Guanidine hydrochloride

Introduction: Guanidine hydrochloride, also known as aminomethylamine hydrochloride, is a white or slightly yellow block substance that is almost insoluble in acetone, benzene, and ether. It can be used as a pharmaceutical, pesticide, dye, and other organic synthesis intermediate. It is an important raw material for manufacturing drugs such as sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, and folic acid. It can also be used as an anti-static agent for synthetic fibers. The preparation method of guanidine hydrochloride has the following process flow: a、 Melting: Place dicyandiamide and ammonium chloride in a reaction vessel with a weight ratio of 1:1.27, and conduct a melting reaction at 170-230 ℃ to obtain crude guanidine hydrochloride; b、 Dissolve crude guanidine hydrochloride in water at a ratio of 1:1 at room temperature, and add ammonium salt removal base to the solution in an amount determined by the ammonium salt content in the crude guanidine hydrochloride; c、 Filtering, removing raw materials and reaction by-products through filtration methods; d、 Dehydration, dehydrate the filtered mother liquor at high temperature; e、 Crystallization, supersaturated solution cooling, and concentrated crystallization resulted in high-purity guanidine hydrochloride. f、 Add the guanidine hydrochloride obtained from step e to the reaction vessel with dicyandiamide, and conduct a melting reaction at 170-230 ℃ for 3-4 hours. Then, proceed with steps b, c, d, and e to obtain higher purity guanidine hydrochloride, which can be repeated multiple times as needed. g、 After step e, the guanidine hydrochloride is subjected to steps b, c, d, and e, where the ratio of guanidine hydrochloride to water in step b is 1.5:1, to obtain higher purity guanidine hydrochloride. This step can be repeated multiple times as needed. The role of urea and guanidine hydrochloride in the purification of inclusion body proteins. Denaturants (urea and guanidine hydrochloride) can break hydrogen bonds in protein structures, increasing the solubility of non-polar molecules, including amino acid side chains, and reducing hydrophobic interactions; Urea can also penetrate deep into the interior of protein molecules and affect their dense packing. In addition, detergents, organic solvents, heavy metals, heat, mechanical forces, freezing, ultrasound, high pressure, radiation, etc. can all cause protein denaturation. These transformations do not destroy the covalent bonds of proteins, but only involve the destruction of secondary bonds such as hydrogen bonds, salt bonds, hydrophobic interactions, and van der Waals interactions. Some denatured proteins can automatically return to their natural state after the denaturation factor is removed. This phenomenon is called protein renaturation, which is also known as refolding in protein folding research. Purification of inclusion body proteins is mostly carried out under pH 8 conditions, using strong denaturing agents such as urea (6-8mol/L) and guanidine hydrochloride (GdnHCl5-8mol/L or 6mol/L) to break various chemical bonds within and between inclusion body protein molecules through ion interactions, causing peptide stretching. Generally speaking, guanidine hydrochloride is superior to urea because it is a stronger denaturing agent that can dissolve inclusion bodies that cannot be dissolved by urea, and the isocyanates decomposed by urea can cause free amino formylation of polypeptide chains, especially when kept at alkaline pH for a long time. Alternatively, descaling agents such as SDS, n-hexadecyltrimethylammonium chloride, Sarkosyl, etc. can be used to break the hydrophobic bonds within proteins and dissolve some inclusion body proteins. Use TritonX-100 to dissolve Zymonasmobilislevansucrase inclusion body protein. In addition, for proteins containing cysteine, the isolated inclusion bodies usually contain some disulfide bonds formed between chains and inactive disulfide bonds within the chains. Organic solvents, alkaline environments (greater than 9), or acids (70% formic acid) can also dissolve it. The role of urea and guanidine hydrochloride in the purification of inclusion body proteins requires the addition of reducing agents such as 2-mercaptoethanol (β - ME), dithiothreitol (DTT), dithioerythritol, reduced glutathione (GST), and cysteine in the denaturing solution. The concentration of reducing agent used is generally 50-100mM β - ME or DTT, and there are also literature using a concentration of 5mM. The concentration of reducing agent used is independent of the number of disulfide bonds in the protein, and the addition or absence of reducing agent has no effect on some proteins without disulfide bonds, such as the solubilization of bovine growth hormone inclusion bodies. For the solubilization of certain inclusion bodies in target proteins without disulfide bonds, the use of reducing agents is sometimes necessary, possibly due to the influence of impurities containing disulfide bonds on the dissolution of inclusion bodies. At the same time, metal chelating agents such as EDTA or EGTA need to be added to chelate metal ions such as Cu2+and Fe2+to prevent oxidation reactions between the already reduced thiol groups. After the denaturing agent dissolves, the protein loses its biological activity.