Chemical synthesis of polypeptide

From the simplest virus to human, the complex protein structure in all organisms is composed of the same 20 kinds of amino acids, which constitutes a variety of protein world. Biologists in the process of in-depth study of proteins, found a class of amino acids, but different from protein intermediates, this kind of substance is called polypeptide.

Polypeptide is a kind of compound which is simpler than protein and has smaller molecular weight and is connected by amino acid through peptide bond. Polypeptides have the dual effects of regulating the physiological function and providing nutrition for the body.

It affects almost all metabolism and synthesis of human body. A peptide containing less than 10 amino acids is called oligopeptide, and the excess is called polypeptide; the polypeptide with more than 50 amino acids is a familiar protein.

The value of peptide synthesis

Up to now, more than 100 kinds of peptides have been found and isolated from human body, and the research and utilization of peptides have witnessed unprecedented prosperity. The total synthesis of peptides is not only of great theoretical significance, but also of important application value. Through the total synthesis of polypeptide, we can:

1. To verify the structure of a new polypeptide;
2. Design new peptides to study the relationship between structure and function;
3. Provide important information for the mechanism of peptide biosynthesis;
4. Establish model enzyme and synthesize new peptide drugs. Chemical synthesis of polypeptide

There are two main ways of peptide synthesis: chemical synthesis and biosynthesis.

Chemical synthesis is mainly realized by amino acid condensation reaction. In order to obtain synthetic peptides with a specific sequence, when the synthetic raw materials contain amino acid monomers with a functional degree greater than 2, the groups that do not need reaction should be protected temporarily, and then the ligation reaction should be carried out to ensure the directional synthesis.

Generally speaking, these protective groups are stable in the process of synthesis without side reaction, and can be completely removed after the synthesis. 1. The common amino protecting groups can be divided into alkoxycarbonyl group, acyl group and alkyl group.

Among them, alkoxycarbonyl group can prevent racemization, so it is widely used. The most commonly used are Z, Fmoc, and BOC. The Z group can be removed by catalytic hydrogenation of palladium black and 5% – 20% palladium carbon. BOC Group has different chemical properties from Z group, so it can not be removed by catalytic hydrogenation, but it is easy to be removed by acid hydrolysis.

It can be used with Z group selectively. Fmoc group is stable to acid and can be removed by alkali. Therefore, it is particularly suitable for the synthesis of peptides containing Trp, met, Cys and other acid unstable peptides. A kind of

Compared with amino group, there are fewer kinds of carboxyl protecting groups, which are usually in the form of salt or ester. Salt is the temporary protection of carboxyl group, commonly used are potassium salt, sodium salt, triethylamine salt and tributylamine salt.

Common esters are methyl ester, ethyl ester, benzyl ester and tert butyl ester. Tert butyl ester is the most commonly used carboxyl protection group in recent years, which can be removed by acid under mild conditions. 3. Side chain protecting groups: in order to avoid side effects, the side chain functional groups of some amino acids need to be protected by appropriate protective groups.

There are many different protective groups in the same side chain, which can be selectively removed under different conditions, which is of great significance in the modification of cyclic peptides and peptides, and the side chain protection groups are closely related to the selected synthesis methods. A kind ofThe main difference between liquid phase synthesis and solid phase synthesis is whether solid phase carrier is used or not.

A kind of Liquid phase synthesis of polypeptides:

there are two main strategies for liquid-phase synthesis of polypeptides: Step-by-Step synthesis and fragment combination.

Step by step synthesis is simple and rapid, and is used for the synthesis of various bioactive polypeptides. Fragment combination method provides the most promising route for the synthesis of peptides containing more than 100 amino acids, and a variety of bioactive peptides have been successfully synthesized In the pure system.

Liquid phase synthesis mainly adopts BOC and Z protection methods, and now it is mainly used in the synthesis of short peptides, such as aspartame, peptide, oxytocin, etc.

compared with solid-phase synthesis, it has many advantages, such as more choice of protective groups, low cost and easy scale-up of synthesis.

However, compared with solid-phase synthesis, the main disadvantage of liquid-phase synthesis is that the scope of synthesis is small, generally concentrated in peptide synthesis within 10 amino acids, and the intermediate needs to be purified in the synthesis, which takes a long time and heavy workload.

Solid phase synthesis of polypeptides in 1963, R. B. Merrifield fixed the C-terminal of amino acids on an insoluble resin, and then condensed amino acids on the resin successively to extend the peptide chain and synthesize polypeptides.

After continuous improvement and improvement, solid-phase method has become a common technology in peptide and protein synthesis, showing the incomparable advantages of classical liquid-phase synthesis.

A kind of The basic principle is:

firstly, the hydroxyl group of the hydroxyl terminal amino acid of the peptide chain to be synthesized is connected with an insoluble polymer resin in the structure of covalent bond, and then the amino acid combined with the solid carrier is taken as the amino component, and then the peptide chain is extended by removing the amino protection group and reacting with excessive activated carboxyl group component.

Repeat the operation (condensation washing deprotection neutralization and washing next round condensation) to reach the length of peptide chain to be synthesized. Finally, the peptide chain is split from the resin and purified to obtain the desired peptide.

After each step of the reaction, the purification can be achieved by simply washing the resin, which overcomes the problem that each step of the classical liquid phase synthesis needs to be purified.

There are two main strategies in solid phase peptide synthesis: BOC and Fmoc. In the synthesis process of BOC method, TFA needs to be repeatedly used to remove BOC, and HF is required to be used when cutting from the resin.

Since HF must be operated with special instruments, and side reactions are easy to occur in the cutting process, its use is limited by experimental conditions, and the use is gradually reduced.

Fmoc method has been widely used because of its mild reaction conditions and general experimental conditions.   

2.1 selection of resin solid phase carrier for peptide synthesis must meet the following requirements:

A. Reaction sites (or reaction groups) must be included so that the peptide chain can be on these sites and then removed

B. It must be stable to the physical and chemical conditions in the synthesis process;

C. The carrier must allow rapid and unimpeded contact between the growing peptide chain and the reagent

D. In addition, the carrier must be allowed to provide enough connection points to give a useful yield of peptide per unit volume of carrier, and the interaction between peptide chains bound by the carrier must be minimized. A kind of

There are three kinds of polymer carriers used in the solid-state synthesis of polypeptides: Polystyrene polyethylene crosslinking resin, polyacrylamide, polyethylene glycol resin and derivatives. These resins can be directly linked to amino acids only by introducing reaction groups.

According to the different reaction groups introduced, these resins can be divided into chloromethyl resin, carboxyl resin, amino resin or hydrazide resin. Chloromethyl resin is usually selected for BOC synthesis, and carboxyl resin such as Wang’s resin is usually selected for Fmoc synthesis.    

2.2 solid phase synthesis and cutting method: after the solid phase synthesis is completed, the peptide must be cut off from the resin with appropriate cutting reagent, and then precipitated by ice ether, centrifuged and collected, separated and purified by HPLC, and then freeze-dried to obtain the final product. Because of the different resin and amino acid sequence, the cutting method is not the same. A kind of

Generally, acid cutting is selected. For PAM and mbha resin, HF cutting is generally used. In the process of cutting, p-cresol, p-mercaptophenol, anisole and other reagents need to be added. For Wang, rink amide and TRT resins, TFA is generally used for cutting. During the cutting process, ethyldithiol, benzyl sulfide, water, triisopropylsilane, phenol and so on are added.

A kind of These additives are mainly used as carbon positive ion capture reagents, which are generated during the capture cutting reaction

Carbon positive ions, reduce the side effects caused by the attack of these carbon positive ions on the side chain of some amino acids. The amino acids that are easy to produce side reactions are: Trp, Tyr. The amount of cutting reagent is general 10-15ml/gResin.

The commonly used cutting ratio is HF / p-cresol / P-thiocresol (90 / 5 / 5), TFA / tis / EDT / H2O (94 / 1 / 2.5 / 2.5), and the reaction is generally 2 H-4 h at room temperature. A kind of

2.3 detection of free amino groups in solid-phase peptide synthesis, the connection rate is mainly determined by detecting the free amino groups on the resin. The detection method is called Kaiser method.

The judgment standard is that if there are free amino groups, the solution or resin will show blue or reddish brown.

A kind of Kaiser reagents include:

A. Alcohol solution of 6% ninhydrin

B. Ethanol solution of 80% phenol

C. In the preparation of 2% 0.001M KCN pyridine solution, pyridine needs to be treated with ninhydrin and then re evaporated before use. When testing, take a small amount of resin, add 2-3 drops of a, B, C each, and heat it at 100 ℃ for 1-2min.

If the solution or resin appears blue or purple brown, it indicates that there is free amino group, otherwise it indicates complete connection.

D. Other detection methods of free amino groups are: trinitrobenzene sulfonic acid method, picric acid method, bromophenol blue method

A kind of Development prospects

Solid phase peptide synthesis has a history of several decades. However, up to now, people can only synthesize some short polypeptides, not to mention protein.

At the same time, the toxicity of reagents, high cost, reaction by-products and so on have been the problems faced by researchers. In vivo, the rate and yield of peptides synthesized from ribosomes are amazing.

Therefore, whether we can get some enlightenment from the principle of protein synthesis in organisms and apply them to solid-phase synthesis or develop new synthetic methods may be the development direction of peptide synthesis.

For sale, buy peptides, purchase peptides, and buy peptides online, you can find the Chinese peptide company:

Shengnuo Biotechnology is a high-tech China polypeptide manufacturers,who has over 19 years experience for the biotechnology .Our Products involve peptide raw materials,polypeptide drug.

Our 0 defect has passed the FDA certification, and has become the first-class professional polypeptide drug and product development and large-scale production and export industry in China .

Our service scope：peptide synthesis、peptide hormones、peptide medicine、beauty peptide、peptide medicine product、peptide technology transfer、peptide technology service、peptide large-scale production、export of peptide

Thymopentin for Injection、 Thymalfasin for Injection 、 Bivalirudin 、Bulk Drug、 Liraglutide ……