Chemical Modification Of Tyrosine Residues Of Silk Protein And The Effect On Its Light Stabilization

The light stability of silk is poor. In order to enhance the light stability and its wet fastness of silk, the benzotriazole structure was synthesized based chemical modification of tyrosine residues of fibroin protein through coupling and reductive cyclization reaction processes in this thesis. The main research work was to reveal the factor and regulatory law of benzotriazole structure synthesized based chemical modification of tyrosine residues of fibroin protein, and to clarify the light stability and other properties of chemical modified silk protein and its mimetic.At the same time, a preliminary study of solvent and substituent effects of benzotriazole structure synthesized were researched to clarify further the factor and mechanism to the light stability property of the modified silk protein mimetic and silk fabric. The main contents and conclusions of the thesis were as follows.Part 1 was the research on light-yellowing properties of silk under characteristic bands of ultraviolet irradiation. The ultraviolet bond is a relatively broad range of wavelengths. The aim of this part research was to determine a suitable light aging test condition for silk fabric, as well as to verify the effect of different ultraviolet band on silk light-yellowing properties. The results showed that there are huge differences of different bands of ultraviolet on the light-yellowing properties of silk fabric, and the UVB range of ultraviolet radiation has resulted in the occurrence of light-yellowing of silk easily, and the light-yellowing degree of silk will be aggravated with UV irradiance increasing and irradiation time extending. In this part research,the standard curve for L-tyrosine quantitative analysis has been drafted based on HPLC test technology, which was used to analysis the tyrosine content of light-yellowed silk fabric. The tyrosine content analysis result showed that tyrosine is the vital amino acid composition during the photo chemical reaction of silk protein, and the tyrosine content will be decreased as aggravation of light-yellowing of silk.Part 2 was the research on chemical modification of tyrosine to synthesis benzotriazole structure. In order to study the chemical modification reaction and the modified target product,the chemical modification of free tyrosine as silk protein mimetic to synthesis benzotriazole structure has been carried out firstly in the thesis. The first step of tyrosine chemically modified process was to make the diazonium salt of o-nitroaniline, at the same time, preparation of the diazonium salt and its stability has been researched. The second step was to generate azo intermediate through the coupling reaction between o-nitroaniline diazonium salt and tyrosine.The modified target product of tyrosine has been synthesized in the third step with the ring closure reaction of azo intermediate using thiourea dioxide as reducing agent. The azo intermediate and modified target product of tyrosine have been purified by column separationand HPLC testing technology respectively, and each structure of them have been confirmed through UV, IR, MS, NMR spectrum analyzed. The above results illustrated the feasibility of the chemical modified reaction of tyrosine to synthetic benzotriazole structure. The pure target modified product of tyrosine has been prepared and which laid the foundation for the next analysis of chemically modified silk protein tyrosine residues.Part 3 was the research on chemical modification of tyrosine residues to synthesis benzotriazole structure. The degummed silk fabric has been modified according the tyrosine modified process mentioned previously, and the modified product of silk tyrosine residues has been confirmed at first in this research part through HPLC, MS and UV spectrum of the hydrolyzate of modified silk fabric. The standard curve for target modified product of tyrosine quantitative analysis has been drafted based on HPLC test technology, which was used to analysis silk protein tyrosine residues that involved in the synthesis of benzotriazole structure in this chemically modification.In order to investigate factors and regulatory law of the chemical modification of silk tyrosine residues to synthesis benzotriazole structure, the experimental modification process of tyrosine residues has been optimized. The K/S value of coupling modified silk fabric has been used as the measure to optimize the coupling modification parameters, and the optimization coupling modified process parameters of silk fabric were 2/3:1 mole ratio of o-nitroaniline diazonium salt to tyrosine content in the silk fabric, 5-10℃ of coupling temperature, 6.8 of coupling bath pH value, 45 min of coupling time and 1:45 of bath ratio. There will be 50.89% tyrosine constituent of silk protein involved in the coupling chemical modification under this optimization process.The ratio of tyrosine residue involved in the synthesis of benzotriazole structure during the ring closure reaction of coupling modified silk has been used as the measure to optimize the reduction process. The optimized ring closure reaction of coupling modified silk was realized by the one-bath two-step reduction process of glucose and thiourea dioxide. In the glucose reduction process, the mole ratio of glucose, to sodium carbonate, and to tyrosine content in the silk fabric was set at 3:5:1, the glucose reduction was continued for 60 min at 60 °C, and the liquor-to-goods ratio was 40:1. After the glucose reduction, thiourea dioxide and more sodium carbonate will be added into the reduction bath directly to begin the second step reduction. The optimized molar ratio of thiourea dioxide, to sodium carbonate(including the sodium carbonate added at the first glucose reduction step) and to tyrosine content in the silk fabric was set at 4:10.5:1, and the thiourea dioxide reduction was continued for 3h at 90°C. There will be 22.27% tyrosine constituent of silk protein involved in the benzotriazole structure synthesis during the ring closure reaction under this optimization process. Meanwhile, the influence of the chemicalmodification to the performances of silk fabric has been analyzed systematically through fabric appearance, UV-visible transmittance spectra, breaking strength, morphology, aggregation structure, dyeing performance and other aspects. Results showed that the modified silk fabric can maintain good fabric appearance, the UV transmittance of modified silk fabric decreased significantly, and the whiteness and reflectance both decreased. Although the chemical modification has little effect on aggregation structure of modified silk, silk fiber will be swelled for base, reducing agent and heat process factors in the modified reaction, and slightly fibrillation was appeared on the modified silk fibers. The breaking strength of modified silk has decreased 14.29% comparing to untreated silk fabric. Besides there is no significant difference in the dyeing and color indexes between the modified silk and silk fabric except the yellow hue dyes.Part 4 was the research on the light stability performance of modified tyrosine and modified silk fabric. The light stability of target modified product of tyrosine and modified silk fabric have been researched systematically through several testing and characterization techniques, such as HPLC, light-yellowing course, light degradation rate and etc. Results showed that both the tyrosine and tryptophan have poor light stability in dissolved state, which will be prone to light degradation along with the light-yellowing under ultraviolet irradiation. Tryptophan has the worst light stability among tyrosine, tryptophan and other amino acids. Compared with tyrosine,the target modified product of tyrosine has good light stability, and its light degradation rate has slowed down significantly. Meanwhile, the target modified product of tyrosine can not only improve the light stability of tryptophan, tyrosine, phenylalanine, histidine in the same dissolved bath, and reduce the light degradation rate of each amino acid, but also improve the light stability of acid dye red 182 molecule. Compared with untreated silk fabric, the light stability of modified silk fabric has been greatly improved. The light yellowing rate, light tender rate and tyrosine degradation rate of modified silk fabric have been decreased, while the light stability of the modified silk fabrics have a high wet fastness. It is the chemical modification reaction of tyrosine residues to synthesis benzotriazole structure, which has weakened the damaging effects of ultraviolet irradiation on the silk fiber morphology and aggregation, as well as enhanced the role of color fastness on dyed silk fabric.Part 5 was the research on the solvent effects and structure-activity relationship of benzotriazole structure synthesized in the chemically modified reaction. The aim of the solvent effects research of target modified product of tyrosine was to investigate the factors which will be effect on the light-tautomeric formation of benzotriazole structure synthesized in the modified reaction. The results showed that acidic and weakly polar solvent system will be conducive tothe occurrence of light-tautomeric formation of benzotriazole structure when excited by ultraviolet irradiation. Meanwhile, the fluorescence spectrum showed the target modified product of tyrosine will be excited by ultraviolet irradiation and will emit energy of the visible light photons, which can be achieved the transformation of the ultraviolet irradiation energy. The research also proved that fluorescence properties of the target modified product of tyrosine will be enhanced when dissolved in weak polar solvent. At the same time, the fluorescence properties and light stability of the target product of the modified tyrosine was positively correlated. The results of structure-activity relationship studies showed that the UV-visible spectrum and molar absorption coefficient of the modified tyrosine product will be influenced by the different substituent of diazo component. Substituent with the electron-withdrawing effect will increase the molar absorption coefficient of modified tyrosine product. The result also showed that diazo component with different substituents has different effects on the fluorescence spectra and color index of modified silk. More detailed substituent effects will be informed in further research.In summary, the research in this thesis has expanded the scientific research means and methods on chemical modification of proteins and material light stability studies, and provided a new way to resolve the old problem of silk with poor light stability. The thesis research showed that the benzotriazole structure synthesized in the chemically modified reaction attached to the protein tyrosine residues with covalent bond, the light stability of the modified silk and its wet fastness and durability have been enhanced obviously. The quantitative analysis methods of tyrosine and tyrosine residues involved in the chemically modified of silk proteins have been built. Base on the quantitative analysis methods, the variation of tyrosine ingredient during the yellowing process of silk has been disclosed, and effect factors of tyrosine residues involved in the chemically modified process have been elucidated, and thus the optimum chemically modified process parameters of silk tyrosine residues has been obtained. The mechanism of benzotriazole structure synthesized in the chemically modified reaction to enhance the light stability of modified protein has been clarified further through research on solvent effects and structure-activity relationship. This thesis will also provide experimental and theoretical basis for the study of protein chemical modification and directed evolution, particularly to the development of new light stabilization fibroin materials.