Cyclic peptide synthesis

The importance of polypeptide drugs in treatment has attracted more and more attention from pharmaceutical workers. According to the composition of the peptide chain, polypeptides can be divided into two categories: Homomeric and Heteromeric, the former is completely composed of amino acids, and the latter is composed of amino acid parts and non-amino acid parts, such as glycopeptides. According to the structure of the peptide bond, it can be divided into straight chain peptide and cyclic peptide. Among them, the research of linear chain peptide is the most extensive and in-depth, especially in the synthesis technology of linear chain peptide, whether it is liquid phase or solid phase method has been mature. Although many straight-chain peptides have good biological activity and stability in vitro, the activity quickly disappears after entering the body. Because of the complex environment in the body, there are a variety of enzymes. Straight-chain peptides degrade quickly under the action of enzymes, resulting in loss of activity [1-2]. In addition, the conformational flexibility of linear peptides in the liquid phase makes it difficult to meet the conformational requirements of receptors. These unfavorable factors cause many problems to be solved for polypeptide drugs. In order to obtain peptides with excellent biological activity and long half-life and high receptor selectivity, many peptide modification methods have been reported in the literature, including the modification of straight-chain peptides into cyclic peptides [3-8]. This macrocyclic molecule has a definite fixed conformation [9], which can fit well with the receptor, and the absence of free ammonia and carboxypeptidase in the molecule greatly reduces the sensitivity to aminopeptidase and carboxypeptidase [10-12]. Generally speaking, the metabolic stability and bioavailability of cyclic peptides are much higher than that of straight-chain peptides [13]. In view of the many advantages of cyclic peptides, the focus of peptide research has shifted to the synthesis and biological evaluation of cyclic peptides in recent years.

According to the bicycling mode of cyclic peptides, they can be divided into Head-to-tail, Sidechain-to-sidechain [14], Sidechain-to-end and sidechain-to-end [15], and Disufide- containing cyclic peptides bridge) [16-18], and cyclic peptides containing other bridging structures [19-23]. In terms of synthesis methods, the synthesis of end-to-end cyclic peptides is the most difficult. Because the peptide bond of the precursor of cyclic peptide - straight chain peptide has a strong p bond, the molecule prefers to form a trans-conformation, showing an extended state, resulting in a far distance between the carboxyl group and the amino group belonging to the end group of the reaction center, which is not conducive to intramolecular condensation reaction, but is conducive to intermolecular condensation.
End-to-end cyclic peptides are usually N-terminal and C-terminal free straight-chain peptides synthesized in dilute solution (10-3~10-4M) from carboxyl groups and amino groups to form an amide bond. The type and number of amino acids in the straight-chain precursor play an important role in the degree of ring formation and the yield of cyclic peptide. Glycine, proline or D-configurational amino acids can induce b-Turn, which is often considered to increase the possibility and yield of ring formation [24-25].

1. The classical method of synthesis of head-tail catenated peptides
The classical method of synthesis of cephalo-tail phase catenopeptide is to selectively activate and cyclize the protected linear precursor in a dilute solution (10-3~10-4M). Active ester method and azide method are commonly used.
1.1 Active ester method
The activated carboxyl group and cyclization reaction in active ester method are carried out in two steps. Active esters are relatively stable and generally do not need to be purified and can be directly used in the ring reaction. Almost all active esters that can be used for coupling reactions can be used to synthesize cyclic peptides, mainly p-nitrophenol ester, n-hydroxysuccinimide ester, pentafluorophenyl ester and 2,4, 5-trichlorophenol ester. The C-terminal carboxyl group of linear polypeptides reacts with p-nitrophenol, n-hydroxysuccinimide, pentafluorophenol or 2,4, 5-trichlorophenol at low temperature in the presence of DCC or other reducing agents to obtain corresponding active esters. This active ester, usually with BOC or Z protection at the n-end, removes the protective group under acidic conditions to form a hydrohalide of active ester, and maintains pH 8 to 9 in a weakly alkaline dilute solution, such as in a solvent with a large dielectric constant such as pyridine, DMF or dioxane, and is heated (60 to 100°C) or stirred at room temperature for several hours to a few days, and finally obtains a cyclic peptide.

1.1.1 p-nitrophenol ester method
The general formula for the synthesis of cyclic peptides by p-nitrophenol ester is as Scheme 1[26]

In the synthesis of Cyclo(b-Ala-Phe-Pro), the p-nitrophenol ester method was used. Boc-b-ala-ph-pro-oh was mixed with 1.5 moles of p-nitrophenol in ethyl acetate with DCC as the shrinking agent to obtain BOC-B-Ala-Ph-pro-ONP. Boc was removed by TFA with 0.1M NaHCO3 and 0.1M Na2CO3 as the base and dioxane as the solvent. At room temperature, a cyclotripeptide with a yield of 32% was obtained [27]. The advantage of p-nitrophenol ester method is that p-nitrophenol is cheap and easy to obtain, the disadvantage is that excessive p-nitrophenol is not easy to completely remove, the product is not easy to purify, and the color is yellow.

1.1.2 N-hydroxysuccinimide method:
The principle of the method is the same as that of the p-nitrophenol method. The only difference is that the C-terminal carboxyl group of the linear polypeptide is condensed with n-hydroxysuccinimide (HONSu) in the presence of the condensation agent EDC to form the n-hydroxysuccinimide of the straight-chain polypeptide. Using this method, Toshihisa et al. synthesized cyclo (Pro-Val-Pro-Val) and cyclo (Pro-D-Val-Pro-D-Val) in pyridine solution with yields of 15% and 12% respectively. The two are diastereoisomers, and the former has the effect of inhibiting plant growth, while the latter has the effect of promoting plant growth [28].

1.1.3 Pentafluorophenol ester method:
The use of these active esters in the synthesis of cyclic peptides has only recently been developed. Joullie used pentafluorophenol ester method in the cyclation step when synthesizing the natural cyclic peptide alkaloid Sanjoinine G1 and its C11 enantiomer. Firstly, D-serine was used as raw material to obtain the cyclization precursor through multi-step reaction. The carboxyl group was activated by pentafluorophenol, and n-terminal benzyl oxycarbonyl group was liberated by hydrogen. Then, 4-pyrrolidine pyridine was used as catalyst to reflux in dioxane, and two isomers of each other were finally obtained, with yields of 27% and 22% respectively [29-30].

In addition, pentafluorophenate method was also applied for the cyclization step of Marine cyclic peptide Patellamine B and Cyclotheonamide A, a cyclic peptide with strong inhibition of fibrinase and serine protease, with yields of 20% and 53% respectively [31].
1.2 Azide method

In peptide synthesis, azide method is another classical method, which has the advantage that it rarely causes racemization reactions. It was first used for the synthesis of straight-chain peptides, and now it is often used for the synthesis of cyclic peptides [32-34]. The specific method is to produce hydrazide by hydrazine hydrolysis of methyl ester, ethyl ester, benzyl ester, substituted benzyl ester or other more active ester, dissolved in acetic acid or hydrochloric acid-acetic acid mixed solution, at a temperature of about -5°C to add 1M sodium nitrite solution, the resulting nitrite reaction with hydrazide to form azide. Cyclic peptides can be obtained by stirring N-terminal free straight-chain peptide azide at 4°C for one day and then heating to room temperature. The general formula is Scheme 4
Scheme 4. Peptide Cyclization via the acyl azide (X=Z or Boc, R=Me, Et, Bzl)
Bodansky was the first to synthesize cyclo(D-Ala-D-Ala-Val-D-Leu-Ile) by azide method. Although the above-mentioned cyclic peptide does not have the biological activity of its parent compound malformin, its synthesis opens up the prospect for the synthesis of cyclic peptides by azide method [35].

Another successful example of cyclopeptide synthesis using azide is the synthesis of endothelin antagonists. Endothelin(ET) is a highly effective vasoconstrictor composed of 21 amino acid residues [36]. The synthesis process of cyclo(D-Trp-D-Asp(OtBu)Fmoc-Ser-D-Val-Leu), one of its receptor antagonists, can be described as follows [37] : endothelin (ET) :

DPPA is a stable liquid with a boiling point of 157°C, which can be easily obtained by the reaction of diphenylphosphoyl chloride and NaN3 in acetone at room temperature, and can be directly used as a shrinking agent for polypeptide coupling [38]. In recent years, it has been widely used in the synthesis of cyclic peptides [39-41].
Arg-Gly-Asp (RGD) is a key sequence recognized by integrins when various extracellular proteins interact with integrins, and there have been many reports on the synthesis of cyclic peptides containing this sequence [42-45]. Kessler et al. synthesized 13 linear hexapeptides containing RGD sequence and seven linear pentapeptides containing RGD sequence with solid phase synthesizer SP650. The linear chain peptides with free N and C terminals were synthesized in dilute solution with DPPA as the condensation agent at pH 8.5-9 for 4 days, and the corresponding cyclic hexapeptides and cyclic pentapeptides were obtained with the yield between 15% and 50%. The bioactivity experiments showed that the inhibition of all cyclohexapeptides on cell adhesion was significantly lower than that of linear peptide GRGDS. Among cyclic pentapeptides, only Cyclo(RGDdFV) and Cyclo(RGDFd-V) have significant inhibitory effects on cell adhesion of Laminin P1 [46-48].

For some objects that are easily decomposed under alkaline conditions, inert gases are used to protect the reaction process, for example, linear polypeptide H-Asp(Fmoc) -D-SER-PHE - D-Phe- Arg-Gly-OH is added to anhydrous DMF with 5 times the amount of NaHCO3 and 10 times the amount of DPPA. After reaction for 66 hours, Cyclo(ASP-D-ser-phy-D-phy-arg-Gly), a fibrinogen receptor antagonist with yield of 3%, was obtained. If the dosage of NaHCO3 and DPPA is changed, and the reaction process is protected by argon gas for three days, the yield of the above cyclic peptide can be obtained as high as 39%. The main reason for such a large difference in yield between the two conditions is that the target substance is easily decomposed under alkaline conditions [40].
In addition to NaHCO3 and Na2CO3 as inorganic bases, KH2PO4 is often used in the synthesis of cyclic peptides with DPPA. For example, in the synthesis of Try-C[D-A2bu-phe -(L or D)-Leu], a cyclic peptide that can bind to opium receptors, a weak base such as KH2PO4 is used to catalyze the synthesis, and the yield is as high as 75%[49-50]. Cyclo(Lys-Phit-d-TRp-Lys-thy-Phe), an analogue of Somatostatin, was synthesized by the same method, and the yield reached 42%[51].
When DPPA is used as a shrinking agent to synthesize cyclic peptides, organic bases commonly used triethylamine [52] (Et3N), N-methylmorpholine (NMM) and diisopropylethylamine (DIEA), these three weak bases can be miscible with organic solvents, the amount is much less than NaHCO3 and KH2PO4, and NMM and DIEA are not easy to cause racemization.

2. New shrinkage mixture in cyclic peptide synthesis
2.11-hydroxy-7-azepentriazole (HOAt) derivatives

In recent years, HoAt polypeptide synthetic binding agents have developed rapidly, which include TAPipU[O-(7-azabenzotriazol-1-yl)-1,1,3,3-penta-methyl enuronium tetrafluo roborate]、HAPyU[O-(7-azabenzo triazol-1-yl)-1,1,3,3-bis(tetra methylene) uronium hexafluoro phosphate]、PyAOP(7-azabenzo triazol -1- yloxyl - trispyrrolidino phosphonium hexa fluorophosphate)和HATU [O - (7 - azabenzotriazol -1-yl)-1,1,3,3-tetramethyluronium hexafluoro-phosphateThe use of these condensation agents not only has a fast reaction speed, but also the chirality is not damaged [53-55].

Ehrlich et al. investigated the effects of different shrinking agents on the cyclation reaction of GnRH derivative decapeptide H-Nal-d-Cpa-d-Pal-Glu-Tyr-d-Arg-Leu-Arg-Pro-Lys(Ac)-OH, and found that HAPyU and TAPipU were very effective shrinking agents in the synthesis of cyclic peptides. When the concentration of linear chain peptide was 1.5mmol/L, the cyclization reaction was completed within 30 minutes. In the reaction, the mixture usually needs to be excess by 10% to ensure the reaction is complete. Increasing the concentration of linear polypeptides in the solution can cause the cyclization reaction to occur more quickly. For example, when the concentration of linear decapeptide is 0.1M, the above two shrinkage agents are added, and cyclization occurs within two minutes; Furthermore, surprisingly, no intermolecular condensation reaction occurred even at linear polypeptide concentrations up to 0.2M, suggesting that dilute solutions may not be necessary in both tail and side chain reactions. By comparing the effects of HAPyU and TAPipU on the racemization of cyclic peptides, it was found that HAPyU caused less racemization. When TAPipU was used as a shrinking mixture to synthesize Cyclo[Val-Arg-Lys(Ac)-Ala-Val-Tyr], the yield was 25%, and the racemization of terminal tyrosin was 8%. When HAPyU is used as the condensation mixture, 55% of the cyclic hexapeptide can be obtained within 30 minutes, and the D-tyrosine-isomer is less than 0.5%[56-57].
In order to further verify the advantages of HoAt binding agents in the synthesis of cyclic peptides, Ehrlich chose thymopentin analogues, which are difficult to obtain with ordinary binding agents, as the research object. HAPyU, PyAOP and HATU were compared with each other on the yield of cyclization, dimerization and configuration change of C-terminal tyrosine residues during cyclization. The results showed that when the concentration of linear peptide was 0.1mmol/L, DIEA was three times excessive, and HAPyU was a shrinking agent, the yield of monomer cyclic peptide was the highest, reaching 82%, and no D-Tyr- isomer and dimer were detected, indicating that this condition was the most suitable condition for synthesizing thymopentin analog. In one case, the yield is low, even no reaction, and in the other case, tyrosine racemization is caused. Although prolonging the time can increase the yield, it is followed by an increase in tyrosine racemization [58].
Phakellistatin 5[59] is a cyclic heptaceptide extracted from sponges. Pettit et al obtained the straight-chain precursor by solid phase method and used PyAOP as the shrink mixture to obtain R-Asn-Phakellistatin 5 with a yield of 28%.
Mink et al. used N-terminal BOC-protected aspartic acid and benzyl serine as raw materials to obtain straight-chain precursors with multiple oxazole structures through a multistep liquid phase reaction. After hydrolysis and acid deprotection of N-terminal and C-terminal, HATU was used as a shrink agent to obtain planar Dolastatin E analogized analogs. Such compounds with multiple functional groups can be used in supramolecular chemistry and combinatorial chemistry [60-62].
In addition, the Dolastatin D analogue Cyclo[Arg-Gly-Asp-D-Phe-Lys(or Tyr)], which functions as a carrier for assisting the entry of radioisotopes (111In and 125I) into platelets [63], The B-chain sequence analogue of platelet growth factor Cyclo(Arg-Lys-Iles-Gla-Ile-Val-Arg-Lys-Lys-Cys), which has an antagonistic effect on platelet growth factor, was also synthesized by cycle-binding reaction using HATU as the contraction agent [64].
From the examples given above, it can be seen that the condensation mixture of cyclic peptide synthesis is not as universally applicable as DCC. The synthesis of different cyclic peptides requires different binding agents.

2.2 TBTU和HBTU

TBTU/HOBt和DPPA/NaHCO3复合缩合剂在这种环六肽合成中的应用，结果表明前者反应速度非常快是后者的5~70倍[66]。TBTU and HBTU were originally benzotriazole compounds for the synthesis of linear dipeptides and tripeptides. In the process of application, it was found that these two compounds showed the advantages of fast and efficient synthesis of some cyclic peptides. Knorr et al. [65] obtained Cyclo(Tyr-asp-Ph-Ph-ser /Phr-Ala) in DMF by using TBTU/HoBt as the condensation agent. Zimmer et al. used HPLC technology to compare the application of TBTU/HOBt and DPPA/NaHCO3 complex shrinking mixture in the synthesis of this cyclohexapeptide, and the results showed that the reaction rate of the former was very fast and 5-70 times that of the latter [66].
phakellistation2 isolated from Marine sponges can inhibit the proliferation of mouse P388 lymphocytes and human cancer cells. Due to the difficulty of artificially extracting this cyclic peptide from sponges, Pettit et al. conducted a complete synthesis of this compound in order to further study its biological activity. The key cycle-binding steps were TBTU, BP-Cl, PyBrOP and TBTU/HOBt, respectively. After a few days (4-14 days), the yield of the products varied. The yield of TBTU was the highest, reaching 55%. Although the synthetic cyclic peptide has the same chemical structure as the natural peptide, the biological activity of the two is very different. The inhibitory effect of the synthetic cyclic peptide on P388 leukemia lymphocytes is much lower than that of the natural cyclic peptide, which may be due to their different conformation [67].
HBTU fully demonstrated its high efficiency in the synthesis of MK-678:Cyclo[Phe-(N-Me)Ala-Tyr-D-Trp-Lys-Val], a cyclic hexapeptide with high affinity for growth hormone release inhibitory factor (SRIH) [68-69]. Another cyclohexapeptide Cyclo[hCys-(N-Me) Ph-Tyr-D-TRp-LYS-VAL] is also synthesized using HBTU as a condensation agent, and the reaction route is as follows:
(SRIH)具有高效亲和能力的环六肽MK-678:Cyclo[Phe-(N-Me)Ala-Tyr-D-Trp-Lys-Val]的合成中HBTU充分发挥了其高效的特点[68-69]。另一种环六肽Cyclo[hCys-(N-Me)Phe -Tyr-D-Trp-Lys-Val]的合成也是应用HBTU为缩合剂，反应路线如下：

2.3 Bop method
McMurray et al. [70-72] took Cyclo(Asp-Asn-Glu-Tyr-Ala-Ala-Arg-Gln-D-Phe-Pro)(Tyr I+1) as the research object in order to determine which positions of Tyr I+1 were essential for protein tyrosine kinase affinity. And which amino acids contributed the most to the activity. A series of Tyr I+1 analogs were synthesized using Bop/HOBt as a complex shrinkage mixture at the concentration of 1mmol/L. After phosphorylation and affinity experiments, 6 analogs were found. Cyclo(ASP-ASN-GLN-Tyr-Ala-Ph-Ph-GLn-D-Ph-Pro), the aromatic amino acid, has the strongest activity at position 7 [73].
Previous studies on peptides with RGD sequence were mostly focused on anti-platelet aggregation. With further studies on the biological activities of these compounds, the excitement of peptides containing RGD sequence has gradually shifted to anti-adhesion, anti-angiogenesis and osteoporosis. Two RGD-containing Cyclo(RGDRGD) and Cyclo(RGd RGD) (d= D-ASP) and linear peptide RGDRGD selectively bind to AVB3-vitreous adhesin and show moderate activity in bone regeneration experiments. The synthesis of two cyclic peptides is based on Bop/HOBt as a complex binding agent. The yield was as high as 80% in the presence of 6.2 times excess DIEA [74].


3. Cyclopeptide was synthesized by solid phase method
The solid phase method can effectively avoid the side reactions such as dimerization and polymerization. As early as the 1960s, Fridkin et al. used polymer carriers to synthesize cyclic peptides [75]. The C-terminal carboxyl group of the linear polypeptide forms an ester bond with the resin, and the linear peptide is hung on the resin. After removing the N-terminal protective group, the linear peptide is neutralized with triethylamine. After 12 hours at room temperature, the cyclic peptide with a yield of 60%~80% is obtained.

In recent years, the strategy of synthesizing cyclic peptides by connecting amino acid side chains with resins has been widely used in cyclic peptide synthesis [76]. For linear polypeptides with aspartate or glutamic acid residues, the side chain carboxyl group of these two acidic amino acid residues can be selected as the C-terminal, condensed with PAC(alkoxy-benzyl alcohol) or PAL(alkoxy-benzylamine) or other types of resin, and the linear polypeptide is hung on the resin. The main chain carboxyl group is protected by allyl group. The N-terminal and C-terminal protective groups were removed after the completion of the stepwise peptide grafting, and the cyclization product was obtained by adding the shrinking agent. Finally, the cyclic peptide was cut from the resin with a mixture of trifluoroacetic acid, anisyl sulfide, B-mercaptoethanol, anisole, and other side chain protection groups were removed. Cyclo(Ala-Ala-Arg-D-Phe-Pro-Glu-asp-Asn-Tyr-glu) was synthesized with this strategy, and the yield was 71%[77]. The limitation of this approach is that the linear polypeptide precursor must contain aspartate or aspartamide, glutamate or glutamine [78-80].


P-nitrophenylketoxime polymers are initially DeGrado and Kaiser as solid phase carriers for solid phase synthesis of polypeptides [83]. Peptidoxime esters are stable under acidic conditions, but very unstable under ammonolysis. Using the characteristics of oxime esters that can ammonolysis, Ospay et al. used this method to synthesize straight chain decapeptide on cyclodecapeptide Tyrocidine A (TA) [84] on oxime resin. The n-end was removed by TFA and neutralized with DIEA to free the amino group. After 24 hours of mixing at room temperature, side chain protected cyclic peptide was obtained, the protective group was removed and purified. The yield of TA is as high as 55%.

4. Enzymatic synthesis of cyclic peptides:
Synthesis of cyclic peptides using proteases in buffers is also one of the developing methods. Jackson et al. [85] reported that several cyclic peptides containing 12-25 amino acid residues were synthesized by enzyme-catalyzed ring formation using linear polypeptide ester derivatives as substrates. The cyclization enzyme Subtiligase was the product of the mutation of Subtiligase, and the catalytic reaction system was a buffer solution with pH=8. The yield was between 30% and 80% by HPLC. The cyclization efficiency is related to the sequence and length of the peptide. The minimum length of a linear peptide required to synthesize a cyclic peptide using Subtiligase is 12 amino acid residues, below which a hydrolytic product or a linear peptide dimerization product is obtained. It may be that the head-to-tail spatial conformation formed by peptide substrates with less than 12 residues does not match the active center of the enzyme.


5. Other methods for the synthesis of cyclic peptides
Several special cyclic peptide synthesis methods are described below:
Meuterman[86-87] et al. cleverly fused light-sensitive AIDS into the cyclic peptide synthesis process. This strategy, which is different from conventional synthesis methods, not only enriches the content of cyclic peptide synthesis methodology, but also provides imagination space for other synthesis workers. The linear pentapeptides H-Ala-Phe- Leu-Pro-Ala-OH and H-Phe -Leu-Pro-D-Ala-OH dissolve in DMF under normal conditions, making them 10-3~10-4 M solutions. By adding 3 times the amount of Bop as the condensation agent and 5 times the amount of DIEA as the base and catalyst, no monomers were obtained, only cyclodimers and cyclotrimers were obtained. Photosensitive structures such as 5-nitro-2-hydroxy-benzyl, 6-nitro-2-hydroxy-benzyl, and mercaptoethyl were introduced into the n-terminal of linear peptide by the method of photosensitive auxiliary agents. After the hydroxyl or sulfhydryl groups in these structures were ester with the carboxyl group of C-terminal, the N-terminal and C-terminal were closer in spatial position, and the ring was reduced by acyl transfer to obtain a cyclic peptide with photosensitive auxiliary agents on the N-terminal. Finally, the photosensitive auxiliary agent was removed by photolysis reaction, and the end-to-end cyclic pentapeptide was obtained with a yield of 20%. Taking the synthesis of Cyclo(Ala-Phit-Leu-pro-ALA) as an example, the specific process is as follows:

In the traditional cyclic peptide synthesis method, not only the amino acid side chain of the linear peptide precursor generally needs protection, but also requires the reactant to be in a highly diluted state in solution. The non-protected amino acid cyclization is different from the traditional cyclization method both in concept and mechanism. The main characteristics are: (1) The amide bond is formed through intramolecular acyl group transfer in the absence of activator; (2) The reversible reaction of the two reaction end groups in the buffer causes the structural tautometry of the ring-chain, regulating and controlling the formation of the ring. The synthetic method of non-protective cyclic peptide avoids the cumbersome protection and deprotection steps and the requirement of high dilution of reaction solution, and the final product can be directly used for biological activity experiments.
Jame P. Tram et al. [88-90] established a method for the preparation of non-protective cyclic peptides by intramolecular transfer thiolide esterification and Ag+ ion-assisted cyclization. For a linear polypeptide with n-terminal cysteine and C-terminal thioester, in a phosphate buffer of pH=7, sulfyl and thioester groups form covalent thiolactone, which spontaneously passes through the S atom to the N atom acyl group to form a cyclic peptide, as shown in the figure:

A series of Cyclo(Cys-Tyr-Gly-Xaa-Yaa-Leu) with n-terminal cysteine were synthesized by the above method. In order to prevent the formation of disulfide Bridges and accelerate the cyclization reaction, TCEP(tricarboethyl phosphine) was added during the reaction. The reaction time was about 4 hours, and the yield was between 78% and 92%. No side reactions and oligomers were detected by HPLC.
For the cysteine-free cysteine-free linear peptides, the N-terminal amino group of the flexible linear peptides is assisted by the sulfurophilic Ag+ ion to form a ring intermediate with the C-terminal thioester, and the intramolecular cyclization is promoted by entropy activation. Similar to the principle of thiolactone cyclization, Ag+ ions promote intramolecular cyclization through a non-classical ring-chain structure tautomization. The annular intermediates are shown as follows:

The synthesis of Cyclo(Ala-Lys-Try-Gly-Gly-Phe-Leu) is an example of the synthesis of cyclo. 10% DMSO was added to the acetic acid buffer solution of pH5.7 as a cosolvent, and the yield of 67% was obtained after reaction for 5 hours.

6. Synthesis of cyclic dipeptides:
Cyclodipeptide (2, 5-piperazine dione) is the smallest cyclic peptide, and many natural cyclodipeptide compounds have clear biological activities, such as antibiotics, bitters, plant growth inhibitors, and hormone release inhibitors [91-92]. The special structure of cyclic dipeptide makes the synthesis of this kind of compound self-contained system, usually by the N-terminal free straight chain peptide ester in polar solvent reflux, can easily obtain the object. Fischer obtained cyclic dipeptide by ammonolysis of linear dipeptide methyl ester in methanol ammonia, but found that this method was prone to racemization. Nitecki[93] proposed that the synthesis of cyclodipeptide from N-terminal free linear dipeptide methyl ester in a mixture of butanol and toluene would not result in racemization. Ueda[94] also obtained cyclodipeptides with good yield by using methanol as solvent for reflux. Cook et al. used 1, 2-ethylene glycol as the reaction solvent to obtain two diastereoisomer cyclic dipeptides with a total yield of 64.5%[95]. Recently, Wang Youzhu reported that a series of cyclic dipeptides were synthesized according to Ueda and Cook's methods, and the yield was between 55% and 99%. Through biological activity experiments, it was found that Cyclo(PH-Pro), Cyclo(Ile-Ile) and Cyclo(Met-Met) had slight calcium antagonistic effect. Cyclo(Ala-Ala) and Cyclo(Pro-Pro) have shown the contractile effect of enhanced potassium [96-97].


The above describes the methods of synthesis of head-tail catenated peptides so far. Because the number and types of amino acids contained in the cyclopeptide precursors - straight chain peptides are very different, the synthesis methods of cyclic peptides are diversified. Reagents and methods that exhibit efficient, rapid condensation for one type of straight-chain peptide may become inefficient or ineffective for another. Therefore, it is necessary to search for the corresponding cyclic peptide synthesis method according to the sequence of target cyclic peptide through careful exploration and hard efforts.