Preparation And Bioactivities Of Chitosan Derivatives Bearing Urea Groups

Chitosan,mainly derived from the deacetylation of chitin,is the most abundant biological polysaccharide on the earth after cellulose.Chitosan has been widely used in field of biopolymer materials due to the advantages of non-toxicity,excellent biocompatibility,natural biodegradability,and good biological activity.However,the further commercial applications of chitosan are limited by its characteristics of poor water solubility,narrow antibacterial spectrum,and relatively weak biological activity.Chemical modification can change the chemical structure of chitosan and enhance its mechanical,chemical,and biological properties.Meanwhile,urea groups have attracted numerous attention due to its prominent biological activity.Therefore,the urea groups can be introduced into the structure of chitosan through chemical modification to obtain several chitosan derivatives with good water solubility and strong biological activity.In this thesis,four series of chitosan derivatives bearing urea groups,including N,O-(3-(ureido)-pyridyl)acetyl chitosan derivatives,N-ureido chitosan derivatives,N-ureido-O-(3-(ureido)-pyridyl)acetylchitosanderivatives,and N,N,N-trimethyl-O-(3-(ureido)-pyridyl)acetyl chitosan derivatives,were prepared by different chemical modification methods.FTIR and ~1H NMR techniques were applied for the confirmation of chemical structures of chitosan derivatives.Meanwhile,the degrees of substitution of these derivatives were calculated by elemental analysis.The effects of the introduction of active urea groups on the biological activity of chitosan derivatives were studied by antifungal as well as antioxidant activity test,and the structure-activity relationship was also investigated.In addition,the in vitro CCK-8assay was used to test the cytotoxicity of all samples at different concentrations on L929 cells.Firstly,eight N,O-(3-(ureido)-pyridyl)acetyl chitosan derivatives containing benzene ring and azole were synthesized via intermediate chloroacetyl chitosan.The results showed that the introduction of urea groups significantly improved the antifungal activity of chitosan derivatives by more than 25%.Meanwhile,the antifungal property of the derivatives was positively correlated with electronegativity.The antioxidant results indicated that the antioxidant capacity of the synthesized chitosan derivatives bearing urea groups was also significantly enhanced compared with pristine chitosan and intermediate chloroacetyl chitosan.Especially,the scavenging values of all derivatives against DPPH-radical,superoxide-radical,and hydroxyl-radical enhanced by about 60%at 1.6 mg/m L.The results of cytotoxicity test showed that most samples had decreased cytotoxicity and some samples even could promote cell growth.With a large excess of methyl chloroformate and primary amine,the amino group in chitosan could be converted to urea structure.Based on this mechanism,we synthesized N-pyridylureido chitosan derivatives,quaternized N-pyridylureido chitosan derivatives,N-benzylureido chitosan derivatives,and N-azolureido chitosan derivatives,secondly.As shown by antioxidant test of N-pyridylureido chitosan derivatives,the derivatives possessed higher antioxidant property than that of pristine chitosan and methoxyformylated chitosan.Moreover,after quaternization with iodomethane,quaternized N-pyridylureido chitosan derivatives immediately exhibited improved antioxidant ability compared with N-pyridylureido chitosan derivatives.The antifungal results of N-benzylureido chitosan derivatives and N-azolureido chitosan derivatives showed that although the activity was improved compared with chitosan,the improvement was not significant.It was found from the antioxidant results that their antioxidant activity was greatly improved.In particular,when the concentration was 0.8 mg/m L,the DPPH-radical scavenging rate of N-triazolureido chitosan derivative could reach to 98.90%.The cytotoxicity test showed that the survival rates of L929 cells treated with N-ureido chitosan derivatives were above 60%at the concentration of 500?g/m L.Thirdly,in order to study the effects of the introduction of urea groups on the biological activity of N-ureido chitosan derivatives,eight N-ureido-O-(3-(ureido)-pyridyl)acetyl chitosan derivatives were designed and synthesized.The results showed that the antifungal activity of these derivatives was significantly improved compared with N-ureido chitosan derivatives,but the antioxidant capacity was not significantly enhanced by the introduction of substituted urea groups.Therefore,it seems that the active urea groups play a pivotal role in improving the antifungal activity of chitosan derivatives,and the formed urea structure is more advantageous in improving the antioxidant activity.Finally,eight N,N,N-trimethyl-O-(3-(ureido)-pyridyl)acetyl chitosan derivatives were synthesized to investigate the effects of trimethylammonium group on the biological activity of chitosan derivatives bearing urea group.This series of derivatives had a significant inhibitory effect on the tested fungi,especially on Botrytis cinerea.When the concentration was 1.0 mg/m L,the inhibitory rates of most samples were more than 80%.Furthermore,derivatives with stronger electron-withdrawing ability showed better antifungal property.The combination of trimethylammonium group and urea group could also significantly enhance the antioxidant activity of chitosan derivatives.And these chitosan derivatives still showed the rule that samples with stronger electron-withdrawing ability showed better antioxidant activity.Among all chitosan derivatives bearing urea groups synthesized in this thesis,N,N,N-trimethyl-O-(3-(ureido)-pyridyl)acetyl chitosan derivatives possessed the best antifungal and antioxidant activities.Cytotoxicity tests also confirmed the low toxicity of these derivatives.Therefore,this series of derivatives were characterized by good water solubility,strong antifungal ability,excellent antioxidant activity,as well as low toxicity and had a good application prospect.This thesis systematically studied the preparation method,structure characterization,as well as in vitro bioactivity test of chitosan derivatives bearing urea groups,and screened out N,N,N-trimethyl-O-(3-(ureido)-pyridyl)acetyl chitosan derivatives,which with promising antifungal and antioxidant properties.This research will broaden the application of chitosan in biopolymer materials.