NMR investigation of chitosan derivatives formed by the reaction of chitosan with levulinic acid

Chitosan derivatives are obtained by reaction of chitosan with a low degree of acetylation and levulinic acid under different experimental conditions. The chemical structure of the different derivatives obtained is determined using ¹H and ¹³C NMR spectroscopies. The intrinsic viscosity is used to follow the molecular weight evolution. Finally, conditions are described in which water-soluble N-carboxybutylchitosan is obtained. In particular, the time of the reduction step and the ratio between reagents are investigated. Under mild conditions and short times of reduction there is a very low degree of substitution and only the monocarboxybutylchitosan is formed. The dicarboxylated form is never observed. The cyclic derivative (5-methylpyrrolidinone chitosan) is obtained when the reducing agent is added slowly to the reactants.     

Preparation of N-vanillyl chitosan and 4-hydroxybenzyl chitosan and their physico-mechanical, optical, barrier, and antimicrobial properties

Chitosan derivatives such as N-vanillyl chitosan and 4-hydroxybenzyl chitosan were prepared by reacting chitosan with 4-hydroxy-3-methoxybenzaldehyde (vanillin) and 4-hydroxybenzaldehyde. Amino groups on chitosan reacts with these aldehydes to form a Schiff base intermediate, which is later on converted into N-alkyl chitosans by reduction with sodium cyanoborohydride. The chemical reaction was monitored by ¹H NMR spectroscopy and the absence of aldehydic proton at 9.83 ppm in NMR spectra was observed for both the modified chitosan derivatives confirming the reaction. Modified chitosan films were later prepared by solution casting method and their physico-mechanical, barrier, optical and thermal properties were studied. The results clearly indicated significant change in tensile strength, water vapour transmission rate, and haze properties of modified chitosans. Modified chitosan films were also studied for their antimicrobial activity against Aspergillus flavus. The results showed a marked reduction of aflatoxins produced by the fungus in the presence of the N-vanillyl chitosan and 4-hydroxybenzyl chitosan film discs to 98.9% and non-detectable levels, respectively.     

Enzymatic grafting of carboxyl groups on to chitosan--to confer on chitosan the property of a cationic dye adsorbent

Chitosan (CTS) is a good adsorbent for dyes but lacks the ability to adsorb cationic dyes. In this study, chitosan was modified to possess the ability to adsorb cationic dyes from water. Four kinds of phenol derivatives: 4-hydroxybenzoic acid (BA), 3,4-dihydroxybenzoic acid (DBA), 3,4-dihydroxyphenyl-acetic acid (PA), hydrocaffeic acid (CA) were used individually as substrates of tyrosinase to graft onto chitosan. FTIR analysis provided supporting evidence of phenol derivatives being grafted. The grafting amounts of these phenol derivatives onto chitosan were examined by the adsorption of an anionic dye (amaranth) and reached a plateau value. The final contents of carboxyl groups in chitosan (mmol carboxyl groups per kg chitosan) were measured as 46.36 for BA, 70.32 for DBA, 106.44 for PA, and 113.15 for CA. These modified chitosans were used in experiments on uptake of the cationic dyes crystal violet (CV) and bismarck brown Y (BB) by a batch adsorption technique at pH 7 for CV and at pH 9 for BB and 30 degrees C. Langmuir type adsorption was found, and the maximum adsorption capacities for both dyes were increased with the following order CTS-CA>CTS-PA>CTS-DBA>CTS-BA.     

Chemical modifications of carboxylated chitosan

C-6-carboxylated chitosan obtained by oxidation of chitosan was selectively modified in order to obtain derivatives similar to bacterial antigens. Selective O-acetylation of 6-carboxyl chitosan afforded a modified polysaccharide with the 2-amino group available for further modifications to create carbonyl groups. A deaminative degradation reaction allowed the formation of oligosaccharides with terminal aldehyde groups. Reductive alkylation with lactose introduced lactityl branches which were oxidized with galactose oxidase to give aldehyde groups in its -galactose residues.     

Surface modification of chitosan films. Effects of hydrophobicity on protein adsorption

The surface of chitosan films was modified using acid chloride and acid anhydrides. Chemical composition at the film surface was analyzed by attenuated total reflectance Fourier-transform infrared spectroscopy (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS). ATR-FTIR data verified that the substitution took place at the amino groups of chitosan, thus forming amide linkages, and the modification proceeded to the depth at least 1 microm. Choices of molecules substituted at the amino groups of the glucosamine units did affect the hydrophobicity of the film surface, as indicated by air-water contact angle analysis. The surface became more hydrophobic than that of non-modified film when a stearoyl group (C(17)H(35)CO-) was attached to the films. The reaction of chitosan films with succinic anhydride or phthalic anhydride, however, produced more hydrophilic films. Selected modified films were subjected to protein adsorption study. The amount of protein adsorbed, determined by bicinchoninic acid (BCA) assay, related to the types of attached molecules. The improved surface hydrophobicity affected by the stearoyl groups promoted protein adsorption. In contrast, selective adsorption behavior was observed in the case of the chitosan films modified with anhydride derivatives. Lysozyme adsorption was enhanced by H-bonding and charge attraction with the hydrophilic surface. While the amount of albumin adsorbed was decreased possibly due to negative charges that gave rise to repulsion between the modified surface and albumin. This study has demonstrated that it is conceivable to fine-tune surface properties which influence its response to bio-macromolecules by heterogeneous chemical modification.     

Studies on 2-aziridinecarboxylic acid peptides: Their reaction and uses

The reaction of 2-aziridinecarboxylic acid derivatives with several protic reagents was used to synthesize depsipeptides, dehydroamino acid derivatives, diaminopropionic acid derivatives, and phospho peptide derivatives. The reaction of N-aminoacyl-2-aziridinecarboxylic acid benzyl ester with amino acid ester induced stereoselective transacylation.     

Obtaining and study of monosaccharide derivatives of low-molecular-weight chitosan

The possibility of obtaining monosaccharide derivatives of low-molecular-weight chitosan with the use of the Maillard reaction was studied. Chitosan derivatives (molecular weight, 24 and 5 kDa) obtained with glucosamine, N-acetyl galactosamine, galactose, and mannose with a substitution degree of 4-14% and a yield of 60-80% were obtained. Some physicochemical and biological properties of these derivatives were studied. We showed that monosaccharide derivatives of low-molecular-weight chitosan exhibited antibacterial activity. Chitosan at a concentration of 0.01% caused 100% death of bacteria B. subtilis and E. coil. The strongest antibacterial effect was exhibited by 24-kDa derivatives: only 0.02-0.08% of cells survived. These derivatives were two orders of magnitude more effective than the 5-kDa chitosan modified with galactose.     

Chemical modification of chitosan. Part 15: synthesis of novel chitosan derivatives by substitution of hydrophilic amine using N-carboxyethylchitosan ethyl ester as an intermediate

The Michael type reaction of chitosan with ethyl acrylate has been investigated. Although this reaction was quite slow in the case of chitosan, the reiteration of the reaction was an effective means for increasing the degree of substitution (DS) of ethyl ester. The N-carboxyethylchitosan ethyl ester as an intermediate was successfully substituted with various hydrophilic amines, although the simultaneous hydrolysis of the ester to carboxylic acid also occurred. Water-soluble chitosan derivatives were obtained by substitution with hydroxyalkylamines and diamines.     

6-Amino-6-deoxy-chitosan. Sequential chemical modifications at the C-6 positions of N-phthaloyl-chitosan and evaluation as a gene carrier

The C-6 positions of chitosan were successively modified in a highly regioselective manner. The starting material, N-phthaloyl-chitosan, was successfully converted into the corresponding 6-deoxy-6-halo derivatives by reaction with N-halosuccinimides and triphenylphosphine in N-methyl-2-pyrrolidone. The resulting chloride and bromide derivatives were then substituted with azido groups by reaction with sodium azide at 120 and 80 degrees C, respectively. The azido groups were then reduced to amines via formation of the triphenylphosphinimine intermediate followed by hydrolysis using aqueous hydrazine, which also led to the removal of the N-phthaloyl groups at the C-2 positions. This sequence gave 6-amino-6-deoxy-chitosan, which, unlike chitosan, is soluble in water at neutral pH. The synthesized 6-amino-6-deoxy-chitosan derivative was evaluated as a gene carrier, and the transfection efficiency for COS-1 cells was shown to be superior to chitosan. In addition, the cytotoxicity was similar to chitosan.     

Trinitrophenylation of the bilirubin binding site of human serum albumin.

Human serum albumin has been modified with 2,4,6-trinitrobenzenesulphonic acid and picryl chloride in low ratios of reagents/albumin. The derivatives have been investigated by spectrophotometry and by thin layer chromatography of the hydrolysates in order to assess the specificity of the reagents. The same reaction conditions were used to modify albumin previously complexed with bilirubin in the ratio of 1:1. The affinity of bilirubin to the modified albumins was estimated by an improved perozidase method. It is concluded that TNBS and picryl chloride react almost quantity with epsilon-amino groups of lysine on the albumin molecule. The results also suggest that at least on TNBS reactive amino group and at least one picryl chloride reactive amino group are located in or near the high-affinity bilirubin binding site.     

羧甲基壳聚糖的制备及其在保鲜中的应用

研究了在碱性条件,无溶剂体系下壳聚糖羧甲基化制备工艺,通过IR对反应前后壳聚糖结构进行了表征。最佳反应条件：2g壳聚糖,在NaOH加入量为5g、碱化时间为6h、氯乙酸用量为6g、3％KI、反应时间为8h、反应温度60℃,产品取代度为1．27。同时研究了羧甲基壳聚糖对辣椒、猪肉涂膜保鲜实验,结果显示对辣椒、猪肉具有较好的涂膜保鲜作用。     

Preparation of soluble p-aminobenzoyl chitosan ester by Schiff's base and antibacterial activity of the derivatives

Schiff's base of chitosan (BCTS) was obtained by the reaction of chitosan (CTS) and benzaldehyde. Then BCTS reacted with acyl chloride which was synthesized by p-aminobenzoic acid and thionyl chloride to get N-benzoyl-O-aminobenzoyl chitosan ester (BABCTSE), removing the groups of amino protection of BABCTSE to get the final product (ABCTSE). The structures of the derivatives were characterized by FT-IR, (1)H NMR, (13)C NMR and elemental analysis. The elemental analysis results indicated that the degrees of substitution (DS) of the products were 16.8% and 40.4%. The synthesized compounds exhibited an excellent solubility in organic solvents. TG and DTG results showed that thermal stability of the derivatives was lower than that of chitosan. In addition, the existence of two different amido in the molecular structures contributed to forming more -NH(3)(+) in the acid solution which could make the derivatives have a greater advantage in the field of bacteriostasis.     

Obtaining and study of monosaccharide derivatives of low-molecular-weight chitosan

The possibility of obtaining monosaccharide derivatives of low-molecular-weight chitosan with the use of the Maillard reaction was studied. Chitosan derivatives (molecular weight, 24 and 5 kDa) obtained with glucosamine, N-acetyl galactosamine, galactose, and mannose with a substitution degree of 4–14% and a yield of 60–80% were obtained. Some physicochemical and biological properties of these derivatives were studied. We showed that monosaccharide derivatives of low-molecular-weight chitosan exhibited antibacterial activity. Chitosan at a concentration of 0.01% caused 100% death of bacteria B. subtilis and E. coli. The strongest antibacterial effect was exhibited by 24-kDa derivatives: only 0.02–0.08% of cells survived. These derivatives were two orders of magnitude more effective than the 5-kDa chitosan modified with galactose.     

Preparation and important functional properties of water-soluble chitosan produced through Maillard reaction

The objective of this research was to improve the solubility of chitosan at neutral or basic pH using the Maillard-type reaction method. To prepare the water-soluble chitosans, various chitosans and saccharides were used under various operating conditions. Biological and physicochemical properties of the chitosan-saccharide derivatives were investigated as well. Results indicated that the solubility of modified chitosan is significantly greater than that of native chitosan, and the chitosan-maltose derivative remained soluble when the pH approached 10. Among chitosan-saccharide derivatives, the solubility of chitosan-fructose derivative was highest at 17.1 g/l. Considering yield, solubility and pH stability, the chitosan-glucosamine derivative was deemed the optimal water-soluble derivative. Compared with the acid-soluble chitosan, the chitosan-glucosamine derivative exhibited high chelating capacity for Zn(2+), Fe(2+) and Cu(2+) ions. Relatively high antibacterial activity against Escherichia coli and Staphylococcus aureus was noted for the chitosan-glucosamine derivative as compared with native chitosan. Results suggest that the water-soluble chitosan produced using the Maillard reaction may be a promising commercial substitute for acid-soluble chitosan.     

The preparation and antioxidant activity of the sulfanilamide derivatives of chitosan and chitosan sulfates

Chitosan (CS) and chitosan sulfates (CSS) with different molecular weight (Mw) were reacted with 4-acetamidobenzene sulfonyl chloride to obtain sulfanilamide derivatives of chitosan and chitosan sulfates (LSACS, HSACS, LSACSS, HSACSS). The preparation conditions such as different reaction time, temperature, solvent, and the molar ratio of reaction materials are discussed in this paper. Their structures were characterized by FTIR spectroscopy and elemental analyses. The antioxidant activities of the derivatives were investigated employing various established in vitro systems, such as hydroxyl-radical ((*)OH) superoxide anion (O2(*-)) scavenging and reducing power. All kinds of the compounds (HCS, LCS, HCSS, LCSS, HSACS, LSACS, HSACSS, LSACSS) showed stronger scavenging activity on hydroxyl radical than ascorbic acid (Vc). The inhibitory activities of the derivatives toward superoxide radical by the PMS-NADH system were obvious. The experiment showed that the superoxide radical scavenging effect of sulfanilamide derivatives of chitosan and chitosan sulfates was stronger than that of original CS and CSS. All of the derivatives were efficient in the reducing power. The results indicated that the sulfanilamide group were grafted on CS and CSS increased the reducing power of them obviously.     

Synthesis and biological evaluation of 9-substituted tetracycline derivatives

The synthesis of 9-substituted tetracycline derivatives has been accomplished by the reaction of C9 diazonium tetrafluoroborate tetracycline salts with organotin reagents under modified Stille coupling conditions. Several of these unreported derivatives show promising in vitro biological activity against tetracycline resistant and antibiotic resistant bacteria.     

Enzymatic grafting of a natural product onto chitosan to confer water solubility under basic conditions

Chitosan is a natural biopolymer whose rich amine functionality confers water solubility at low pH. At higher pH's (greater than 6. 5), the amines are deprotonated and chitosan is insoluble. To attain water solubility under basic conditions we enzymatically grafted the hydrophilic compound chlorogenic acid onto chitosan. Despite its name, chlorogenic acid is a nonchlorinated phenolic natural product that has carboxylic acid and hydroxyl functionality. The enzyme in this study was tyrosinase, which converts a wide range of phenolic substrates into electrophilic o-quinones. The o-quinones are freely diffusible and can undergo reaction with the nucleophilic amino groups of chitosan. Using slightly acidic conditions (pH = 6), it was possible to modify chitosan under homogeneous conditions. When the amount of chlorogenic acid used in the modification reaction exceeded 30% relative to chitosan's amino groups, the modified chitosan was observed to be soluble under both acidic and basic conditions, and to have a pH window of insolubility at near neutral pH. 1H NMR spectra confirmed that chitosan was chemically modified, although the degree of modification was low. Copyright 1999 John Wiley & Sons, Inc.     

壳聚糖载体的改性及其用于固定化漆酶的研究

利用有机酸改性壳聚糖,并用交联法制备酸化的壳聚糖载体,然后用改性壳聚糖载体固定漆酶。甲酸、乙酸改性壳聚糖的最适条件:壳聚糖与甲酸、乙酸的质量摩尔比(g/mol)分别为100∶1、100∶1.5,戊二醛的质量分数为1%,缓冲溶液的pH分别是4.4、5.0,反应时间为3h;壳聚糖与酒石酸、草酸的质量摩尔比(g/mol)分别为100∶0.5、100∶2,戊二醛的质量分数为2%,缓冲溶液的pH分别是3.6、4.2,反应时间为4.5h。不同有机酸改性的壳聚糖用于漆酶的固定,其酶活都有不同程度的提高,尤其用酒石酸改性的壳聚糖载体效果最好,其酶活提高了57%。     

低聚壳聚糖与α-丙氨酸/天冬酰胺的美拉德反应及其衍生物的抗氧化性能研究

研究低聚壳聚糖(COS)与α-丙氨酸/天冬酰胺的美拉德反应,考察了两个体系(低聚壳聚糖的羰基与氨基的物质量比均为1∶1)的pH、吸光度和荧光值的变化。醇沉法提取低聚壳聚糖衍生物CA和CN。对两种衍生物进行红外表征和分子量测定,并研究其对超氧阳离子O2-.、DPPH自由基的清除能力以及还原能力。结果显示:抗氧化能力强弱次序为CA>CN>COS,即美拉德反应后低聚壳聚糖衍生物抗氧化能力得到显著提高,且CA的抗氧化活性优于CN,表明与小分子氨基酸进行美拉德反应制得的壳聚糖衍生物具有更好的抗氧化性。     

Synthesis of organosoluble chitosan derivatives with polyphenolic side chains

A one-pot synthesis was used to produce chitosan derivatives with polyphenolic side chains via a regioselective phenolic coupling reaction. Under Mannich reaction conditions, treatment of chitosan with formaldehyde and methyl 2,4-dihydroxybenzoate gave N-(2,6-dihydroxy-3-methoxycarbonylphenyl)methylated chitosan in good yield (87%). Formation of a CC bond occurred regioselectively at the C(3) position of methyl 2,4-dihydroxybenzoate. Chitosan derivatives having various phenolic compounds as a side chain were easily synthesized in a similar manner. The chitosan derivatives showed good biodegradability and improved their solubility in methanol (9.8mgmL(-1)) and 2-methoxyethanol (> 10mgmL(-1)). The UV protection provided by the derivatives with phenolic benzophenone side chain was evaluated using UV spectra of polyethylene terephthalate and poly(vinyl butyral-co-vinyl alcohol-co-vinyl acetate) films coated with the derivatives and the derivatives absorbed effectively in the UV-A region (<60%). Self-aggregation of the chitosan derivatives with the phenolic side chain was observed by using a fluorescent probe in aqueous solution.