JP7640994B2 - Peptide synthesis - Google Patents

Description

The present invention relates to a novel method for peptide synthesis. More specifically, it relates to a method for extending a peptide chain from the N-terminus to the C-terminus via a thiocarboxylic acid intermediate.

Medium-sized peptide compounds are attracting attention as candidates for the next generation of pharmaceuticals. In the exploration stage, they are supplied by solid-phase synthesis, but when considering large-scale trials and commercialization, the supply is insufficient, making it necessary to supply them in large quantities by liquid-phase synthesis.

Conventionally, the mainstream chemical synthesis method that can freely incorporate various amino acids, including unnatural amino acids, has been the method of extending from the C-terminus to the N-terminus (C → N-terminus extension method), but it is necessary to use more than an equivalent amount of condensation agent and protected amino acid elements that are expensive and have low atom efficiency. This has led to the problem of producing large amounts of waste from the main chain amino group protecting groups and condensation agents, which has led to increased costs and environmental burden in the peptide supply process (see Figure 1).

On the other hand, a method of extending from the N-terminus to the C-terminus in the opposite direction (N→C-terminus extension method) has the potential to solve this problem, but epimerization occurs at the same time, making it difficult to realize a synthesis method that produces products with excellent purity.

Holanders, K.; Mars, B. U. W.; Ballet, S. Synthesis 2019, 51, 2261. de Figueiredo, R. M.; Suppo, J.-S.; Campagne, J.-M. Chem. Rev. Crich, D.; Sana, K.; Guo, S. Org. Lett. 2007, 9, 4423. Crich, D.; Sharma, I. Angew. Chem. Int. Ed. 2009, 48, 7591. Wu, W.; Zhang, Z.; Liebeskind, L. S. J. Am. Chem. Soc 2011, 133, 14256-14259. Mali, S. M.; Jadhav, S. V.; Gopi, H. N. Chem. Commun. 2012, 48, 7085. Wang, P.; Danishefsky, S. J. J. Am. Chem. Soc. 2015, 137, 13167. Review: Narenda N, Thimmalapura VM, Hosamani B, Prabhu G, Kumar LR, Sureshbabu VV. Org. Biomol. Chem. 2018, 16, 3524.

The present invention aims to provide a novel peptide synthesis method that can avoid the use of condensation agents and main chain protecting groups in the peptide supply process, suppress epimerization, and further improve the solubility of unprotected amino acid elements and accelerate the reaction.

As a result of extensive investigations, the present inventors have found that the above-mentioned problems can be solved by a novel peptide synthesis method comprising the following three elements, and have thus completed the present invention.
(1) Peptide chain elongation from the N to C terminal direction via peptide thiocarboxylic acid.
(2) A newly discovered epimerization inhibitor.
(3) A solubilizer for unprotected amino acid elements.

（式中、

は、
保護基を表すか、Ｎ末端が保護基で保護されたアミノ酸又はペプチドを表し、
Ｒ_１は、α－アミノ酸の側鎖であり、当該側鎖は保護基で保護されていてもよい。）

（式中、Ｒ_２は、α－アミノ酸の側鎖であり、当該側鎖は保護基で保護されていてもよい。）

（式中、
Ｌは、エステル基（－ＣＯ_２Ｒ；Ｒは炭素数１～３０のアルキル基）、置換又は無置換のアリ－ル基、置換又は無置換のベンゾイル基、及びアミノ基で置換されていてもよいアミド基（－ＣＯＮＲ’Ｒ’’；Ｒ’、Ｒ’’は、各々独立に、水素原子又は炭素数１～４の置換又は無置換のアルキル基であり、Ｒ’及びＲ’’の少なくとも一方はアミノ基で置換されているアルキル基である）、及びアシル基（－ＣＯＲ_ａ’； Ｒ_ａ’は炭素数１～４のアルキル基）からなる群から選択され、但し、Ｒ_３及びＲ_４の少なくとも１つがエステル基（－ＣＯ_２Ｒ_ａ；Ｒ_ａは炭素数１～４のアルキル基）である場合は、Ｌは水素（Ｈ）であってもよく、
Ｒ_３及びＲ_４は、各々独立に、水素、炭素数１～４のアルキル基、ハロゲン又はエステル基（－ＣＯ_２Ｒ_ａ；Ｒ_ａは炭素数１～４のアルキル基）を表す。）

（式中、Ｒ_５及びＲ_６は、各々独立に、炭素数１～４のアルキル基、炭素数１～４のアルコキシ基、置換基を有していてもよいベンジルオキシ基又はヒドロキシ基を表し（但し、Ｒ_５及びＲ_６の両方がヒドロキシ基になることはない）、
Ｒ_５及びＲ_６は一緒になってＲ_５及びＲ_６が結合しているリン原子を含む４～７員の置換又は無置換のヘテロシクリルを形成してもよい。）

（式中、

、Ｒ_１及びＲ_２は、上記で定義した通りである。）
［２］反応溶媒として、ＤＭＳＯとトルエンの混合溶媒を用いる、［１］に記載の方法。
［３］前記カップリング反応の前に、以下の式（VI）で表される化合物を、以下の式（１）の化合物と反応させることにより、式（I）で表される化合物を調製する工程を含む、［１］又は［２］に記載の方法。

（式中、Ｒ_１は、式（I）で定義した通りである。）

（式中、Ｒ_７は、炭素数１～４のアルキル基又は置換又は無置換のアリ-ル基である。）
［４］ＰＧ－（ＡＡ）_ｎ－ＳＨ：
（ＰＧは、Ｎ末端の保護基を表し、
ＡＡは、任意のアミノ酸残基を表し、各出現において同一又は異なっていてもよく、アミノ酸残基の一部又は全ての側鎖は保護基で保護されていてもよく、
ｎは、１～４の整数である。）
で表される化合物と、
Ｈ－（ＡＡ’）_ｍ－ＯＨ：
（ＡＡは、任意のアミノ酸残基を表し、各出現において同一又は異なっていてもよく、アミノ酸残基の一部又は全ての側鎖は保護基で保護されていてもよく、
ｍは、１～４の整数である。）
で表されるアミノ酸又はペプチドを、
以下の式（III）で表される化合物及び以下の式（IV）で表される化合物の存在下で反応させることにより、ＰＧ－（ＡＡ）_ｎ（ＡＡ’）_ｍ－ＯＨで表される化合物を調製する方法。

（式中、
Ｌは、エステル基（－ＣＯ_２Ｒ；Ｒは炭素数１～３０のアルキル基）、置換又は無置換のアリ－ル基、置換又は無置換のベンゾイル基、及びアミノ基で置換されていてもよいアミド基（－ＣＯＮＲ’Ｒ’’；Ｒ’、Ｒ’’は、各々独立に、水素原子又は炭素数１～４の置換又は無置換のアルキル基であり、Ｒ’及びＲ’’の少なくとも一方はアミノ基で置換されているアルキル基である）、及びアシル基（－ＣＯＲ_ａ’； Ｒ_ａ’は炭素数１～４のアルキル基）からなる群から選択され、但し、Ｒ_３及びＲ_４の少なくとも１つがエステル基（－ＣＯ_２Ｒ_ａ；Ｒ_ａは炭素数１～４のアルキル基）である場合は、Ｌは水素（Ｈ）であってもよく、
Ｒ_３及びＲ_４は、各々独立に、炭素数１～４のアルキル基、ハロゲン又はエステル基（－ＣＯ_２Ｒ_ａ；Ｒ_ａは炭素数１～４のアルキル基）を表す。）

（式中、Ｒ_５及びＲ_６は、各々独立に、炭素数１～４のアルキル基、炭素数１～４のアルコキシ基、置換基を有していてもよいベンジルオキシ基又はヒドロキシ基を表し（但し、Ｒ_５及びＲ_６の両方がヒドロキシ基になることはない）、
Ｒ_５及びＲ_６は一緒になってＲ_５及びＲ_６が結合しているリン原子を含む４～７員の置換又は無置換のヘテロシクリルを形成してもよい。）
［５］［１］～［３］のいずれか１項に記載の方法に続けて、
（ｉ）以下の式（V）で表される化合物を、以下の式（１）の化合物と反応させることにより、式（VII）で表される化合物を調製する工程：

（式中、

、Ｒ_１、Ｒ_２は、式（I）及び（II）で定義した通りである。）

（式中、Ｒ_７は、炭素数１～４のアルキル基又は置換又は無置換のアリ-ル基である。）

（ｉｉ）式（VII）で表される化合物と、以下の式（VIII）で表されるアミノ酸を、以下の式（III）で表される化合物及び以下の式（IV）で表される化合物の存在下で反応させる工程：
を含む、式（VIIII）の化合物を調製する方法。

（式中、Ｒ_３ａは、α-アミノ酸の側鎖であり、当該側鎖は保護基で保護されていてもよい。）

（式中、
Ｌは、エステル基（－ＣＯ_２Ｒ；Ｒは炭素数１～３０のアルキル基）、置換又は無置換のアリ－ル基、置換又は無置換のベンゾイル基、及びアミノ基で置換されていてもよいアミド基（－ＣＯＮＲ’Ｒ’’；Ｒ’、Ｒ’’は、各々独立に、水素原子又は炭素数１～４の置換又は無置換のアルキル基であり、Ｒ’及びＲ’’の少なくとも一方はアミノ基で置換されているアルキル基である）、及びアシル基（－ＣＯＲ_ａ’； Ｒ_ａ’は炭素数１～４のアルキル基）からなる群から選択され、但し、Ｒ_３及びＲ_４の少なくとも１つがエステル基（－ＣＯ_２Ｒ_ａ；Ｒ_ａは炭素数１～４のアルキル基）である場合は、Ｌは水素（Ｈ）であってもよく、
Ｒ_３及びＲ_４は、各々独立に、炭素数１～４のアルキル基、ハロゲン又はエステル基（－ＣＯ_２Ｒ_ａ；Ｒ_ａは炭素数１～４のアルキル基）を表す。）

（式中、Ｒ_５及びＲ_６は、各々独立に、炭素数１～４のアルキル基、炭素数１～４のアルコキシ基、置換基を有していてもよいベンジルオキシ基又はヒドロキシ基を表し（但し、Ｒ_５及びＲ_６の両方がヒドロキシ基になることはない）、
Ｒ_５及びＲ_６は一緒になってＲ_５及びＲ_６が結合しているリン原子を含む４～７員の置換又は無置換のヘテロシクリルを形成してもよい。）

（式中、

、Ｒ_１～Ｒ_２、Ｒ_３ａは、上記で定義した通りである。）
［６］以下の式（IV）で表される化合物をペプチド合成において可溶化剤として使用する方法。

（式中、Ｒ_５及びＲ_６は、各々独立に、炭素数１～４のアルキル基、炭素数１～４のアルコキシ基、置換基を有していてもよいベンジルオキシ基又はヒドロキシ基を表し（但し、Ｒ_５及びＲ_６の両方がヒドロキシ基になることはない）、
Ｒ_５及びＲ_６は一緒になってＲ_５及びＲ_６が結合しているリン原子を含む４～７員の置換又は無置換のヘテロシクリルを形成してもよい。）
を提供するものである。 That is, the present invention provides:
[1] A method for preparing a compound of formula (V) by reacting a compound of formula (I) below with an amino acid of formula (II) below in the presence of a compound of formula (III) below and a compound of formula (IV) below.

(Wherein,

teeth,
represents a protecting group, or represents an amino acid or peptide whose N-terminus is protected with a protecting group;
_R1 is a side chain of an α-amino acid, and the side chain may be protected with a protecting group.

(In the formula, _R2 is a side chain of an α-amino acid, and the side chain may be protected with a protecting group.)

(Wherein,
L is selected from the group consisting of an ester group (-CO ₂ R; R is an alkyl group having 1 to 30 carbon atoms), a substituted or unsubstituted aryl group, a substituted or unsubstituted benzoyl group, an amide group which may be substituted with an amino group (-CONR'R'', R' and R'' are each independently a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 4 carbon atoms, and at least one of R' and R'' is an alkyl group substituted with an amino group), and an acyl group (-COR _a '; R _a ' is an alkyl group having 1 to 4 carbon atoms), provided that when at least one of R ₃ and R ₄ is an ester group (-CO ₂ R _a ; R _a is an alkyl group having 1 to 4 carbon atoms), L may be hydrogen (H);
_R3 and _R4 each independently represent a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, a halogen _atom , or an ester group ( _-CO2Ra ; _Ra is an alkyl group having 1 to 4 carbon atoms).

(In the formula, R ₅ and R ₆ each independently represent an alkyl group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, a benzyloxy group which may have a substituent, or a hydroxy group (however, R ₅ and R ₆ cannot both be a hydroxy group);
_R5 and _R6 may join together to form a 4-7 membered substituted or unsubstituted heterocyclyl containing a phosphorus atom to which _R5 and _R6 are bonded.

(Wherein,

, _R1 and _R2 are as defined above.
[2] The method according to [1], wherein a mixed solvent of DMSO and toluene is used as a reaction solvent.
[3] The method according to [1] or [2], further comprising, prior to the coupling reaction, a step of reacting a compound represented by the following formula (VI) with a compound represented by the following formula (1) to prepare a compound represented by the following formula (I).

(In the formula, _R1 is as defined in formula (I).)

(In the formula, _R7 is an alkyl group having 1 to 4 carbon atoms or a substituted or unsubstituted aryl group.)
[4]PG-(AA) _n -SH:
(PG represents an N-terminal protecting group,
AA represents any amino acid residue, which may be identical or different in each occurrence, some or all of the side chains of the amino acid residue may be protected with protecting groups;
n is an integer from 1 to 4.
A compound represented by the formula:
H-(AA') _m -OH:
(AA represents any amino acid residue, which may be the same or different in each occurrence, some or all of the side chains of the amino acid residue may be protected with protecting groups;
m is an integer from 1 to 4.
An amino acid or peptide represented by
A method for preparing a compound represented by PG-(AA) _n (AA') _m -OH by reacting a compound represented by the following formula (III) and a compound represented by the following formula (IV):

(Wherein,
L is selected from the group consisting of an ester group (-CO ₂ R; R is an alkyl group having 1 to 30 carbon atoms), a substituted or unsubstituted aryl group, a substituted or unsubstituted benzoyl group, an amide group which may be substituted with an amino group (-CONR'R'', R' and R'' are each independently a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 4 carbon atoms, and at least one of R' and R'' is an alkyl group substituted with an amino group), and an acyl group (-COR _a '; R _a ' is an alkyl group having 1 to 4 carbon atoms), provided that when at least one of R ₃ and R ₄ is an ester group (-CO ₂ R _a ; R _a is an alkyl group having 1 to 4 carbon atoms), L may be hydrogen (H);
_R3 and _R4 each independently represent an _alkyl group having 1 to 4 carbon atoms, a halogen, or an ester group ( _-CO2Ra ; _Ra is an alkyl group having 1 to 4 carbon atoms).

(In the formula, R ₅ and R ₆ each independently represent an alkyl group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, a benzyloxy group which may have a substituent, or a hydroxy group (however, R ₅ and R ₆ cannot both be a hydroxy group);
_R5 and _R6 may join together to form a 4-7 membered substituted or unsubstituted heterocyclyl containing a phosphorus atom to which _R5 and _R6 are bonded.
[5] Following the method according to any one of [1] to [3],
(i) reacting a compound represented by the following formula (V) with a compound represented by the following formula (1) to prepare a compound represented by formula (VII):

(Wherein,

, R ₁ and R ₂ are as defined in formulas (I) and (II).

(In the formula, _R7 is an alkyl group having 1 to 4 carbon atoms or a substituted or unsubstituted aryl group.)

(ii) reacting a compound represented by formula (VII) with an amino acid represented by the following formula (VIII) in the presence of a compound represented by the following formula (III) and a compound represented by the following formula (IV):
A method for preparing a compound of formula (VIIII), comprising:

(In the formula, R _3a is a side chain of an α-amino acid, and the side chain may be protected with a protecting group.)

(Wherein,
L is selected from the group consisting of an ester group (-CO ₂ R; R is an alkyl group having 1 to 30 carbon atoms), a substituted or unsubstituted aryl group, a substituted or unsubstituted benzoyl group, an amide group which may be substituted with an amino group (-CONR'R'', R' and R'' are each independently a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 4 carbon atoms, and at least one of R' and R'' is an alkyl group substituted with an amino group), and an acyl group (-COR _a '; R _a ' is an alkyl group having 1 to 4 carbon atoms), provided that when at least one of R ₃ and R ₄ is an ester group (-CO ₂ R _a ; R _a is an alkyl group having 1 to 4 carbon atoms), L may be hydrogen (H);
_R3 and _R4 each independently represent an _alkyl group having 1 to 4 carbon atoms, a halogen, or an ester group ( _-CO2Ra ; _Ra is an alkyl group having 1 to 4 carbon atoms).

(In the formula, R ₅ and R ₆ each independently represent an alkyl group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, a benzyloxy group which may have a substituent, or a hydroxy group (however, R ₅ and R ₆ cannot both be a hydroxy group);
_R5 and _R6 may join together to form a 4-7 membered substituted or unsubstituted heterocyclyl containing a phosphorus atom to which _R5 and _R6 are bonded.

(Wherein,

, R ₁ to R ₂ , and R _3a are as defined above.)
[6] A method of using a compound represented by the following formula (IV) as a solubilizing agent in peptide synthesis.

(In the formula, R ₅ and R ₆ each independently represent an alkyl group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, a benzyloxy group which may have a substituent, or a hydroxy group (however, R ₅ and R ₆ cannot both be a hydroxy group);
_R5 and _R6 may join together to form a 4-7 membered substituted or unsubstituted heterocyclyl containing a phosphorus atom to which _R5 and _R6 are bonded.
This provides:

The present invention provides a novel peptide synthesis method that can avoid the use of condensation agents and main chain protecting groups in the peptide supply process, suppress epimerization, and further improve the solubility of unprotected amino acid elements and accelerate the reaction.

Schematic diagram of a conventional C→N terminal elongation method using a coupling agent. Basic reaction scheme of the peptide synthesis method of the present invention. Summary of reaction schemes for non-limiting methods of obtaining compounds of formula (I).

1. Peptide synthesis method One embodiment of the present invention is a method for preparing a compound of formula (V) by coupling reaction of a compound of formula (I) with an amino acid of formula (II) in the presence of a compound of formula (III) and a compound of formula (IV) (hereinafter also referred to as the "peptide synthesis method of the present invention").

The basic reaction scheme of the peptide synthesis method of the present invention is shown in Figure 2. In the present invention, it is important to have three elements: (1) a peptide chain elongation method in the N→C-terminal direction via peptide thiocarboxylic acid, (2) a newly discovered epimerization inhibitor, and (3) a solubilizer for main-chain unprotected amino acid elements. Below, the peptide synthesis method of the present invention will be explained in detail, including these three elements.

(1) N→C-Terminal Peptide Chain Elongation Method via a Thiocarboxylic Acid Compound The peptide synthesis method of the present invention employs an N→C-terminus peptide chain elongation method via a peptide thiocarboxylic acid, which is a compound represented by formula (I).

は、
保護基を表すか、Ｎ末端が保護基で保護されたアミノ酸又はペプチドを表す。 In formula (I),

teeth,
It represents a protecting group, or represents an amino acid or peptide whose N-terminus is protected with a protecting group.

Protecting groups include those commonly used as protecting groups for the N-terminus of amino acids, such as tert-butoxycarbonyl group (Boc), benzyloxycarbonyl group (Cbz), 9-fluorenylmethoxycarbonyl group (Fmoc), acetyl group (Ac), and benzoyl group (Bz).

An amino acid whose N-terminus is protected with a protecting group means an amino acid whose N-terminus is protected with the above-mentioned protecting group.
The amino acid can be used without any particular limitation. Examples include α-amino acids and β-amino acids. Examples of α-amino acids include, but are not limited to, leucine, isoleucine, valine, lysine, threonine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, serine, histidine, phenylalanine, alanine, glycine, tryptophan, tyrosine, cysteine, methionine, proline, hydroxyproline, ornithine, citrulline, N-methylglycine (sarcosine), N-methylleucine, 2,3-diaminopropanoic acid, 2,4-diaminobutyric acid, α-hydroxyleucine, homoserine, homocysteine, tert-leucine, and α-aminoisobutyric acid. Examples of β-amino acids include, but are not limited to, β-alanine.
The above amino acids may be either L- or D-optical isomers.
When the side chain of these amino acids has a functional group, R ₁ may be protected with a protecting group as described below.

A peptide whose N-terminus is protected with a protecting group means a peptide whose N-terminus is protected with the above-mentioned protecting group. Here, the peptide has a structure in which 2 to 9, preferably 2 to 6, and more preferably 2 to 4 of the above-mentioned amino acid residues are bonded.

_R1 is the side chain of an α-amino acid. _R1 is determined by the type of α-amino acid residue bonded to SH. For example, if the amino acid residue is glycine, _R1 is hydrogen, and if the amino acid residue is alanine, R1 is a methyl group.
That is, the α-amino acid residue bound to SH in formula (I), NH-CH(R ₁ )-C(═O), may be any α-amino acid residue other than phenylglycine. Examples of α-amino acids include, but are not limited to, leucine, isoleucine, valine, lysine, threonine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, serine, histidine, phenylalanine, alanine, glycine, tryptophan, tyrosine, cysteine, methionine, proline, hydroxyproline, ornithine, citrulline, N-methylglycine (sarcosine), N-methylleucine, 2,3-diaminopropanoic acid, 2,4-diaminobutyric acid, α-hydroxyleucine, homoserine, homocysteine, tert-leucine, and α-aminoisobutyric acid. R ₁ is the side chain of an amino acid selected from these.
The above α-amino acids may be either L- or D-optical isomers.

The side chain of the above α-amino acid residue represented by _R1 may be protected with a protecting group.
For example, when R ₁ is a lysine side chain (having an amino group), the protecting group can be a tert-butoxycarbonyl group (Boc), a benzyloxycarbonyl group (Cbz), or a 9-fluorenylmethoxycarbonyl group (Fmoc).
When R ₁ is a glutamic acid side chain or an aspartic acid side chain having a carboxy group, a benzyl ester (Bzl) or a tert-butyl ester (t-Bu) can be used as a protecting group.
Furthermore, when R ₁ is a serine or threonine side chain (having a hydroxy group), a benzyl group or a tert-butyl group can be used as a protecting group.
When R ₁ is a tyrosine side chain (having a phenolic hydroxy group), 2-bromobenzyloxycarbonyl (Z(2Br)) or a tert-butyl group can be used as a protecting group.
When _R1 is a cysteine side chain (having a sulfhydryl group), the protecting group can be 4-methylbenzyl (Bzl(4Me)), trityl (Trt), tert-butyl, or N-(acetyl)aminomethyl (Acm).
When R ₁ is an arginine side chain (having a guanidino group), a protecting group such as p-toluenesulfonyl group (p-Ts) can be used.
Furthermore, when _R1 is a histidine side chain (having an imidazole ring), the π-nitrogen can be protected with a benzyloxymethyl group (Bom) or a tert-butoxymethyl group (Bum), and the τ-nitrogen can be protected with a 2,4-dinitrophenyl group (Dnp), a trityl group, or the like.

The thiocarboxylic acid compound represented by formula (I) can be obtained using the one-step conversion conditions reported by the present inventors' group (Chem. Commun. 2018, 54, 1222) or any existing method.

式（１）中、Ｒ_７は、炭素数１～４のアルキル基又は置換又は無置換のアリ-ル基であり、好ましくはメチル基又はエチル基であり、更に好ましくはメチル基である。 In one preferred aspect of the present invention, the compound represented by formula (I) can be obtained by reacting a compound represented by the following formula (VI) with a compound represented by the following formula (1).

In formula (1), R ₇ is an alkyl group having 1 to 4 carbon atoms or a substituted or unsubstituted aryl group, preferably a methyl group or an ethyl group, and more preferably a methyl group.

は、
保護基を表すか、Ｎ末端が保護基で保護されたアミノ酸又はペプチドを表し、Ｒ_１は、α－アミノ酸の側鎖であり、これらの詳細については式（I）において説明したのと同様である。 In formula (VI),

teeth,
R1 represents a protecting group, or represents an amino acid or peptide whose N-terminus is protected with a protecting group, and _R1 is the side chain of an α-amino acid, and the details thereof are the same as those explained in relation to formula (I).

The outline of the reaction scheme for obtaining the compound represented by formula (I) above is shown in Figure 3. In this reaction, acetyl sulfide is used as a catalyst.
This reaction is carried out using DMF as a solvent, and the reaction temperature is usually from 0° C. to room temperature.

In the peptide synthesis method of the present invention, a thiocarboxylic acid compound represented by formula (I) is reacted with a compound represented by the following formula (II).

The compound represented by formula (II) is an unprotected α-amino acid, and in addition to natural amino acids, any α-amino acid can be used as the α-amino acid, including, but not limited to, leucine, isoleucine, valine, lysine, threonine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, serine, histidine, phenylalanine, alanine, glycine, tryptophan, tyrosine, cysteine, methionine, ornithine, 2,3-diaminopropanoic acid, 2,4-diaminobutyric acid, α-hydroxyleucine, tert-leucine, phenylglycine, and α-aminoisobutyric acid.
The above α-amino acids may be either L- or D-optical isomers.

_R2 in formula (II) is the side chain of the α-amino acid and is determined by the type of the α-amino acid, for example, when the amino acid is glycine, _R2 is hydrogen, and when the amino acid is alanine, R2 is a methyl group.

The side chain of the α-amino acid represented by _R2 may be protected with a protecting group, which may be the same as that described for _R1 in formula (I).

In the peptide synthesis method of the present invention, by converting a peptide or amino acid to be elongated into a thiocarboxylic acid as represented by formula (I), it becomes possible to distinguish, to the minimum extent possible, between the C-terminus of the starting compound of formula (I) and the C-terminus of the unprotected amino acid element of formula (II), and it is possible to elongate the peptide one amino acid at a time by selectively activating the C-terminus of the starting compound.
In addition, by using a peptide chain elongation method in the N→C-terminal direction via thiocarboxylic acid, an equivalent amount or more of a condensation agent is not required, and waste products generated in the elongation process can in principle be reduced to only solvent and elemental sulfur. In addition, inexpensive unprotected amino acids can be used as synthetic building blocks. Therefore, a significant reduction in waste products and costs can be expected compared to conventional peptide elongation methods.
In addition, since a peptide chain with an unprotected C-terminus is obtained as the product, it is possible to rapidly elongate the peptide chain by repeating the thiocarboxylic acid conversion-peptide chain linking process.

(2) Epimerization Inhibitor In the peptide synthesis method of the present invention, a novel epimerization inhibitor, which is a compound represented by the following formula (III), is used.

When N→C-terminal elongation is performed according to conventional thinking, epimerization (loss of stereochemistry) occurs via a cyclized intermediate, making it impossible to supply a product with excellent purity. The inventors synthesized and screened various additives that are expected to have epimerization suppression capabilities, and found that N-hydroxypyridone-type additives solve the epimerization problem in the peptide synthesis method of the present invention.

In formula (III), L is selected from the group consisting of an ester group (-CO ₂ R; R is an alkyl group having 1 to 30 carbon atoms), a substituted or unsubstituted aryl group, a substituted or unsubstituted benzoyl group, an amide group which may be substituted with an amino group (-CONR'R'', R' and R'' each independently represent a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 4 carbon atoms, and at least one of R' and R'' is an alkyl group substituted with an amino group), and an acyl group (-COR _a '; R _a ' is an alkyl group having 1 to 4 carbon atoms).

The ester group is represented by —CO ₂ R, where R is an alkyl group having 1 to 30 carbon atoms. The alkyl group may be unsubstituted or may have a substituent.
The alkyl group may be either a linear alkyl group or a branched alkyl group. In the case of a branched alkyl group, there is an advantage that the additive can be easily recovered from the product by recrystallization even if the total number of carbon atoms is as large as about 30.
As the branched alkyl group, a branched alkyl group of -( _CH2CH2CH ( _CH3 ) _CH2 ) _s -H (s is 1 to 4 ₎ is preferred from the viewpoint of increasing the recovery rate of the additive from the product by recrystallization. In the case of a linear alkyl group, even if the carbon number is large, the yield and degree of epimerization are good, but the degree of crystallization increases and recovery from the product may become difficult. As for the linear alkyl group, an alkyl group having 1 to 4 carbon atoms is preferred from the viewpoints of the yield, degree of epimerization, and ease of recovery of the additive from the product, and a methyl group (i.e., as an ester group, a methyl _ester group ( _-CO2CH3 )) is particularly preferred.

The amido group which may be substituted with an amino group is preferably a dimethylaminoethylamido group (-CONHC ₂ H ₄ N(CH ₃ ) ₂ ).

The aryl group may be a phenyl group, which may be substituted or unsubstituted. The substituent of the aryl group may be an alkyl group, an ester group, etc., and is preferably an ester group (e.g., a methyl ester group (—CO ₂ CH ₃ )).

Substituents for the benzoyl group include alkyl groups and amino groups, with amino groups (e.g., dimethylamino groups) being preferred.

The acyl group is represented by --COR _a ' (R _a ' is an alkyl group having 1 to 4 carbon atoms), and is preferably an acetyl group.

L is preferably selected from the groups listed above, but when at least one of R ₃ and R ₄ is an ester group (-CO ₂ R _a ; R _a is an alkyl group having 1 to 4 carbon atoms), L may be hydrogen (H). In this case, the degree of epimerization tends to be slightly higher than when L is a group such as an ester group, but it is possible to obtain a product in high yield.

In formula (III), _R3 and _R4 each independently represent a hydrogen atom, an alkyl group having 1 to 4 _carbon atoms, a halogen, or an ester group ( _{-CO2R a} ; R _a is an alkyl group having 1 to 4 carbon atoms). Chlorine is preferred as the halogen. A methyl ester group ( _-CO2CH3 ) is preferred as the ester group.

In one preferred aspect of the invention, both _R3 and _R4 are hydrogen atoms.
In another preferred aspect of the present invention, one of _R3 and _R4 is chlorine, and the other is a hydrogen atom.
In another preferred aspect of the present invention, one of _R3 and _R4 is a methyl ester group, and the other is a hydrogen atom.

(3) Solubilizing Agent for Main Chain Unprotected Amino Acids In the peptide synthesis method of the present invention, a solubilizing agent which is a compound represented by formula (IV) is used.

Amino acid elements (raw materials) with unprotected main chains generally do not require organic solvents, so peptide elongation methods using them have poor reaction efficiency and require a long time to complete one step. The inventors searched for additives that solubilize unprotected amino acid elements and found that phosphite-based additives contribute to improving the solubility of unprotected amino acid elements, improving reaction efficiency in the liquid phase and making it possible to shorten reaction times.

In formula (IV), _R5 and _R6 each independently represent an alkyl group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, a benzyloxy group which may have a substituent, or a hydroxy group, provided that both _R5 and _R6 are not hydroxy groups.
The alkyl group is preferably an n-butyl group, and the alkoxy group is preferably a methoxy group, an ethoxy group, an i-propoxy group, or an n-butoxy group.

In one preferred aspect of the present invention, the compound of formula (IV) is represented by the following formula (IVa):

In formula (IVa), R ₅ ' and R ₆ ' each independently represent an alkyl group having 1 to 4 carbon atoms, a benzyl group which may have a substituent, or hydrogen, provided that R ₅ ' and R ₆ ' are not both hydrogen.

In one preferred aspect of the invention, both R ₅ ' and R ₆ ' are methyl groups.
In addition, in one preferred aspect of the present invention, both R ₅ ' and R ₆ ' are ethyl groups.
Also, in one preferred aspect of the invention, both R ₅ ' and R ₆ ' are i-propyl groups.
In addition, in one preferred aspect of the present invention, both R ₅ ' and R ₆ ' are n-butyl groups.
Also, in one preferred aspect of the invention, both R ₅ ' and R ₆ ' are benzyl groups.

In formula (IV), R ₅ and R ₆ may join together to form a 4- to 7-membered substituted or unsubstituted heterocyclyl containing a phosphorus atom to which R ₅ and R ₆ are attached.
Such a heterocyclyl is preferably a 5- or 6-membered ring structure, more preferably a 6-membered ring structure.
The substituents include alkyl groups having a number of 1 to 4, and the number of the substituents may be 1 or 2 or more. The alkyl groups are preferably methyl and ethyl groups.

Non-limiting examples of formula (IV) that may be used in the method of the present invention are shown below, but are not limited to these.

According to the peptide synthesis method of the present invention, a compound represented by the following formula (V) can be obtained.

、Ｒ_１及びＲ_２は、式（I）、（II）について説明した通りである。 In formula (V),

, _R1 and _R2 are as described above with respect to formulae (I) and (II).

In the peptide synthesis method of the present invention, the compound of formula (I) is usually reacted with 1 to 2 equivalents of the compound of formula (II), 1 equivalent of the compound of formula (III), and 1 equivalent of the compound of formula (IV).

In the peptide synthesis method of the present invention, a solvent usually used in liquid phase peptide synthesis can be used, but a DMSO-based solvent is preferred because it can efficiently activate the thioacid. Furthermore, a mixed solvent such as DMSO and toluene is preferred because it can reduce polarity and effectively suppress epimerization.
In one preferred aspect of the method of the present invention, a mixed solvent of DMSO:toluene=1:1 (volume ratio) is used.

The peptide synthesis method of the present invention is usually carried out by reacting at about room temperature for 3 to 6 hours.

As explained above, the peptide synthesis method of the present invention is based on a peptide chain elongation method in the N→C-terminal direction via a thiocarboxylic acid compound of a peptide or amino acid, but the method of the present invention can also be applied to peptide fragment coupling and iterative coupling.

（式中、
Ｌは、エステル基（－ＣＯ_２Ｒ；Ｒは炭素数１～３０のアルキル基）、置換又は無置換のアリ-ル基、置換又は無置換のベンゾイル基、アミノ基で置換されていてもよいアミド基（－ＣＯＮＲ’Ｒ’’；Ｒ’、Ｒ’’は、各々独立に、水素原子又は炭素数１～４の置換又は無置換のアルキル基であり、Ｒ’及びＲ’’の少なくとも一方はアミノ基で置換されているアルキル基である）、アシル基（－ＣＯＲ_ａ’； Ｒ_ａ’は炭素数１～４のアルキル基）からなる群から選択され、但し、Ｒ_３及びＲ_４の少なくとも１つがエステル基（－ＣＯ_２Ｒ_ａ；Ｒ_ａは炭素数１～４のアルキル基）である場合は、Ｌは水素（Ｈ）であってもよく、
Ｒ_３及びＲ_４は、各々独立に、炭素数１～４のアルキル基、ハロゲン又はエステル基（-ＣＯ_２Ｒ_ａ；Ｒ_ａは炭素数１～４のアルキル基）を表す。）

（式中、Ｒ_５及びＲ_６は、各々独立に、炭素数１～４のアルキル基、炭素数１～４のアルコキシ基、置換基を有していてもよいベンジルオキシ基又はヒドロキシ基を表し（但し、Ｒ_５及びＲ_６の両方がヒドロキシ基になることはない）、
Ｒ_５及びＲ_６は一緒になってＲ_５及びＲ_６が結合しているリン原子を含む４～７員の置換又は無置換のヘテロシクリルを形成してもよい 2. Fragment Coupling Another embodiment of the present invention is
PG-(AA) _n -SH:
(PG represents an N-terminal protecting group,
AA represents any amino acid residue, which may be identical or different in each occurrence, some or all of the side chains of the amino acid residue may be protected with protecting groups;
n is an integer from 1 to 4.
A compound represented by the formula:
H-(AA') _m -OH:
(AA' represents any amino acid residue, which may be the same or different in each occurrence, and some or all of the side chains of the amino acid residue may be protected with protecting groups;
m is an integer from 1 to 4.
The peptide represented by
This method (hereinafter also referred to as the "fragment coupling of the present invention") involves reacting a compound represented by formula (III) below with a compound represented by formula (IV) below to prepare a compound represented by PG-(AA) _n (AA') _m -OH.

(Wherein,
L is selected from the group consisting of an ester group (-CO ₂ R; R is an alkyl group having 1 to 30 carbon atoms), a substituted or unsubstituted aryl group, a substituted or unsubstituted benzoyl group, an amide group which may be substituted with an amino group (-CONR'R'', R' and R'' are each independently a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 4 carbon atoms, and at least one of R' and R'' is an alkyl group substituted with an amino group), and an acyl group (-COR _a '; R _a ' is an alkyl group having 1 to 4 carbon atoms), provided that when at least one of R ₃ and R ₄ is an ester group (-CO ₂ R _a ; R _a is an alkyl group having 1 to 4 carbon atoms), L may be hydrogen (H);
_R3 and _R4 each independently represent an _alkyl group having 1 to 4 carbon atoms, a halogen, or an ester group ( _-CO2Ra ; _Ra is an alkyl group having 1 to 4 carbon atoms).

(In the formula, R ₅ and R ₆ each independently represent an alkyl group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, a benzyloxy group which may have a substituent, or a hydroxy group (however, R ₅ and R ₆ cannot both be a hydroxy group);
R ₅ and R ₆ may be joined together to form a 4-7 membered substituted or unsubstituted heterocyclyl containing a phosphorus atom to which R ₅ and R ₆ are attached.

As the N-terminal protecting group of PG, the same N-terminal protecting group as explained in the peptide synthesis method of the present invention can be used.
The arbitrary amino acid residues of AA and AA' include α-amino acid and β-amino acid residues, and the α-amino acid residues include amino acid residues such as leucine, isoleucine, valine, lysine, threonine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, serine, histidine, phenylalanine, alanine, glycine, tryptophan, tyrosine, cysteine, methionine, proline, ornithine, N-methylleucine, 2,3-diaminopropanoic acid, 2,4-diaminobutyric acid, and α-hydroxyleucine. The β-amino acid is an amino acid residue such as β-alanine. However, the amino acid residue bound to SH among the amino acid residues of AA is any amino acid residue other than phenylglycine, and the amino acid residue at the N-terminus among the amino acid residues of AA' is an amino acid residue other than proline and N-methylamino acid.
When the side chains of these amino acid residues are protected, the same protecting groups as those explained in the peptide synthesis method of the present invention can be used.
Any amino acid residue in AA and AA' may be either the L- or D-optical isomer.

In the fragment coupling of the present invention, n and m are preferably the same. n and m are preferably 2 or 3.

In the fragment coupling of the present invention, the reaction conditions such as solvent, temperature, and time described in the peptide synthesis method of the present invention can be used in the same manner.

（式中、

、Ｒ_１、Ｒ_２は、式（I）及び（II）で定義した通りである。）

（ｉｉ）式（VII）で表される化合物と、以下の式（VIII）で表されるアミノ酸を、以下の式（III）で表される化合物及び以下の式（IV）で表される化合物の存在下で反応させる工程：
を含む、式（VIIII）の化合物を調製する方法である（以下「本発明の反復カップリング」ともいう）。

（式中、Ｒ_３ａは、α－アミノ酸の側鎖であり、当該側鎖は保護基で保護されていてもよい。）

（式中、Ｌ、Ｒ_３～Ｒ_４は、上記で定義した通りである。）

（式中、Ｒ_５～Ｒ_６は、上記で定義した通りである。）

（式中、

、Ｒ_１～Ｒ_２、Ｒ_３ａは、上記で定義した通りである。） 3. Iterative Coupling Another embodiment of the present invention comprises carrying out the peptide synthesis method of the present invention as described above, followed by:
(i) reacting a compound represented by the following formula (V) with a compound represented by the following formula (1) to prepare a compound represented by formula (VII):

(Wherein,

, R ₁ and R ₂ are as defined in formulas (I) and (II).

(ii) reacting a compound represented by formula (VII) with an amino acid represented by the following formula (VIII) in the presence of a compound represented by the following formula (III) and a compound represented by the following formula (IV):
(hereinafter also referred to as "the iterative coupling of the present invention")

(In the formula, R _3a is a side chain of an α-amino acid, and the side chain may be protected with a protecting group.)

(In the formula, L, R ₃ to R ₄ are as defined above.)

(In the formula, R ₅ to R ₆ are as defined above.)

(Wherein,

, R ₁ to R ₂ , and R _3a are as defined above.)

In formula (VIII), R _3a is the side chain of an α-amino acid, which is the same as that explained for the amino acid of formula (II). The side chain may be protected with a protecting group, which is the same as that explained for formula (II).

The iterative coupling of the present invention can be used repeatedly to extend a peptide chain by repeating the method described above. The peptide chain can be extended until a peptide having usually 2 to 5 amino acid residues is obtained.

In the iterative coupling of the present invention, the reaction conditions such as solvent, temperature and time described in the peptide synthesis method of the present invention can be used in the same manner.

The peptide synthesis method of the present invention, the fragment coupling of the present invention, and the iterative coupling of the present invention described above can be applied to the liquid phase synthesis of peptides.

（式中、Ｒ_５及びＲ_６は、各々独立に、炭素数１～４のアルキル基、炭素数１～４のアルコキシ基、置換基を有していてもよいベンジルオキシ基又はヒドロキシ基を表し（但し、Ｒ_５及びＲ_６の両方がヒドロキシ基になることはない）、
Ｒ_５及びＲ_６は一緒になってＲ_５及びＲ_６が結合しているリン原子を含む４～７員の置換又は無置換のヘテロシクリルを形成してもよい。） Another aspect of the present invention is the use of a compound represented by the following formula (IV) as a solubilizing agent in peptide synthesis.

(In the formula, R ₅ and R ₆ each independently represent an alkyl group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, a benzyloxy group which may have a substituent, or a hydroxy group (however, R ₅ and R ₆ cannot both be a hydroxy group);
_R5 and _R6 may join together to form a 4-7 membered substituted or unsubstituted heterocyclyl containing a phosphorus atom to which _R5 and _R6 are bonded.

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

1. General Methods (1) General Electrospray ionization (ESI) mass spectra were measured on a Shimadzu LCMS-2020 spectrometer (for LRMS). Analytical HPLC charts were obtained using a JASCO HPLC system equipped with a UV-2075 spectrometer, a PU-2080 pump, a DG-2080-54 degasser, and an MX-2080-32 mixer.

(2) HPLC for analysis
Peptide composition was assessed by analytical reverse-phase HPLC using a gradient of acetonitrile vs. 0.1% trifluoroacetic acid (TFA) in water.
Reverse-phase analytical HPLC was performed as follows: a YMC-Triart- _C18 (4.6 mm ID x 150 mm) column was used with a linear gradient of 0-100% acetonitrile in 0.1% TFA in water at room temperature over 40 min at a flow rate of 1 mL/min ^-1 . The eluent was monitored by absorbance at 230 nm.

(3) Preparative HPLC
Longer peptides were purified by preparative reversed-phase HPLC using a gradient of acetonitrile vs. 0.1% TFA in water. Preparative HPLC was performed as follows: YMC-Triart C18 (20 nm i.d. x 250 mm) column at 40°C with a flow rate of 10.0 mL ^min-1 . Gradient conditions were modified for each peptide. The eluent was monitored by absorbance at 230 nm.

(4) Preparation of a calibration curve for peptides Authentic peptides (5 μmol) were dissolved in DMSO (250 μL) to prepare a 20 mM peptide solution. This solution was diluted to 15 mM, 10 mM, 8 mM, 5 mM, and 2 mM. For peptides containing Trt or Pbf, authentic peptides (1 μmol) were dissolved in DMSO (500 μL) to prepare a 2 mM peptide solution, which was then diluted to 1 mM, 0.5 mM, 0.25 mM, and 0.125 mM, and these solutions were then analyzed by analytical HPLC using absorbance at 230 nm. For each solution, the peak area corresponding to the peptide was measured, and a calibration curve was prepared from these values.

(5) Calculation method for the epimerization level of thioacids Peptide thioacids were converted to p-methoxybenzyl thioesters with p-methoxybenzyl chloride, and the reaction mixtures containing the thioesters were analyzed by normal-phase chiral HPLC to measure the peak areas corresponding to the thioester isomers, and the epimerization rate was calculated by the following formula:

このＡＥＬは基質として用いたペプチドチオ酸のエピマ-化レベルを含む。したがって、チオ酸（ＴＥＬ）のエピマ-化レベルを用いて以下の式に基づいて計算したカップリング反応においてエピマ－化レベルが生じた。

(6) Calculation method of epimerization level of peptide after coupling reaction After the peptide coupling reaction, the reaction mixture was analyzed by reversed-phase HPLC. The peak areas corresponding to LLL isomers and LDL isomers were measured, and the apparent epimerization level (AEL) was calculated based on the following formula:

This AEL includes the epimerization level of the peptide thioacid used as the substrate. Therefore, the epimerization level of the thioacid (TEL) produced in the coupling reaction was calculated based on the following formula:

Synthesis Examples
1. Preparation of peptide thioacids
The peptide thioacids were obtained by the method described in Chem. Commun., 2018, 54, 12222. The preparation method of each peptide thioacid is described below.

(1) Synthesis of Cbz-Phe-Val-SH To a frame-dried flask, Cbz-Phe-Val-OH (1.19 g, 3.0 mmol) and DMF (20 mM) were added under argon atmosphere. AcSK (3.43 g, 30 mmol) and Ac ₂ S (161 μL, 1.5 mmol) were then added to the solution. After the resulting mixture was stirred at 0° C. for 3 h, the reaction mixture was extracted with ethyl acetate and the combined organic layer was concentrated under reduced pressure. The residue was dissolved in acetonitrile and purified by automated RP-column chromatography to give Cbz-Phe-Val-SH (870 mg, 70%) as a yellow solid. MS (ESI): m/z 415.53 (calcd [M+H] ⁺ =415.10). Retention time: 29.75 min.

(2) Synthesis of Cbz-Phe-Phe-SH To a frame-dried flask, Cbz-Phe-Phe-OH (446 mg, 1.0 mmol) and DMF (10 mM) were added under argon atmosphere. AcSK (342 mg, 3.0 mmol) and Ac ₂ S (21 μL, 0.2 mmol) were then added to the solution. After the resulting mixture was stirred at 0° C. for 3 h, the reaction mixture was extracted with ethyl acetate and the combined organic layer was concentrated under reduced pressure. The residue was dissolved in acetonitrile and purified by automated RP-column chromatography to give Cbz-Phe-Phe-SH (254 mg, 55%) as a white solid. MS (ESI): m/z 485.15 (calcd [M+Na] ⁺ =485.55). Retention time: 31.67 min.

(3) Synthesis of Boc-Phe-Val-SH To a frame-dried flask, Boc-Phe-Val-OH (109 mg, 0.3 mmol) and DMF (3 mM) were added under argon atmosphere. AcSK (171 mg, 1.5 mmol) and Ac ₂ S (18 μL, 0.15 mmol) were then added to the solution. After the resulting mixture was stirred at 0° C. for 3 h, the reaction mixture was extracted with ethyl acetate and the combined organic layer was concentrated under reduced pressure. The residue was dissolved in acetonitrile and purified by preparative HPLC to give Boc-Phe-Val-SH (57 mg, 50%) as a white solid. MS (ESI): m/z 403.15 (calcd [M+Na] ⁺ =403.49). Retention time: 30.16 min.

(4) Synthesis of Cbz-Phe-Cys(Trt)-SH In a frame-dried test tube, Cbz-Phe-Cys(Trt)-OH (129 mg, 0.2 mmol) and DMF (2 mM) were added under argon atmosphere. Then, AcSK (114 mg, 1 mmol) and Ac ₂ S (10 μL, 0.1 mmol) were added to the solution. After the resulting mixture was stirred at 0° C. for 3 h, the reaction mixture was extracted with ethyl acetate and the combined organic layer was concentrated under reduced pressure. The residue was dissolved in acetonitrile and purified by automated RP-column chromatography to give Cbz-Phe-Cys(Trt)-SH (25 mg, 19%) as a yellow solid. MS (ESI): m/z 683.15 (calcd [M+Na] ⁺ =683.84). Retention time: 37.89 min.

(5) Synthesis of Cbz-Phe-Lys(Boc)-SH To a frame-dried test tube, Cbz-Phe-Lys(Boc)-OH (105 mg, 0.2 mmol) and DMF (2 mM) were added under argon atmosphere. AcSK (68 mg, 0.6 mmol) and Ac ₂ S (5 μL, 40 μmol) were then added to the solution. After the resulting mixture was stirred at 0° C. for 3 h, the reaction mixture was extracted with ethyl acetate and the combined organic layer was concentrated under reduced pressure. The residue was dissolved in acetonitrile and purified by automated RP-column chromatography to give Cbz-Phe-Lys(Boc)-SH (30 mg, 28%) as a yellow solid. MS (ESI): m/z 566.10 (calcd [M+Na] ⁺ =566.67). Storage time: 31.80 min.

(6) Synthesis of Cbz-Phe-Ala-Val-SH To a frame-dried test tube, Cbz-Phe-Ala-Val-OH (47 mg, 0.1 mmol) and DMF (1 mM) were added under argon atmosphere. AcSK (57 mg, 0.5 mmol) and Ac ₂ S (5 μL, 50 μmol) were then added to the solution. After the resulting mixture was stirred at 0° C. for 3 h, the reaction mixture was extracted with ethyl acetate and the combined organic layer was concentrated under reduced pressure. The residue was dissolved in acetonitrile and purified by preparative HPLC to give Cbz-Phe-Ala-Val-SH (11 mg, 25%) as a yellow solid. MS (ESI): m/z 508.15 (calcd [M+Na] ⁺ =508.59). Retention time: 29.34 min.

2. Preparation of N-hydroxy-pyridone additive (1) Methyl 1-hydroxy-6-oxo-1,6-dihydropyridine-3-carboxylate (Compound 1)
Compound 1 was synthesized according to the following synthetic scheme.

This compound was synthesized as described in the literature (Ando, M.; Sato, N.; Nagase, T.; Nagai, K.; Ishikawa, S.; Takahashi, H.; Ohtake, N.; Ito, J.; Hirayama, M.; Mitobe, Y.; et al. Bioorganic Med. Chem. 2009, 17, 6106-6122.). To a stirred solution of S1 (10.2 g, 60 mmol) and DCM (80 mL), urea hydrogen peroxide (12.0 g, 122 mmol) and TFAA (17.1 mL, 120 mmol) were added dropwise successively at 0° C., and the reaction mixture was allowed to warm to room temperature and stirred for 3 h. After cooling to 0° C., aqueous Na ₂ S ₂ O ₃ was added to the reaction mixture and concentrated. The mixture was extracted with DCM, washed with aqueous NaHCO ₃ and brine, and dried over Na ₂ SO ₄ . The organic layer was concentrated to give crude S2. The crude mixture was purified by flash column chromatography (ethyl acetate/hexanes=3:1) to give S2 (7.8 g, 70%) as a yellow solid.

To a stirred solution of S2 (7.8 g, 41.7 mmol) and MeCN (3 mL) was added TFAA ( ₇ ml, 50 mmol) at room temperature, and the mixture was stirred for 3 h. The reaction mixture was concentrated under reduced pressure. To the residue was added solid NaHCO3 and methanol. After filtration, the filtrate was concentrated to give crude product 1. The crude product was purified by flash column chromatography (DCM/MeOH=19:1) to give 1 (3.0 g, 43%) as a pale yellow solid.
MS (ESI) ^: m/ _z 170.10 (calcd [M+H] ⁺ = 170.14).

(2) 1-hydroxy-5-phenylpyridin-2(1H)-one (Compound 2)
Compound 2 was synthesized according to the following synthetic scheme.

To a stirred solution of S3 (241 μL, 2 mmol) and DCM (10 mL) was added mCPBA (986 mg, 4 mmol, containing 30% H ₂ O) at 0° C., and the mixture was stirred for 2 h. 1 M aqueous Na ₂ S ₂ O ₃ was added to the reaction mixture, and the mixture was extracted with DCM. The combined organic layers were washed with aqueous NaHCO ₃ and brine, and dried over Na ₂ SO _4. After filtration, the organic layers were concentrated to give crude product S4. Crude product S4 was purified by flash column chromatography (DCM/MeOH=19:1) to give S4 (320 mg, 79%) as a white solid.

To a stirred solution of S4 (50 mg, 0.25 mmol) and DMF (500 μL), phenylboronic acid (45 mg, 0.375 mmol), Pd(PPh ₃ ) ₄ (14 mg, 2.5 μmol) and K ₂ CO ₃ (69 mg, 0.5 mmol) were added and stirred at 100° C. overnight, the reaction mixture was extracted with DCM and the combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give crude product S5. The crude mixture was purified by flash column chromatography (DCM/MeOH=19:1) to give S5 (34 mg, 67%) as a white solid.

To a stirred solution of S5 (20 mg, 0.1 mmol) and dry DCM (500 μL) was added BCl ₃ (1 M in heptane, 150 μL, 0.15 mmol) dropwise under argon atmosphere at −10° C., and the reaction mixture was allowed to warm to room temperature and stirred overnight. After addition of MeOH (1 mL), the reaction mixture was concentrated to give crude product 2. The crude product was purified by flash column chromatography (ethyl acetate/hexanes=1:3) to give 2 (5 mg, 27%) as a white solid.
¹ H NMR (500 MHz, CDCl ₃ ): δ 6.84 (1H, d, J = 9.2), 7.36 (1H, m), 7.44 (4H, m), 7.67 (1H, d, J = 10.3), 8.02 (1H, s); MS (ESI): m/z 188.10 (calcd [M+H] ⁺ =188.21).

(3) Methyl 1-hydroxyl-2-oxo-5-phenyl-1,2-dihydropyridine-3-carboxylate (Compound 3)
Compound 3 was synthesized according to the following synthetic scheme.

To a frame-dried 50 mL flask, Na (340 mg, 14 mmol) and MeOH (10 mL) were added at 0° C. under argon atmosphere, and S6 (1.0 g, 4 mmol) was added to the solution, and the mixture was stirred for 3 h. Acetic acid was added to the reaction mixture until neutralization, and the mixture was concentrated under reduced pressure. Ethyl acetate and H ₂ O were added to the residue, and the product was extracted with ethyl acetate. The organic layer was washed with H ₂ O and brine, dried over Na ₂ SO ₄ , and concentrated to give crude product S7. The crude mixture was purified by flash column chromatography (ethyl acetate/hexanes=1:3) to give S7 (800 mg, 77%) as a colorless oil.

_In a 30 mL flask, S7 (390 mg, 1.5 mmol), phenylboronic acid (272 mg, 2.25 mmol), Pd( _PPh3 ) ₄ (86 mg, 75 μmol), _K2CO3 (414 mg, 3 mmol), and DMF (8 mL) were added under argon atmosphere, and the mixture was stirred at 100° _C. overnight. Ethyl acetate and _H2O were added to the reaction mixture, and the product was extracted with ethyl acetate. The combined organic layers were washed with _H2O , dried over _Na2SO4 , and concentrated to crude product S8. The crude mixture was purified by flash column chromatography (ethyl acetate/hexane=1:3) to give S8 (280 mg, 77%) as a white solid.

A test tube was charged with S8 (120 mg, 0.5 mmol), urea hydrogen peroxide (294 mg, 6.1 mmol), and MeCN (2 mL). TFAA (450 μL, 6 mmol) was added dropwise to the 0° C. solution, the reaction was allowed to warm to room temperature, and the mixture was stirred overnight. Aqueous Na ₂ S ₂ O ₃ was added to the reaction mixture, and the mixture was concentrated. Ethyl acetate and H ₂ O were added to the residue, and the product was extracted with ethyl acetate. The organic layer was washed with brine, dried over Na ₂ SO ₄ , and concentrated to give crude product S9. The crude mixture was purified by flash column chromatography (ethyl acetate/hexanes=3:1) to give S9 (50 mg, 38%) as a white solid.

To a test tube was added S9 (25 mg, 0.1 mmol) and DCM (500 μL) under argon atmosphere. AcCl (72 μL, 1 mmol) was added to the solution and the mixture was heated to reflux for 1 h. The reaction mixture was concentrated. Methanol was added and the mixture was stirred at room temperature overnight. The mixture was concentrated to give crude product 3. The crude mixture was purified by flash column chromatography (DCM/MeOH=19:1) to give 3 (11 mg, 45%) as a white solid.
¹ H NMR (500 MHz, CDCl ₃ ): δ3.97 (3H, s), 7.39 (2H, m), 7.46 (3H, m), 8.26 (1H, s), 8.47 (1H, s); MS (ESI): m/z 246 (calcd [M+H] ⁺ = 246.24).

(4) Methyl 2-(1-hydroxy-6-oxo-1,6-dihydropyridin-3-yl)benzoate (Compound 4)
Compound 4 was synthesized according to the following synthetic scheme.

In a 100 mL flask, S3 (482 mg, 4 mmol), 2-(methoxycarbonyl)-phenylboronic acid (1074 mg, 6 mmol), Pd(PPh ₃ ) ₄ (231 mg, 0.2 mmol), K ₂ CO ₃ (1100 mg, 12 mmol) and DMF (20 mL) were added under argon atmosphere, and the mixture was stirred at 100° C. overnight. Ethyl acetate and H ₂ O were added to the reaction mixture, and the product was extracted with ethyl acetate. The combined organic layers were washed with H ₂ O and brine, dried over Na ₂ SO ₄ and concentrated to give crude product S10. The crude mixture was purified by flash column chromatography (ethyl acetate/hexane=1:3) to give S10 (800 mg, 82%) as a white solid.

To S10 (600 mg, 2.5 mmol) in DCM (10 mL) in a 50 mL flask was added mCPBA (1.85 g, 7.5 mmol, containing _{30 % H2O} ) at 0°C, and the mixture was allowed to warm to room temperature and stirred overnight. 1M _Na2S2O3 aqueous solution was added to the reaction mixture, and the mixture was extracted with DCM. The combined organic layers were washed with _NaHCO3 aqueous solution and brine, _and dried over _Na2SO4 . The organic layers were concentrated to give the crude product S11. The crude mixture was purified by flash column chromatography (DCM/MeOH = 19:1) to give S11 (580 mg, 90%) as a white solid.

The reaction mixture was concentrated, methanol was added and stirred at room temperature overnight. The mixture was concentrated to give crude product 3. The crude mixture was purified by flash column chromatography (DCM/MeOH=19:1) to give 4 (125 mg, 51%) as a white solid.
¹ H NMR (500 MHz, CDCl ₃ ): δ 3.65 (3H, s), 6.79 (1H, s), 7.41 (4H, m), 7.84 (1H, d, J = 8.0), 8.10 (1H, s); MS (ESI): m/z 246 (calcd [M+H] ⁺ = 246.24).

(5) Methyl 1-hydroxy-2-oxo-1,2-dihydropyridine-3-carboxylate (Compound 5)
Compound 5 was synthesized according to the following synthetic scheme.

This compound was prepared using the procedure described for compound 1 as a yellow solid (72 mg, 85%).
(500 MHz, (CD ₃ ) ₂ SO): δ 3.74 (3H, s), 6.25 (1H, dd, J = 6.7, 7.4), 7.96 (1H, d, J = 7.4), 8.23 (1H, d, J = 6.7); MS (ESI): m/z 170.10 (calcd [M+H] ⁺ = 170.14).

(6) N-(2-(dimethylamino)ethyl)-1-hydroxy-6-oxo-1,6-dihydropyridine-3-carboxamide (Compound 6)
Compound 6 was synthesized according to the following synthetic scheme.

To a stirred solution _of compound 1 (3.0 g, 17.75 mmol), _K2CO3 (7.3 g, 53.25 mmol), and DMF (40 mL) was added BnBr (2.5 mL, 21.3 mmol), stirred at 80°C for 3 hours, and the reaction mixture was concentrated. Ethyl acetate and _H2O were added to the resulting mixture, and the product was extracted with ethyl acetate. The organic layer was washed with brine, dried over _Na2SO4 , filtered, and concentrated to give crude product S13. The crude mixture was purified by flash column chromatography ₍ ethyl acetate/hexanes = 1:3) to give S13 (2.0 g, 44%) as a white solid.

To a stirred solution of S13 (2.0 g, 8 mmol) and MeOH (8 mL) was added 3M aqueous NaOH (8 mL, 24 mmol) and the mixture was stirred overnight. 1M aqueous HCl was added to the reaction mixture until neutralization. The product was concentrated. Ethyl acetate and 1M aqueous HCl were added _to the resulting residue and the product was extracted with ethyl acetate. The organic layer was washed with brine, dried over _Na2SO4 , filtered and concentrated to give S14 (1.7 g, 87%) as a white solid.

To a stirred solution of S14 (49 mg, 0.2 mmol), N,N-dimethylethylenediamine (43 μL, 0.4 mmol), and DCM (1 mL) were added WSCI (76 mg, 0.4 mmol), and HOBt (54 mg, 0.4 mmol) at 0° C., and the reaction mixture was warmed to room temperature and stirred for 3 h. To the resulting mixture were added DCM and aqueous NaHCO ₃ solution. The product was extracted with DCM, and the organic layer was washed with brine, dried over Na ₂ SO ₄ , filtered, and concentrated to give the crude product S15. This crude mixture was used in the next reaction without further purification.

To a stirred solution of crude S15 and DCM (1 mL) was added BCl ₃ (240 μL of a 1 M solution in heptane, 0.24 mmol) and the mixture was stirred overnight. The reaction mixture was concentrated to give crude 6. The crude mixture was purified by preparative HPLC to give 6 (11 mg, 24%, 2 steps) as a white solid.
¹ H NMR (500 MHz, CDCl ₃ ): δ 2.25 (6H, s), 2.44 (2H, t, J = 5.7), 3.39 (2H, t, J = 5.7), 6.67 (1H, d, J = 9.7), 7.58 (1H, d, J = 9.7), 7.88 (1H, s); MS (ESI): m/z 259 (calcd [M+H] ⁺ = 259.28).

(7) 5-(3-(dimethylamino)benzyl)-1-hydroxypyridin-2(1H)-one (Compound 7)
Compound 7 was synthesized according to the following synthetic scheme.

To a stirred solution of S14 (490 mg, 2.4 mmol) and p-toluenethiol (298 mg, 2.4 mmol) in DCM (10 mL) was added WSCI (458 mg, 2.4 mmol) and HOBt (270 mg, 2 mmol) at 0 °C, and the reaction mixture was warmed to room temperature and stirred for 3 h. The resulting mixture was concentrated. Ethyl acetate and aqueous NaHCO ₃ were added, and the product was extracted with ethyl acetate. The organic layer was washed with brine, dried over Na ₂ SO ₄ , filtered, and concentrated to give crude product S16. The crude mixture was purified by flash column chromatography (ethyl acetate/hexanes=1:3) to give S16 (480 mg, 68%) as a white solid.

To a stirred solution of S16 (70 mg, 0.2 mmol) and THF (2 mL) was _{added 2-dimethylaminophenylboronic acid (99 mg, 0.6 mmol), Pd2(dba)3.CHCl3 ( 5} mg, 4 μmol), tri(2-furyl)phosphine (3.5 mg, 15 μmol), and CuTC (115 mg, 0.6 mmol). The reaction mixture was heated to reflux and stirred overnight. After filtration through Celite, the filtrate was concentrated to give S17. This crude mixture was used in the next reaction without further purification.

To a stirred solution of S17 and DCM (1 mL) was added BCl ₃ (690 μL, 0.7 mmol) and the mixture was stirred overnight. The reaction solution was concentrated to give the crude product 7. The crude mixture was purified by preparative HPLC to give 7 (6 mg, 12%, 2 steps) as a colorless oil.
¹ H NMR (500 MHz, CDCl ₃ ): δ 3.28 (6H, s), 6.73 (1H, s), 7.28-7.43 (4H, m), 7.75 (1H, d, J = 8.0), 7.99 (1H, d, J = 8.0); MS (ESI): m/z 259 (calcd [M+H] ⁺ = 259.28).

(8) Synthesis of Compound 8 Compound 7 was synthesized according to the following synthetic scheme.

3,7,11,15-Tetramethylhexadecan-1-ol (10.8 mL, 30.2 mmol) and triethylamine (7.2 mL, 37.8 mmol) were added to a mixture of S18 (4 g, 25.2 mmol), WSCI (7.2 g, 37.8 mmol), DMAP (4.6 g, 37.8 mmol) and DCM (80 mL) at room temperature, and the mixture was stirred at room temperature for 25 hours under an argon atmosphere, and the solvent was removed under reduced pressure. Ethyl acetate and 1M aqueous HCl were added to the residue, and the mixture was extracted with ethyl acetate. The organic layer was washed twice with saturated sodium bicarbonate water, washed with brine, dried over Na2SO4, filtered, and the solvent was removed under reduced pressure to obtain the crude product S19. The crude mixture was purified by silica gel column chromatography (ethyl acetate/hexane = 20:80 → 40:60) to obtain S19 (8.85 g, 80%).

Trifluoroacetic anhydride (5.7 mL, 40.4 mmol) was added dropwise to a mixture of S19 (8.85 g, 20.2 mmol), urea hydrogen peroxide (3.95 g, 42.4 mmol) and DCM (50 mL) at 0° C., and the mixture was stirred at room temperature for 16 hours under an argon atmosphere. The mixture was cooled to 0° C., and saturated aqueous sodium bisulfite solution (5.5 mL) was added to the reaction solution, and the reaction mixture was extracted with ethyl acetate. The organic layer was washed twice with saturated aqueous sodium bicarbonate, washed with brine, dried over Na ₂ SO ₄ , filtered, and the solvent was removed under reduced pressure to obtain the crude product S19. The crude mixture was purified by silica gel column chromatography (ethyl acetate/hexane=10:90 → 100:0) to obtain S20 (7.51 g, 82%).

To a stirred solution of S20 (7.51 g, 16.5 mmol) and acetonitrile (15 mL) was added TFAA (30 ml) at room temperature and stirred under argon atmosphere at room temperature for 14 h. The solvent was removed under reduced pressure, and solid _NaHCO3 and chloroform were added to the residue. After filtration, the solvent was removed under reduced pressure to give the crude product 8. The crude product was purified by neutral silica gel column chromatography (ethyl acetate/hexane = 40:60 → 100:0) to give a yellow liquid 8 (5.82 g, 81%).
¹ H NMR (500 MHz, CD ₃ OD): δ 0.83-0.89 (12H, m), 0.96 (3H, d, J = 6.3 Hz), 1.02-1.44 (20H, m), 1.45-1.66 (3H, m), 1.70-1.82 (1H, m), 4.24-4.40 (2H. m), 6.63 (1H, d, J = 9.2 Hz),7.92 (1H, dd, J = 2.3, 9.7 Hz), 8.52 (1H, d, J =2.9 Hz); MS (ESI): m/z 436.30 (calcd [M+H] ⁺ = 436.34).

(9) Synthesis of Compound 9 Compound 9 was synthesized according to the following synthetic scheme.

This compound was prepared using the procedure described for compound 8 to give a yellow liquid 9 (1.53 g, 96%).
¹ H NMR (500 MHz, CDCl ₃ ): δ0.87 (6H, d, J = 6.3 Hz), 0.94 (3H, d, J = 6.3 Hz), 1.08-1.20 (3H, m), 1.23-1.38 (3H, m), 1.44-1.64 (3H, m), 1.70-1.82 (1H, m), 4.28-4.38 (1H, m), 6.73 (1H, d, J =9.7 Hz), 7.96 (1H, dd, J =2.9, 9.7 Hz), 8.55 (1H, d, J =2.3 Hz); MS (ESI): m/z 296.20 (calcd [M+H] ⁺ = 296.19).

(10) Synthesis of Compound 10 Compound 10 was synthesized according to the following synthetic scheme.

This compound was prepared using the procedure described for compound 8 to give a yellow solid 10 (205 mg, 92%).
¹ H NMR (500 MHz, CD ₃ OD): δ0.91 (3H, t, J =7.4 Hz), 1.30-1.38 (4H, m), 1.40-1.46 (2H, m),1.70-1.76 (2H, m), 4.26 (3H, t, J =6.9 Hz), 6.61 (1H, d, J = 9.2 Hz), 7.90-7.92 (1H, m), 8.48-8.54 (1H, m); MS (ESI): m/z 240.15 (calcd [M+H] ⁺ = 240.12).

(11) Synthesis of Compound 11 Compound 11 was synthesized according to the following synthetic scheme.

This compound was prepared using the procedure described for compound 8 to give 11 (131 mg, 48%) as a yellow liquid.
¹ H NMR (500 MHz, CD ₃ OD): δ0.93 (6H, t, J = 7.4 Hz), 1.60-1.75 (4H, m), 4.90-4.95 (1H. m), 6.63 (1H, d, J = 9.2 Hz), 7.93 (1H, dd, J = 2.3 Hz, 9.2 Hz), 8.53 (1H, d, J = 2.3 Hz); MS (ESI): m/z 226.40 (calcd [M+H] ⁺ =226.11).

(12) Synthesis of Compound 12 Compound 12 was synthesized according to the following synthetic scheme.

A mixture of S18 (632 mg, 4 mmol), Boc ₂ O (1.31 g, 6 mmol), DMAP (48.9 mg, 0.4 mmol) and tetrahydrofuran (10 mL) was stirred under argon atmosphere with heating under reflux for 3 hours, and the solvent was removed under reduced pressure. Diethyl ether and water were added to the residue, and the mixture was extracted with diethyl ether. The organic layer was washed with saturated sodium bicarbonate water and saturated saline in this order, dried over Na2SO4, filtered, and the solvent was removed under reduced pressure to obtain crude product S27. The crude mixture was purified by silica gel column chromatography (ethyl acetate/hexane = 5:95 → 30:70) to obtain S27 (622 mg, 73%).

Trifluoroacetic anhydride (374 μL, 2.66 mmol) was added dropwise to a mixture of S27 (285 mg, 1.33 mmol), urea hydrogen peroxide (263 mg, 2.80 mmol) and DCM (13.3 mL) at 0° C., and the mixture was stirred at room temperature for 7 hours under an argon atmosphere. The mixture was cooled to 0° C., and saturated aqueous sodium bisulfite and water were added to the reaction solution, and the reaction mixture was extracted with chloroform. The organic layer was washed twice with saturated aqueous sodium bicarbonate, washed with brine, dried over Na ₂ SO ₄ , filtered, and the solvent was removed under reduced pressure to obtain the crude product S28. The crude mixture was purified by silica gel column chromatography (ethyl acetate/hexane=20:90 → 100:0) to obtain S28 (280 mg, 92%).

To a stirred solution of S28 (280 mg, 1.22 mmol) and acetonitrile (12.2 mL), trifluoroacetic anhydride (344 μL, 2.44 mmol) was added at room temperature, and the reaction solution was stirred for 1 hour and 20 minutes under argon atmosphere. Trifluoroacetic anhydride (175 μL, 1.22 mmol) was added to the reaction solution at room temperature, and the reaction solution was stirred for 14 hours and 30 minutes at room temperature. The mixture was stirred for 1 hour and 20 minutes. Trifluoroacetic anhydride (175 μL, 1.22 mmol) was added to the reaction solution at room temperature, and the reaction solution was stirred for 2 hours at room temperature. The solvent was removed under reduced pressure, and solid NaHCO ₃ and chloroform were added to the residue. After filtration, the solvent was removed under reduced pressure to obtain crude product 12. The crude product was purified by neutral silica gel column chromatography (ethyl acetate/methanol=100:0 → 85:15) to obtain white solid 12 (119 mg, 46%).
¹ H NMR (500 MHz, CD ₃ OD): δ1.56 (9H,s), 6.60 (1H, d, J = 9.2 Hz), 7.84--7.90 (1H, m), 8.43 (1H, d, J =2.3 Hz); MS (ESI): m/z 240.12 (calcd [M+H] ⁺ = 240.15).

Ｈ-ＡＡ-ＯＨは任意のアミノ酸を示す。 [Synthesis Example 1]
Condensation of dipeptide thioacids with various amino acids.
Condensation of dipeptide thioacids with various amino acids was carried out according to the following reaction scheme.

H-AA-OH represents any amino acid.

N-hydroxypyridone additive, secondary phosphorous acid, and peptide thioacid were used as stock solutions. N-hydroxypyridone additive, secondary phosphorous acid, and amino acid were added to a 1.5 mL microtube. Then, a solution of peptide thioacid was added and stirred at 30°C for a given reaction time. The reaction mixture was diluted with 1% TFA in DMSO and analyzed by HPLC. The yield was determined by a calibration curve.

The results (yield, epimerization degree) are shown in Table 1 below.

The characteristics of the peptide obtained above were examined, and the results are described below.
Cbz-Phe-Val-Gly-OH MS (ESI): m/z 478.15 (calcd [M+Na] ⁺ = 478.50)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 25.10 min
Cbz-Phe-Val-Ala-OH MS (ESI): m/z 470.10 (calcd [M+H] ⁺ = 470.23)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 24.69 min
Cbz-Phe-Val-Val-OH MS (ESI): m/z 520.10 (calcd [M+Na] ⁺ = 520.24)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 26.72 min
Cbz-Phe-Val-Ile-OH MS (ESI): m/z 512.20 (calcd [M+H] ⁺ = 512.28)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 28.56 min
Cbz-Phe-Val-Phe-OH MS (ESI): m/z 568.15 (calcd [M+Na] ⁺ = 568.24)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 28.21 min
Cbz-Phe-Val-Met-OH MS (ESI): m/z 552.10 (calcd [M+Na] ⁺ = 552.21)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 27.59 min
Cbz-Phe-Val-Pro-OH MS (ESI): m/z 518.10 (calcd [M+Na] ⁺ = 518.23)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 25.52 min
Cbz-Phe-Val-Trp(Boc)-OH MS (ESI): m/z 707.20 (calcd [M+Na] ⁺ = 707.31)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 33.68 min
Cbz-Phe-Val-Lys(Boc)-OH MS (ESI): m/z 649.25 (calcd [M+Na] ⁺ = 649.32)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 28.58 min
Cbz-Phe-Val-Tyr(O ^t Bu)-OH MS (ESI): m/z 584.40 (calcd [M+Na] ⁺ = 584.24)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 30.46 min
Cbz-Phe-Val-Cys(Trt)-OH MS (ESI): m/z 766.20 (calcd [M+Na] ⁺ = 766.29)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 34.59 min
Cbz-Phe-Val-Asp( ^t Bu)-OH MS (ESI): m/z 592.10 (calcd [M+Na] ⁺ = 592.64)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 28.25 min
Cbz-Phe-Val-Asn(Trt)-OH MS (ESI): m/z 777.25 (calcd [M+Na] ⁺ = 777.87)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 33.25 min
Cbz-Phe-Val-Ser(O ^t Bu)-OH MS (ESI): m/z 564.20 (calcd [M+Na] ⁺ = 564.27)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 29.08 min
Cbz-Phe-Val-Thr(O ^t Bu)-OH MS (ESI): m/z 500.10 (calcd [M+Na] ⁺ = 500.24)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 29.47 min
Cbz-Phe-Val-Arg(Pbf)-OH MS (ESI): m/z 807.05 (calcd [M+H] ⁺ = 807.37)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 29.87 min
Cbz-Phe-Val-His(Trt)-OH MS (ESI): m/z 778.20 (calcd [M+H] ⁺ = 778.93)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 30.87 min
Cbz-Phe-Val-tertLeu-OH MS (ESI): m/z 512.10 (calcd [M+H] ⁺ = 512.63)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 28.53 min
Cbz-Phe-Val-Aib-OH MS (ESI): m/z 484.15 (calcd [M+H] ⁺ = 484.57)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 25.31 min
Cbz-Phe-Val-Pg-OH MS (ESI): m/z 532.15 (calcd [M+H] ⁺ = 532.62)

[Synthesis Example 2]
Consideration of Protecting Groups
The study was carried out using terminal Val dipeptides having Fmoc and Boc, which are widely used in peptide synthesis. The results are shown below.

The characteristics of the peptide obtained above were examined, and the results are described below.
Fmoc-Phe-Val-Ala-OH MS (ESI): m/z 580.15 (calcd [M+Na] ⁺ = 580.64)
Purity: >95% (HPLC analysis at 230 nm) Retention time 29.83 min
Boc-Phe-Val-Ala-OH MS (ESI): m/z 458.15 (calcd [M+Na] ⁺ = 458.51)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 24.62 min

As shown in Table 2, the reaction proceeded with high yields and low epimerization rates, similar to when Cbz was used.

[Synthesis Example 3]
Examination of C-Terminal Residue Examination was carried out using dipeptide thioacids whose C-terminal residue was other than Val. The results are shown below.

The characteristics of the peptide obtained above were examined, and the results are described below.
Cbz-Phe-Phe-Ala-OH MS (ESI): m/z 540.05 (calcd [M+Na] ⁺ = 540.21)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 27.56 min
Cbz-Phe-Phe-Val-OH MS (ESI): m/z 568.10 (calcd [M+Na] ⁺ = 568.24)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 29.28 min
Cbz-Phe-Lys(Boc)-Ala-OH MS (ESI): m/z 621.20 (calcd [M+Na] ⁺ = 621.69)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 27.95 min
Cbz-Phe-Lys(Boc)-Val-OH MS (ESI): m/z 649.25 (calcd [M+Na] ⁺ = 649.74)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 29.53 min
Cbz-Phe-Cys(Trt)-Ala-OH MS (ESI): m/z 738.15 (calcd [M+Na] ⁺ = 738.85)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 34.15 min
Cbz-Phe-Cys(Trt)-Val-OH MS (ESI): m/z 766.10 (calcd [M+Na] ⁺ = 766.91)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 35.33 min

When the hydrophobic amino acid Phe was used as the C-terminus, or when the hydrophilic amino acid Lys(Boc) was used, the reaction proceeded with a high yield, and the epimerization rate was suppressed to less than 1%. On the other hand, when Cys(Trt), which is considered to be prone to epimerization, was used, epimerization was observed at about 4% when coupled with Ala and about 2% when coupled with valine.

[Synthesis Example 4]
Study of additives (epimerization inhibitors) (1)
In the following condensation reaction of Cbz-Phe-Val-SH with alanine, the type of additive was changed, and the results are summarized below.

The methyl ester moiety was made into an amide structure, and the basic site pyridine was added, probably because deprotonation was promoted at the active ester stage, and the reactivity of the active ester was also lower than that of the ester type, resulting in poor results in both yield and epimerization rate. The chlorine-substituted product had a low nucleophilicity of the hydroxy group, and the active ester formation did not occur at an appropriate rate, so the yield tended to decrease.

[Synthesis Example 5]
Study of additives (epimerization inhibitors) (2)
In the following condensation reaction of Cbz-Phe-Val-SH with alanine, the type of additive was changed, and the results are summarized below.

The results (yield, degree of epimerization) when the reaction time was set to 22 hours and various additives (epimerization inhibitors) were used are shown below.

Compound 5, in which R ₄ is an ester group and L is hydrogen in the general formula (III), has a slightly higher epimerism than compound 1 and the like, but the product could be obtained in a high yield.

The results (yield, epimerization rate) when the reaction time was set to 6 hours and various additives were used are shown below.

The results (yield, epimerization degree) when H-Al-OH was 2 equivalents and the additive was 2 equivalents were used with various additives are shown below.

The product in which the alkyl chain of the methyl ester portion was extended had good yields and epimerization rates. However, compound 11, which has an alkyl chain of 6 carbon atoms in the ester portion, had a high degree of crystallization and was difficult to separate from product 2ba. In contrast, compounds 12 and 13, which have branched alkyl chains but a larger number of carbon atoms than compound 11, were able to recover 76% and 73% of the additive, respectively, by recrystallization.

[Synthesis Example 6]
Condensation of dipeptide thioacids with various amino acids.
Condensation of dipeptide thioacids with various amino acids was carried out according to the following reaction scheme.

N-hydroxypyridone (78.4 mg, 0.18 mmol), dipeptide thioacid (50 mg, 0.12 mmol), and L-valine (28.1 mg, 0.24 mmol) were added to a test tube. Toluene (600 μL) and dimethyl sulfoxide (600 μL) were then added, and the reaction solution was stirred at 30°C for 6 hours. The reaction solution (12 μL) was diluted with 1% trifluoroacetic acid in dimethyl sulfoxide (68 μL) and analyzed by HPLC. The yield and epimerism were determined by a calibration curve (yield <99%, epimerism <1%). The solvent of the remaining reaction solution was removed under reduced pressure, and ethyl acetate and 1M HCl aqueous solution were added to the residue, followed by extraction with ethyl acetate. The mixture was dried over Na2SO4, filtered, and the solvent was removed under reduced pressure to obtain the crude tripeptide product. The crude product was purified by silica gel column chromatography (ethyl acetate/hexane = 30:70 → 100:0) to obtain the tripeptide (50 mg, 84%).

[Synthesis Example 7]
Examination of solubilizers
In the following condensation reaction of Cbz-Phe-Val-SH with alanine, the type of solubilizer was changed, and the results are summarized below.

Dialkyl phosphites gave good results, and the highest reaction acceleration effect was observed when diethyl phosphite was used. The yield increased from 59% without addition to 84% 6 hours after the start of the reaction. When dialkyl phosphites with functional groups bulkier than ethoxy groups were used, the yield decreased, suggesting that the steric properties of the phosphite contribute to the reactivity. In addition, the reactivity decreased when phosphites with alkyl groups directly introduced onto phosphorus were used, suggesting that in addition to the steric effect, the electronic state also contributes to the reactivity. In addition, when diethylthiophosphite was used, the system became complicated, probably due to the direct reaction between thioacid and thiophosphite, and the yield of tripeptide also decreased to 34%.

[Synthesis Example 8]
General method for fragment coupling reactions
N-hydroxypyridone additive 1 (12 μmol), diethyl phosphite (12 μmol), DIPEA (24 μmol) and peptide thioacid (12 μmmol) were used as stock solutions, respectively. To a 1.5 mL microtube, peptide fragment (14.4 μmol) was added. Then, stock solutions of N-hydroxypyridone additive 1 (12 μmol in DMSO, 12 μL), diethyl phosphite (12 μmol in DMSO, 12 μL) and DIPEA (12 μmol in DMSO, 12 μL) were added to the microtube. Finally, a solution of peptide thioacid (12 μmol in DMSO, 48 μL) was added and stirred at 30°C for the indicated reaction time. The reaction mixture was diluted with 1% TFA solution in DMSO (680 μL) and analyzed by HPLC. The yield was determined by HPLC based on the calibration curve.

[Synthesis Example 9]
Sequential peptide synthesis
(1) Cbz-Tyr(O ^t Bu)-OH -> Cbz-Tyr(O ^t Bu)-SH
To a frame-dried test tube, Cbz-Tyr(O ^t Bu)-OH (74 mg, 0.2 mmol) and DMF (2 mL) were added under argon atmosphere. Then, AcSK (114 mg, 1.0 mmol) and Ac ₂ S (12 μL, 0.1 mmol) were added to the solution. The reaction mixture was stirred at 0° C. for 5 h, the mixture was extracted with ethyl acetate, and the combined organic layer was washed with 1M aqueous HCl and brine, then dried over Na ₂ SO _4. After filtering off the drying agent, the filtrate was concentrated under reduced pressure. The residue was lyophilized and used in the next reaction without further purification.

(2) Cbz-Tyr(O ^t Bu)-SH -> Cbz-Tyr(O ^t Bu)-Gly-OH
Cbz-Tyr(O ^t Bu)-Gly-SH and a mixed solvent of DMSO:toluene=1:1 (2 mL) were added to a test tube. N-hydroxypyridone additive 1 (34 mg, 0.2 mmol), Gly (18 mg, 0.24 mmol), and diethyl phosphite (26 μL) were then added and stirred at 30° C. for 6 hours. The reaction mixture was diluted to 1.5 mM and analyzed by analytical HPLC, and the HPLC yield was 89%. Insoluble materials were then removed using a syringe filter, and the reaction mixture was purified by preparative HPLC. 66 mg (77%) of Cbz-Tyr(O ^t Bu)-Gly-OH was obtained.
The properties of Cbz-Tyr(O ^t Bu)-Gly-OH were examined, and the results are as follows.
MS (ESI): m/z 451.15 (calcd [M+Na] ⁺ = 451.47)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 26.87 min

(3) Cbz-Tyr(O ^t Bu)-Gly-OH -> Cbz-Tyr(O ^t Bu)-Gly-SH
To a frame-dried test tube, Cbz-Tyr(O ^t Bu)-Gly-OH (85 mg, 0.2 mmol) and DMF (2 mL) were added under argon atmosphere. Then, AcSK (114 mg, 1.0 mmol) and Ac ₂ S (12 μL, 0.1 mmol) were added to the solution. The reaction mixture was stirred at 0° C. for 5 h, the mixture was extracted with ethyl acetate, and the combined organic layer was washed with 1M aqueous HCl and brine, then dried over Na ₂ SO _4. After filtering off the drying agent, the filtrate was concentrated under reduced pressure. The residue was lyophilized and used in the next reaction without further purification.

(4) Cbz-Tyr(O ^t Bu)-Gly-SH -> Cbz-Tyr(O ^t Bu)-Gly-Gly-OH
Cbz-Tyr(O ^t Bu)-Gly-SH and a mixed solvent of DMSO:toluene=1:1 (2 mL) were added to a test tube. Then, N-hydroxypyridone additive 1 (34 mg, 0.2 mmol), Gly (18 mg, 0.24 mmol), and diethyl phosphite (26 μL) were added and stirred at 30° C. for 6 hours. The reaction mixture was diluted to 1.5 mM and analyzed by analytical HPLC, and the HPLC yield was 94%. Then, insoluble materials were removed with a syringe filter, and the reaction mixture was purified by preparative HPLC. 71 mg (73%) of Cbz-Tyr(O ^t Bu)-Gly-Gly-OH was obtained.
The properties of Cbz-Tyr(O ^t Bu)-Gly-Gly-OH were examined, and the results are as follows.
MS (ESI): m/z 508.15 (calcd [M+Na] ⁺ = 508.21)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 26.87 min

(5) Cbz-Tyr(O ^t Bu)-Gly-Gly-OH -> Cbz-Tyr(O ^t Bu)-Gly-Gly-SH
To a frame-dried test tube, Cbz-Tyr(O ^t Bu)-Gly-Gly-OH (49 mg, 0.1 mmol) and DMF (1 mL) were added under argon atmosphere. Then, AcSK (34 mg, 0.3 mmol) and Ac ₂ S (2.1 μL, 0.02 mmol) were added to the solution. The reaction mixture was stirred at 0° C. for 3 h, the mixture was extracted with ethyl acetate, and the combined organic layers were washed with 1M aqueous HCl and brine, then dried over Na ₂ SO _4. After filtering off the drying agent, the filtrate was concentrated under reduced pressure. The residue was lyophilized and used in the next reaction without further purification.

(6) Cbz-Tyr(O ^t Bu)-Gly-Gly-SH -> Cbz-Tyr(O ^t Bu)-Gly-Gly-Phe-OH
Cbz-Tyr(O ^t Bu)-Gly-Gly-SH and a mixed solvent of DMSO:toluene=1:1 (1 mL) were added to a test tube. Then, N-hydroxypyridone additive 1 (17 mg, 0.1 mmol), Phe (20 mg, 0.12 mmol), and diethyl phosphite (13 μL) were added and stirred at 30° C. for 6 hours. The reaction mixture was diluted to 1.5 mM and analyzed by analytical HPLC, and the HPLC yield was 87%. Then, insoluble materials were removed with a syringe filter, and the reaction mixture was purified by preparative HPLC. 40 mg (63%) of Cbz-Tyr(O ^t Bu)-Gly-Gly-Phe-OH was added.
The properties of Cbz-Tyr(O ^t Bu)-Gly-Gly-Phe-OH were examined, and the results are as follows.
MS (ESI): m/z 655.25 (calcd [M+Na] ⁺ = 655.27)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 28.39 min

(7) Cbz-Tyr(O ^t Bu)-Gly-Gly-Phe-OH -> Cbz-Tyr(O ^t Bu)-Gly-Gly-Phe-SH
Cbz-Tyr(O ^t Bu)-Gly-Gly-Phe-OH (13 mg, 20 μmol) and DMF (200 μL) were added to a frame-dried test tube under an argon atmosphere. Then AcSK (11 mg, 100 μmol) and Ac ₂ S (1 μL, 10 μmol) were added to the solution. The reaction mixture was stirred at 0° C. for 5 h, the mixture was extracted with ethyl acetate, and the combined organic layer was washed with 1M aqueous HCl and brine, then dried over Na ₂ SO _4. After filtering off the drying agent, the filtrate was concentrated under reduced pressure. The residue was lyophilized and used in the next reaction without further purification.

(8) Cbz-Tyr(O ^t Bu)-Gly-Gly-Phe-SH -> Cbz-Tyr(O ^t Bu)-Gly-Gly-Phe-Leu-OH
Cbz-Tyr(O ^t Bu)-Gly-Gly-Phe-SH and a mixed solvent of DMSO:toluene=1:1 (200 μL) were added to a test tube. N-hydroxypyridone additive 1 (4 mg, 20 μmol), Leu (4 mg, 24 μmol), and diethyl phosphite (13 μL) were then added and stirred at 30° C. for 6 hours. The reaction mixture was diluted to 1.5 mM and analyzed by analytical HPLC, and the HPLC yield was 54%. Insoluble materials were then removed with a syringe filter, and the reaction mixture was purified by preparative HPLC. 7 mg (47%) of Cbz-Tyr(O ^t Bu)-Gly-Gly-Phe-Leu-OH was obtained.
The properties of Cbz-Tyr(O ^t Bu)-Gly-Gly-Phe-LeuOH were examined, and the results are as follows.
MS (ESI): m/z 768.25 (calcd [M+Na] ⁺ = 768.36)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 30.00 min

Claims (6)

A method for preparing a compound of formula (V) by reacting a compound of formula (I) below with an amino acid of formula (II) below in the presence of a compound of formula (III) below and a compound of formula (IV) below.

(Wherein,

teeth,
represents a protecting group, or represents an amino acid or peptide whose N-terminus is protected with a protecting group;
wherein the peptide has a structure in which 2 to 9 amino acid residues derived from the same or different amino acids selected from the group consisting of α-amino acids and β-amino acids are bound together;
_R1 is a side chain of an α-amino acid, and the side chain may be protected with a protecting group.

(In the formula, _R2 is a side chain of an α-amino acid, and the side chain may be protected with a protecting group.)

(Wherein,
L is selected from the group consisting of an ester group (-CO ₂ R; R is a linear alkyl group having 1 to 4 carbon atoms or a branched alkyl group of -(CH ₂ CH ₂ CH(CH ₃ )CH ₂ ) _s -H (s is 1 to 4) ), a substituted or unsubstituted phenyl group (the substituent of the phenyl group is an alkyl group or an ester group) , a substituted or unsubstituted benzoyl group (the substituent of the benzoyl group is an alkyl group or an amino group) , a dimethylaminoethylamide group (-CONHC ₂ H ₄ N(CH ₃ ) ₂ ) , and an acyl group (-COR _a '; R _a ' is an alkyl group having 1 to 4 carbon atoms), with the proviso that when at least one of R ₃ and R ₄ is an ester group (-CO ₂ R _a ; R _a is an alkyl group having 1 to 4 carbon atoms), L may be hydrogen (H);
_R3 and _R4 each independently represent hydrogen, an _alkyl group having 1 to 4 carbon atoms, chlorine , or an ester group ( _-CO2Ra ; _Ra is an alkyl group having 1 to 4 carbon atoms).

(In the formula, _R5 and _R6 each independently represent an n-butyl group, a methoxy group, an ethoxy group, an i-propoxy group, an n-butoxy group , a benzyloxy group which may have a substituent, or a hydroxy group (provided that both _R5 and _R6 are not hydroxy groups),
_R5 and _R6 may join together to form a 5- or 6-membered substituted or unsubstituted heterocyclyl containing a phosphorus atom to which _R5 and _R6 are bonded (the substituent of the heterocyclyl is an alkyl group having 1 to 4 carbon atoms) .

(Wherein,

, _R1 and _R2 are as defined above. The method according to claim 1, wherein a mixed solvent of DMSO and toluene is used as the reaction solvent. 3. The method according to claim 1 or 2, comprising, prior to the coupling reaction, a step of preparing a compound represented by formula (I) by reacting a compound represented by formula (VI) below with a compound represented by formula (1) below.

(In the formula, _R1 is as defined in formula (I).)

(In the formula, _R7 is an alkyl group having 1 to 4 carbon atoms or a substituted or unsubstituted aryl group.) PG-(AA) _n -SH:
(PG represents an N-terminal protecting group,
AA represents any amino acid residue, which may be identical or different in each occurrence, some or all of the side chains of the amino acid residue may be protected with protecting groups;
n is an integer from 1 to 4.
A compound represented by the formula:
H-(AA') _m -OH:
(AA' represents any amino acid residue, which may be the same or different in each occurrence;
Some or all of the side chains of the amino acid residues may be protected with protecting groups;
m is an integer from 1 to 4.
An amino acid or peptide represented by
A method for preparing a compound represented by PG-(AA) _n (AA') _m -OH by reacting a compound represented by the following formula (III) and a compound represented by the following formula (IV):

(Wherein,
L is selected from the group consisting of an ester group (-CO ₂ R; R is a linear alkyl group having 1 to 4 carbon atoms or a branched alkyl group of -(CH ₂ CH ₂ CH(CH ₃ )CH ₂ ) _s -H (s is 1 to 4) ), a substituted or unsubstituted phenyl group (the substituent of the phenyl group is an alkyl group or an ester group) , a substituted or unsubstituted benzoyl group (the substituent of the benzoyl group is an alkyl group or an amino group) , a dimethylaminoethylamide group (-CONHC ₂ H ₄ N(CH ₃ ) ₂ ) , and an acyl group (-COR _a '; R _a ' is an alkyl group having 1 to 4 carbon atoms), with the proviso that when at least one of R ₃ and R ₄ is an ester group (-CO ₂ R _a ; R _a is an alkyl group having 1 to 4 carbon atoms), L may be hydrogen (H);
_R3 and _R4 each independently represent an _alkyl group having 1 to 4 carbon atoms, or chlorine or an ester group ( _-CO2Ra ; _Ra is an alkyl group having 1 to 4 carbon atoms).

(In the formula, _R5 and _R6 each independently represent an n-butyl group, a methoxy group, an ethoxy group, an i-propoxy group, an n-butoxy group , a benzyloxy group which may have a substituent, or a hydroxy group (provided that both _R5 and _R6 are not hydroxy groups),
_R5 and _R6 may join together to form a 5- or 6-membered substituted or unsubstituted heterocyclyl containing a phosphorus atom to which _R5 and _R6 are bonded (the substituent of the heterocyclyl is an alkyl group having 1 to 4 carbon atoms) . The method according to any one of claims 1 to 3, followed by
(i) reacting a compound represented by the following formula (V) with a compound represented by the following formula (1) to prepare a compound represented by formula (VII):

(Wherein,

, R ₁ and R ₂ are as defined in formulas (I) and (II).

(In the formula, _R7 is an alkyl group having 1 to 4 carbon atoms or a substituted or unsubstituted aryl group.)

(ii) reacting a compound represented by formula (VII) with an amino acid represented by the following formula (VIII) in the presence of a compound represented by the following formula (III) and a compound represented by the following formula (IV):
A method for preparing a compound of formula (VIIII), comprising:

(In the formula, R _3a is a side chain of an α-amino acid, and the side chain may be protected with a protecting group.)

(Wherein,
L is selected from the group consisting of an ester group (-CO ₂ R; R is a linear alkyl group having 1 to 4 carbon atoms or a branched alkyl group of -(CH ₂ CH ₂ CH(CH ₃ )CH ₂ ) _s -H (s is 1 to 4) ), a substituted or unsubstituted phenyl group (the substituent of the phenyl group is an alkyl group or an ester group) , a substituted or unsubstituted benzoyl group (the substituent of the benzoyl group is an alkyl group or an amino group) , a dimethylaminoethylamide group (-CONHC ₂ H ₄ N(CH ₃ ) ₂ ) , and an acyl group (-COR _a '; R _a ' is an alkyl group having 1 to 4 carbon atoms), with the proviso that when at least one of R ₃ and R ₄ is an ester group (-CO ₂ R _a ; R _a is an alkyl group having 1 to 4 carbon atoms), L may be hydrogen (H);
_R3 and _R4 each independently represent an _alkyl group having 1 to 4 carbon atoms, chlorine , or an ester group ( _-CO2Ra ; _Ra is an alkyl group having 1 to 4 carbon atoms).

(In the formula, _R5 and _R6 each independently represent an n-butyl group, a methoxy group, an ethoxy group, an i-propoxy group, an n-butoxy group , a benzyloxy group which may have a substituent, or a hydroxy group (provided that both _R5 and _R6 are not hydroxy groups),
_R5 and _R6 may join together to form a 5- or 6-membered substituted or unsubstituted heterocyclyl containing a phosphorus atom to which _R5 and _R6 are bonded (the substituent of the heterocyclyl is an alkyl group having 1 to 4 carbon atoms) .

(Wherein,

, R ₁ to R ₂ , and R _3a are as defined above.) A method for preparing a compound of formula (V) as defined in claim 1, comprising using a compound of formula (IV) below as a solubilizing agent:

(In the formula, _R5 and _R6 each independently represent an n-butyl group, a methoxy group, an ethoxy group, an i-propoxy group, an n-butoxy group , a benzyloxy group which may have a substituent, or a hydroxy group (provided that both _R5 and _R6 are not hydroxy groups),
_R5 and _R6 may join together to form a 5- or 6-membered substituted or unsubstituted heterocyclyl containing a phosphorus atom to which _R5 and _R6 are bonded (the substituent of the heterocyclyl is an alkyl group having 1 to 4 carbon atoms) .

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