Study On Colorized Modification And Its Mechanism Of Silk Fibroin With Aromatic Primary Amine Dye Based On Mannich Reaction

Due to the shortcomings of the existing chemical modification methods for silk fibroin protein side groups,in order to find a suitable chemical modification method for silk fibroin protein side groups,with that the efficient modification of silk fibroin protein can be realized while maintaining its natural quality.In this study,the modification and its mechanism of silk fibroin protein with aromatic primary amine dyes based on Mannich reaction will be systematically studied.The main research work in the paper is focus on the following fouraspects:To explore various influencing factors,rules and regulatory means of aromatic primary amine compounds in modifying silk fibroin protein side groups based on Mannich reaction;To expound the structure-activity relationship of aromatic primary amine dyes to colorized modification of silk fibroin protein side group by Mannich reaction;To reveal the chemical mechanism of aromatic primary amine compounds on the side group modification of silk fibroin protein;Finally,to establish a complete method and theoretical system for modification of silk fibroin protein by aromatic primary amine compound based on Mannich reaction.The main research contents and results are listed as following:(1)The modification characteristics of aromatic primary amine dyes for silk fibroin protein based on Mannich reaction was researched as followed.In the research of silk fibroin protein with aromatic primary amine dyes based on Mannich reaction,modification characteristics becomes thathasto be solved at first.It can provide references for the extensive applications of modification with aromatic primary amine compounds based on Mannich reaction.Therefore,in order to obtain a simple and visual modification effect for silk fibroin protein,we adopts the aromatic primary amine dyes as the modification agent and degummed silk fabric as the modified substance to carry out various research work.The results showed that the appropriate modification conditions for Mannich reaction were 30℃,pH value 4.0,10h,and the dosage of aromatic primary amine dye and formaldehyde was 1:3(molar ratio).Among various aldehydes components,formaldehyde has the most prominent effect.Aromatic primary amine dyes have a high fastness to silk fibroin protein,which can withstand DMF stripping without falling off.And aromatic primary amine dye modification based on Mannich reaction causes extremely little damage to silk fibroin protein.(2)The structure-activity relationship of aromatic primary amine dyes was studied as followed.A series of 14 aromatic primary amine dyes were obtained though synthesis and screening.And a simple visual characterization method was developed to evaluate the modification efficiency on silk fabrics by Mannich reaction.Furthermore,the structure-activity relationship of aromatic primary amine dyes was revealed by using this method.The results show that,for the aniline dyes,the amino group in the para-position and meta-position of azo substituent can produce covalent bond with silk protein,and the color depth of the para-position dye is significantly higher than that of the meta-position dye.However,when the amino group is located in the ortho-position of azo substituent,the modification effect cannot be produced due to the preferential decoloration reaction with formaldehyde.When the Mulliken charge number of the amino group on the designed dye molecule is appropriate,it helps to achieve a higher color fixation effect.For the naphthylamine dyes,the amino group should be kept away from the hydroxyl group on the naphthalene ring in order to avoid the occurrence of intramolecular reaction and reduce the modification efficiency of Mannich reaction.Finally,by using a series of aromatic primary amine dyes,the modification effect can be visualized,and the Mannich reaction modification degree of silk fibroin protein can be clearly determined.In a word,the Mannich reaction modification of silk fibroin protein with appropriate aromatic primary amine compounds should meet the following requirements:The amine groups should have appropriate nucleophilic propertiesandthere is no center of high electron density around the amino group.(3)The aromatic primary amine dyes modification mechanism of silk fibroin protein based on Mannich reaction was researched.By means of amino acid simulation and polypeptide simulation,combined with TLC and ESI-MS,the aromatic primary amine dyes modification mechanism of silk fibroin protein based on Mannich reaction was clarified.Specific results are as follows:Through single amino acid modification,it was found that tryptophan could exhibit Mannich reaction,while tyrosine and other amino acids could not undergo Mannich reaction modification.The results of polypeptide simulation showed that both tyrosine residues and tryptophan residues in the polypeptide molecules could be used as binding sites for silk protein modification by aromatic primary amine compounds in Mannich reaction,but tryptophan residues were preferentially modified by Mannich reaction of aromatic primary amine compounds.The amino acid sequence of the polypeptide has a significant effect on the Mannich reaction reactivity of tyrosine residues.On the whole,tryptophan has good Mannich reactivity,and its activity is higher than tyrosine.It is also found that the primary amino group in the polypeptide is easy to react with formaldehyde to form schiff base.The imino in the polypeptide molecules cannot undergo Mannich reaction.When the polypeptide contains more than one arginine residue,the cross-linking reaction is easy to occur,which reduces the localization and modification efficiency of the aromatic primary amine compounds on tyrosine residue based on Mannich reaction.(4)The intramolecular Mannich reaction of aromatic primary amine dyes with formaldehyde and its application was researched.The decoloration phenomenon of the ortho-aniline aromatic azo dyes which was found in study of structure-activity of aromatic primary amine dyewas analyzed using FTIR,ESI-MS,1H NMR and Gaussian computation,and the results indicated that an intramolecular Mannich reaction occurred between ortho-amino aromatic azo dye and formaldehyde.In addition,a secondary effect of reduction of dyes by formaldehyde took place,in which the conjugated chromogenic system of the dye molecules was destroyed,producing benzimidazole,o-phenylenediamine and nitrogen-containing heterocyclic substances with polymethyl substitution.This decolorization of dye was studied as a detection or monitoring method for formaldehyde,and it showed advantages of sensitive response,clear visual indication,and no volatilization of organic solvent.The method was applied to the monitoring of formaldehyde in modification products and residual liquid after aromatic primary amine dyes modification for silk fibroin protein by Mannich reaction.which can achieve the consistent results as that of acetylacetone method.It is of great significance to help construct a method system for the elimination and monitoring of formaldehyde residue added in Mannich reaction modification.(5)The adsorption characteristics of silk fibroin protein modified by aromatic primary amine dyes based on Mannich reaction was researched.In order to improve further the theoretical system of aromatic primary amine dyes modification method based on Mannich reaction,the adsorption characteristics of aromatic primary amine dyes on silk fibroin protein were discussed based on the apparent adsorption characteristics of dyes on silk.The conclusions are as follows:Through the adsorption kinetics study,it was found that the structure of aromatic primary amine dyes had a significant effect on the modification effect,which may be caused by the comprehensive effect of Mannich reaction and dye molecular penetration.The modification of fibroin protein by the aromatic primary amine compounds based on Mannich reaction was more in line with the quasi-secondary kinetic model.And the method is more suitable to be used at lower temperature,the degree of conformity of quasi-secondary kinetic model will be reduced inhigher temperature.As the temperature increases,the reaction efficiency between the dye and silk protein decreases,and the diffusion coefficient decreases with the temperature.Due to the different dominant factors in different stages,the diffusion coefficient tends to increase first and then decrease as time goes by.According to the study of thermodynamics,the adsorption rate and adsorption amount of aromatic primary amine dyes on silk show a decreasing trend with the increase of temperature,indicating that the chemical activity of Mannch reaction is a key factor to control the modification process.Through thermodynamic study,it was found that the adsorption rate and adsorption amount of aromatic primary amine dyes on silk tended to decrease with the increase of temperature,indicating that the chemical activity of Mannch reaction was a key factor to control the modification process.The Freundlich model and the Langmuir model were used to study the adsorption thermodynamics,and it was found that the adsorption law of aromatic primary amine dye was in line with the Langmuir model.In summary,the research results of this paper show that method of the aromatic primary amine compounds modification for silk fibroin protein based on the Mannich reaction can realize the firm and efficient selective binding between the aromatic primary amine compound and the silk fibroin protein,and the damage of this method to the protein is very slight.The binding sites between aromatic primary amine compounds and silk fibroin protein are located on tyrosine and tryptophan residues.When designing the modification molecules of aromatic primary amine,the primary amino should have the appropriate nucleophilic property,and there is no high electron cloud density around it.The method can realize the visualization,convenient and accurate detection of formaldehyde by using the decoloration phenomenon of the intramolecular Mannich reaction between ortho-aniline aromatic azo dyes and formaldehyde.