Peptide synthesis, also known as peptide chain synthesis, is a solid-phase synthesis sequence from the N-terminal (ammonia end) to the C-terminal (carboxyl end). In the past, peptide synthesis was carried out in solution, which is called liquid phase synthesis. The synthesis of polypeptide can be divided into two ways: chemical synthesis and biological synthesis. The principle of peptide synthesis peptide synthesis is how to connect all kinds of amino acid units according to the amino acid sequence and connection mode of natural substances. Because amino acids exist in the form of intramolecular zwitterions (H3 + NCH (R) COO -) under neutral conditions, the direct condensation of amino acids to form amide bonds is difficult to carry out under general conditions.

The reaction activity of amino acid ester is higher. When heated at 100 ℃ or placed at room temperature for a long time, peptide ester can be polymerized to form peptide ester, but the reaction has no directionality A1a1... A2a1... A mixture of any order.

In order to obtain synthetic peptides with specific sequence, it is not feasible to use any polymerization method, but only the step-by-step condensation method. Generally, it is shown in the following formula, that is, the amino or carboxyl groups that do not need to be reacted are temporarily protected with appropriate groups, and then the connection reaction is carried out to ensure the directional synthesis of polypeptide.

In the formula, X and Q are the protective groups of amino group and carboxyl group respectively, which can not only prevent the occurrence of random side reactions, but also eliminate the form of amphoteric ions of amino acids and make them easily soluble in organic solvents.

In some cases, Q may not be a covalently linked group, but an organic cation composed of a strong organic base (such as triethylamine) and a carboxyl hydrogen ion of an amino acid. Y is a strong electron absorption group, which can activate the carboxyl group, and is beneficial to the free amino group of another amino acid. The carboxyl carbon atom of the activated carboxyl group is nucleophilic attacked to form amide bond.

The binding product is a protective peptide with protective groups at both the N-terminal and the C-terminal. If the peptide chain is not so far, but needs to be extended from the N-terminal or the C-terminal, it can selectively remove X or Q, and then connect with the new N-protected amino acid (or peptide) or C-protected amino acid (or peptide) for a second time, and repeat until the required peptide chain length.

For peptide synthesis of long peptides, there are generally two ways to extend peptide chain: gradual growth and fragment condensation. The former is started by the initial amino acid (or peptide). Each time, an amino acid is added, and the latter is a new long peptide chain with the length added by condensation of N-protected peptide and C-protected peptide.

For peptides containing glutamic acid, aspartic acid, lysine, arginine, histidine, cysteine and other amino acids with side chain functional groups in peptide synthesis, in order to avoid side reactions caused by side chain functional groups, it is generally necessary to temporarily protect the side chain groups with appropriate protective groups. There are two main ways to synthesize peptides: chemical synthesis and biological synthesis.

Chemical synthesis is mainly in the form of condensation between amino acids. In the synthesis of peptides with a specific sequence, because the raw materials for peptide synthesis contain amino acid monomers with a functional degree greater than 2, when peptide synthesis, the groups that do not need to be reacted should be temporarily protected before peptide synthesis, so as to ensure the directionality of the target product of peptide synthesis. The chemical synthesis of polypeptide can be divided into liquid phase synthesis and solid phase synthesis.

The liquid-phase synthesis of polypeptide can be divided into two strategies: Step-by-Step synthesis and fragment combination. It can be used in the synthesis of various bioactive peptide fragments. Fragment combination method mainly includes natural chemical connection and Staudinger connection. In recent years, peptide liquid-phase fragment synthesis has developed rapidly, and has made a significant breakthrough in the field of peptide and protein synthesis. In peptide synthesis, according to the chemical specificity or chemical selectivity of peptide fragments, the peptide fragments can spontaneously connect to get the target peptide. Because the peptide fragment contains relatively few amino acid residues, it has high purity and is easy to purify.

In 1963, Merrifield, a famous biochemist in the United States, proposed a solid-phase synthesis method to carry out the solid-phase synthesis of polypeptide, that is, the C-terminal (carboxyl terminal) of amino acid is connected to the insoluble resin, and then the amino acid is condensed and the peptide chain is extended successively on the resin. The solid-phase synthesis method can be divided into two methods: the BOC method and the Fmoc method. Based on the liquid-phase and solid-phase synthesis methods of polypeptides, NCA and combinatorial chemistry methods of amino acids have been developed.

With the development of bioengineering technology, gene engineering, which is dominated by DNA recombination technology, has been applied to peptide synthesis.

The solid-phase synthesis of polypeptides is a process of repeated addition of amino acids, usually from the C-terminal to the N-terminal (ammonia base terminal). The principle of peptide solid-phase synthesis is to connect the C-terminal of the first amino acid of the target peptide with the solid-phase carrier through covalent bond, and then take the N-terminal of the amino acid as the starting point for synthesis. After removing the amino protection group and excessive activated second amino acid, the peptide chain is extended, repeated operation, and the ideal length of the synthetic peptide chain is achieved. Finally, the peptide chain is broken down from the resin, and purified To obtain the target peptide.

\1. BOC peptide synthesis

BOC method is a classical solid-phase peptide synthesis method. BOC is used as the protection group of amino acid α - amino group, benzyl alcohol as the side chain protection group, and TFA is usually used to remove BOC. In peptide synthesis, n-α-amino acids which have been protected by BOC are covalently cross-linked to the resin, the BOC protecting group is removed by TFA, and the N-terminal is neutralized by weak base.

The peptide chain was extended by DCC activation and coupling, and the target peptide was finally dissociated from the resin by HF or tfmsa. In the BOC peptide synthesis method, in order to facilitate the next step of peptide synthesis, acid is repeatedly used for deprotection, and some side reactions are brought into the experiment, such as peptide is easy to be removed from resin, amino acid side chain is unstable in acid conditions, etc.

\2. Fmoc peptide synthesis

Based on BOC peptide synthesis, carpino and Han developed a new solid-phase peptide synthesis method, Fmoc peptide synthesis.

In the synthesis of Fmoc polypeptide, Fmoc is used as the protective group of α - amino acid. Its advantage is that it is stable under acid conditions, not affected by TFA and other reagents, and can be deprotected by mild alkali treatment, so the side chain can be protected by BOC protecting group which is easy to be removed by acid.

TFA / dichloromethane (DCM) can be used to quantitatively remove the peptide from the resin, avoiding the use of strong acid. At the same time, compared with BOC method, Fmoc method has mild reaction conditions, less side reactions and high yield, and Fmoc group itself has characteristic UV absorption, which is easy to monitor and control the reaction. Fmoc method is widely used in the field of peptide solid-phase synthesis. With the development of peptide synthesis, liquid-phase peptide synthesis (i.e. the synthesis method of peptide fragments spontaneously joining long peptides in solution according to their chemical specificity or chemical selectivity) plays a more and more important role in the field of peptide synthesis. It is characterized in that it can be used for the synthesis of long peptides with high purity and easy purification.

The synthesis of polypeptide in liquid phase is mainly divided into natural chemical connection and stoodinger connection. Natural chemical linkage is the basic method of peptide synthesis, which is limited in that the synthesized peptide must contain Cys residues, so the application of natural chemical linkage is limited. The extension of natural chemical connection methods includes chemical region selective connection, removable auxiliary base connection and light sensitive auxiliary base connection.

Staudinger connection is another basic method of fragment connection, which opens up a wider way for peptide fragment connection. The orthogonal chemical connection method is the extension of the stoodinger connection method, which can improve the condensation rate between segments by simplifying the phosphine sulfur ester auxiliary group. Other peptide synthesis methods 1. Carboxylic anhydride method of amino acids (NCA)

The amino protecting group of carboxylic anhydride of amino acid can also activate the carboxyl group.

Principle of NCA: under alkaline condition, amino acid anion forms a more stable carbamate ion with NCA, which loses carbon dioxide and generates dipeptide during acidification. The dipeptides were combined with other NCAs and repeated.

NCA is suitable for peptide synthesis of short chain peptide fragments. Its cycle is short, operation is simple, cost is low, and the molecular weight of the product is high.

\2. Combinatorial chemistry

In the 1980s, based on the solid-phase peptide synthesis, a combinatorial chemistry method was proposed, that is, the construction units of amino acids were connected through combination to synthesize a chemical library containing a large number of compounds, from which a set of peptide synthesis strategies and screening programs with certain physical and chemical properties or pharmacological activities were screened.

The peptide synthesis strategies of combinatorial chemistry mainly include: mix and average method, iterative method, light controlled location combinatorial library method, tea bag method, etc. The biggest advantage of combinatorial chemistry is that it can synthesize many kinds of compounds at the same time, and it can screen all kinds of new compounds and their isomers to the greatest extent.

\3. Enzymolysis

\4. Enzymolysis is the use of biological enzymes to degrade plant protein and animal protein to obtain small molecular polypeptides. Because of its low output, large investment, long cycle and serious pollution, enzymatic hydrolysis could not achieve industrial production. The peptide obtained by enzymolysis can retain the original nutritional value of the protein, and can obtain more functions, more green and more healthy than the original protein.

\4. Genetic engineering

Gene engineering is mainly based on DNA recombination technology, through the appropriate DNA template to control the peptide sequence synthesis. Some researchers have obtained the quasi elastin polyvaline proline glycine valine glycine peptide (VPGVG) by genetic engineering.

The active peptides produced by genetic engineering technology include peptide antibiotics, interferon, interleukin, growth factors, tumor necrosis factor, human growth hormone, blood coagulation factor, erythropoietin, tissue non protein plasminogen, etc.

The synthesis of polypeptides by genetic engineering has the advantages of strong expression orientation, safety and health, wide source of raw materials and low cost, but it is difficult to achieve large-scale production due to the problems of high-efficiency expression, difficult separation and low yield.

\5. Fermentation

Fermentation is a method to obtain polypeptides from microbial metabolites. Although the cost of fermentation is low, its application scope is narrow, because the only polyaminoacids that can be independently synthesized by microorganisms are ε - poly lysine (ε - PL), γ - poly glutamic acid (γ - PGA) and cyanobacterial peptide.

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Chengdu Shengnuo Biotechnology Co., Ltd. has "Chengdu polypeptide drug engineering technology research center" in Chengdu, mainly engaged in polypeptide, polypeptide drug and beauty peptide research. Our zero defect has passed the FDA certification, and now it has become the first-class professional peptide drug and product development, technology transfer, technical service and peptide drug industry in the scale production and export of China's parks.