concentrated phosphoric acid at a minimum concentration of 75% readily dissolves -chitin at room temperature. A maximum concentration of 3% (w/v) can be obtained. The initially high viscosity of these solutions decreases quickly during the first 12h. After one week in phosphoric acid, chitin yields mainly a sugar phosphate. Under the same experimental conditions, N-acetyl-d-glucosamine, the monomer of chitin, has been shown to be esterified at its anomeric position by a phosphate group. During the first hours in a phosphoric acid solution, regenerated chitins are not modified chemically although their molecular weights are reduced. Concentrated phosphoric acid is shown to be useful solvent for the preparations of regenerated chitins having an average DP from 1000 to 100.     

Hydrogels of N-acylchitosans and their cellulose composites generated from the aqueous alkaline solutions

Hydrogels of N-acetyl and N-propionylchitosans were prepared form aqueous solutions of sodium N-acylchitosan salts (alkaline N-acylchitosans) and sodium N-acylchitosan xanthate [O-(sodium thio)thiocarbonyl N-acylchitosan], respectively, by standing at room temperature and on heating. Novel hydrogels of N-acetylchitosan-cellulose and N-propionylchitosan-cellulose composites were also prepared from sodium cellulose xanthate [O-(sodiumthio)thiocarbonyl cellulose] solutions mixed with sodium N-acylchitosan salts and with sodium N-acylchitosan xanthates, respectively.     

Succinoylation of sago starch in the N,N-dimethylacetamide/lithium chloride system

The modification reaction of sago starch with succinic anhydride (SA) using pyridine (PY) and/or 4-dimethyaminopyridine (DMAP) as catalyst and N,N-dimethylacetamide (DMA)/lithium chloride (LiCl) system as solvent was studied. A series of succinylated starch derivatives were prepared with a degree of substitution (DS) ranging from 0.14 to 1.54. The structure of the resulting polymers determined by means of ¹³C NMR spectroscopy indicated that substitution preferably occurs at the C₂ and C₆ hydroxyl groups. The thermal stability of the material was decreased by chemical modification. Effects of reactant molar ratio, reaction time, and the concentrations of DMAP and LiCl on the reaction efficiency are discussed.     

X-ray crystal structure of Cu2(medpco-2H)Cl2, (medpco = N,N′-bis(2-N,N-dimethylaminoethyl)pyridine-2,6-dicarboxamide 1-oxide. Copper(II) complexes of a deprotonated binucleating pyridine N-oxide ligand

The X-ray structure is reported for the complex Cu₂(medpco-2H)Cl₂, (medpco = N,N′-bis-N,N-dimethylaminoethyl)pyridine-2,6-dicarboxamide 1-oxide. The complex is triclinic, , a=8.313(4), B=11.403(5), C=11.611(3) Å, =91.66(3), β=108.99(4), γ=109.60(3)° and Z=2. The deprotonated ligand (medpco-2H)²⁻ acts as a binulceating ligand, producing an N-oxide-bridged complex. Each copper in Cu₂(medpco-2H)Cl₂ is five-coordinate, being coordinated by a bridging N-oxide oxygen, a deprotonated amide nitrogen, a tertiary amine nitrogen and two bridging chlorides. The complex does not exhibit significant magnetic interaction, and this may be the result of distortion of the bridging geometry from planarity. A range of other, apparently N-oxide-bridged, complexes of the type Cu₂(medpco-2H)X₂ is reported. The complex Cu₂(medpco-2H)Br₂·H₂O is strongly antiferromagnetic, with magnetic data closely fitting the expected binuclear structure.     

Osteogenesis promoted by calcium phosphate N,N-dicarboxymethyl chitosan

The effects of N,N-dicarboxymethyl chitosan (DCMC) on the precipitation of insoluble calcium salts, namely phosphate, sulfate, oxalate, carbonate, bicarbonate and fluoride, and magnesium salts, namely phosphate and carbonate, were studied. Results indicated that the chelating ability of DCMC interfered effectively with the well-known physico-chemical behaviour of magnesium and calcium salts. Dicarboxymethyl chitosan formed self-sustaining gels upon mixing with calcium acetate, as a consequence of calcium chelation. DCMC mixed with calcium acetate and with disodium hydrogen phosphate in appropriate ratios (molar ratio Ca/DCMC close to 2.4) yielded a clear solution, from which, after dialysis and freeze-drying, an amorphous material was isolated containing an inorganic component about one half its weight. This compound was used for the treatment of bone lesions in experimental surgery and in dentistry. Bone tissue regeneration was promoted in sheep, leading to complete healing of otherwise non-healing surgical defects. Radiographic evidence of bone regeneration was observed in human patients undergoing apicectomies and avulsions. The DCMC–CaP chelate favoured osteogenesis while promoting bone mineralization.     

Potential roles of hypochlorous acid and N-chloroamines in collagen breakdown by phagocytic cells in synovitis

We have tested the effects of the neutrophil/macrophage products, hypochlorous acid (HOCl) and N-chloroamines, on the structural integrity and proteolytic susceptibility of collagen to determine if these agents could play a role in inflammatory joint destruction. Rates of HOCl reaction with collagen, and collagen gelation were monitored by spectrophotometric methods. Direct fragmentation, and degradation by collagenase were measured by the release of acid-soluble counts from ³H-collagen. Physiologically relevant concentrations of HOCl (5–50 μM) reacted rapidly and quantitatively at several sites in the collagen polypeptide chain, causing extensive protein fragmentation and preventing collagen gelation. In contrast, reaction with (5–50 μM) N-chloroalanine induced little direct collagen fragmentation. Oxidative damage by N-chloroamines was, however, evident because collagen displayed greatly increased proteolytic susceptibility following N-chloroamine treatment. Collagen degradation by collagenase increased as much as 3-fold after exposure to N-chloroamine treatment. Collagen degradation by collagenase increased as much as 3-fold after exposure to N-chloroalanine. N-chloroleucine caused a small increase in proteolytic susceptibility, but N-chlorotaurine had no effect. Collagen fragmentation by HOCl, inhibition of gelation by HOCl, and N-chloroalanine-induced proteolytic susceptibility, all increased with linear kinetics at oxidant concentrations of 5 μM to 1.0 mM. In synovitis, phagocytes expose collagen to HOCl, N-chloroamines, and collagenase. It is known that HOCl can activate neutrophil procollagenase. Based on our new findings, we propose a model of inflammatory joint destruction that also includes collagen fragmentation, and increased susceptibility of collagen to degradation by collagenase. It may also be possible that taurine exerts a protective effect against HOCL/OCL⁻ damage by reacting to form what appears to be essentially an inert N-chloroamine. The validity of this model must now be tested.