Several Main Synthetic Methods Of Peptide Bond

1. Acyl azide method

The acyl azide method was introduced into peptide chemistry as early as 1902.Therefore, this method is one of the oldest condensation methods.Amino acids are seldom racemized by azide method during peptide reduction reaction, so this method is widely adopted by peptide synthesizers.Recently, Li Qinggeng, a Chinese scholar, successfully synthesized a series of glutamine antitumor compounds by this method.The grafting of azide peptides is divided into three steps.First is a peptide whose carboxyl group is partially protected by methyl ester, ethyl ester, benzyl ester, etc.Hydrazide hydrolysis was carried out at room temperature, and then hydrazide reacted with nitrite to produce azide under low temperature (below -15 C) and acidic conditions. Finally, the acidity and alkalinity were adjusted to 8-9 with organic bases, and amino components were added to complete the peptide grafting reaction.The mechanism is as follows.

In addition, a phosphate condensation reagent DPPA was found, which can directly change from carboxyl group to azide without hydrazinolysis.Compared with the traditional azide method, it does not require hydrazide intermediates, simplifies the reaction steps, and the reaction conditions are milder, so it is welcomed by people.The mechanism is as follows.

2. Mixed anhydride method

This method refers to the method of preparing the carboxyl group of the protected amino acid into a highly active mixed anhydride and then condensating with the amino acid to produce a peptide.The mixed anhydride method was developed in the early 1950s.Diphenylphosphoric acid is the main reagent for the preparation of mixed anhydrides, followed by Benzoic acid.Alkyl chloroformate esters are now widely used, mainly ethyl chloroformate, isobutyl chloroformate and isopropyl chloroformate.Mixed anhydride method has many advantages, such as fast reaction speed, high product purity, high yield and good economy, so it is popular among pharmaceutical workers nowadays.Mi Pengcheng et al. of Nanjing Agricultural University successfully synthesized Fmoc-Tyr(t-Bu)-WangResin, an intermediate of thymopentin (TP5), in 2007.During the reaction, the carboxyl component reacts with isobutyl chloroformate at low temperature (-15 C) to produce mixed anhydride, and then reacts with the amino component under alkaline conditions in the presence of tertiary amines to produce peptides.The mechanism is as follows.

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3. carbodiimide method

During the development of the carbodiimide method, dicyclohexyl carbodiimide (DCC) was first used in the synthesis of peptides and was widely used.DCC is relatively inexpensive and soluble in commonly used peptide synthesis reagents.However, when DCC is used as a condensant, DCU produced by DCC has a certain solubility in water and organic solvents, which makes it difficult to eliminate it during product purification.Therefore, water-soluble carbodiimide EDC has been developed to replace DCC, which can remove urea produced after the reaction only by washing with acid or water, effectively solving this problem.Liu Guodu et al. also synthesized active tripeptide Cystin by this method.The mechanism of carbodiimide condensation reagent is as follows.The condensation reagent reacts with the carboxyl component to form an active intermediate, and then reacts with the amino component under alkaline conditions to complete the peptide chain growth reaction.

4. Acyl halide method

This method is similar to organic chemistry, where activation of the carboxyl group with acyl chloride followed by amide formation at room temperature or low temperature is a commonly used method.However, this method is only used for Fmoc-protected amino acids in reagent applications, because Fmoc is very stable under acidic conditions, and amino acids protected by other protecting groups are rarely synthesized by this method, because most scholars believe that the acyl chloride method is "too active" and prone to many side reactions such as racemization.

5. Benzotriazole type (blowout preventer series)

It can be said that BOP is the first generation of benzotriazole condensation reagents, a revolution of condensation reagents, because its peptide grafting efficiency is much higher than DCC, and the risk of racemization is significantly reduced.Later, the same type of condensation reagents HBTU, ByBOP, TBTU and so on were found.The common point is that the condensation reagent reacts with the carboxylic component in the presence of organic bases to generate the active intermediate of benzotriazole ester of carboxylic acid, and then reacts with the amino component to generate the target polypeptide, as shown in the following figure.

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