Principles and steps of peptide synthesis
Introduction: Polypeptide synthesis is a process of repeating the addition of amino acids. The solid phase synthesis sequence is generally synthesized from the C-terminal (carboxy terminal) to the N-terminal (amino terminal). Past peptide synthesis is carried out in solution called liquid phase synthesis. Medium ancient biological microbial cell
1.1 The basic principle of peptide synthesis:
First, the hydroxyl group of the hydroxy terminal amino acid of the peptide chain to be synthesized is linked to an insoluble polymer resin by a covalent bond structure, and then the amino acid bonded to the solid phase carrier is used as an amino component through a deamination protecting group. Excessive activated carboxyl component reacts to lengthen the peptide chain. Repeat (condensation → washing → deprotection → neutralization and washing → next round condensation) operation to achieve the length of the peptide chain to be synthesized, and finally the peptide chain is cleaved from the resin, and after purification and the like, the desired polypeptide is obtained. The α-amino group is protected by BOC (tert-butoxycarbonyl), which is called BOC solid phase synthesis, and the α-amino group is protected by FMOC (9-fluorenylmethoxycarbonyl), which is called FMOC solid phase synthesis.
1.2 steps of solid phase peptide synthesis:
A, resin selection and amino acid immobilization
There are three main types of carriers for the synthesis of polypeptides: crosslinked polystyrene; polyamide: polyethylene monoethylene glycol lipid resin. The immobilization of the amino acid is mainly achieved by forming a covalent bond between the carboxyl group of the protected amino acid and the reactive group of the resin.
B, protection and removal of amino groups, carboxyl groups and side chains
In order to successfully synthesize a polypeptide having a specific amino acid sequence, it is necessary to protect the amino group and the carboxyl group which are not involved in the formation of the amide bond, and also to protect the active group on the side chain of the amino acid, and then remove the protective group after completion of the reaction. In recent years, FMOC synthesis has been widely used. The carboxyl group is usually protected by a method of forming an ester group. Methyl esters and ethyl esters are common methods for protecting carboxyl groups in a stepwise synthesis.
C, peptide reaction
The peptide-forming reaction in the solid phase generally involves placing two corresponding amino-protected and carboxyl-protected amino acids in a solution without forming a peptide bond. To form an amide bond, a commonly used means is to activate the carboxyl group and become A method of forming an amide bond by mixing an acid anhydride, an active ester, an acid chloride or a strong condensing agent such as carbodiimide to form a symmetric acid anhydride.
D, purification of cleavage and synthetic peptide chains
The BOC method uses TFA+HF to cleave and deprotect the side chain, and the FMOC method directly uses TFA. Sometimes, depending on the conditions, other deprotection methods such as alkali, photolysis, fluoride ion and hydrogenolysis are also employed. Further purification, separation and purification of the synthetic peptide chain are usually carried out by high performance liquid chromatography, affinity chromatography, capillary electrophoresis or the like.
TCT
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