KR101692992B1 - The synthesis method of cyclic peptide by pre-activation cyclization and cyclic peptide synthesized thereby - Google Patents
The synthesis method of cyclic peptide by pre-activation cyclization and cyclic peptide synthesized thereby Download PDFInfo
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- KR101692992B1 KR101692992B1 KR1020150070578A KR20150070578A KR101692992B1 KR 101692992 B1 KR101692992 B1 KR 101692992B1 KR 1020150070578 A KR1020150070578 A KR 1020150070578A KR 20150070578 A KR20150070578 A KR 20150070578A KR 101692992 B1 KR101692992 B1 KR 101692992B1
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Abstract
The present invention relates to a method for synthesizing a cyclic peptide and a cyclic peptide synthesized thereby, comprising the steps of: (A) forming a linear peptide having an amino group at both terminals and having an amino acid of 20 to 50; (B) protecting one terminal amine terminal group of the linear peptide with a protecting group, and mixing another amine terminal group and a succinylated compound to form a carboxyl group; (C) reacting a carboxyl group formed on the linear peptide with a hydroxyl group-containing cyclic compound to form an ester group; (D) removing a protecting group protecting the terminal amine terminal group of the linear peptide; And (E) reacting an amine having a protected group on one side of the linear peptide and an ester group on the other side to form a cyclic peptide, thereby preparing a cyclic peptide having at least 20 amino acid peptides Peptide. ≪ / RTI >
Description
The present invention relates to a method for synthesizing macrocyclic peptides using 20 or more amino acid giant linear peptides, which have been difficult to synthesize in the past, and to the thus synthesized cyclic peptides.
The cyclic peptide has many advantages over conventional linear peptides in the development of drugs and new materials. In particular, it has a high resistance to proteolytic enzymes and is structurally stable due to limited stereoselectivity.
This structural stiffness of the cyclic peptide further aids in mimicking the stabilized state of the folded protein and minimizing entropic penalties associated with binding of the target. For this reason, cyclic peptides are considered as promising high molecular weight drug candidates.
In addition, the cyclic peptides demonstrate the properties of building blocks for self-assembly and can be used as self-assembled peptide nanostructures with a stable secondary structure, abnormally high thermal stability, and well-controlled morphological characteristics. Lt; / RTI >
Conventionally, many synthetic methods for peptide cyclization have been developed, but most of them relate to a cyclization reaction using a relatively low molecular weight peptide, for example, less than 20 amino acids, and the production of such a low molecular weight cyclic peptide Can be performed simply and easily.
However, macrocyclic peptides with more than 20 amino acids are very difficult to synthesize and synthesis yields are too low to synthesize as much as needed for research, but mass production for commercialization is almost impossible.
Peptides or proteins can be cyclized by biological methods. However, the production of nonpolar, amphipathic and chemically modified peptides is limited.
Specifically, both ends of the linear peptide are placed very close for cyclization between the molecules, and for the reaction between the molecules, the peptide cyclization reaction is carried out under high heptic state. The intermolecular cyclisation reaction is much slower than the general intermolecular reaction, and it is typically very difficult to synthesize macrocyclic peptides by cyclization by both ends placed close enough together, typically with reduced interactions.
Among the cyclic peptide synthesis methods, the amide bond between the protected peptide fragment ends is one of the most common methods for cyclization. However, the first problem of the above method is that most of the coupling reagents used for amide formation are decomposed during the long-term cyclization reaction. Therefore, the above coupling reagent can not be used for the cyclization reaction using the macro linear peptide which lasts for several days.
The second problem is that nucleophilic amines in macrocyclic peptides can undergo slow cyclization and end-capping by coupling reagents during the carboxyl activation step, with the probability that the cyclic reaction time is long The higher molecular weight peptides (macrolide peptides) are higher than the lower molecular weight peptides. Guanidino formation is a common example of end-capping with permanently amine-terminated reactions. When macrocyclic peptides are end-capped, they often exhibit residence times similar to those of the cyclized peptides on high performance liquid chromatography (HPLC).
Thus, considering the many by-products formed during the reaction, the presence of end-capped peptides often requires many purification steps, so that the desired cyclic peptides are obtained in very low yields.
Thus, there is a need for a method for producing macrocyclic peptides at a rapid and high yield using 20 or more amino acid giant linear peptides.
It is an object of the present invention to provide a synthesis method capable of producing an ancient cyclic peptide using 20 or more amino acid giant linear peptides which have not been possible in the past.
Another object of the present invention is to provide a macrocyclic peptide synthesized according to the above synthesis method.
In order to accomplish the above object, the present invention provides a method of synthesizing a macrocyclic peptide comprising the steps of: (A) forming a linear peptide having an amino group at both terminals and having 20 to 50 amino acids;
(B) protecting one side amine terminal group of the linear peptide with a protecting group and reacting another amine terminal group with a succinylation compound to form a carboxyl group;
(C) reacting a carboxyl group formed on the linear peptide with a hydroxyl group-containing cyclic compound to form an ester group;
(D) removing a protecting group protecting one terminal amine group of the linear peptide; And
(E) forming an annular peptide by reacting an amine group on one side of the linear peptide with a protecting group removed and an ester group on the other side.
In step (A), one of the amine groups at both terminals may be formed by a bond of lysine or ornithine.
In the step (B), the succinylated compound may be at least one member selected from the group consisting of succinic anhydride, succinic acid and succinic acid monomethyl ester.
In step (B), another unprotected amine group of the linear peptide is mixed with a succinylation compound, N-methyl-2-pyrrolidone (NMP) and diisopropylamine (DIPEA) and reacted at room temperature for 2 to 8 hours To form a carboxyl group.
The linear peptide, succinylated compound and diisopropylamine (DIPEA) may be mixed in a molar ratio of 1: 5-20: 5-20.
In the step (C), the hydroxyl group-containing cyclic compound is selected from the group consisting of N-hydroxysuccinimide (NHS) and N-hydroxysulfosuccinimide (N-hydroxysulfosuccinimide) It may be more than one kind.
In the step (C), the carboxyl group formed on the linear peptide may be reacted at room temperature for 2 to 8 hours to form an ester group by mixing with a hydroxyl group-containing cyclic compound and N, N' -diisopropylcarbodiimide.
The linear peptide, the hydroxyl group-containing cyclic compound and the N, N' -diisopropylcarbodiimide may be mixed in a molar ratio of 1: 2-10: 1-5.
In step (D), the protecting group may be removed with a protecting group removing solution mixed with acetic acid, trifluoroethanol (TFE) and methylene chloride in a weight ratio of 1: 1-5: 5-15. Alternatively, in the step (D), the protecting group may be removed with a protecting group removing solution mixed with trifluoroacetic acid, triisopropylsilane and methylene chloride in a weight ratio of 1: 10: 10-30.
In step (E), the cyclic peptide can be formed by reacting the linear peptide with N-methyl-2-pyrrolidone (NMP) and diisopropylamine (DIPEA) at room temperature for 2 minutes to 72 hours.
After the step (E), the linear peptide is reacted with a mixture of 1,2-ethanedithiol, thioanisole and trifluoroacetic acid at room temperature for 1 to 3 hours and then treated with tert -butyl methyl ether (TBME) Can be added.
The mixed solution may be a mixture of 1,2-ethanedithiol, thioanisole, and trifluoroacetic acid in a weight ratio of 1: 1: 30-40.
The macrocyclic peptide of the present invention may be a macrocyclic peptide having 20 to 50 amino acids synthesized according to the above synthetic method.
The macrocyclic peptide of the present invention can carry out a macropeptide cyclization reaction with high efficiency and a fast reaction rate by using 20 or more amino acid giant linear peptide, thereby mass-producing the macrocyclic peptide. Specifically, when 1 mg of a giant linear peptide having 25 amino acids is reacted, a macrocyclic peptide of 0.01 mg to 0.9 mg is obtained.
In addition, the present invention can completely eliminate the side reaction called amine end-capping, which has been a problem in the synthesis of the cyclic peptide.
The macrocyclic peptide of the present invention is strong against a target due to its constrained structure and proteolytic stability, and can be selectively bound, thus being highly valuable as a drug. In addition, it is very suitable for a target having a wide action site such as protein-protein interaction. The above-mentioned macromolecule peptides have very high marketability as a drug since the drug-targeting of macromolecules such as protein-protein interactions is a modern trend of drug development.
In addition, the macrocyclic peptide of the present invention has very good physical properties as a material, and has good characteristics as a self-assembly material. The self-assembled macrocyclic peptide as described above can be used in a wide variety of fields such as drugs, drug delivery systems, gene delivery materials, electronic materials, and sensor materials.
Figure 1 is a graph of matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) measurements of giant linear peptides with activated ester groups prepared according to the present invention.
Figure 2 is a graph of matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) measurements of macrocyclic peptides prepared according to the present invention.
FIG. 3 is a graph showing RP-HPLC of a macrocyclic peptide prepared using a) S N 2 reaction according to Comparative Example 1 and b) a pre-activated NHS ester reaction according to one embodiment of the present invention.
4 is a graph showing the results of a) the S N 2 reaction according to Comparative Example 1 and b) the macrocyclic peptide prepared using the pre-activated NHS ester reaction according to one embodiment of the present invention by MALDI-TOF MS .
5 is a graph showing RP-HPLC measurement of a) 3 minutes and b) 6 hours after the cyclization reaction according to the synthesis method of one embodiment of the present invention.
The present invention relates to a synthetic method capable of producing macrocyclic peptides using 20 or more amino acid giant linear peptides which have not been possible in the past, and a cyclic peptide thus synthesized.
Hereinafter, the present invention will be described in detail.
The method for synthesizing the cyclic peptide of the present invention comprises the steps of (A) forming a high molecular weight linear peptide comprising an amine group at both terminals; (B) protecting one side amine terminal group of the linear peptide with a protecting group and reacting another amine terminal group with a succinylation compound to form a carboxyl group; (C) reacting a carboxyl group formed on the linear peptide with a hydroxyl group-containing cyclic compound to form an ester group; (D) removing a protecting group protecting one terminal amine group of the linear peptide; And (E) reacting an amine having a protected group on one side of the linear peptide and an ester group on the other side to form a cyclic peptide.
The method of synthesizing the cyclic peptide of the present invention can be represented by the following Reaction Scheme 1, and the linear peptide and the cyclic peptide shown in Scheme 1 are representative examples for showing a large peptide. But is not limited thereto.
[Reaction Scheme 1]
First, in step (A), a giant linear peptide having an amino group at both terminals and having amino acids of 20 to 50 is formed like the compound represented by formula (4).
In order to form a cyclic peptide, an amine group is provided at both ends of the linear peptide, wherein the amine group at one end of the two terminals is an amine group formed by a bond of lysine or ornithine, The group is an amine group that is basically an amino acid in the linear peptide.
The linear peptide used in the present invention is a macromolecule containing 20 to 50 amino acids which are conventionally large enough not to be produced in a cyclic form.
Next, in step (B), the amine side group of one side of the linear peptide is protected with a protecting group such as the compound represented by the general formula (3), and the other side amine end group not protected by a protecting group is reacted with a succinylated compound, A carboxyl group is formed at the terminal of the peptide.
The amine group protected by the protecting group is an amine group formed by the linkage of lysine and protects the amine group formed by the linkage of lysine or ornithine to facilitate the reaction when cyclizing.
Specifically, the amine group at the other end of the linear peptide is mixed with a succinylation compound, N-methyl-2-pyrrolidone (NMP) and diisopropylamine (DIPEA) And reacted for 72 hours to form a carboxyl group. The N-methyl-2-pyrrolidone (NMP) is used as a solvent.
The linear peptide, succinylated compound and diisopropylamine (DIPEA) are mixed in a molar ratio of 1: 5-20: 5-20, preferably 1: 8-12: 8-12.
When the content of the succinylated compound and / or the diisopropylamine (DIPEA) is less than the lower limit based on the linear peptide, the reaction between the amine group and the succinylated compound does not proceed easily, so that a large amount of a compound having no carboxyl group can be formed If it exceeds the upper limit value, side reactions may occur.
In particular, it is preferable that the succinylated compound and diisopropylamine (DIPEA) are mixed at a molar ratio of 1: 1. If the molar ratio is out of the range, the yield may be significantly lowered due to side reactions.
The succinylated compound may be at least one selected from the group consisting of succinic anhydride, succinic acid, and succinic acid monomethyl ester.
Next, in step (C), a carboxyl group formed on the linear peptide and a hydroxyl group-containing cyclic compound are reacted in step (B) to form an activated ester group like the compound represented by the formula (2).
By reacting a carboxyl group formed on the linear peptide with a hydroxyl group-containing cyclic compound to form an activated ester group, the amine group and the ester group provided at the other end of the linear peptide are easily reacted to cause cyclization.
Specifically, the carboxyl group formed on the linear peptide is mixed with a hydroxyl group-containing cyclic compound and N, N' -diisopropylcarbodiimide and reacted at room temperature of 23 to 26 ° C for 2 to 8 hours to form an activated ester group do.
The linear peptide, the hydroxyl group-containing cyclic compound and the N, N' -diisopropylcarbodiimide are used in a molar ratio of 1: 2-10: 1-5, preferably 1: 3-6: 1-2 Mixed.
When the content of the hydroxyl group-containing cyclic compound and / or N, N' -diisopropylcarbodiimide is less than the lower limit value based on the linear peptide, the reaction between the carboxyl group and the hydroxyl group-containing cyclic compound may not be performed smoothly If the upper limit is exceeded, the yield may be lowered due to the side reaction.
The hydroxyl group-containing cyclic compound may be at least one selected from the group consisting of N-hydroxysuccinimide (NHS) and N-hydroxysulfosuccinimide (N-hydroxysulfosuccinimide) .
Next, in step (D), a protecting group for protecting the terminal amine terminal group of the linear peptide is removed.
By treating the linear peptide with the protecting group removing solution 5 to 15 times, the protecting group protecting the amine group of the lysine or ornithine is removed.
The protecting group removing solution is a solution in which acetic acid, trifluoroethanol (TFE) and methylene chloride are mixed in a weight ratio of 1: 1-5: 5-15, preferably 1: 1-3: 5-8; Or trifluoroacetic acid, triisopropylsilane and methylene chloride are mixed in a weight ratio of 1: 1-10: 10-30, preferably 1: 1-5: 12-20, Solution.
If the content of triflouro ethanol (TFE) and / or methylene chloride is less than the lower limit based on the acetic acid, the cyclized peptide can not be obtained because the protecting group is not removed. If the content exceeds the upper limit, Can cause.
When the content of triisopropylsilane and / or methylene chloride is less than the lower limit of the above-mentioned trifluoroacetic acid, the cyclic peptide can not be obtained because the protecting group is not removed. If the content of triisopropylsilane and / or methylene chloride is higher than the upper limit, It can cause damage.
Next, in step (E), a macromolecule-like peptide is formed by reacting an amine group having a protected group on one side of the linear peptide and an ester group on the other side to form a compound represented by Chemical Formula 1.
Specifically, the linear peptide is mixed with N-methyl-2-pyrrolidone (NMP) and diisopropylamine (DIPEA) and reacted at room temperature for 2 minutes to 72 hours, preferably 2 minutes to 30 hours To form macrocyclic peptides.
Since synthesis of the macrocyclic peptide is carried out using a linear peptide coupled with a resin, after the step (E), a step of dissolving 1,2-ethanedithiol, thioanisole and trifluoroacetic acid , And the mixture was stirred at room temperature for 1 to 3 hours. Then, to remove the trifluoroacetic acid, a process of treating the peptide with tert -butyl methyl ether (TBME) in which only the trifluoroacetic acid was dissolved Can be performed.
The mixed solution is mixed with 1,2-ethanedithiol, thioanisole and trifluoroacetic acid in a weight ratio of 1: 1: 30-40, preferably 1: 1: 35-38. If the content of trifluoroacetic acid based on 1,2-ethanedithiol and thioanisole is below the lower limit, the resin may not be cleaved.
The present invention is characterized in that it is cyclized at a high yield and a fast reaction rate using a high molecular weight macroscopic linear peptide having an amino acid of 20 to 50. However, the present invention is not limited thereto, and the present invention can also be used as a method of cyclizing low molecular weight linear peptides.
It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the present invention. Such variations and modifications are intended to be within the scope of the appended claims.
Example 1. Synthesis of macrocyclic peptide
Peptides were synthesized on a Rink amide MBHA resin LL (Novabiochem) according to the standard Fmoc protocol using a Tribute peptide synthesizer (Protein Technologies, Inc). Except that lysine was used as a standard amino acid protecting group, and the lysine was protected with an acid labile methoxytrityl (Mmt) group to prepare a linear peptide.
In order to cyclize the linear peptide, the linear peptide-attached resin (one side N-terminal protected by Mmt and the other N-terminal involved in the reaction, peptide: 20 mmol) and 1 mL N -methyl 2-pyrrolidone (NMP), succinic anhydride (20 mg, 200 mmol) and DIPEA (35.4 mL, 200 mmol) were mixed and stirred at room temperature for 4 hours to form a carboxyl group at the N-terminus of the peptide. After the reaction was completed, the carboxylated peptide was reacted with methylene chloride (MC), N -hydroxysuccinimide (NHS) (9.2 mg, 80 mmol) and N, N'- diisopropylcarbodiimide (DIC; , 40 mmol) were mixed and stirred at room temperature for 4 hours to form an activated ester group. After the reaction was completed, the reaction mixture was washed with MC and dimethylformamide (DMF) to completely remove the coupling agent capable of causing amine end-capping, and 10% of the peptide-bound resin was removed to remove the Mmt group contained in the lysine side chain. (1 min X 10) with acetic acid solution (AcOH: TFE: MC = 1: 2: 7 weight ratio).
The peptide-bound resin from which the protecting group had been removed, NMP (3 mL) and 2% DIPEA were mixed and stirred at room temperature to prepare a macrocyclic peptide.
In order to cut the resin, the macroreticular peptide to which the resin was attached was treated with a mixture solution (trifluoroacetic acid: 1,2-ethanedithiol: thioanisole = 95: 2.5: 2.5) for 2 hours, Was dissolved in tert -butyl methyl ether (TBME) to remove the rosacet acid.
The macrocyclic peptide prepared according to Example 1 is shown in Fig.
Comparative Example One. Bromo Using acetic acid
Peptides were synthesized on a Rink amide MBHA resin LL (Novabiochem) according to the standard Fmoc protocol using a Tribute peptide synthesizer (Protein Technologies, Inc). Except that cysteine was used as a standard amino acid protecting group, and the cysteine was protected with an acid labile methoxytrityl (Mmt) group, respectively, to prepare a linear peptide.
In order to cyclize the linear peptide, the linear peptide-attached resin (one side N-terminal protected with Mmt and the other N-terminal involved in the reaction, peptide: 20 mmol) and N-methyl- -Pyrrolidone (NMP) for 30 minutes, and the bromoacetic acid was bound to the N-terminal portion of the peptide immobilized on the resin. Thereafter, bromoacetic acid (28 mg, 200 mol) and N, N-diisopropylcarbodiimide (15.5 uL, 100 umol) were mixed in N-methyl-2-pyrrolidone The solution was incubated for 10 minutes to activate the carboxyl group, added to the resin, placed in a 6 mL polypropylene tube containing a frit, and the reaction was allowed to proceed at room temperature for 1 hour. It was then washed with N-methyl-2-pyrrolidone and dichloromethane (DCM). To remove the Mmt protecting group from cysteine, the resin was treated with 1% trifluoroacetic acid (TFA) in dichloromethane for at least 7 minutes each for 1 minute. Thereafter, the mixture was stirred at room temperature overnight in 3 mL of 1% diisopropylethylamine (DIPEA) in N-methyl-2-pyrrolidone to proceed an intramolecular cyclization reaction.
< Test Example >
For the production of the macrocyclic peptides of the present invention, a long linear peptide consisting of 25 amino acids was used. Comparative Example 1 also used a long linear peptide consisting of 25 amino acids.
Test Example One. MALDI - TOF MS measurement
Figure 1 shows the measurement of matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS; Microflex LRF20, Bruker) for macroscopic linear peptides with activated ester groups, The matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) was measured.
As shown in FIG. 1, it was confirmed that the change from the carboxyl group to the NHS ester group was smoothly performed, and that the NHS ester group was stable even in the removal of the protecting group and ionization.
In addition, as shown in Fig. 2, it was confirmed that the cyclized peptide was successfully formed by the mass spectrometry data, and the end-capped product having a higher molecular weight than the linear or cyclic peptide was not detected.
Test Example 2. Comparison of cyclization efficiency with Comparative Example 1
FIG. 3 is a graph showing RP-HPLC of a macrocyclic peptide prepared using a) S N 2 reaction according to Comparative Example 1 and b) the pre-activated NHS ester reaction according to Example 1; Macrocyclic peptides are indicated by arrows.
4 is a graph of a macrocyclic peptide prepared using a) S N 2 reaction according to Comparative Example 1 and b) the pre-activated NHS ester reaction according to Example 1 by MALDI-TOF MS. Macrocyclic peptides are indicated by arrows.
As shown in Figs. 3 and 4, both Example 1 and Comparative Example 1 were used for the cyclization reaction using linear peptides having the same length and sequence. The thiol-mediated S N 2 reaction of Comparative Example 1 is a commonly used method for peptide cyclization and the untreated macrocyclic peptide has a small peak in the thiol-mediated S N 2 reaction (FIG. In addition, the MALDI spectrum shows that many by-products were involved which would require multiple purification steps to obtain the pure product (FIG. 4A).
On the other hand, it was confirmed that macrocyclic peptides appeared as major peaks in the preparation according to Example 1 (Fig. 3B). In addition, it was confirmed that the MALDI spectral analysis also included macromolecule peptides in an almost pure state (Fig. 4B).
Test Example 3. Measurement of reaction rate
5 is a graph showing RP-HPLC measurement of a) 3 minutes and b) 6 hours after the cyclization reaction according to the synthesis method of Example 1. Macrocyclic peptides are indicated by arrows.
As shown in FIG. 5, the macrocyclic peptide appeared after 3 minutes, and the peak intensity against the macrocyclic peptide remained almost constant even after 6 hours.
Thus, it was confirmed that the cyclization reaction was carried out at a very high speed and that a macroreticular peptide close to complete in a few minutes was obtained with respect to the 25-mer peptide used in the above reaction.
The synthetic method according to the present invention was also applied to various other peptides containing 22-28 amino acids including an amphipathic peptide capable of self-assembly into a nanostructure in aqueous solution. Peptides containing 25 amino acids can be considered hydrophilic.
Strategies using preactivated NHS esters are well formed with amphipathic peptides with a fast reaction rate and a highly efficient conversion to the cyclization product.
Amide bonds in the macrocyclic peptide synthesis of the present invention are achieved using amino acid active esters formed by species or in situ activation, and include hydroxyl group containing cyclic compounds such as N -hydroxysuccinimide ( N -hydroxysuccinimide, NHS) is used to activate the carboxylic acid.
In addition, NHS ester formation completely eliminated the problem of amine end-capping in a subsequent step involving coupling agent removal, deamination and cyclization according to the present invention.
Although simple, these new methods can perform large peptide cyclization reactions with high efficiency and fast reaction rates, which are suitable for large-scale production of macrocyclic peptides.
Such a synthesis method of the present invention is effective not only for peptides having a large number of amino acids but also for peptides having a small number of amino acids.
- Substance -
Fmoc-amino acids and coupling agents were purchased from Novabiochem (Germany) and AnaSpec (U.S.A.), and general chemicals for peptide cyclization reactions were purchased from Sigma-Aldrich (U.S.A.) and Merck (Germany).
In addition, HPLC solvents were purchased from Fisher Scientific (USA), succinic anhydride and N - was purchased from hydroxy succinimide (N -hydroxysuccinimide, NHS) is a Sigma-Aldrich (USA).
Claims (17)
(B) protecting the amine terminal group of the side chain of the linear peptide with a protecting group, and reacting the amine terminal group of the other unprotected side with a succinylidene compound, N-methyl-2-pyrrolidone (NMP) Propylamine (DIPEA) to form a carboxyl group;
(C) reacting a carboxyl group formed on the linear peptide with a hydroxyl group-containing cyclic compound and N, N' -diisopropylcarbodiimide to form an ester group;
(D) selectively removing a protecting group protecting the amine terminal group of the side chain in the linear peptide; And
(E) reacting an amine group from which the protecting group of the side chain of the linear peptide is removed and an ester group provided on the other side of the linear peptide to form a cyclic peptide having a resin on one side thereof. .
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