阅读说明：本技术 用于合成含有n-取代氨基酸的肽的方法 (Method for synthesizing peptides containing N-substituted amino acids ) 是由 野村研一 村冈照茂 棚田干将 江村岳 于 2018-06-08 设计创作，主要内容包括：生产本发明的含有N-取代的氨基酸或N-取代的氨基酸类似物的肽的方法,包括以下步骤：制备Fmoc保护的氨基酸、Fmoc保护的氨基酸类似物或Fmoc保护的肽；通过使用碱,使具有Fmoc保护的氨基酸等的Fmoc骨架的保护基脱保护；和通过添加新的Fmoc保护的氨基酸等,形成酰胺键；以及当通过固相方法生产肽时,在比TFA弱的酸度条件下将获得的肽从固相上切下。此外,所获得的肽的至少一个侧链具有保护基,该保护基在碱性条件下不被脱保护,并且在比TFA弱的酸性条件下被脱保护。(A method for producing a peptide of the present invention comprising an N-substituted amino acid or an N-substituted amino acid analog, comprising the steps of: preparing Fmoc-protected amino acids, Fmoc-protected amino acid analogs, or Fmoc-protected peptides; deprotecting a protecting group of Fmoc skeleton of an Fmoc-protected amino acid or the like by using a base; and forming an amide bond by adding a new Fmoc-protected amino acid or the like; and when the peptide is produced by a solid phase method, cleaving the obtained peptide from the solid phase under a condition of weaker acidity than TFA. Further, at least one side chain of the obtained peptide has a protecting group which is not deprotected under basic conditions and deprotected under acidic conditions weaker than TFA.)

1. A method of producing a peptide comprising at least one N-substituted amino acid or N-substituted amino acid analog, wherein said method comprises the steps of:

1) preparing a peptide comprising one or both of the following functional groups i) and ii) (Fmoc-protected amino acids), an amino acid analog comprising at least one of the following i) and ii) (Fmoc-protected amino acid analog), or an Fmoc-protected amino acid and Fmoc-protected amino acid analog (Fmoc-protected peptide):

i) a backbone amino group protected by at least one protecting group having an Fmoc backbone; and

ii) at least one free carboxylic acid group or a reactive esterified carboxylic acid group;

2) supporting the Fmoc-protected amino acid, Fmoc-protected amino acid analog, or Fmoc-protected peptide prepared in step 1) onto a solid phase;

3) deprotecting a protecting group having an Fmoc skeleton of an Fmoc-protected amino acid, an Fmoc-protected amino acid analog or an Fmoc-protected peptide supported on a solid phase by using a base to expose an amino group thereof;

4) forming an amide bond by adding a new Fmoc-protected amino acid, a new Fmoc-protected amino acid analog, or a new Fmoc-protected peptide; and

5) the peptide obtained in step 4) was cleaved from the solid phase under acidic conditions weaker than TFA.

2. The production method according to claim 1, wherein at least one side chain of an amino acid or an amino acid analog constituting the peptide obtained in step 4) is protected by a protecting group which is not protected under basic conditions but deprotected by a first acid, and wherein the method further comprises a step of deprotecting the protecting group using the first acid before or after step 5); and

wherein in step 5) the peptide is cleaved off using a second acid, and

wherein the first acid and the second acid are both weaker acids than TFA, and the acidity of the first acid is higher than the acidity of the second acid.

3. A method of producing a peptide comprising at least one N-substituted amino acid or N-substituted amino acid analog, wherein said method comprises the steps of:

1) preparing a peptide comprising one or both of the following functional groups i) and ii) (Fmoc-protected amino acids), an amino acid analog comprising at least one of the following functional groups i) and ii) (Fmoc-protected amino acid analog), or an Fmoc-protected amino acid and Fmoc-protected amino acid analog (Fmoc-protected peptide):

i) a backbone amino group protected by at least one protecting group having an Fmoc backbone; and

ii) at least one free carboxylic acid group or a reactive esterified carboxylic acid group;

2) deprotecting a protecting group having an Fmoc backbone of an Fmoc-protected amino acid, an Fmoc-protected amino acid analog or an Fmoc-protected peptide by using a base to expose an amino group thereof;

3) forming an amide bond by adding a new Fmoc-protected amino acid, a new Fmoc-protected amino acid analog, or a new Fmoc-protected peptide, wherein at least one side chain of the amino acid or amino acid analog constituting the peptide obtained in this step has a protecting group which is not deprotected under basic conditions and deprotected under conditions having a weaker acidity than TFA; and

4) deprotecting the protecting group of the side chain under conditions less acidic than TFA.

4. The production method of claim 3, wherein the peptide production is carried out by a solid phase method.

5. The production method according to claim 4, further comprising a step of cleaving the peptide obtained in step 3) from the solid phase under a weaker condition than the weak acidic condition used in step 4), before or after step 4).

6. The production method of claim 3, wherein the peptide production is carried out by a liquid phase method.

7. The production method according to any one of claims 1 to 6, wherein the step 4) of claim 1 or the step 3) of claim 3 further comprises the steps of:

deprotecting a protecting group having an Fmoc backbone on the newly added Fmoc-protected amino acid, the newly added Fmoc-protected amino acid analog or the newly added Fmoc-protected peptide by using a base to expose the amino group thereof;

by further adding a new Fmoc-protected amino acid, a new Fmoc-protected amino acid analogue or a new Fmoc-protected peptide, an amide bond is formed,

and wherein these steps are repeated one or more times.

8. The production method according to any one of claims 1 to 7, wherein the prepared peptide comprises an amino acid residue or an amino acid analog residue containing one reactive site on the C-terminal side thereof and an amino acid residue, an amino acid analog residue or a carboxylic acid analog containing another reactive site on the N-terminal side thereof.

9. The production method of claim 8, further comprising a step of bonding the reactive site and the other reactive site to cyclize the peptide.

10. The production method of claim 9, wherein the amino acid residue, amino acid analog residue, or carboxylic acid analog having the other reactive site is at the N-terminus, and the linkage is an amide linkage.

11. The production method of claim 9, wherein the amino acid residue, amino acid analog residue, or carboxylic acid analog having the other reactive site is at the N-terminus, and the bond is a carbon-carbon bond.

12. The production method as claimed in any one of claims 1 to 11, wherein the step carried out under acidic conditions having a weaker acidity than TFA is carried out using a weakly acidic solution which is contained in a solution having an aqueous pKa value of 5 to 14 and its ionization capacity value Y_OTsA weak acid having an aqueous pKa value of 0 to 9 in a positive solvent.

13. The production method according to claim 12, wherein the solvent is a fluoroalcohol.

14. The production process according to claim 13, wherein the fluoroalcohol is TFE or HFIP.

15. The production method of any one of claims 2 to 14, wherein the side chain protecting group is a protecting group deprotected in a range of pH1 to pH7, or a protecting group deprotected in TFA at a concentration of 10% or less.

16. The production process according to any one of claims 2 to 15, wherein the side chain protecting group is selected from the following a) to d):

a) when the side chain protecting group is a protecting group for side chain hydroxyl group of Ser, Thr, Hyp and derivatives thereof, any one protecting group selected from MOM skeleton, Bn skeleton, Dpm skeleton, Trt skeleton, silyl skeleton and Boc skeleton represented by the following general formula;

b) when the side chain protecting group is a protecting group for side chain hydroxyl group of Thr and its derivatives, any one protecting group selected from MOM skeleton, Bn skeleton, Dpm skeleton, Trt skeleton, silyl skeleton, Boc skeleton and tBu skeleton represented by the following general formula;

c) when the side chain protecting group is a protecting group for side chain imidazole ring of His and its derivatives, any one protecting group selected from MOM skeleton, Bn skeleton and Trt skeleton represented by the following general formula;

d) when the side chain protecting group is a protecting group for a side chain carboxylic acid group of Asp, Glu and derivatives thereof, any one protecting group selected from MOM skeleton, Bn skeleton, Dpm skeleton, Trt skeleton, tBu skeleton, phenyl-EDOTn skeleton and orthoester skeleton in which a carbon atom of a carboxylic acid group to be protected is substituted with three alkoxy groups, represented by the following general formula:

< protecting group having MOM skeleton >

(wherein

R1 is H, R2 is H, and X is methyl, benzyl, 4-methoxybenzyl, 2, 4-dimethoxybenzyl, 3, 4-dimethoxybenzyl or 2-trimethylsilylethyl;

r1 is methyl, R2 is H, X is ethyl;

r1, R2 and R3 are all methyl; or

R1 and X together form-CH₂-CH₂-CH₂-or-CH₂-CH₂-CH₂-CH₂-, and R2 is H,

wherein when any of R1, R2 and X is methyl or ethyl, these groups may be further substituted with alkyl, benzyl or aryl);

< protecting group having Bn skeleton >

(wherein

R1 to R5 are each independently H, alkyl, aryl or halogen, R6 and R7 are alkyl;

r1, R2, R4 and R5 are each independently H, alkyl, aryl or halogen, R3 is methoxy, R6 and R7 are H;

r1 and R3 are methoxy, R2, R4 and R5 are each independently H, alkyl, aryl or halogen, R6 and R7 are H; or

R1, R4 and R5 are each independently H, alkyl, aryl or halogen, and R2 and R3 together form-O-CH₂-O-)；

< protecting group having Dpm skeleton >

(wherein

R1 to R10 are each independently H, alkyl, aryl, alkoxy, or halogen; or

R1 to R4 and R7 to R15 are each independently H, alkyl, aryl, alkoxy or halogen, and R5 and R6 together form-O-or-CH₂-CH₂-)；

< protecting group having Trt skeleton >

(wherein

R1 to R15 are each independently H, alkyl, aryl, alkoxy, or halogen;

r1, R2 and R4 to R15 are each independently H, alkyl, aryl, alkoxy or halogen, and R3 is methyl or methoxy;

r1 is Cl and R2 to R15 are each independently H, alkyl, aryl, alkoxy, or halogen; or

R1 to R4 and R7 to R15 are each independently H, alkyl, aryl, alkoxy, or halogen, and R5 and R6 together form-O-);

< protecting group having silyl skeleton >

(wherein R1 to R3 are each independently alkyl or aryl);

< protecting group having Boc skeleton >

(wherein R1 to R9 are each independently H, alkyl, or aryl);

< protecting group having tBu skeleton >

(wherein

R1 to R9 are each independently H, alkyl or aryl); and

< protecting group having phenyl-EDOTn skeleton >

(wherein each of R1 to R3 is independently H or methoxy).

Technical Field

The present invention relates to a novel method for synthesizing a peptide, which allows synthesis to be performed with high purity and high synthesis efficiency in synthesizing a peptide comprising an N-substituted amino acid.

Background

Peptides are a very valuable chemical species, and 40 or more types of peptides have been put on the market as drugs (NPL 1). Among them, for cyclic peptides and N-methylated (or N-alkylated) non-natural peptides, it has been expected to improve membrane permeability to improve lipophilicity and metabolic stability to obtain resistance to hydrolytic enzymes (hydrolases) (NPL 2). Recently, studies on cyclic peptides of drug-like (drug-like: preferably, it is shown that both membrane permeability and metabolic stability characteristics are achieved) which is the key to achieving intracellular transfer and allowing formulation into oral agents are under development (NPL3 and 4). Further, a patent document (PTL1) that clarifies conditions necessary for drug-like cyclic peptides is also disclosed, and the importance and understanding of these peptides in drug development is increasing.

On the other hand, development of a method for synthesizing a peptide comprising many unnatural amino acids (e.g., amino acids represented by N-alkyl amino acids) has been relatively narrow. In most cases, established techniques for native peptides have been applied directly to non-native peptides.

The Fmoc method and the Boc method are widely known peptide synthesis methods, and most of the findings about these methods have been obtained from the development of methods for synthesizing natural peptides. The Fmoc group is stable to acid; thus, when the N-terminal amino group is protected by Fmoc group, deprotection reaction thereof is carried out using a base such as DBU and piperidine. Thus, for example, a (deprotectable) protecting group which can be deprotected by an acid is used as a protecting group for a peptide side chain functional group, and a peptide chain is extended by selectively deprotecting an N-terminal amino group. As commonly used protecting groups, tert-butyl (tBu), trityl (Trt) and such groups that can be deprotected by acid at the level of trifluoroacetic acid (TFA) can be used to protect amino acid side chains in the Fmoc process, and the steps of cleaving the peptide from the resin and deprotecting the protecting groups for the side chain functions can be performed under milder conditions compared to the Boc process.

However, even in the solid-phase synthesis method by Fmoc method, which allows cleavage from the resin and deprotection of the protecting group of the side chain functional group under relatively mild conditions, the following problems have been revealed in the synthesis of N-alkylated peptides during the step of cleavage from the resin or deprotection of the protecting group on the side chain functional group using TFA.

However, it is known that in the case of N-methylated peptides, particularly in the case of peptides having a sequence of consecutive N-methyl amino acids, side reactions occur in which acid hydrolysis is performed via oxazolium (oxolonium) and the peptide chain is cleaved (NPL5 and 6). furthermore, in these steps using TFA, in addition to acid hydrolysis, N-to-O-acyl transfer reactions are also known to occur as side reactions, which lead to the formation of depsipeptides (depsipeptides) (NPL7 and 8).

In the cleavage step and deprotection step using an acid, measures to avoid the hydrolysis problem, such as using a low concentration TFA solution and controlling a short reaction time, are being taken. For example, according to Albericio et al, in the solid phase synthesis of a peptide named NMe-IB-01212, peptide degradation at the N-Me site was observed when the Boc group on the amino group included in the N-methylated cyclohexapeptide was deprotected in a TFA-DCM (1:1) solution. Although attempts have been made to improve to avoid degradation by using lower concentrations of TFA and minimizing reaction time, no adequate improvement has been obtained (NPL 9). First, protecting groups widely used in conventional peptide synthesis have shown a case where a deprotection step using a low concentration TFA solution causes a deprotection reaction on a side chain to proceed very slowly or not, while a cleavage reaction of a peptide from a resin proceeds at a satisfactory rate.

Furthermore, in order to prevent cleavage of the N-terminal Ac-MePhe by the same reaction mechanism as hydrolysis of highly N-methylated peptides, Fang et al used TFA and lowered the reaction temperature to 4 ℃ to deprotect the Pbf group, which is a protecting group for the Arg side chain (NPL 10). However, even by using such a method of lowering the temperature, it is difficult to completely prevent Ac-MePhe cleavage, and the method can only stop the reaction when the production level of the desired product reaches a maximum.

In addition to the problems in the deprotection process, the problem of low reactivity in the extension step is also known. When the N-methyl amino acid reaches the N-terminus of the newly formed amide bond, the amide formation reaction (extension reaction) with the subsequent amino acid may not proceed sufficiently due to the bulky secondary amine thereof (NPLs 2 and 5).

For this problem in the extension step, a measure has been taken to reduce unreacted sites by repeating the same reaction conditions two or more times (the method of repeating twice is called double coupling). Furthermore, with regard to the activation of the amino acid to be condensed, efforts have been made to improve the condensation efficiency, for example, by changing to a highly active acid halide (NPL 11). However, repeating the same reaction conditions as for the double coupling would double or more the time and reagent costs spent; and condensation with acid halides would require the preparation of the acid halide at the time of use and also adds to the question of no concern as to whether the acid halide produced during the peptide synthesis step can be stable. Furthermore, HCl and HF generated by this reaction raise a possible concern that deprotection reactions may proceed unintentionally.

Other measures for improving the low reactivity in the extension step have been tried, such as decreasing the density of peptide chains on the solid phase by decreasing the load on the resin and increasing the condensation efficiency by increasing the concentration of the reaction solution (NPL 9). Recently, efforts have been made to improve the condensation efficiency by increasing the reaction temperature via microwave irradiation (NPLs 12 and 13).

However, in the synthesis of N-methylated peptides, there is no report on a basic solution for the reduction in purity and yield of the peptide to be synthesized, and the desired product cannot be obtained at all in some cases.

[ Prior art reference ]

[ patent document ]

[PTL 1]WO 2013/100132 A1

[ non-patent document ]

[ NPL 1] S.R. Gracia et al, Synthesis of chemical modified biologicalpeptides: recourse enhancements, changements and requirements for media chemistry, future Med. chem.,2009,1,1289.

Chatterjee et al, N-Methylation of peptides, A new permselective media chemistry, Acc. chem. Res.,2008,41,1331.

[ NPL 3] J.E.bock et al, mounting in Shape: Controlling Peptide Bioactivityand Bioavailability Using structural constraints ACS chem.biol.,2013,8,488.

Jpsepthson et al, mRNA display from basic principles reverse display drive data, DOI 10.1016/j. drive.2013.10.011

[ NPL 5] M.teixido et al, Solid-phase synthesis and catalysis of N-methyl-rich peptides.J.peptide Res.,2005,65,153.

[ NPL 6] J.Urban et al, laboratory of N-alkylated peptides handbags TFAcleave. int.J.Pept.prot.Res.,1996,47,182.

[ NPL 7] L.A. Carpino et al, Dramatic enhanced N → O acyl migratory acid-based detection step in colloidal peptide synthesis, tetrahedron Lett.,2005,46,1361.

Eberhard et al, N → O-Acyl shift in Fmoc-based synthesis of phosphopeptides, org.biomol.chem.,2008,6,1349.

Marcucci et al, Solid-Phase Synthesis of NMe-IB-01212, a HighlyN-Methylated Cyclic peptide.

[NPL 10]Fang et al, Deletion of Ac-NMePhe¹From[NMePhe¹]arodyn UnderAcidic Conditions,Part 1:Effects of Cleavage Conditions and N-TerminalFunctionality.Peptide Science Vol.96,97

[ NPL11 ] L.A.Carpino et al, Stepwise Automated Solid Phase Synthesis of Naturally aerobic solids Using FMOC Amino Acid fluorides.J.Org.chem.,1995,60,405.

[ NPL12 ] H.Rodriguez et al, A conditional microwave-enhanced soluble-phase synthesis of short chain N-methyl-rich peptides.J.Pept.Sci.,2010,16,136.

[ NPL 13] R.Roodben et al, Microwave Heating in the Solid-Phase synthesis N-Methylated Peptides, where Is a Room Temperature setter? Eur.j.org.chem.,2012,7106.

Summary of The Invention

[ problem to be solved by the invention ]

Specifically, the present inventors found that, in the case of a peptide containing an N-alkylated amino acid, it is difficult to obtain a desired peptide because, during the reaction under acidic conditions using TFA as a step for cleaving from a solid phase or for deprotecting a protecting group for a side chain functional group, a side reaction in which a peptide chain is cleaved becomes a main reaction, when an amino acid having β -hydroxyl group is included in the peptide, under acidic conditions using TFA, it is also possible to perform an N-to-O-acyl transfer reaction during the reaction, it is difficult to obtain a desired peptide, it is further found that, in the case of a peptide containing an N-alkylated amino acid, under conditions using TFA compound having β -hydroxyl group, it is difficult to obtain a desired peptide, under conditions using TFA compound having β -hydroxyl group, it is not necessary to observe a problem in the known peptide, but the present inventors have newly observed a problem in the case of esterifying a peptide containing an N-alkylated amino acid, in addition to the problems of using TFA backbone, such as a TFA compound having β, and the following problems.

Furthermore, the present inventors found that, when considering the industrialization of peptide synthesis containing N-alkylated amino acids which may become drug-like peptides, the conventional deprotection method using TFA has considerable difficulty in achieving industrialization not only from the point of view of the deprotection reaction or extension reaction itself but also from the point of view of the subsequent post-treatment process and large-scale synthesis. For example, when the solvent of the TFA/DCM solution is removed by condensation, the TFA concentration increases as the condensation proceeds, and problems such as hydrolysis and N-to-O-acyl transfer occur simultaneously with the condensation. This may result in failure to obtain the desired compound or result in a significant reduction in yield. The condensation step needs to be performed at low temperature. Furthermore, even with low concentrations of TFA, a large excess of TFA compared to the target molecule is included; therefore, in order to stop the reaction by neutralizing TFA, a large excess of base needs to be added, which results in a large excess of salt remaining with the desired peptide, which may be troublesome to the purification step. Furthermore, although TFA itself is an effective solvent for dissolving the peptide, with only a low concentration of TFA solution, the solubility of the peptide will be low. Regarding solubility, not only when considering industrialization, but also in parallel synthesis where many different peptide compounds are handled at once, it is necessary to select a solvent having high solubility for a group of peptides.

In addition, the present inventors have looked at improving reactivity by reducing the steric size of the Protecting group of Fmoc-Amino acids having a Protecting group-carrying functional group on the side chain, which has not been done with much effort so far, for example, threonine (Thr) has a hydroxyl group, which is necessary for the subsequent acylation reaction to occur selectively on the Amino group, whereas, since Thr has a branched secondary alcohol as its side chain functional group at position β, the condensation efficiency of protected Thr is relatively low due to its bulky volume, the Protecting groups commonly used for Thr in peptide synthesis include acetyl (Ac), tBu, Trt, benzyl (Bn) and t-butyldimethylsilyl (TBS) (Albert Isido-Llobet al, Amino acid-Protecting groups, Chem.2012.rev., 2009,109,2455.; Watanabe Industries, Ltd, D, Amino acids & Protecting groups, L-serine & t, which are easily accessible to the deprotection of serine-Amino acids, which has been found to be easily accessible to the deprotection of Amino groups, even if the steric deprotection efficiency is not reduced by using a large number of Amino acids, and also the steric deprotection of Amino acids, which are found to be easily accessible to the steric deprotection of Amino acids, which is not found to be suitable for the problems, and also to be easily accessible to be able to be removed by acid-to be found to be easily accessible under the steric deprotection conditions which are not found to be suitable for the aforementioned steric deprotection conditions, which are not to be suitable for the high, which are generally, which are not to be used, which are not suitable for the high, which are not found to be used, which are not to be suitable for the steric deprotection conditions, in general, which are not to be suitable for the high, in general, which are not to be suitable for the deprotection conditions, which are found to be suitable for the deprotection conditions, which are not found to be used, which are not to be used.

More specifically, the object of the present invention is to find a novel reaction method which can reduce the problems of side reactions such as hydrolysis of peptide acid and N-to O-acyl transfer, and TFA esterification of hydroxyl group in the deprotection step using TFA, which are found to become significant during the parallel synthesis of peptides containing N-substituted amino acids, and which can also ensure the solubility of peptides. Further, an object of the present invention is to provide a method for obtaining a peptide comprising an N-substituted amino acid with high purity and high synthetic yield by using an appropriate protecting group for a side chain functional group (which is appropriate from the viewpoint of reducing the volume of the protecting group to improve low reactivity during extension and is deprotectable under the deprotection conditions of the present invention).

Specifically, the object was to perform parallel synthesis of peptide compounds containing N-substituted amino acids having various sequences:

(1) it was found that the reaction conditions required to inhibit hydrolysis, in particular hydrolysis originating from an N-substituted amino group, during acid addition (during cleavage reactions from the solid phase and during side chain deprotection reactions);

(2) reaction conditions under which actual post-treatment can be carried out at the time of acid addition were found;

(3) considering the characteristic solubility of the non-native peptide compound, reaction conditions were found to include a solvent; and

(4) when the non-natural peptide compound contains a functional group such as a hydroxyl group, side reactions after deprotection (N-to O-acyl transfer and side reactions between the hydroxyl group and a reactive agent, for example, TFA acylation reaction when TFA is used as a reagent) are suppressed.

In addition, the present invention has an object to find a protecting group which satisfies the above four conditions for various functional groups in the side chain of an amino acid.

Furthermore, in view of the industrial production of peptide compounds comprising N-substituted amino acids, it is an object of the present invention to find an optimized production method suitable for a specific sequence.

[ means for solving problems ]

In order to achieve efficient synthesis of cyclic peptides comprising N-substituted amino acids, the present inventors have found a novel method that can solve many problems other than those observed when using conventional peptide synthesis methods that use TFA in order to synthesize compounds described in known documents, such as inhibition of progress of hydrolysis and N-to-O-acyl transfer, establishment of actual post-treatment methods, inhibition of TFA ester formation when a hydroxyl group is present, and selection of solvents that can ensure peptide solubility, which are not sufficiently solved by commonly performed modification methods (such as a method of reducing TFA concentration or a method of reducing reaction temperature). In the new method, TFA used in conventional peptide synthesis was not used at all, and the present inventors succeeded in obtaining a target molecule with high selectivity.

In one embodiment of the invention, TFA is not used in the step of cleavage from the solid phase, but a weaker acid, such as 2,2, 2-Trifluoroethanol (TFE) or hexafluoro-2-propanol (HFIP), is used. In addition, in another embodiment of the present invention, a protecting group for a side chain functional group which is not deprotected in the cleavage step is used. In the cleavage step using an acid weaker than TFA, such as TFE or HFIP, the rate of side reactions such as hydrolysis of an amide bond is sufficiently low, unlike the case of using TFA, even in the concentration step after the reaction. In particular, when an acid weaker than TFA, such as TFE or HFIP, is used, the rate of side reactions is low even for peptides containing highly N-substituted amino acids and cyclic peptides sensitive to side reactions. Thus, the desired compound can be obtained as a main product. In another embodiment of the invention, reagents are used in the cleavage step that satisfy the following conditions: (1) the reaction of cleavage from the solid phase proceeds smoothly while side reactions (such as hydrolysis) of the peptide are suppressed; (2) the rate of side reactions is sufficiently slow even when post-treatments such as concentration are performed; (3) high solubility is also ensured for highly fat-soluble non-native peptides; and (4) can be cleaved while retaining the protecting group of the side chain functional group. By using reagents satisfying these conditions, synthesis of peptides comprising a number of N-substituted amino acids, in particular, synthesis of pharmaceutical-like peptides comprising a number of N-alkyl groups becomes possible. Reagents satisfying these conditions can be used not only during parallel synthesis but also when industrially synthesizing a specific peptide.

Embodiments of the present invention provide a peptide synthesis method that can suppress hydrolysis and N-to O-acyl transfer, and can deprotect a side chain protecting group, thereby promoting a main reaction, i.e., a desired deprotection reaction. The acid strength (proton concentration) alone may be important for the hydrolysis and the N-to-O-acyl transfer to proceed. Also, the present inventors have found that the progress of hydrolysis and N-to-O-acyl transfer can be suppressed by using a weak acid having weak acidity in place of a strong acid such as TFA. In addition, in order to carry out the desired deprotection, a step of cleaving a protecting group as a cation species (carbocation or oxonium cation) from the protected functional group may be important in addition to the acid strength (proton concentration). Therefore, as a solvent for promoting the step of dissociation of the protecting group as a cation species, the present inventors have found that deprotection of the weak acid can be promoted by using a solvent having an ionization ability.

In addition, in order to establish a highly efficient method for synthesizing the drug-like peptide described in PTL1, the present inventors found that a protecting group of an amino acid side chain functional group having a side chain with a small degree of ionization under neutral conditions, which protecting group is not deprotected under the weak acidic conditions used when cleaving a peptide from a resin, but can be deprotected under the above weak acidic conditions, and that the functional group is, for example, a hydroxyl group, which is a side chain functional group of amino acids such as Ser and Thr; an alkyl alcohol group having a hydroxyl group in a side chain; a phenol group which is a side chain functional group of an amino acid such as Tyr; imidazolyl, which is a side chain functional group of an amino acid such as His; side chain carboxylic acids, which are side chain functional groups of amino acids such as Asp and Glu; and backbone carboxylic acids of peptides or amino acids.

Furthermore, the present inventors have found that, in the case where an amino acid having low reactivity during the extension reaction, such as β -hydroxy- α -amino acid (e.g., Thr, Ser, and derivatives thereof), has a protecting group, the protecting group can be deprotected under the above-mentioned weak acid conditions and can improve low reactivity during the extension reaction.

More specifically, the present invention is:

[1] a method of producing a peptide comprising at least one N-substituted amino acid or N-substituted amino acid analog, wherein said method comprises the steps of:

1) preparing a peptide comprising one or both of the following functional groups i) and ii) (Fmoc-protected amino acids), an amino acid analog comprising at least one of the following i) and ii) (Fmoc-protected amino acid analog), or an Fmoc-protected amino acid and Fmoc-protected amino acid analog (Fmoc-protected peptide):

i) a backbone amino group protected by at least one protecting group having an Fmoc backbone; and

ii) at least one free carboxylic acid group or a reactive esterified carboxylic acid group;

2) supporting the Fmoc-protected amino acid, Fmoc-protected amino acid analog, or Fmoc-protected peptide prepared in step 1) onto a solid phase;

3) deprotecting a protecting group having an Fmoc skeleton of an Fmoc-protected amino acid, an Fmoc-protected amino acid analog or an Fmoc-protected peptide supported on a solid phase by using a base to expose an amino group thereof;

4) forming an amide bond by adding a new Fmoc-protected amino acid, a new Fmoc-protected amino acid analog, or a new Fmoc-protected peptide; and

5) cleaving the peptide obtained in step 4) from the solid phase under acidic conditions weaker than TFA;

[2] [1] the production method, wherein at least one side chain of an amino acid or an amino acid analog constituting the peptide obtained in step 4) is protected by a protecting group which is not protected under basic conditions but is deprotected by a first acid, and wherein the method further comprises a step of deprotecting the protecting group using the first acid before or after step 5); and

wherein in step 5) the peptide is cleaved off using a second acid, and

wherein the first acid and the second acid are both weaker acids than TFA, and the acidity of the first acid is higher than the acidity of the second acid;

[3] a method of producing a peptide comprising at least one N-substituted amino acid or N-substituted amino acid analog, wherein said method comprises the steps of:

1) preparing a peptide comprising one or both of the following functional groups i) and ii) (Fmoc-protected amino acids), an amino acid analog comprising at least one of the following functional groups i) and ii) (Fmoc-protected amino acid analog), or an Fmoc-protected amino acid and Fmoc-protected amino acid analog (Fmoc-protected peptide):

i) a backbone amino group protected by at least one protecting group having an Fmoc backbone; and

ii) at least one free carboxylic acid group or a reactive esterified carboxylic acid group;

2) deprotecting a protecting group having an Fmoc backbone of an Fmoc-protected amino acid, an Fmoc-protected amino acid analog or an Fmoc-protected peptide by using a base to expose an amino group thereof;

3) forming an amide bond by adding a new Fmoc-protected amino acid, a new Fmoc-protected amino acid analog, or a new Fmoc-protected peptide, wherein at least one side chain of the amino acid or amino acid analog constituting the peptide obtained in this step has a protecting group which is not deprotected under basic conditions and deprotected under conditions having a weaker acidity than TFA; and

4) deprotecting the protecting group of the side chain under conditions less acidic than TFA;

[4] [3] the production method, wherein the peptide production is carried out by a solid phase method;

[5] the production method of [4], further comprising a step of cleaving the peptide obtained in step 3) from the solid phase under a weaker condition than the weak acidic condition used in step 4), before or after step 4);

[6] [3] the production method, wherein the peptide preparation is carried out by a liquid phase method;

[7] the production process of any one of [1] to [6], wherein the step 4) of [1] or the step 3) of [3] further comprises the steps of:

deprotecting a protecting group having an Fmoc backbone on the newly added Fmoc-protected amino acid, the newly added Fmoc-protected amino acid analog or the newly added Fmoc-protected peptide by using a base to expose the amino group thereof;

by further adding a new Fmoc-protected amino acid, a new Fmoc-protected amino acid analogue or a new Fmoc-protected peptide, an amide bond is formed,

and wherein these steps are repeated one or more times;

[8] the production method of any one of [1] to [7], wherein the produced peptide comprises an amino acid residue or an amino acid analog residue containing one reactive site on the C-terminal side thereof and an amino acid residue, an amino acid analog residue or a carboxylic acid analog containing the other reactive site on the N-terminal side thereof;

[9] [8] the production method further comprising a step of bonding the reactive site and the other reactive site to cyclize the peptide;

[10] [9] the production method, wherein the amino acid residue, amino acid analog residue or carboxylic acid analog having the other reactive site is at the N-terminus, and the linkage is an amide linkage.

[11] [9] the production method, wherein the amino acid residue, amino acid analog residue or carboxylic acid analog having the other reactive site is at the N-terminus, and the bond is a carbon-carbon bond;

[12][1]to [11]]The production method of any one of, wherein the step carried out under acidic conditions having a weaker acidity than TFA is carried out using a weakly acidic solution containing a compound having an aqueous pKa value of 5 to 14 and an ionization capacity value Y thereof_OTsA weak acid having an aqueous pKa value of 0 to 9 in a positive solvent;

[13] [12] the production method, wherein the solvent is a fluoroalcohol;

[14] [13] the production process wherein the fluoroalcohol is TFE or HFIP.

[15] [2] to [14], wherein the side chain protecting group is a protecting group deprotected in a range of pH1 to pH7, or a protecting group deprotected in TFA at a concentration of 10% or less; and

[16] [2] to [15], wherein the side chain protecting group is selected from the following a) to d):

a) when the side chain protecting group is a protecting group for side chain hydroxyl group of Ser, Thr, Hyp (hydroxyproline) and derivatives thereof, any one protecting group selected from MOM skeleton, Bn skeleton, Dpm skeleton, Trt skeleton, silyl skeleton and Boc skeleton represented by the following general formula;

b) when the side chain protecting group is a protecting group for side chain hydroxyl group of Thr and its derivatives, any one protecting group selected from MOM skeleton, Bn skeleton, Dpm skeleton, Trt skeleton, silyl skeleton, Boc skeleton and tBu skeleton represented by the following general formula;

c) when the side chain protecting group is a protecting group for side chain imidazole ring of His and its derivatives, any one protecting group selected from MOM skeleton, Bn skeleton and Trt skeleton represented by the following general formula;

d) when the side chain protecting group is a protecting group for a side chain carboxylic acid group of Asp, Glu and derivatives thereof, any one protecting group selected from MOM skeleton, Bn skeleton, Dpm skeleton, Trt skeleton, tBu skeleton, phenyl-EDOTn skeleton, and orthoester skeleton (in which carbon atoms of a carboxylic acid group to be protected are substituted with three alkoxy groups) represented by the following general formula:

< protecting group having MOM skeleton >

(wherein

R1 is H, R2 is H, and X is methyl, benzyl, 4-methoxybenzyl, 2, 4-dimethoxybenzyl, 3, 4-dimethoxybenzyl or 2-trimethylsilylethyl;

r1 is methyl, R2 is H, X is ethyl;

r1, R2 and R3 are all methyl; or

R1 and X together form-CH₂-CH₂-CH₂-or-CH₂-CH₂-CH₂-CH₂-, and R2 is H,

wherein when any of R1, R2 and X is methyl or ethyl, these groups may be further substituted with alkyl, benzyl or aryl);

< protecting group having Bn skeleton >

(wherein

R1 to R5 are each independently H, alkyl, aryl or halogen, R6 and R7 are alkyl;

r1, R2, R4 and R5 are each independently H, alkyl, aryl or halogen, R3 is methoxy, R6 and R7 are H;

r1 and R3 are methoxy, R2, R4 and R5 are each independently H, alkyl, aryl or halogen, R6 and R7 are H; or

R1, R4 and R5 are each independently H, alkyl, aryl or halogen, and R2 and R3 together form-O-CH₂-O-)；

< protecting group having Dpm skeleton >

(wherein

R1 to R10 are each independently H, alkyl, aryl, alkoxy, or halogen; or

R1 to R4 and R7 to R10 are each independently H, alkyl, aryl, alkoxy or halogen, and R5 and R6 together form-O-or-CH₂-CH₂-)；

< protecting group having Trt skeleton >

(wherein

R1 to R15 are each independently H, alkyl, aryl, alkoxy, or halogen;

r1, R2 and R4 to R15 are each independently H, alkyl, aryl, alkoxy or halogen, and R3 is methyl or methoxy;

r1 is Cl and R2 to R15 are each independently H, alkyl, aryl, alkoxy, or halogen; or

R1 to R4 and R7 to R15 are each independently H, alkyl, aryl, alkoxy, or halogen, and R5 and R6 together form-O-);

< protecting group having silyl skeleton >

(wherein R1 to R3 are each independently alkyl or aryl);

< protecting group having Boc skeleton >

(wherein R1 to R9 are each independently H, alkyl, or aryl);

< protecting group having tBu skeleton >

(wherein

R1 to R9 are each independently H, alkyl or aryl); and

< protecting group having phenyl-EDOTn skeleton >

(wherein each of R1 to R3 is independently H or methoxy).

Effects of the invention

By the present invention, a peptide comprising an N-substituted amino acid can be obtained with high synthesis efficiency and high purity.

For example, in the case where the peptide sequence contains an amino acid having a protecting group on its side chain,

(1) the combination of an acid weaker than TFA and a solvent exhibiting ionization capability discovered by the present invention allows for run-off deprotection while minimizing acid hydrolysis of the peptide chain, and minimizing N-to O-acyl transfer, TFA esterification, and this can occur in sequences containing β -hydroxy- α -amino acids (e.g., Ser, Thr, and derivatives thereof), and

(2) when the amino acid is elongated by the amide bond formation reaction, the reaction rate and the reaction efficiency can be improved as compared to when the amino acid has a protecting group for general peptide synthesis.

Brief Description of Drawings

FIG. 1 shows the basic synthetic route for cyclic peptides comprising N-methyl amino acids in their sequence.

FIG. 2 shows the desired peptide (compound 131), the target molecule hydrolysate (TM + H) by LCMS analysis under deprotection conditions using 0.1M tetramethylammonium hydrogen sulfate/HFIP solution (2% TIPS)₂O) and the detection of the solvolysis product of the target molecule by HFIP (TM + HFIP).

FIG. 3 shows the desired peptide (compound 131), the target molecule hydrolysate (TM + H) by LCMS analysis under deprotection conditions using 0.05M tetramethylammonium hydrogen sulfate/HFIP solution (2% TIPS)₂O) and solvolysis product of the target molecule by HFIP (TM + H)FIP) was detected.

FIG. 4 shows the detection of the desired peptide (compound 133) and the N-to O-acyl transfer product of the desired product by LCMS analysis under deprotection conditions using 0.05M tetramethylammonium bisulfate/HFIP solution (2% TIPS).

FIG. 5 shows the desired peptide (Compound 131), the target molecule hydrolysis product (TM + H) by LCMS analysis under deprotection conditions using 0.05M oxalic acid/HFIP solution (2% TIPS)₂O) and the detection of the solvolysis product of the target molecule by HFIP (TM + HFIP).

FIG. 6 shows the desired peptide (Compound 131), the target molecule hydrolysis product (TM + H) by LCMS analysis under deprotection conditions using 0.05M maleic acid/HFIP solution (2% TIPS)₂O) and the detection of the solvolysis product of the target molecule by HFIP (TM + HFIP).

FIG. 7 shows the results of detection of N-to O-acyl transfer products of the desired peptide (compound 133) and target molecule by LCMS analysis under deprotection conditions using 0.05M oxalic acid/HFIP solution (2% TIPS).

FIG. 8 shows the detection of the N-to O-acyl transfer product of the desired peptide (compound 133) and target molecule by LCMS analysis under deprotection conditions using 0.05M maleic acid/HFIP solution (2% TIPS).

FIG. 9 shows the results of detection by LCMS analysis of the desired peptide (compound 137) and target molecule by the solvolysis product of HFIP (product in which any one of the amide bonds has been solvated by HFIP) under deprotection conditions using 0.05M tetramethylammonium hydrogen sulfate/HFIP (2% TIPS).

Fig. 10 shows the results of detection of the desired peptide (compound 137) and target molecule by solvolysis of TFE (a product in which any one of the amide bonds has been solvolyzed by TFE) by LCMS analysis under deprotection conditions using 0.05M tetramethylammonium hydrogen sulfate/TFE (2% TIPS).

FIG. 11 shows the results of detection of the desired peptide (compound 135) by LCMS analysis when a 0.1M tetramethylammonium hydrogensulfate/HFIP solution (2% TIPS) was used as a deprotection condition and a base (DIPEA) was added to the solution to stop the reaction.

FIG. 12 shows the results of detection of the desired peptide (Compound 133) by LCMS analysis when a 0.1M tetramethylammonium hydrogensulfate/HFIP solution (2% TIPS) was used as the deprotection condition and a base (DIPEA) was added to the solution to stop the reaction.

FIG. 13 shows the results of detection of the desired peptide (compound 112) and the product of Thr removal from the desired peptide (compound 113) by LCMS analysis when Fmoc-Thr (Trt) -OH was added.

FIG. 14 shows the results of detection of the desired peptide (compound 114) by LCMS analysis when Fmoc-Thr (THP) -OH was added. No product (Compound 113) lacking Thr from the desired peptide (Compound 114) was detected.

FIG. 15 shows the results of detection of the desired peptide (compound 115) and the product of deletion of MeSer from the desired peptide (compound 116) by LCMS analysis when synthesized using Fmoc-MeSer (DMT) -OH.0.75DIPEA.

FIG. 16 shows the results of detection of the desired peptide (compound 115) and the product of deletion of MeSer from the desired peptide (compound 116) by LCMS analysis when synthesized using Fmoc-MeSer (THP) -OH (compound 6).

FIG. 17 shows the hydrolysis product of the desired peptide (compound 131) and the target molecule (TM + H) by LCMS analysis under deprotection conditions using 5% TFA/DCE (5% TIPS)₂O) is detected.

FIG. 18 shows the results of detection of the desired peptide (compound 133), the N-to O-acyl transfer product of the target molecule, a compound in which one hydroxyl group of the target molecule is esterified with TFA, and a compound in which two hydroxyl groups of the target molecule are esterified with TFA, by LCMS analysis under deprotection conditions using 5% TFA/DCE (5% TIPS).

FIG. 19 shows the results of detection of the desired peptide (compound 133), the N-to O-acyl transfer product of the target molecule, a compound in which one hydroxyl group of the target molecule is esterified with TFA, and a compound in which two hydroxyl groups of the target molecule are esterified with TFA, by LCMS analysis under deprotection conditions using 5% TFA/DCE (5% TIPS) (0 ℃).

FIG. 20 shows the results of detection of the desired peptide (compound 133), the N-to O-acyl transfer product of the target molecule, a compound in which one hydroxyl group of the target molecule is esterified with TFA, and a compound in which two hydroxyl groups of the target molecule are esterified with TFA, by LCMS analysis under deprotection conditions using 5% TFA/DCE (5% TIPS) (25 ℃).

FIG. 21 shows a synthetic method involving an extension reaction in the liquid phase.

Modes for carrying out the invention

In certain embodiments, the present invention relates to a method of producing a peptide comprising at least one N-substituted amino acid or N-substituted amino acid analog, wherein the method comprises the steps of:

1) preparing an amino acid having at least one of the following functional groups i) and ii) (Fmoc-protected amino acid), an amino acid analog having at least one of the following i) and ii) (Fmoc-protected amino acid analog), or a peptide comprising one or both of Fmoc-protected amino acid and Fmoc-protected amino acid analog (Fmoc-protected peptide):

i) a backbone amino group protected by at least one protecting group having an Fmoc backbone; and

ii) at least one free carboxylic acid group or a reactive esterified carboxylic acid group;

2) supporting the Fmoc-protected amino acid, Fmoc-protected amino acid analog, or Fmoc-protected peptide prepared in step 1) onto a solid phase;

3) deprotecting a protecting group having an Fmoc skeleton of an Fmoc-protected amino acid, an Fmoc-protected amino acid analog or an Fmoc-protected peptide supported on a solid phase by using a base to expose an amino group thereof;

4) forming an amide bond by adding a new Fmoc-protected amino acid, a new Fmoc-protected amino acid analog, or a new Fmoc-protected peptide; and

5) the peptide obtained in step 4) was cleaved from the solid phase under acidic conditions weaker than TFA.

In another embodiment, the present invention relates to a method of producing a peptide comprising at least one N-substituted amino acid or N-substituted amino acid analog, wherein said method comprises the steps of:

1) preparing an amino acid having at least one of the following functional groups i) and ii) (Fmoc-protected amino acid), an amino acid analog having at least one of the following functional groups i) and ii) (Fmoc-protected amino acid analog), or a peptide comprising one or both of Fmoc-protected amino acid and Fmoc-protected amino acid analog (Fmoc-protected peptide):

i) a backbone amino group protected by at least one protecting group having an Fmoc backbone; and

ii) at least one free carboxylic acid group or a reactive esterified carboxylic acid group;

2) deprotecting a protecting group having an Fmoc backbone of an Fmoc-protected amino acid, an Fmoc-protected amino acid analog or an Fmoc-protected peptide by using a base to expose an amino group thereof;

3) forming an amide bond by adding a new Fmoc-protected amino acid, a new Fmoc-protected amino acid analog, or a new Fmoc-protected peptide, wherein at least one side chain of the amino acid or amino acid analog constituting the peptide obtained in this step has a protecting group which is not deprotected under basic conditions and deprotected under conditions having a weaker acidity than TFA; and

4) deprotecting the protecting group of the side chain under conditions less acidic than TFA;

the production of the above-mentioned peptide can be carried out by a solid phase method or a liquid phase method.

The "peptide" in the present invention is not particularly limited as long as it is a peptide formed by amide bonding or ester bonding of an amino acid and/or an amino acid analog, and is preferably a peptide of 5 to 30 residues, more preferably a peptide of 7 to 15 residues, and even more preferably a peptide of 9 to 13 residues. The peptides synthesized in the present invention comprise at least one or more amino acids or amino acid analogs that have been N-substituted (also referred to as N-substituted amino acids), preferably two or more, more preferably three or more, even more preferably five or more N-substituted amino acids in a single peptide. These N-substituted amino acids may be present in the peptide either continuously or discontinuously.

The peptide in the present invention may be a linear peptide or a cyclic peptide, preferably a cyclic peptide.

The "cyclic peptide" in the present invention can be obtained by synthesizing a linear peptide according to the method of the present invention and then cyclizing it. The cyclization may be in any form, for example, cyclization through a carbon-nitrogen bond such as an amide bond, cyclization through a carbon-oxygen bond such as an ester bond or an ether bond, cyclization through a carbon-sulfur bond such as a thioether bond, cyclization through a carbon-carbon bond or cyclization through the construction of a heterocyclic ring. Although not particularly limited, the cyclization is preferably performed by covalent bonding such as carbon-carbon bonding or amide bonding, and particularly preferably performed by amide bonding formed by a side chain carboxylic acid group and an N-terminal main chain amino group. The position of the carboxylic acid group, amino group, or the like used in the cyclization may be on the main chain or on the side chain, and is not particularly limited as long as they are at positions where cyclization is possible.

Specific examples of the N-substituted amino acid include amino acids or amino acid analogs in which the main chain amino group of the "amino acid" or "amino acid analog" described later is N-substituted, preferably N-alkylated (e.g., N-methylated) amino acids or amino acid analogs, specific examples of the N-substituted amino acid include amino acids or amino acid analogs in which the main chain amino group is an NHR group, wherein R is an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, an optionally substituted aryl group, an optionally substituted heteroaryl group, an optionally substituted aralkyl group or an optionally substituted cycloalkyl group, or alternatively, those in which a carbon atom bonded to the N atom forms a ring with a carbon atom at position α, such as proline.

Specifically, for such an N-substituted amino acid, an alkyl group, an aralkyl group, a cycloalkyl group, or the like is preferably used.

"amino acids" in the present invention are α -, β -and γ -amino acids, and are not limited to natural amino acids, and may be unnatural amino acids (in the present invention, "natural amino acids" refer to 20 kinds of amino acids contained in proteins, specifically, they refer to Gly, Ala, Ser, Thr, Val, Leu, Ile, Phe, Tyr, Trp, His, Glu, Asp, gin, Asn, Cys, Met, Lys, Arg and Pro.) in the case of α -amino acids, they may be L-amino acids or D-amino acids, or may also be α -dialkyl amino acids, selection of amino acid side chains is not particularly limited, but examples include hydrogen atom, alkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, aralkyl group and cycloalkyl group.

The term "amino acid analog" in the present invention preferably means α -hydroxycarboxylic acid, like amino acids, the side chain of α -hydroxycarboxylic acid is also not particularly limited, and examples thereof include hydrogen atom, alkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, aralkyl group and cycloalkyl group. α -hydroxycarboxylic acid may have a steric structure corresponding to the L-or D-form of amino acid.

The "amino acids" or "amino acid analogs" that constitute the peptides synthesized in the present invention include all of their respective corresponding isotopes. An isotope in an "amino acid" or "amino acid analog" refers to an isotope in which at least one atom is replaced with an atom of the same atomic number (the same proton number) and different mass numbers (different in the sum of the proton number and the neutron number). Examples of isotopes contained in "amino acids" or "amino acid analogs" constituting the peptide compound of the present invention include hydrogen atoms, carbon atoms, nitrogen atoms, oxygen atoms, phosphorus atoms, sulfur atoms, fluorine atoms and chlorine atoms, and specific examples include²H、³H、¹³C、¹⁴C、¹⁵N、¹⁷O、¹⁸O、³¹P、³²P、³⁵S、¹⁸F and³⁶Cl。

the amino acid or amino acid analog may have one or two or more substituents. Examples of such substituents include those derived from an O atom, an N atom, an S atom, a B atom, a P atom, an Si atom, and a halogen atom.

Examples of halogen-derived substituents include fluorine (-F), chlorine (-Cl), bromine (-Br), and iodine (-I).

Examples of the substituent derived from an O atom include a hydroxyl group (-OH), an oxy group (-OR), a carbonyl group (-C ═ O-R), a carboxyl group (-CO)₂H) An oxycarbonyl group (-C ═ O-OR), a carbonyloxy group (-O-C ═ O-R), a thiocarbonyl group (-C ═ O-SR), a carbonylthio group (-S-C ═ O-R), an aminocarbonyl group (-C ═ O-NHR), a carbonylamino group (-NH-C ═ O-R), an oxycarbonylamino group (-NH-C ═ O-OR), a sulfonylamino group (-NH-SO)₂-R), aminosulfonyl (-SO)₂-NHR), sulfamoylamino (-NH-SO)₂-NHR), thiocarboxyl (-C (═ O) -SH) and carboxycarbonyl (-C (═ O) -CO₂H)。

Examples of the oxy group (-OR) include alkoxy, cycloalkoxy, alkenyloxy, alkynyloxy, aryloxy, heteroaryloxy and aralkyloxy groups.

Examples of the carbonyl group (-C ═ O-R) include formyl group (-C ═ O-H), alkylcarbonyl group, cycloalkylcarbonyl group, alkenylcarbonyl group, alkynylcarbonyl group, arylcarbonyl group, heteroarylcarbonyl group, and aralkylcarbonyl group.

Examples of the oxycarbonyl group (-C ═ O-OR) include an alkoxycarbonyl group, a cycloalkoxycarbonyl group, an alkenyloxycarbonyl group, an alkynyloxycarbonyl group, an aryloxycarbonyl group, a heteroaryloxycarbonyl group, and an aralkoxycarbonyl group.

(-C＝O-OR)

Examples of the carbonyloxy group (-O-C ═ O-R) include alkylcarbonyloxy, cycloalkylcarbonyloxy, alkenylcarbonyloxy, alkynylcarbonyloxy, arylcarbonyloxy, heteroarylcarbonyloxy and aralkylcarbonyloxy.

Examples of thiocarbonyl (-C ═ O-SR) include alkylthiocarbonyl, cycloalkylthiocarbonyl, alkenylthiocarbonyl, alkynylthiocarbonyl, arylthiocarbonyl, heteroarylthiocarbonyl and aralkylthiocarbonyl.

Examples of carbonylthio (-S-C ═ O-R) include alkylcarbonylthio, cycloalkylcarbonylthio, alkenylcarbonylthio, alkynylcarbonylthio, arylcarbonylthio, heteroarylcarbonylthio and aralkylcarbonylthio.

Examples of aminocarbonyl (-C ═ O-NHR) include alkylaminocarbonyl, cycloalkylaminocarbonyl, alkenylaminocarbonyl, alkynylaminocarbonyl, arylaminocarbonyl, heteroarylaminocarbonyl and aralkylaminocarbonyl. Further examples include compounds produced by further substituting an H atom bonded to an N atom in-C ═ O-NHR with an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or an aralkyl group.

Examples of carbonylamino (-NH-C ═ O-R) include alkylcarbonylamino, cycloalkylcarbonylamino, alkenylcarbonylamino, alkynylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino and aralkylcarbonylamino. Further examples include compounds produced by further substituting an H atom bonded to an N atom in-NH-C ═ O-R with an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or an aralkyl group.

Examples of the oxycarbonylamino (-NH-C ═ O-OR) include alkoxycarbonylamino, cycloalkoxycarbonylamino, alkenyloxycarbonylamino, alkynyloxycarbonylamino, aryloxycarbonylamino, heteroaryloxycarbonylamino and aralkyloxycarbonylamino. Further examples include compounds produced by further substituting an H atom bonded to an N atom in-NH-C ═ O-OR with an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, OR an aralkyl group.

Sulfonylamino (-NH-SO)₂Examples of-R) include alkylsulfonylamino, cycloalkylsulfonylamino, alkenylsulfonylamino, alkynylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino and aralkylsulfonylamino. Additional examples include by further reacting with-NH-SO₂-a compound wherein the H atom bonded to the N atom in R is substituted with an alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl or aralkyl group.

Aminosulfonyl (-SO)₂Examples of-NHR) include alkanesAlkylaminosulfonyl, cycloalkylaminosulfonyl, alkenylaminosulfonyl, alkynylaminosulfonyl, arylaminosulfonyl, heteroarylaminosulfonyl and aralkylaminosulfonyl. Additional examples include by further reacting with-SO₂-NHR wherein the N-bonded H atom is substituted with an alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl or aralkyl group.

Sulfamoylamino (-NH-SO)₂-NHR) includes alkylsulfamoylamino, cycloalkylsulfamoylamino, alkenylsulfamoylamino, alkynylsulfamoylamino, arylsulfamoylamino, heteroarylsulfamoylamino and aralkylsulfamoylamino. In addition, -NH-SO₂-two H atoms bonded to the N atom in the NHR may be substituted by substituents independently selected from the group consisting of alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl and aralkyl; or the two substituents may form a ring.

Examples of the substituent derived from the S atom include thiol (-SH), thio (-S-R), sulfinyl (-S-O-R), sulfonyl (-S (O))₂-R) and a sulfo group (-SO)₃H)。

Examples of thio (-S-R) are selected from alkylthio, cycloalkylthio, alkenylthio, alkynylthio, arylthio, heteroarylthio, aralkylthio and the like.

Examples of sulfinyl (-S ═ O-R) include alkylsulfinyl, cycloalkylsulfinyl, alkenylsulfinyl, alkynylsulfinyl, arylsulfinyl, heteroarylsulfinyl and aralkylsulfinyl.

Sulfonyl (-S (O)₂Examples of-R) include alkylsulfonyl, cycloalkylsulfonyl, alkenylsulfonyl, alkynylsulfonyl, arylsulfonyl, heteroarylsulfonyl and aralkylsulfonyl.

For substituents derived from the N atom, examples include azides (-N)₃Also known as "azido"), cyano (-CN), primary amino (-NH)₂) (ii), a secondary amino group (-NH-R), a tertiary amino group (-NR (R')), an amidino group (-C (═ NH) -NH)₂) Substituted amidino (-C (-NR) -NR' R "), guanidino (-NH-C (-NH) -NH)₂) Substituted, byGuanidino (-NR-C (═ NR ' ") -NR ' R") and aminocarbonylamino (-NR-CO-NR ' R ").

Examples of secondary amino groups (-NH-R) include alkylamino, cycloalkylamino, alkenylamino, alkynylamino, arylamino, heteroarylamino and aralkylamino.

Examples of tertiary amino groups (-NR (R')) include amino groups having any two substituents, such as alkyl (aralkyl) amino groups, each substituent being independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl; and the two optional substituents may form a ring.

Examples of substituted amidino groups (-C (═ NR) -NR 'R ") include groups in which the three substituents R, R' on the N atom and R" are each independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl; and examples thereof include alkyl (aralkyl) (aryl) amidino groups.

Examples of substituted guanidino (-NR-C (═ NR '") -NR' R") include those wherein R, R ', R ", and R'" are each independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl; or groups in which they form a ring.

Examples of aminocarbonylamino (-NR-CO-NR 'R') include those wherein R, R 'and R' are each independently selected from the group consisting of hydrogen atom, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl and aralkyl; or groups in which they form a ring.

Examples of substituents derived from a B atom include a boryl group (-BR (R ')) and a dioxyboryl group (-B (OR)) (OR')). The two substituents R and R' are each independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl and aralkyl; or they may form a ring.

Thus, the amino acid or amino acid analog of the present invention may have one or two or more of various substituents commonly used for small molecule compounds, including an O atom, an N atom, an S atom, a B atom, a P atom, an Si atom, and a halogen atom. These substituents may be further substituted with other substituents.

Herein, "amino acid" and "amino acid analog" constituting the peptide synthesized in the present invention are also referred to as "amino acid residue" and "amino acid analog residue", respectively.

In the present invention, "Fmoc-protected amino acids" and "Fmoc-protected amino acid analogs" are amino acids and amino acid analogs having at least one of the following functional groups i) and ii), respectively:

i) a backbone amino group protected by at least one protecting group having an Fmoc backbone; and

ii) at least one free carboxylic acid group or a reactive esterified carboxylic acid group.

The "protecting group having Fmoc skeleton" in the present invention refers to an Fmoc group or a group formed by introducing an arbitrary substituent at an arbitrary position constituting the Fmoc skeleton of the Fmoc group. Specific examples of the protecting group having Fmoc skeleton include a 9-fluorenylmethoxycarbonyl (Fmoc) group, a 2, 7-di-t-butyl-Fmoc (Fmoc @) group, a 2-fluoro-Fmoc (2F)) group, a 2-monoisooctyl-Fmoc (Mio-Fmoc) group and a 2, 7-diisooctyl-Fmoc (Dio-Fmoc) group. In the present invention, a protecting group which can be deprotected (deprotectable) under basic conditions or by a nucleophile showing basicity (such as piperidine or hydrazine) may also be used in place of the protecting group having an Fmoc skeleton. Specific examples of such protecting groups include a 2- (4-nitrophenylsulfonyl) ethoxycarbonyl (Nsc) group, a (1, 1-dioxobenzo [ b ] group]Thien-2-yl) methoxycarbonyl (Bsmoc) radicals, (1, 1-dioxonaphtho [1,2-b ]]Thien-2-yl) methoxycarbonyl (α -Nsmoc) group, 1- (4,4-dimethyl-2, 6-dioxocyclohex-l-yl) -3-methylbutyl (l- (4,4-dimethyl-2, 6-dioxacyclohex-l-ylidine) -3-methylbutylyl) (ivDde) group, Tetrachlorophthaloyl (TCP) group, 2- [ phenyl (methyl) sulfonium group]Ethoxycarbonyltetrafluoroborate (Pms) group, ethylsulfonylethoxycarbonyl (Esc) group, 2- (4-sulfophenylsulfonyl) ethoxycarbonyl (Sps) group. In addition, protecting groups deprotectable by means other than acid or base may also be used. Specific examples of such protecting groups include benzyloxycarbonyl (Z) groups deprotectable by hydrogenation in the presence of a transition metal catalyst such as palladium, by a combination of a palladium catalyst and a scavenger (e.g., tetrakis (triphenylphosphine) palladium (0) (Pd (PPh)₃)₄) And phenylsiliconesA combination of alkanes) an allyloxycarbonyl (Alloc) group that can be deprotected, an o-nitrobenzenesulfonyl (oNBS, Ns) group that can be deprotected by a combination of an alkyl or aryl thiol and a base, a 2, 4-dinitrobenzenesulfonyl (dNBS) group and a dithiosuccinoyl (Dts) group, and by a reducing agent such as sodium dithionite (Na) or sodium dithionite (dTS)₂S₂O₄) Or a p-nitrobenzyloxycarbonyl group (pNZ) deprotectable by reduction by hydrogenation in the presence of a transition metal catalyst (reference: amino Acid-Protecting Groups, chem. Rev.2009,109, 2455-2504).

In the present invention using the Fmoc method, Fmoc-protected amino acids or Fmoc-protected amino acid analogs that can be preferably used are, for example, those in which the main chain amino group is protected by an Fmoc group, the side chain functional group is protected, if necessary, by a protecting group that is not cleaved with a base such as piperidine or DBU, and the main chain carboxylic acid group is not protected. It is also preferred to use Fmoc-protected amino acids or Fmoc-protected amino acid analogs having an amino group protected by a protecting group having an Fmoc backbone and a carboxylic acid group not having a protecting group.

In the present invention, when the Fmoc-protected amino acid, Fmoc-protected amino acid analog or Fmoc-protected peptide has side chain functional groups, these functional groups are preferably protected by protecting groups. When the side chain functional group is protected with a protecting group, a well-known protecting group which can be deprotected under the selected conditions can be used. Such protecting groups are preferably protecting groups which do not cleave under basic conditions and deprotect under acidic conditions which are weaker than TFA. Protecting groups which can be deprotected under acidic conditions include those which can be deprotected in the range of, for example, pH1 to pH7, preferably pH2 to pH 6. Alternatively, a protecting group which can be deprotected by 10% or less of TFA, or a protecting group having a structure described later can be used. In the present invention, a known protecting group can be used as the side chain protecting group. For example, among the protecting groups described in the following documents i) and ii), those satisfying the above conditions can be used as side chain protecting groups.

NPL i) Green's Protective Groups in Organic Synthesis, fourth edition

NPL ii)Chemical Reviews,2009,109(6),2455-2504

The method of the invention can be used for peptide synthesis by parallel synthesis. In this case, a protecting group is not always necessary for an amino acid side chain, but when a protecting group is necessary for a side chain, it is preferable to deprotect the protecting group used rapidly under the deprotection conditions of the present invention. Preferably, the side chain protecting groups are deprotected in 50% in 24 hours or less, and particularly preferably, the side chain protecting groups are deprotected in 90% in 4 hours or less. As a protecting group satisfying these conditions, a protecting group having a Trt skeleton, a THP skeleton, a THF skeleton, or a TBS skeleton described later is preferable. In order to enable easy deprotection with an acid and ensure high reactivity during extension, a protecting group having at least one hydrogen atom substituent on the protecting group atom directly bonded to the functional group (three-dimensional volume smaller than that of a protecting group having a Trt skeleton) is preferable. Among them, a protecting group in which a substituent other than hydrogen forms a ring is more preferable, and THP and THF are particularly preferable.

The method of the present invention can also be used for industrial peptide synthesis. In this case, the amino acid side chains are not always required to have a protecting group as in the case of parallel synthesis, but when the side chains have a protecting group, they preferably have the same protecting group as in parallel synthesis. For the sequence of the peptide to be synthesized, if there is no problem of hydrolysis and N-to-O-acyl transfer during deprotection, but the extension reaction is problematic due to the bulky size of the protecting group, a strong acid such as TFA, which is generally used for deprotection, can be used for deprotection. Furthermore, bulky protecting groups can also be used when there is no problem with the extension reaction of the peptide to be synthesized.

In the present invention, "the acidic condition weaker than TFA" preferably includes a condition of using a weakly acidic solution which is contained in a solution having an aqueous pKa value of 5 to 14 and an ionization capacity value Y thereof_OTsIs a weak acid having an aqueous pKa value of 0 to 9 in a positive solvent.

"weak acid having an aqueous pKa value of from 0 to 9" is more preferably a weak acid having an aqueous pKa of from 1 to 5. These weak acids specifically include tetramethylammonium hydrogen sulfate (aqueous pKa ═ 2.0), oxalic acid (aqueous pKa ═ 1.23), and maleic acid (aqueous pKa ═ 1.92). The concentration of the weak acid dissolved in the solvent may be any concentration as long as the condition that shows weaker acidity than TFA is satisfied.

"has an aqueous pKa value of 5 to 14 and its ionization capacity value Y_OTsThe "positive solvent" preferably includes a fluoroalcohol. Fluoroalcohol is a general term for alcohols in which a fluorine atom is bonded to a carbon atom other than the carbon atom bonded to a hydroxyl group among the carbon atoms constituting the alcohol. In the present invention, an alcohol in which a hydroxyl group is bonded to an aromatic ring, such as 2,3,4,5, 6-pentafluorophenol, is also included in the fluoroalcohol. Preferred fluoroalcohols are 2,2, 2-Trifluoroethanol (TFE) and hexafluoro-2-propanol (HFIP).

In the present invention, other organic solvents (e.g., dichloromethane or 1, 2-dichloroethane), cation scavengers (e.g., triisopropylsilane), and the like may be further added to the above weakly acidic solution as long as the conditions for generating a weaker acidity than TFA are satisfied.

In the present invention, when the Fmoc-protected amino acid or Fmoc-protected amino acid analog has a protecting group on its side chain, preferable examples of such side chain protecting groups are as described below.

When the side chain protecting groups are those for the hydroxyl group of Ser, Thr, Hyp and derivatives thereof, protecting groups having MOM skeleton, Bn skeleton, Dpm skeleton, Trt skeleton, silyl skeleton or Boc skeleton, each of which is represented by the following general formula, are preferable.

Representative examples of protecting groups having a MOM skeleton include MOM (R1 ═ H, R2 ═ H, X ═ Me), EE (R1 ═ Me, R2 ═ H, X ═ Et), MIP (R1 ═ Me, R2 ═ Me, X ═ Me), THP (R2 ═ H, a cyclic structure having four carbon atoms through R1 and X), THF (R2 ═ H, a cyclic structure having three carbon atoms through R1 and X), and SEM (R1 ═ H, R2 ═ H, X ═ 2-trimethylsilylethyl). With respect to the Me and Et substituents on the backbone, one can use a backbone substituted with other substituents such as alkyl, benzyl, and aryl.

Representative examples of the protecting group having a Bn skeleton include Pis (R6 ═ Me, R7 ═ Me, and other R ═ H), PMB (R3 ═ OMe, and other R ═ H), and DMB (R1 ═ OMe, R3 ═ OMe, and other R ═ H). Instead of Me substituents, another alkyl group may be used. Further, the benzene ring may have a substituent such as an alkyl group, an aryl group and a halogen group.

Representative examples of protecting groups having a tpm backbone include tpm (all R ═ H). The aromatic ring may have a substituent such as an alkyl group, an aryl group, an alkoxy group, and a halogen group.

Groups in which R5 and R6 are bridged can also be used, for example a Xan group in which R5 and R6 are bridged by an oxygen atom or a dibenzosuberyl group in which R5 and R6 are bridged by two carbon atoms.

Representative examples of protecting groups having a Trt backbone include Trt (all R ═ H), Mmt (R3 ═ Me, other R ═ H), Mtt (R3 ═ OMe, other R ═ H), Dmt (R3 ═ OMe, R8 ═ OMe, other R ═ H), and Clt (R1 ═ Cl, other R ═ H). The aromatic ring may have a substituent such as an alkyl group, an aryl group, an alkoxy group, and a halogen group.

In addition, groups in which R5 and R6 are bridged, such as Pixyl groups in which R5 and R6 are bridged by an oxygen atom, may also be used.

Representative examples of a protecting group having a silyl skeleton include TBS (R1 ═ Me, R2 ═ Me, R3 ═ tBu). Instead of Me and tBu, other groups such as alkyl and aryl may be substituents.

Representative examples of a protecting group having a Boc backbone include Boc (all R ═ H), but it may be substituted with other alkyl groups, aryl groups, and the like.

In addition, the following protecting groups may be used.

Among these protecting groups, THP and Trt are particularly preferable. In addition, when the amino acid residue is Ser, THP and Trt are particularly preferable as side chain protecting groups, and when the amino acid residue is Thr, THP is particularly preferable as a side chain protecting group.

When the side chain protecting groups are those for an amino acid having an aryl group with a hydroxyl group substituent, such as Tyr, D-Tyr or Tyr (3-F), for example, protecting groups having a MOM skeleton, a Bn skeleton, a Dpm skeleton, a Trt skeleton, a silyl skeleton, a Boc skeleton or a tBu skeleton represented by the following general formula are preferable.

Representative examples of protecting groups having a MOM skeleton include MOM (R1 ═ H, R2 ═ H, X ═ Me), BOM (R1 ═ H, R2 ═ H, X ═ Bn), EE (R1 ═ Me, R2 ═ H, X ═ Et), THP (R2 ═ H, a cyclic structure having four carbon atoms through R1 and X), THF (R2 ═ H, a cyclic structure having three carbon atoms through R1 and X), and SEM (R1 ═ H, R2 ═ H, X ═ 2-trimethylsilylethyl). With respect to the Me and Et substituents on the backbone, it is also possible to use a backbone substituted with other substituents such as alkyl, benzyl or aryl groups.

Representative examples of the protecting group having a Bn skeleton include Pis (R6 ═ Me, R7 ═ Me, and other R ═ H), PMB (R3 ═ OMe, and other R ═ H), and DMB (R1 ═ OMe, R3 ═ OMe, and other R ═ H). Other alkyl groups may be used in place of the Me substituents. Further, the benzene ring may have a substituent such as an alkyl group, an aryl group and a halogen group.

Representative examples of protecting groups having a tpm backbone include tpm (all R ═ H). The aromatic ring may have a substituent such as an alkyl group, an aryl group, an alkoxy group, and a halogen group.

Groups in which R5 and R6 are bridged can also be used, for example a Xan group in which R5 and R6 are bridged by an oxygen atom or a dibenzosuberyl group in which R5 and R6 are bridged by two carbon atoms.

Representative examples of protecting groups having a Trt backbone include Trt (all R ═ H), Mmt (R3 ═ Me, other R ═ H), Mtt (R3 ═ OMe, other R ═ H), and Clt (R1 ═ Cl, other R ═ H). The aromatic ring may have a substituent such as an alkyl group, an aryl group, an alkoxy group, and a halogen group.

In addition, groups in which R5 and R6 are bridged, such as Pixyl groups in which R5 and R6 are bridged by an oxygen atom, may also be used.

Representative examples of a protecting group having a silyl skeleton include TBS (R1 ═ Me, R2 ═ Me, R3 ═ tBu). Instead of Me and tBu, other groups such as alkyl and aryl may be substituents.

Representative examples of protecting groups having a Boc backbone include Boc (all R ═ H), but it may be substituted with other groups such as alkyl, aryl.

Representative examples of protecting groups having a tBu backbone include tBu (all R ═ H). It may have a substituent such as alkyl and aryl in place of H.

Among these protecting groups, tBu, Pis, Trt, Clt, THP and THF are particularly preferred. Further, when the amino acid residue is Tyr or D-Tyr, the side chain protecting group is particularly preferably tBu, Trt, Clt or THP, and when the amino acid residue is Tyr (3-F), the side chain protecting group is particularly preferably tBu or Pis.

When the side chain protecting groups are those for amino acids having imidazole in the side chain thereof (e.g., His or MeHis), it is preferable to use a protecting group represented by the following general formula, for example, having MOM skeleton, Bn skeleton or Trt skeleton.

Representative examples of protecting groups having a MOM skeleton include MBom (R1 ═ H, R2 ═ H, X ═ 4-methoxybenzyl), 2,4-DMBom (R1 ═ H, R2 ═ H, X ═ 2, 4-dimethoxybenzyl), 3,4-DMBom (R1 ═ H, R2 ═ H, X ═ 3, 4-dimethoxybenzyl), EE (R1 ═ Me, R2 ═ H, X ═ Et), THP (R2 ═ H, a cyclic structure having four carbon atoms through R1 and X), and THF (R2 ═ H, a cyclic structure having three carbon atoms through R1 and X). With respect to the Me and Et substituents on the backbone, it is also possible to use a backbone having protecting groups substituted with other substituents such as alkyl, benzyl or aryl.

Representative examples of the protecting group having a Bn skeleton include Pis (R6 ═ Me, R7 ═ Me, and other R ═ H), PMB (R3 ═ OMe, and other R ═ H), and DMB (R1 ═ OMe, R3 ═ OMe, and other R ═ H). Other alkyl groups may be used in place of the Me substituents. Further, the benzene ring may have a substituent such as an alkyl group, an aryl group and a halogen group.

Representative examples of protecting groups having a Trt backbone include Trt (all R ═ H), Mmt (R3 ═ Me, other R ═ H), Mtt (R3 ═ OMe, other R ═ H), and Clt (R1 ═ Cl, other R ═ H). The aromatic ring may have a substituent such as an alkyl group, an aryl group, an alkoxy group, and a halogen group.

Among them, Trt is particularly preferable. Further, when the amino acid residue is His or MeHis, the side chain protecting group is particularly preferably Trt.

Further, a protecting group having a MOM skeleton, a Bn skeleton, a Dpm skeleton, a Trt skeleton, a tBu skeleton or a phenyl-EDOTn skeleton represented by the following general formula may be used, for example, as a protecting group for a side chain carboxylic acid group of Asp, Glu and derivatives thereof (when the main chain carboxylic acid group is used as "free carboxylic acid group or active esterified carboxylic acid group"), or as a protecting group for a main chain carboxylic acid group (when the side chain carboxylic acid group of Asp, Glu and derivatives thereof is used as "free carboxylic acid group or active esterified carboxylic acid group"). Furthermore, a protecting group having an orthoester skeleton in which three alkoxy groups are bonded to a carbon atom from which a carboxylic acid group is derived may also be used as a protecting group for a carboxylic acid. The carbon atoms forming such protecting groups may have substitution.

Representative examples of protecting groups having MOM skeleton include BOM (R1 ═ H, R2 ═ H, X ═ Bn), THP (R2 ═ H, a cyclic structure having four carbon atoms through R1 and X), and THF (R2 ═ H, a cyclic structure having three carbon atoms through R1 and X). As the substituent on the skeleton, a skeleton having other substituents such as an alkyl group, a benzyl group or an aryl group may also be used.

Representative examples of protecting groups having a Bn skeleton include Pis (R6 ═ Me, R7 ═ Me, other R ═ H), PMB (R3 ═ OMe, other R ═ H), DMB (R1 ═ OMe, R3 ═ OMe, other R ═ H), and piperonyl (R2 and R3 are both substituted with oxygen atoms, and those oxygen atoms are bridged through a single carbon atom; other R ═ H). Other alkyl groups may be used in place of the Me substituents. Further, the benzene ring may have a substituent such as an alkyl group, an aryl group and a halogen group.

Representative examples of protecting groups having a tpm backbone include tpm (all R ═ H). The aromatic ring may have a substituent such as an alkyl group, an aryl group, an alkoxy group, and a halogen group.

In addition, groups in which R5 and R6 are bridged, such as dibenzosuberyl wherein R5 and R6 are bridged by two carbon atoms, may also be used.

Representative examples of protecting groups having a Trt backbone include Trt (all R ═ H), Mmt (R3 ═ Me, other R ═ H), Mtt (R3 ═ OMe, other R ═ H), and Clt (R1 ═ Cl, other R ═ H). The aromatic ring may have a substituent such as an alkyl group, an aryl group, an alkoxy group, and a halogen group.

In addition, groups in which R5 and R6 are bridged, such as Pixyl groups in which R5 and R6 are bridged by an oxygen atom, may also be used.

Representative examples of protecting groups having a tBu backbone include tBu (all R ═ H) and Mpe (R1 ═ Me, R4 ═ Me, other R ═ H). It may have a substituent such as other alkyl group and aryl group.

phenyl-EDOTn having the following combination of substituents may be used: (i) r1 ═ R2 ═ R3 ═ OMe; (ii) r1 ═ R2 ═ OMe, R3 ═ H; (iii) r1 ═ R2 ═ H, R3 ═ Ome; or (iv) R1 ═ R2 ═ R3 ═ H.

Dicyclopropylmethyl may also be used.

Among them, tBu, Pis and Trt are particularly preferable.

In the present invention, the term "Fmoc-protected peptide" refers to a peptide comprising one or both of the aforementioned "Fmoc-protected amino acid" and "Fmoc-protected amino acid analog". Examples of such peptides include dipeptides and oligopeptides comprising a total of two or more molecules including one or both of the Fmoc-protected amino acids and Fmoc-protected amino acid analogs described above.

In the peptide synthesis by the solid phase method of the present invention, an Fmoc-protected amino acid analog, or an Fmoc-protected peptide (also referred to as Fmoc-protected amino acid, etc.) may be supported on a solid phase using a resin. The groups (resin-bonding groups) for bonding with the Fmoc-protected amino acid or the like in the resin used are not particularly limited as long as they allow the peptide to be cleaved by an acid. The amount and ratio of the Fmoc-protected amino acid or the like to be supported are not particularly limited. In the present invention, for example, trityl chloride resin (Trt resin), 2-chlorotrityl chloride resin (Clt resin), 4-methyltrityl chloride resin (Mtt resin) and 4-methoxytrityl chloride resin (Mmt) can be used. It is particularly preferred that the resin has resin-bonded groups which are described in the handbook of solid phase synthesis (published by Merck co. on, 2002, 5.1) and evaluated as "H (in DCM, < 5% TFA)", and they can be appropriately selected according to the functional groups on the amino acid to be used. For example, when a carboxylic acid (main chain carboxylic acid or side chain carboxylic acid represented by Asp or Glu) or a hydroxyl group on an aromatic ring (phenol group represented by Tyr) is used as the functional group on the amino acid, trityl chloride resin (Trt resin) or 2-chlorotrityl chloride resin (Clt resin) is preferably used as the resin. When an aliphatic hydroxyl group (an aliphatic alcohol group represented by Ser or Thr) is used as the functional group on the amino acid, a trityl chloride resin (Trt resin), a 2-chlorotrityl chloride resin (Clt resin), or a 4-methyltrityl chloride resin (Mtt) is preferably used as the resin.

Further, the type of the polymer constituting the resin is also not particularly limited. For the resin composed of polystyrene, 100 to 200 mesh or 200 to 400 mesh may be used. The crosslinking percentage is also not particularly limited, but those crosslinked with 1% Divinylbenzene (DVB) are preferable.

The Fmoc-protected amino acid, Fmoc-protected amino acid analog or Fmoc-protected peptide is supported onto the resin by a chemical reaction between a bonding group on the resin and a free carboxylic acid group or a reactive esterified carboxylic acid group of the Fmoc-protected amino acid or Fmoc-protected amino acid analog or an amino acid located at the C-terminus of the Fmoc-protected peptide. In this case, the free carboxylic acid may be a main chain carboxylic acid of an amino acid or an amino acid analogue, or a side chain carboxylic acid (Asp, etc.). Instead of a carboxylic acid group, an Fmoc-protected amino acid analogue or a free OH group or a free SH group of the side chain or the main chain of the amino acid located at the C-terminus of the Fmoc-protected peptide may also be used for supporting onto the solid phase.

Deprotecting a protecting group having Fmoc backbone carried by Fmoc-protected amino acid, Fmoc-protected amino acid analogue or Fmoc-protected peptide supported on solid phase by base to expose amino group thereof. The base used herein is not particularly limited, and deprotecting agents commonly used in peptide synthesis (for example, Amino Acid-protecting groups (chem. rev.2009,109,2455-2504)) can be used. Examples of such a deprotecting agent are preferably a secondary amine, a base having an amidine skeleton, and a base having a guanidine skeleton. Specific examples of the secondary amine include piperidine, morpholine, pyrrolidine, and piperidine. Specific examples of the base having an amidine skeleton include 1, 8-diazabicyclo [5.4.0] undec-7-ene (DBU) and 1, 5-diazabicyclo [4.3.0] -5-nonene (DBN). Specific examples of the base having a guanidine skeleton include 1,1,3, 3-tetramethylguanidine.

The exposed amino group described above is condensed with a free or activated esterified carboxylic acid group of a newly added Fmoc-protected amino acid, Fmoc-protected amino acid analog, or Fmoc-protected peptide to form a peptide bond.

The condensing agent used when condensing the amino group and the carboxylic acid group is not particularly limited as long as they can form an amide bond, and a condensing agent commonly used in Peptide synthesis is preferable (for example, Peptide Coupling Reagents, More thana Letter Soup (chem. rev.2011,111, 6557-6602)). Specific examples of such a condensing agent include a condensing agent having a carbodiimide skeleton. For example, condensing agents having a carbodiimide skeleton can be used for the condensation reaction by combining them with a hydroxyl compound that can form an active ester. Examples of the condensing agent having a carbodiimide skeleton include N, N '-Dicyclohexylcarbodiimide (DCC), N, N' -Diisopropylcarbodiimide (DIC) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (WSCI HCl) (see, for example, the catalog of Watanabe Chemical: Amino Acids and chiral Building Blocks to New Medicine). Examples of hydroxy compounds which can form active esters include 1-hydroxy-1H-benzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), ethyl 2-cyano-2- (hydroxyimino) acetate (oxyma), 3, 4-dihydro-3-hydroxy-4-oxo-1, 2, 3-benzotriazine (HOOBt or HODhbt), n-hydroxy-5-norbornene-2, 3-dicarboximide (HONB), 2,3,4,5, 6-pentafluorophenol (HOPfp), N-hydroxysuccinimide (HOSu), and 6-chloro-1-hydroxy-1H-benzotriazole (Cl-HOBt) (see, e.g., the catalog of Watanabe Chemical: Amino Acids and Chiral Building Blocks to New Medicine). In addition, salts having such a backbone, such as K-oxyma, which is a potassium salt of oxyma, may also be used. Among them, HOBt, HOAt, oxyma and HOOBt are particularly preferable. Among them, the combined use of DIC and HOAt or the combined use of DIC and oxyma is preferable. Furthermore, the following reagents may be used in combination in the condensation reaction:

as

Condensing agent and urea

The condensing agent may be any of the following: 0- (1H-benzotriazol-1-yl) -N, N, N ', N' -tetramethylurea

Hexafluorophosphate (HBTU); o- (7-aza-1H-benzotriazol-1-yl) -N, N, N ', N' -tetramethylurea

Hexafluorophosphate (HATU); n- [1- (cyano-2-ethoxy-2-oxoethyleneaminooxy) dimethylamino (morpholino)]UreaHexafluorophosphate (COMU); o- [ (ethoxycarbonyl) cyanomethyleneamino]-N, N, N ', N' -tetramethylurea

Hexafluorophosphate (HOTU); tetrafluoroboric acid O- (1H-benzotriazol-1-yl) -N, N, N ', N' -tetramethylurea

(TBTU); o- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethylurea

Tetrafluoroborate (TATU); hexafluorophosphate 1H-benzotriazol-1-yloxy-tris (pyrrolidinyl)

(PyBOP); 1H-benzotriazol-1-yloxy-tris (dimethylamino)Hexafluorophosphate (BOP); bromo tris (pyrrolidinyl)Hexafluorophosphate (PyBroP); chlorotris (pyrrolidine)

Hexafluorophosphate (PyCloP); (7-azabenzotriazol-1-yloxy) tripyrrolidinyl

Hexafluorophosphate (PyAOP); bromo tri (dimethylamino)

Hexafluorophosphate (Brop); 3- (diethoxyphosphoryloxy) -1,2, 3-benzotriazin-4 (3H) -one (DEPBT); n, N, N ', N' -tetramethyl-O- (N-succinimidyl) ureaTetrafluoroborate (TSTU); n, N, N ', N' -tetramethyl-O- (N-succinimidyl) urea

Hexafluorophosphate (HSTU); o- (3, 4-dihydro-4-oxo-1, 2, 3-benzotriazin-3-yl) -N, N, N ', N' -tetramethylurea

Tetrafluoroborate (TDBTU); tetramethylthiourea S- (1-oxo-2-pyridinyl) -N, N' -tetrafluoroborate (TOTT); and O- (2-oxo-1 (2H) pyridyl) -N, N, N ', N' -tetramethylurea

Tetrafluoroborate (TPTU), and

any one of the bases, N-Diisopropylethylamine (DIPEA); triethylamine (TEA); 2,4, 6-trimethylpyridine (2,4, 6-colidine); and 2,6-lutidine (2, 6-lutidine). The combined use of HATU and DIPEA, or the combined use of COMU and DIPEA is particularly preferred. In addition, N '-Carbonyldiimidazole (CDI), 1' -carbonyl-bis- (1,2, 4-triazole) (CDT), 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylmorpholine hydrochloride (DMT-MM), propylphosphonic anhydride (T3P), and the like can also be used as the condensing agent.

The production method of the present invention further comprises the steps of:

deprotecting the protecting group having the Fmoc backbone of the added new Fmoc-protected amino acid, new Fmoc-protected amino acid analog or new Fmoc-protected peptide by using a base to expose the amino group thereof; and

an amide bond is formed by further adding a new Fmoc-protected amino acid, a new Fmoc-protected amino acid analog, or a new Fmoc-protected peptide.

These steps may be repeated one or more times. Using the method of the present invention, the desired peptide sequence can be obtained by repeating deprotection of the protecting group having Fmoc backbone and condensation reaction with the next new Fmoc-protected amino acid, new Fmoc-protected amino acid analog or new Fmoc-protected peptide.

In carrying out the present invention by the solid phase method, the desired peptide once obtained is cleaved from the solid phase (cleavage step). In addition, structural transformation and cyclization of the peptide may be performed prior to the cleavage step. In the present invention, at the time of cleavage, a side chain functional group protected by a protecting group may or may not be deprotected, and only a part of the protecting group may be deprotected. Preferably, the cleavage step is performed while the side chain functional groups are still protected.

Specifically, the reaction conditions in the cleavage step of the present invention are preferably weakly acidic conditions, and particularly preferably acidic conditions weaker than TFA. In particular, for these weak acids, acids exhibiting higher aqueous pKa values than TFA are preferred. More specifically, acids having a pKa value in the range of 0 to 15 are preferred, and acids having an aqueous pKa value in the range of 6 to 15 are more preferred. Examples of the acid having a weaker acidity than TFA used in this step include TFE and HFIP. Two or more weak acids may be used in combination in any ratio, for example TFE/acetic acid. In addition, solvents such as DCM, DCE and water can be mixed in any ratio. For this combination of weak acid and solvent, the combination of TFE and DCM is particularly preferred. For the solution used for cleavage, other organic solvents and reagents (e.g., DIPEA) and cation scavengers (e.g., triisopropylsilane) can be added.

When the cleavage step is performed before deprotecting the side chain protecting groups of the synthetic peptide, the weak acid used for cleavage is preferably a weaker acid than the acid used for the deprotection reaction. In this case, two acids having different acidity weaker than TFA were prepared in advance, and the weaker acid of the two was used for cleavage.

When the cleavage step is performed after deprotecting the side chain protecting groups of the synthesized peptide, weak acids used for cleavage are not particularly limited as long as they are weaker acids than TFA.

In the deprotection step of the side chain protecting group of the present invention, a desired deprotection reaction can be selectively performed by reducing side reactions such as hydrolysis and transfer of N-to O-acyl. Deprotection of the side chain protecting groups is preferably carried out under acidic conditions weaker than TFA. The deprotection reaction may be carried out at any temperature, and preferably at 0 ℃ to 40 ℃. When deprotection is complete, or when the reaction is stopped during deprotection, bases such as ammonia and primary to tertiary amines may be used. In addition, basic heterocyclic compounds (e.g., pyridine, imidazole, and the like) and the like can also be used.

When the peptide synthesized by the production method of the present invention is further altered or modified, these steps may be performed before or after the cleavage step.

The peptide produced by the production method of the present invention may be a peptide comprising an amino acid residue or an amino acid analog residue having one reactive site on the side chain on the C-terminal side thereof and an amino acid residue, an amino acid analog residue or a carboxylic acid analog having another reactive site on the N-terminal side thereof. For example, such a peptide can be produced by selecting the starting materials Fmoc-protected amino acid, Fmoc-protected amino acid analog and Fmoc-protected peptide such that an amino acid residue or amino acid analog residue having one reactive site on its side chain is contained on the C-terminal side, and an amino acid residue, amino acid analog residue or carboxylic acid analog having another reactive site is contained on the N-terminal side.

The peptide may be cyclized by forming a bond between one reactive site and another reactive site. The production process of the present invention may comprise such a cyclization step. In particular, the cyclisation step may be carried out based on the description in WO 2013/100132.

When the cyclization step is performed after the cleavage step, a concentrated residue obtained from the reaction liquid (cleavage liquid) of the cleavage step under reduced pressure may be used in the cyclization step, or the cleavage liquid may be used in the cyclization step as it is.

In the present invention, "carboxylic acid analogs" include compounds having both an amino group and a carboxyl group and having three or more atoms between the two groups; various carboxylic acid derivatives having no amino group; peptides formed from two to four residues; and an amino acid in which a main chain amino group is chemically modified by forming an amide bond or the like with a carboxylic acid. In addition, "carboxylic acid analogs" may have borate or boronate ester moieties available for cyclization. Further, the "carboxylic acid analog" may be a carboxylic acid having a double bond moiety or a triple bond moiety, or may be a carboxylic acid having a ketone or a halide. In these compounds, a portion other than the specific functional group may be substituted, and for example, such a substituent may be selected from alkyl, aralkyl, aryl, cycloalkyl, heteroaryl, alkenyl, alkynyl and the like (a freely selected substituent).

The cyclization step includes a step of cyclization by formation of, for example, an amide bond, a disulfide bond, an ether bond, a thioether bond, an ester bond, a thioester bond, a carbon-carbon bond, etc. through the above two reaction sites, but is not limited thereto.

Cyclization by an amide bond is, for example, cyclization by formation of an amide bond between a reactive site on an N-terminal amino acid residue, an N-terminal amino acid analog residue, or an N-terminal carboxylic acid analog (a main chain amino group or an amino group present on a side chain) and a reactive site on an amino acid residue or an amino acid analog having one carboxylic acid on a side chain thereof. As the condensing agent used for these reactions, agents similar to those used in the above-mentioned peptide bond bonding can be used. Specifically, for example, the side chain carboxylic acid and the N-terminal main chain amino group, or the side chain amino group and the C-terminal main chain carboxylic acid may be condensed by using a combination of HATU and DIPEA or a combination of COMU and DIPEA. In this case, it is preferable to select the protecting group for the C-terminal side carboxylic acid and the protecting group for the carboxylic acid on the side chain for performing cyclization, or the protecting group for the N-terminal side main chain amino group and the protecting group for the amino group on the side chain for performing cyclization, by taking orthogonality thereof into consideration. Preferred protecting groups in this series of peptide syntheses are as described above.

Cyclization by carbon-carbon bond formation is, for example, cyclization by forming a carbon-carbon bond between a reactive site on an N-terminal amino acid residue, an N-terminal amino acid analog residue, or an N-terminal carboxylic acid analog and a reactive site on an amino acid residue or amino acid analog having one reactive site on its side chain. Specifically, for example, by selecting an alkenyl group as a reaction site on an N-terminal amino acid residue, an N-terminal amino acid analog residue, or an N-terminal carboxylic acid analog, and selecting an alkenyl group as a reaction site on an amino acid residue or an amino acid analog residue having one reaction site on its side chain, the cyclization reaction can be carried out by a transition metal-catalyzed carbon-carbon bonding reaction. In this case, examples of the transition metal used as the catalyst include ruthenium, molybdenum, titanium and tungsten. For example, when ruthenium is used, the cyclization reaction can be carried out by a metathesis reaction. Further, by using a combination of an aryl halide and a boronic acid or a boronic acid analog as a combination of a reactive site on an N-terminal amino acid residue, an N-terminal amino acid analog residue or an N-terminal carboxylic acid analog and a reactive site on an amino acid residue or an amino acid analog residue having one reactive site on its side chain, a cyclization reaction can be carried out by a transition metal-catalyzed carbon-carbon bonding reaction. In this case, the transition metals used as the catalyst include palladium, nickel and iron. For example, when palladium is used, the cyclization reaction can be carried out by a Suzuki reaction. Further, by using a combination of an alkenyl group and an aryl halide or an alkenyl halide as a combination of a reaction site on the N-terminal amino acid residue, the N-terminal amino acid analog residue or the N-terminal carboxylic acid analog and a reaction site on the amino acid residue or the amino acid analog residue having one reaction site on its side chain, the cyclization reaction can proceed by a transition metal-catalyzed carbon-carbon bonding reaction. In this case, the transition metals used as the catalyst include palladium and nickel. For example, when palladium is used, the cyclization reaction can be carried out by a Heck type chemical reaction. Further, by selecting a combination of an ethynyl group and an aryl halide or an alkenyl halide as a combination of a reactive site on the N-terminal amino acid residue, N-terminal amino acid analog residue or N-terminal carboxylic acid analog and a reactive site on the amino acid residue or amino acid analog residue having one reactive site on its side chain, the cyclization reaction can proceed by a transition metal catalyzed carbon-carbon bonding reaction. In this case, transition metals used as the catalyst include palladium, copper, gold, and iron. For example, when a combination of palladium and copper is used, the cyclization reaction can be carried out by the Sonogashira reaction.

In the present invention, the obtained product may be purified as necessary. For example, conventional peptide purification methods such as reverse phase columns or molecular sieve columns may be used. Furthermore, the obtained product can also be purified by crystallization or solidification using a suitable solvent. Concentration under reduced pressure is also possible before purification.

All prior art references cited herein are incorporated by reference into this specification.

Examples

The invention will be further illustrated with reference to the following examples, but is not limited thereto.

The following abbreviations are used in the examples.

DCM dichloromethane

DCE 1, 2-dichloroethane

DMF N, N-dimethylformamide

DIC N, N' -diisopropylcarbodiimide

DIPEA N, N-diisopropylethylamine

DBU 1, 8-diazabicyclo [5.4.0] -7-undecene

NMP N-methyl-2-pyrrolidone

FA formic acid

TFA trifluoroacetic acid

TFE 2,2, 2-trifluoroethanol

HFIP 1,1,1,3,3, 3-hexafluoroisopropanol

HOAt 1-hydroxy-7-azabenzotriazole

HOBt 1-hydroxybenzotriazole

WSCI & HCl 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride

TBME Tert-butyl methyl Ether

TIPS Tri-iso-propylsilane

HATU O- (7-aza-1H-benzotriazol-1-yl) -N, N, N ', N' -tetramethylurea

Hexafluorophosphates

Reaction solvents for peptide synthesis and solid phase synthesis are those used for peptide synthesis (available from WatanabeChemicals and Wako Pure Chemical Industries). Examples include DCM, DMF, NMP, 2% DBU in DMF and 20% piperidine in DMF. In addition, for the reaction without adding water as a solvent, a dehydration solvent, an ultra-dehydration solvent and an anhydrous solvent (available from Kanto Chemical Co, Wako Pure Chemical Industries, etc.) are used.

The conditions for LCMS analysis are shown in table 1.

[ Table 1]

[ example 1]Basic synthetic route to cyclic peptides comprising N-methyl amino acids in their sequence

Solid phase synthesis using Fmoc method for the synthesis of cyclic peptides comprising N-methyl amino acids in their sequence was carried out by the synthetic route shown in fig. 1, which comprises the following five steps:

A) extending the peptide from the N-terminus of Asp supported on 2-chlorotrityl resin through its side chain carboxylic acid by Fmoc method using a peptide synthesizer;

B) cleaving the peptide from the 2-chlorotrityl resin;

C) cyclizing the cleaved peptide by amide bonding by condensation of a side chain carboxylic acid (hollow circular unit) of Asp and an amino group (triangular unit) at the N-terminus of the peptide chain;

D) deprotecting a protecting group of a side chain functional group contained in a peptide chain; and

E) the compound was purified by fractional HPLC.

In the examples, unless otherwise specifically indicated, cyclic peptides are synthesized based on this basic synthetic route.

Fmoc amino acids for peptide synthesis by peptide synthesizer

In the peptide synthesis described in the examples, the following Fmoc amino acids were used for synthesis by a peptide synthesizer (step a above).

Fmoc-Pro-OH, Fmoc-Thr (Trt) -OH, Fmoc-Ile-OH, Fmoc-Trp-OH, Fmoc-D-Tyr (tBu) -OH, Fmoc-D-Tyr (Clt) -OH, Fmoc-Ser (Trt) -OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-His (Trt) -OH, Fmoc-MePhe-OH, Fmoc-MeAla-OH, Fmoc-MeGly-OH, Fmoc-MeLeu-OH, Fmoc-Phe (4-CF3) -OH, Fmoc-b-Ala-OH, Fmoc-b-MeAla-OH, Fmoc-Nle-OH, Fmoc-Met (O2) -OH, Fmoc-Phe (3-Cl) -OH, Fmoc-Leu-OH, Fmoc-MeVal and Fmoc-Val-OH are available from Watanabe Chemical Industries, Chemepp Inc., Chem-Impex International Inc. and the like.

Fmoc-MeSer (DMT) -OH, Fmoc-MePhe (3-Cl) -OH, Fmoc-MeAla (4-Thz) -OH, Fmoc-Hyp (Et) -OH and Fmoc- γ EtAbu-OH, Fmoc-nPrGly-OH were synthesized by methods described in the literature (literature: WO2013/100132A 1).

Fmoc-Ser (THP) -OH (Compound 1), Fmoc-Thr (THP) -OH (Compound 2), Fmoc-MeSer (THP) -OH (Compound 6), Fmoc-MeHis (Trt) -OH (Compound 7), Fmoc-D-Tyr (THP) -OH (Compound 8), Fmoc-D-Tyr (Pis) -OH (Compound 11), Fmoc-Tyr (3-F, tBu) -OH (Compound 13), Fmoc-MePhe (4-Cl) -OH (Compound 16), and Fmoc-Tyr (3-F, Pis) -OH (Compound 22) were synthesized as follows. These synthesized Fmoc amino acids were used not only for the synthesis of peptides, but also for examination of the deprotection of the protecting group for the side chain functional group and the protecting group for the C-terminal carboxylic acid group.