The importance of peptide drugs in treatment has attracted more and more attention of pharmaceutical workers. According to the composition of peptide chain, peptides can be divided into homomeric and heteromeric. The former is composed of amino acids, while the latter is composed of amino acids and non amino acids, such as glycopeptides. According to the structure of peptide bond, it can be divided into straight chain peptide and cyclic peptide. Among them, the research of straight chain peptide is the most extensive and in-depth, especially in the aspect of the synthesis technology of straight chain peptide, whether it is liquid phase method or solid phase method has been mature.

Although many linear chain peptides have good bioactivity and stability in vitro, their bioactivity soon disappears in vivo. Because of the complexity of the internal environment, there are a variety of enzymes. Under the action of enzyme, straight chain peptide is degraded rapidly, resulting in the loss of activity. In addition, the conformational flexibility of the linear peptide in the liquid phase makes it difficult to meet the conformational requirements of the receptor. There are still many problems to be solved.

In order to obtain peptides with excellent biological activity, long half-life and high receptor selectivity, many methods of peptide modification have been reported in the literature, including the modification of straight chain peptides into cyclic peptides. This kind of macrocyclic molecule has a clear fixed conformation and can fit well with the receptor. In addition, there is no free amino terminal and carboxyl terminal in the molecule, which greatly reduces the sensitivity to aminopeptidase and carboxypeptidase. Generally speaking, the metabolic stability and bioavailability of cyclic peptides are much higher than that of linear peptides.

According to the cyclization mode, cyclic peptides can be divided into head to tail, side chain to side chain, side chain to end, disulfide bridge and other bridged cyclopeptides. In terms of synthetic methods, it is the most difficult to synthesize the end-to-end cyclic peptide. Because of the strong p-bond characteristics of the peptide bond between the precursor of cyclic peptide and the straight chain peptide, the molecules prefer to form the trans conformation, which leads to the long distance between the carboxyl group and the amino group of the end group belonging to the reaction center, which is not conducive to the intramolecular condensation reaction and the intermolecular condensation.

The end-to-end cyclic peptides are usually N-terminal and C-terminal free straight chain peptides, which are synthesized in dilute solution (10-3 ~ 10-4m) by acylamine bond formed by carboxyl group and amino group. The type and number of amino acids in straight chain precursors play an important role in the difficulty of cyclization and the yield of cyclic peptides. Glycine, proline or D-amino acids can induce beta-turn, which is often considered to increase the possibility and yield of cyclization.

1. Classical methods for the synthesis of end-to-end cyclic peptides

The classical method for the synthesis of end-to-end cyclic peptides is to selectively activate and cyclize the protected linear precursors in dilute solution (10-3 ~ 10-4m). Active ester method and azide method are commonly used.

In the active ester method, the activation of carboxyl group and cyclization are carried out in two steps. Active esters are relatively stable and can be directly used for cyclization without purification. Almost all the active esters that can be used in coupling reaction can be used to synthesize cyclopeptides, mainly p-nitrophenol ester, N-hydroxysuccinimide ester, pentafluorophenyl ester and 2,4,5-trichlorophenol ester.

Azide method is another classical method in peptide synthesis. The advantage of this method is that it rarely causes racemic reaction. It was first used in the synthesis of linear peptide, but now it is often used in the synthesis of cyclic peptide

2.Solid phase synthesis of cyclic peptides

In recent years, the strategy of synthesizing cyclopeptides by linking amino acid side chains with resins has been widely used in the synthesis of cyclopeptides. For linear polypeptides with aspartic acid or glutamic acid residues, the carboxyl group of the side chain of the two acidic amino acid residues can be selected as the C-terminal, which can be condensed with PAC (alkoxybenzyl alcohol) or pal (alkoxybenzylamine) or other types of resins to hang the linear polypeptides on the resins. The main chain carboxyl group is protected by allyl group. The N-terminal and C-terminal protective groups were removed and the condensation agent was added to obtain the cyclization product attached to the resin. Finally, the cyclopeptide was cut off from the resin with trifluoroacetic acid: anisole sulfide: b-mercaptoethanol: anisole mixed reagent, and other side chain protective groups were removed at the same time.

3.Enzymatic method

It is also one of the developing methods to synthesize cyclopeptides by protease in buffer. It was reported that the enzyme catalyzed the synthesis of several peptides with 8 amino acid residues. The yield was 30% ~ 80% by HPLC. The cyclization efficiency is related to the sequence and length of the peptide. The minimum length of the linear peptide required for the synthesis of cyclic peptide by subtiligase is 12 amino acid residues, below which the hydrolysate or linear peptide dimer will be obtained. It may be that the end-to-end conformation formed by the peptide substrate with less than 12 residues can not match the active center of the enzyme.