JP2748897B2 - Novel arginine derivative and method for producing peptide using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel arginine derivative and a novel method for producing an arginine-containing peptide using the same. More specifically, an arginine derivative in which the side chain guanidino group of arginine is protected with a substituted or unsubstituted dibenzosuberyl group, which is useful in the production of a peptide containing an arginine residue, and an arginine residue characterized by using the same. And a method for producing a peptide comprising:

[0002]

2. Description of the Related Art In chemical production of peptides, a coupling reaction is carried out by protecting an unstable functional group of an amino acid residue, and after all amino acids have been introduced, the protecting group must be removed. No. Protecting groups suitable for such purpose have been developed for many functional groups. For example, in the case of the Fmoc strategy in which a fluorenylmethyloxycarbonyl (Fmoc) group is used as a protecting group for the Nα amino group, and mild piperidine is used for the removal of the protecting group, trifluoroacetic acid is used to protect the functional group of each amino acid side chain. A triphenylmethyl group (Trt group), a tertiary butyl group (tBu), a tertiary butyloxycarbonyl (BOC) group, or the like, which can be removed by (TFA), is used.

However, as a protective group for a guanidino group in the side chain of an arginine residue, a sufficiently satisfactory protective group has not yet been developed. The side chain guanidino group of arginine has strong nucleophilicity and is extremely susceptible to acylation. Therefore, in the production of peptides, protection of the guanidino group on the arginine side chain is essential. However, none of the protective groups for the side chain guanidino group of arginine can be easily removed by TFA as in the case of other functional groups.

For example, a 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr) group (M. Fujino et al.
al., Chem. Pharm. Bull. , 29 , 2825 (1981))
Because it is resistant to acids, it is difficult to remove it. In addition, during the production of a tryptophan-containing peptide, a by-product is generated and the yield is reduced (P. Siber, Tetrahedron Lett., 28 , 1637 (19
87)). In addition, a 2,3,4,5,6-pentamethylbenzenesulfonyl (Pmc) group (R. Ramage
et al., Tetrahedron Lett., 28 , 2287 (1987)), when the guanidino group in the side chain of arginine is protected, it is resistant to acids and has a concentration of 80% or more of TFA.
Requires 2 to 4 hours, and a peptide containing a plurality of arginine residues requires 6 to 8 hours. Therefore, in the production of arginine-containing peptides, the deprotection of the side chain of arginine is rate-determining, and at present, TFA treatment is performed for a long time, or a special reagent having many dangerous side reactions such as trimethylsilyl bromide is used. is there. Thus, a protecting group for the side chain guanidino group of arginine,
There is a need for the development of ones that are easily cleaved by acids.

[0005]

Accordingly, an object of the present invention is to provide a protecting group for the side chain guanidino group of arginine,
It is an unstable substance which is removed by at least as much acid as other side chain protecting groups, and the cation generated upon deprotection is stable, and is easily removed by a combined scavenger. Another object of the present invention is to develop an arginine derivative in which a side chain guanidino group is protected with such a protecting group. Another object of the present invention is to provide a method for efficiently producing an arginine-containing peptide using such a derivative.

[0006]

Means for Solving the Problems In view of the above-mentioned situation, the present inventors diligently studied a protective group for a side chain guanidino group of arginine, and found that a dibenzosuberyl group was a protective group for a side chain guanidino group of arginine. Not only is this excellent, but also because this protecting group is moderately acid-labile and the cation generated upon cleavage of the protecting group by the acid is relatively stable, it is also useful for producing peptides containing tryptophan residues. It has been found that it can be used advantageously. Furthermore, the present inventors have found that when the arginine derivative protected with a dibenzosuberyl group is made into a hydrochloride form, the lone pair electron, which is susceptible to electrophilic attack of the guanidino group, is protonated, and becomes more stable, and the peptide is more advantageously used. It has been found that it can be used for manufacturing.

However, a protecting group in which a side chain guanidino group can be easily cleaved by an acid increases the nucleophilicity of the guanidino group as compared with a conventionally used allylbenzenesulfonic acid derivative protecting group. As a result, when arginine is bound to a peptide, the active ester intermediate of arginine formed undergoes intramolecular ring closure, that is, a lactam bond is formed between the nitrogen of the guanidino group and the carbonyl carbon of the carboxyl group, and cannot react with the peptide. (Organic Preparations and Procedure
s Int. 2 (5), 427-463 (1988)). Therefore, arginine derivatives having a dibenzosuberyl group as a protecting group have been further studied, and arginine is reacted with an active ester-forming reagent in the presence of an amino group involved in peptide bond (free amino group of the peptide), thereby producing a molecule. It has been found that the side reaction of inner lactam formation is suppressed and the target peptide is produced with high purity. The present invention has been completed by further research based on such findings.

That is, the gist of the present invention is to provide (1) an arginine derivative represented by the general formula (1):

[0009]

Embedded image

(Wherein, R _{1 to} R ₈ each independently represent a hydrogen atom, an alkyl group having 1 to 4 carbon atoms or an alkoxy group having 1 to 4 carbon atoms, and both R _{9 and} R ₁₀ represent a hydrogen atom One is a hydrogen atom and the other is a fluorenylmethyloxycarbonyl (Fmoc) group, one is a hydrogen atom and the other is an alkyl group having 1 to 4 carbon atoms, or one is an Fmoc group and the other is an alkyl group having 1 to 4 carbon atoms. (2) The arginine derivative according to the above (1), wherein 4 to 7 of R _{1 to} R ₈ are hydrogen atoms, (3) R _{1 to} R
The arginine derivative according to the above (1), wherein all ₈ are hydrogen atoms; (4) the arginine derivative according to the above (1), wherein one of R _{1 to} R ₈ is a methoxy group and the remaining is a hydrogen atom; The arginine derivative according to the above (1), wherein R ₃ is a methoxy group and the remainder is a hydrogen atom, (6) the hydrochloride of the arginine derivative according to any of the above (1) to (5), and (7) arginine. A method for producing an arginine-containing peptide, which comprises using the arginine derivative according to any one of (1) to (5) or the hydrochloride of the arginine derivative according to (6). .

[0011]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail. The novel arginine derivative of the present invention is represented by the general formula (1).

[0012]

Embedded image

(Wherein, R _{1 to} R ₈ each independently represent a hydrogen atom, an alkyl group having 1 to 4 carbon atoms or an alkoxy group having 1 to 4 carbon atoms, and both R _{9 and} R ₁₀ represent a hydrogen atom One is a hydrogen atom and the other is a fluorenylmethyloxycarbonyl (Fmoc) group, one is a hydrogen atom and the other is an alkyl group having 1 to 4 carbon atoms, or one is an Fmoc group and the other is an alkyl group having 1 to 4 carbon atoms. Represents a group.)

This compound is a novel arginine derivative obtained by protecting the side chain guanidino group of arginine with a substituted or unsubstituted dibenzosuberyl group, and its α-amino group is free and protected with an Fmoc group. May be. In the formula, R _{1 to} R ₈ represent a substituent of a dibenzosuberyl group, a hydrogen atom when unsubstituted, and an alkyl group having 1 to 4 carbon atoms or a carbon atom each independently when substituted. Represents an alkoxy group of 1 to 4; Examples of the alkyl group having 1 to 4 carbon atoms include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, and tert.
-Butyl group. Examples of the alkoxy group having 1 to 4 carbon atoms include a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, a sec-butoxy group, and a tert-butoxy group. Among them, a methoxy group is preferable. Among the above, R _{1 to} R ₈
Are all hydrogen atoms, the case where one of R _{1 to} R ₈ is a methoxy group and the rest are all hydrogen atoms, particularly the case where R ₃ is a methoxy group and the rest are all hydrogen atoms, is particularly advantageously used in the present invention. You.

In the general formula (1), either R ₉ or R ₁₀ is a hydrogen atom, or one of them is a hydrogen atom and the other is Fmoc
A group, one is a hydrogen atom and the other is an alkyl group having 1 to 4 carbon atoms, or the other is an Fmoc group and the other is an alkyl group having 1 to 4 carbon atoms. Here, examples of the alkyl group having 1 to 4 carbon atoms include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, a sec-butyl group, and a tert-butyl group.

The Fmoc group is a protecting group that can be removed under extremely mild conditions and is widely used as a protecting group for an α-amino group in peptide synthesis. The arginine derivative of the present invention is characterized in that the side chain guanidino group of arginine is protected with a substituted or unsubstituted dibenzosuberyl group,
An Fmoc group is preferably used as a protecting group for an α-amino group required for use in a peptide synthesis reaction, but is not limited thereto. Like the Fmoc group, any protecting group that does not require an acid such as TFA for removal thereof and can be removed with piperidine or another base can be used in the present invention instead of the Fmoc group. More generally speaking, any protective group for an α-amino group which can be removed under the condition that the substituted or unsubstituted dibenzosuberyl group protecting the guanidino group in the side chain of arginine is not eliminated is represented by the general formula (1) )) Can be used in the present invention instead of the Fmoc group.

The arginine derivative of the present invention represented by the general formula (1) is preferably used as a hydrochloride form for peptide synthesis. This is to protect the strong basicity of the guanidino group by protonation and protect it from electrophilic attack. When the arginine derivative hydrochloride of the present invention is used for peptide synthesis, 1-hydroxybenzotriazole (HOB)
It is preferable to use 2) to 5 times equivalent of t).

The arginine derivative of the present invention represented by the general formula (1) can be produced as follows.
For example, when one of R _{9 and} R ₁₀ in the general formula (1) is a hydrogen atom and the other is an Fmoc group, it is substituted with Fmoc-arginine (2) in dimethylformamide (DMF) using diisopropyldiethylamine as a base catalyst or The unsubstituted dibenzosuberyl chloride is reacted at room temperature to obtain a compound (3) in which a substituted or unsubstituted dibenzosuberyl group is introduced into a guanidino group and a carboxyl group. Then, only the carboxylic acid ester is hydrolyzed under extremely weak acidic conditions to obtain the desired arginine derivative of the present invention represented by the general formula (4), Fmoc-Arg (Su).
b ') -OH can be obtained. The above reaction is shown in the following chemical reaction formula.

[0019]

Embedded image

The product is isolated from the reaction solution by a conventional method. That is, the target compound is obtained by distilling off the reaction solvent and purifying the residue by silica gel column chromatography or the like using chloroform-methanol as an eluent.

The arginine derivative hydrochloride of the present invention is obtained by adding the arginine derivative obtained as described above to 0.1%.
It can be easily produced by dissolving or suspending in a mixture of 01N HCl-acetonitrile (about 1: 1) and freeze-drying.

Here, as Fmoc-arginine, a commercially available product (for example, manufactured by Senn Chemicals AG, Switzerland) can be used as it is. Unsubstituted or substituted dibenzosuberyl chloride can be produced by reacting commercially available dibenzosuberyl alcohol (manufactured by Aldrich) with thionyl chloride. For example, 3-
Methoxy-5-dibenzosuberyl chloride can be obtained from 3-methoxy-5-dibenzosuberenone (J. Org. Chem. 59,
7969 (1994)) can be easily prepared by reduction with NaBH ₄ to form an alcohol, followed by reaction with thionyl chloride.

Among the arginine derivatives represented by the general formula (1), those having a free position at the Nα-position are obtained by the Fmoc-A represented by the general formula (4) obtained as described above.
It can be obtained by treating rg (Sub ')-OH with piperidine in a conventional manner.

The acid stability of the arginine derivative of the present invention represented by the general formula (1) is determined by high performance liquid chromatography (H
PLC). Fmoc-Arg (Sub) OH which is the arginine derivative of the present invention contains 25%
Fmoc-Arg (Sub-OMe) O in trifluoroacetic acid (TFA) -dichloromethane solution for about 30 minutes
H is observed to undergo almost 100% cleavage of the protecting group in about 5 minutes in a 5% TFA-dichloromethane solution. Therefore, the arginine derivative of the present invention represented by the general formula (1) or a hydrochloride thereof has a higher degree of deprotection under milder acid conditions as compared with a conventionally used arginine derivative protected with a protecting group. It is possible, and even in the production of an arginine-containing peptide, the desired product can be obtained by performing cleavage under mild acid conditions. This indicates that the present invention is also useful for producing an acid-labile tryptophan-containing peptide. When the arginine derivative of the present invention is used, a peptide containing an arginine residue can be produced with high efficiency.

The production of an arginine-containing peptide using the arginine derivative or its hydrochloride form of the present invention can be carried out by a conventional method. It can be used for both the solid phase method and the liquid phase method. In order to produce an arginine-containing peptide by a solid phase method, it is convenient to use an automatic peptide synthesizer model PSSM-8 manufactured by Shimadzu Corporation,
It can also be done manually. Below is PSSM-
8 illustrates the production of an arginine-containing peptide using an automatic synthesizer and the production of an arginine-containing peptide by manual operation.

The protection of the Nα-amino group of an amino acid is determined by Fm
It is efficient to perform with an oc group. In this case, the protection of the side chain functional group is performed, for example, in the case of protecting the carboxyl group of a glutamic acid residue or an aspartic acid residue, as a tert-butyl ester (OtBU), the acid amide group of a glutamine residue or an asparagine residue. In the case of protection, a triphenylmethyl (Trt) group, in the case of protection of a hydroxyl group of a serine residue, threonine residue or tyrosine residue, in the case of a tert-butyl (tBu) group, and in the case of protection of an imidazolyl group of a histidine residue. Is a triphenylmethyl (Trt) group, Lys
Tert-butyloxycarbonyl (Boc) in the case of protecting the side chain amino group of the residue or the indole group of the Trp residue
And in the case of protection of the guanidino group of an arginine residue the substituted or unsubstituted dibenzosuberyl (Su
b ') group. All of these amino acid derivatives are commercially available except for arginine derivatives (for example, SynP
roPep reagent). In addition, for protecting amino acid residues other than arginine residues, it is also possible to use protecting groups other than those described above.

The peptide synthesis reaction is conveniently carried out according to the protocol of PSSM-8 (commercially available). The resin used as the solid phase is a commercially available product such as SynProP
ep Co. TentaGel ^R S-RAM, Tenta
Gel ^R S-CHA or a resin to which the C-terminal amino acid of the target peptide has already been bound, such as Fmoc-Tyr (t
It is convenient to use Bu) -Wang (manufactured by SynProPep).

The synthesis cycle is repeated by the PSSM-8 automatic synthesizer until all necessary amino acids are introduced.
DMF washing, Fmoc group removal (washing with 30% piperidine in DMF), DMF washing, amino acid activation (benzotriazol-1-yl-oxy-
It is convenient to use tris (pyrrolidino) phosphonium hexafluorophosphate (PyBop) (SynProPep reagent). ), Coupling and DMF washing are repeated. After the introduction of the final amino acid is completed, the Fmoc group is removed by washing with DMF, washing with piperidine, washing with DMF, washing with methanol, washing with tertiary butyl methyl ether, and then nitrogen gas is blown and dried. Thus, a peptide resin having a free N-terminus and a protected side chain is obtained.

After the obtained resin is sufficiently dried, a cleaving step is performed. In this step, a side chain protecting group is removed with a TFA scavenger cocktail, and at the same time, the peptide chain is separated from the support resin. This step requires the use of a strong acid or a long-time treatment with a conventional protecting group, which causes a reduction in the yield of the target product due to a side reaction or the like, and is a difficult step requiring skill in the treatment. . However, by using the protecting group of the present invention, this cleavage step is completed only by leaving the cleavage cocktail described below at room temperature for 60 minutes. Although the cleaving time depends on the amino acid sequence of the peptide, even when Pmc, which is conventionally the most excellent protecting group, is used, a peptide containing a plurality of arginine residues requires 4 to 8 hours for cleaving. It costs. Cleavage cocktail: 75% TFA, ethyl methyl sulfide
5%, water 5%, thioanisole 5%, thiophenol 5%, and ethanediol 5%.

After the completion of the cleavage step, the cocktail was transferred to a centrifuge tube under nitrogen pressure, dry ether was added thereto, and a precipitate was obtained by centrifugation. This was dissolved in acetic acid, and further precipitated by adding dry ether. After drying with nitrogen aeration, it is dissolved in dilute hydrochloric acid and freeze-dried to obtain a crude peptide.

The following process is convenient for producing an arginine-containing peptide by manual operation, but is not limited thereto. The amino acid derivative used, the resin used as the solid phase, and the like are as described above. As a method for activating a carboxyl group, PyBop
And HOBT (1-hydroxybenzotriazole)
Is convenient.

One example of a specific operation is as follows. Resin used as a solid phase in a reaction vessel such as Tent
aGel ^R S-CHA was added thereto, an appropriate solvent such as DMF was added thereto, the mixture was swollen with washing, 20% piperidine / DMF was added, and the mixture was shaken repeatedly.
Wash repeatedly. An amino acid derivative is added to this, and P
yBop and HOBT are dissolved in DMF and added. Further, for example, N-methylmorpholine is added as a base catalyst, and the mixture is shaken sufficiently to perform a coupling reaction. This operation is repeated by the number of amino acid residues to be bound to the peptide. In this method, a free amino group is always present when an amino acid is activated, so that the activated amino acid is Fmoc-Arg (Su
b) OH or Fmoc-Arg (Sub-OMe)
Even with OH, intramolecular lactam formation does not occur, and the reaction yield is extremely high. The obtained resin is dried under reduced pressure over phosphorus pentoxide. Cleavage is 50% TFA, 32% methylene chloride, 5% thioanisole, 5% H ₂ O, 3%
% Ethyl methyl sulfide, 3% ethanedithiol, 2%
% Cocktail of thiophenol is added to the synthesized resin and left at room temperature for 1 hour. After Cleavage,
The resin is filtered off, the peptide is precipitated by adding ether to the filtrate and centrifuged. The obtained precipitate is dried under reduced pressure to obtain a crude peptide.

The obtained peptide may be used, if necessary, in reverse phase H.
After purification by PLC and confirmation of a single component by reverse phase HPLC, the target compound can be confirmed by liquid secondary ion mass spectrometry, amino acid sequence analysis by Edman degradation, amino acid composition analysis and the like.

[0034]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, which should not be construed as limiting the present invention.

Example 1 (Fmoc-amino-N ^W -5
Production of dibenzosuberyl arginine) 3.0 g (7.6 mMol) of Fmoc arginine was added to DM
F (20 ml), and dibenzosuberyl chloride (Sub-Cl) (5.2 g, 3 equivalents) was further added.
Diisopropylethylamine (DIPEA) with stirring
1.32 ml (1 equivalent) was added dropwise, and the mixture was stirred at room temperature for 4 hours. 1.32 ml (1 equivalent) of DIPEA was further added dropwise to the reaction solution, and the mixture was left overnight with stirring. The solvent was distilled off under reduced pressure, the residue was dissolved in dichloromethane, and water was added. After neutralizing the aqueous layer with saturated aqueous sodium hydrogen carbonate, the dichloromethane layer was separated, washed with saturated saline, dried, and the solvent was distilled off. Diethyl ether was added to the residue, the insolubles were collected by filtration, washed with diethyl ether, and dried to obtain an intermediate product (ester).

The obtained intermediate product was treated with dioxane (200
ml) and water (100 ml), the pH was adjusted to about 5, and the mixture was stirred for 4 hours. After neutralization, the solvent was distilled off, and the residue was subjected to silica gel column chromatography (developing solvent was CHCl
Purification with _3- MeOH (8: 1 → 5: 1) yields Fmo
c- amino -N ^{W-5-dibenzosuberyl} arginine 2.756G (yield: 62%) was obtained. CHCl ₃ -M
Recrystallization from OH gave colorless crystals (mp 143-146).
° C). Elemental _{analysis: C 36 H 34 O 4 N} 4 · CHCl 3 theoretical: C = 61.06, H = 5.28 , N = 7.91 Analytical values: C = 60.59, H = 5.02 , ^{N = 7.57 1 H-NMR (} DMSO-d 6) δ (ppm): 1.4~
1.8 (6H, m, arginine Cβ-Cγ methylene-
_{(CH 2) 3 -),} 2.9~3.3 (4H, m, dibenzosuberyl methylene C10, C11), 3.65~3.
72 (1H, m, arginine Cα methine), 4.25
(3H, br.s, Fmoc methylene, methine), 6.
32 (1H, br.s, dibenzosuberylmethine CH-
NH), 6.7 (1H, br.s, guanidino NH
=), 7.0-7.9 (16H, m, aromatic ring H), 8.
30 (1H, br.s, arginine NαH), 9.8-
10.8 (1H, br.s, arginine COOH)

Example 2 (Fmoc-amino-N ^W -3-
Production of methoxy-5-dibenzosuberyl arginine) 5.0 g of 3-methoxy-5-dibenzosuberenone (5)
(MMol) was dissolved in isopropyl alcohol (50 ml), and 0.795 g (1 equivalent) of NaBH _{4 was} further added, followed by heating and stirring for 5.5 hours. The solvent was distilled off under reduced pressure, the residue was dissolved in water and a small amount of ethyl acetate, neutralized with dilute hydrochloric acid under ice cooling, extracted with ethyl acetate, and washed with saturated saline.
After drying, the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate: hexane (1: 5)) to obtain 4.272 g (yield: 85%) of 3-methoxy-5-dibenzosuberenol (6). The obtained alcohol was converted to methylene chloride (50
ml), and SOCl ₂ was added dropwise under ice-cooling.
And further stirred at room temperature for 2 hours. The solvent was distilled off under reduced pressure, the residue was washed with petroleum ether, dried and crude 3-methoxy-5-dibenzosuberyl chloride (7) 4.036.
g was obtained.

[0038]

Embedded image

3.0 g of Fmoc arginine (7.6 mM)
ol) was dissolved in DMF (20 ml), and 3-methoxy-5-dibenzosuberyl chloride (Sub (OM)
e) -Cl) (7) 4.9 g (2.5 eq) were added.
Diisopropylethylamine (DIPEA) with stirring
0.662 ml (1 equivalent) was added dropwise, and the mixture was stirred at room temperature for 4 hours. The reaction mixture was further added with DIPEA, 0.662 ml (1
(Equivalent) was added dropwise and left overnight with stirring. The solvent was distilled off under reduced pressure, the residue was dissolved in dichloromethane, and water was added. After neutralizing the aqueous layer with saturated aqueous sodium hydrogen carbonate, the dichloromethane layer was separated. Collected by filtration dichloromethane insoluble crystals to give Fmoc- amino -N ^{W-3-methoxy-5-dibenzosuberyl} arginine 3.0 g.
The filtrate was washed with saturated saline and dried, and the solvent was distilled off. Diethyl ether was added to the residue, the insolubles were collected by filtration, washed with diethyl ether, and the resulting crude crystals were subjected to silica gel column chromatography (developing solvent: CHCl ₃ -Me
OH (8: 1 → 5: 1)) to give 0.97 g of product
I got 3.97 g of Fmoc-amino-N ^W -5
-Dibenzosuberyl arginine was obtained (yield: 85
%). CHCl ₃ was recrystallized from -MeOH, to give colorless crystals (mp150~152 ℃). Elemental analysis: C ₃₇ H ₃₈ O ₅ N ₄ Theoretical: C = 71.82, H = 6.19, N = 9.06 Analysis: C = 71.64, H = 6.16, N = 8 .76 ¹ H-NMR (CD ₃ OD) δ (ppm): 1.4 to 1.9
(6H, m, arginine Cβ-Cγ methylene- (C
_{H 2) 3 -), 2.9~3.4} (4H, m, dibenzosuberyl methylene C10, C11, and IH, m, arginine Cα methine), 3.7 (1H, s, methoxy, OC
_{H 3), 4.0~4.4 (3H,} m, Fmoc methylene, methine), 5.86 (IH, s, dibenzosuberyl Rume Chin, CH-NH), 6.73 ( 1H, br.s, Guanidino NH =), 7.1 to 7.8 (16H, m, aromatic ring H)

[0040] Example 3 (5-preparation of dibenzosuberyl arginine) obtained in Example 1 Fmoc-Amino -N ^{W-5-dibenzosuberyl} arginine 1.0g in DMF solution 10ml containing 30% piperidine Dissolve and leave at room temperature for 2 hours. The solvent was distilled off under reduced pressure, diethyl ether was added to the residue, insolubles were collected by filtration, and the solid was washed with diethyl ether to obtain a crude product. Recrystallization from chloroform-methanol yielded 0.5 g (yield: 80%) of colorless crystalline 5-dibenzosuberylarginine. Elemental analysis: C ₂₁ H ₂₆ O ₂ N ₄ Theoretical: C = 68.83, H = 7.15, N = 15.2
9 Analytical values: C = 68.50, H = 7.01, N = 14.9
9 ¹ H-NMR (DMSO-d ₆ ) δ (ppm): 1.2 to
1.7 (6H, m, arginine Cβ-Cγ methylene-
(CH ₂ ) ₃ −), 2.6 to 2.8 (1H, m, arginine Cα methine), 3.0 to 3.2 (4H, m, dibenzosuberyl methylene C10, C11), 6.35 ( 1
H, br. s, dibenzosuberylmethine CH-NH),
7.0 to 7.25 (6H, m, aromatic ring H), 7.5 to 7.5
7.7 (2H, m, aromatic ring H), and 9.8-10.
7 (1H, br.s, arginine COOH)

Example 4 (Production of Decapeptide Containing Arg and Trp) In the production of a peptide containing both Arg and Trp residues using Fmoc-Arg (Pmc) -OH, the Pmc group released during cleavage was removed. It has been widely recognized that cations caused by Trp attack Trp residues to produce by-products (Tetrahedron Letters Vol. 34, No. 42, 6
661-6664 (1993), Int. J. Peptide Protein Res. 43, 3
1-38 (1994)). Therefore, according to the method of the present invention, Arg and Tr
The following peptides containing both residues of p were produced. H-His-Pro-Ala-Lys-Asp-Asn
-Arg-Trp-Pro-Tyr-OH

For production, Fmoc-Arg (Sub) -OH (Sub: unsubstituted dibenzosuberyl group) of the present invention represented by the general formula (2) was used. Synthesis was performed using benzotriazol-1-yl-oxy-tris (pyrrolidino) phosphonium hexafluorophosphate (PyBo).
p) Activation method using (SynProPep reagent)
A commercially available general protocol for SSM-8 (Tables 1 and 2) was used.

[0043]

[Table 1]

[0044]

[Table 2]

The amino acid derivatives used in each step of the peptide synthesis reaction are as shown in Table 3.

[0046]

[Table 3]

In the above steps, all 10 amino acid residues were introduced, and a peptide resin was obtained in which the N-terminus was free and the side chain functional groups were protected. Resin under reduced pressure P ₂
After sufficiently drying on O ₅ , the step of cleaving was performed. That is, this is a step of removing the side chain protecting group with a TFA scavenger cocktail and simultaneously separating the peptide chain from the support (resin).

About 50 mg of the peptide resin obtained from the synthesizer was dried in the reaction vial of the synthesizer.
0.6 ml of the cleavage cocktail was added thereto, and the mixture was left at room temperature for 60 minutes. The composition of the cocktail is 75% TFA, 5% ethyl methyl sulfide, 5% H ₂ O, 5% thioanisole, 5% thiophenol, 5% ethanedithiol. In addition, 5 mg of 2-peptide was previously added to the peptide resin.
The methylindole was added before the cocktail was added. Next, the cocktail was transferred to a centrifuge tube under nitrogen pressure using a Shimadzu model PSSM-C8 nitrogen pressurized pistol in several tens of seconds, and anhydrous diethyl ether was added to the obtained cocktail, followed by centrifugation (3000 rpm for 5 minutes) to obtain a precipitate. Was.
The supernatant was removed, the precipitate was dissolved again in 0.2 ml of acetic acid, and anhydrous diethyl ether was added to precipitate again. The precipitate was collected by centrifugation, dried with a nitrogen purge for 3 minutes, and then dried with 0.01 N H2.
Cl (1 ml) and lyophilized to give crude peptide 21
mg was obtained.

The purity of the crude peptide was analyzed by analytical HPLC. HPLC used an LC10A system, and the column was SynProPepRPC18 (reverse phase OD).
S silica gel 5μ120Å) (4.6φ × 150mm)
Was used. Eluent: A = 10 mM HCl, B = CH ₃
Linear gradient A = 95% -55% with CN for 40 min, detected at 210 nm (UV). The flow rate is 1.
0 ml / min. The result is shown in FIG. As is clear from this figure, the purity of the crude peptide is extremely high. The main peak eluting at about 15 minutes was collected and analyzed. Mass spectrometry (Liquid Secondary Ion Mass Spectrometry) was performed using Shimadzu-Kratos Model Concept 2H.
Was measured by Theoretical value: M = 1283.4 Experimental value: (M + 1) ⁺ = 1283.6 The amino acid sequence analysis by the Edman method confirmed that the target sequence was obtained.

Example 5 (Production of hexapeptide containing Arg and Trp) Arg performed using Fmoc-Arg (Pmc) -OH
In the production of peptides containing both Trp and Trp residues, Pmc released during cleavage as described above is used.
It is widely accepted that cations due to groups attack Trp residues to produce by-products, and furthermore, the distance between Trp and Arg residues in the product peptide is Pmc
It is known that when a single amino acid residue is present between the Trp residue and the Arg residue, the formation of by-products is remarkable, and the target peptide is hardly obtained. (Int. J. Peptide Protein
Res. 43, 1994 31-38). Therefore, using the method of the present invention,
H-Gly-Ala-Gl which was difficult to synthesize by the conventional method
Production of y-Trp-Ala-Arg-NH ₂ was performed.

The production was PyBOP (benzotriazole-
It was performed manually by an activation method using 1-yl-oxy-tris (pyrrolidino) phosphonium hexafluorophosphate and HOBT (1-hydroxybenzotriazole). The solid phase resin used was Tentagel S-CHA (J. Org. Chem. 59 (26), 7969 (1994)),
The amino acid derivatives are as follows. Molecular weight used amount 1) Tentagel S-CHA (0.26 mMol / g) 28.8 mg 2) Fmoc-Arg (Sub) -OH 588.68 43.8 mg 3) Fmoc-Ala-OH 311.34 25.6 mg 4) Fmoc-Trp (Boc) -OH 526.57 43.0mg 5) Fmoc-Gly-OH 297.31 24.5mg

The reaction vessel was charged with Tentagel S-CHA resin, DMF (0.5 ml) was added thereto, the resin was shaken to wash and swell, and then 20% piperidine /
DMF (0.5 ml) was added and shaken for 4 minutes. This operation was repeated again, and DMF (0.5 ml) was further added, followed by washing five times. Fmoc-Arg (Sub)
-OH was added, and 0.15 ml of a solution containing 0.5 mMol of PyBOP and 0.5 mMol of HOBT in 1 ml of DMF was added, followed by shaking for 5 minutes. N-methylmorpholine (0.012 ml) was added, and the mixture was further shaken for 25 minutes to perform a coupling reaction. DMF (0.5m
After washing in l) (5 times), the operations of Step 1 to Step 6 of the reaction operation shown in Table 4 were repeated (5 times). The final step was performed as shown in Table 5. The resulting resin was P ₂ O ₅
It was dried under reduced pressure in the presence.

Cleavage consisted of 50% TFA, 32% methylene chloride, 5% thioanisole, 5% H ₂ O,
% Ethyl methyl sulfide, 3% ethanedithiol, 2%
% Thiophenol mixed cocktail (0.5ml)
Was added to the resin after synthesis, and left at room temperature for 1 hour. After the cleavage, the resin was removed by filtration, and the peptide was precipitated by adding ether to the obtained filtrate and centrifuged.

The obtained residue was dried under reduced pressure to obtain 4.6 mg of a crude peptide. About the obtained crude peptide, HP
Purity analysis by LC was performed. HPLC was Shimadzu LC-1
An OAS, SynProPepRPC18 (reverse phase ODS silica gel 5μ120Å, 4.6φ × 150mm) column was used. Eluent: A: 0.01N HCl, B: CH
Linear gradient with ₃ CN, A: 100-60%, 4
0 min, flow rate 1 ml / mm, 210 nm (UV)
Was detected. FIG. 2 shows the obtained results. As apparent from FIG. 2, the purity of the obtained crude peptide was extremely high,
No by-products were found. The main peak was separated by HPLC and subjected to mass spectrometry (LSIMS). Shimadzu-Kvatos Concept 2H Measurement result: Theoretical value: 615.69 Experimental value: 616.3 The amino acid sequence analysis by Edman method confirmed that the target sequence was obtained.

[0055]

[Table 4]

[0056]

[Table 5]

Example 6 (Fmoc-Arg (Sub-O
H-Gly-Ala-Gly by using Me) -OH
Production of _{-Trp-Ala-Arg-NH 2} ) Fmoc-Arg (Sub-OMe) used -OH H
-Gly-Ala-Gly-Trp-Ala-Arg-
Production of NH ₂ was also performed. The preparation is PyBOP (benzotriazol-1-yl-oxy-tris (pyrrolidino) phosphonium hexafluorophosphate and HOB
The activation was carried out manually using T (1-hydroxybenzotriazole). The solid-phase resin used was Tentagel S-CHA (J. Org. Chem. 59 (26), 7969).
(1994)), and the amino acid derivatives are as follows. Molecular weight used 1) Tentagel S-CHA (0.26 mMol / g) 28.8 mg 2) Fmoc-Arg (Sub-OMe) -OH 618.70 46.4 mg 3) Fmoc-Ala-OH 311.34 25.6 mg 4) Fmoc-Trp (Boc) -OH 526.57 43.0 mg 5) Fmoc-Gly-OH 297.31 24.5 mg Fmoc-Arg (Sub-OMe) -OH instead of 43.8 mg of Fmoc-Arg (Sub) -OH
Exactly the same operation was performed except that 4 mg was used, whereby the desired hexapeptide containing Arg and Trp could be produced. The result by HPLC is shown in FIG. As is clear from FIG. 3, the purity of the obtained crude peptide was extremely high, and no by-product was observed.

[0058]

Industrial Applicability The novel arginine derivative of the present invention provides an excellent starting compound for producing an arginine-containing peptide. Further, by using the novel arginine derivative of the present invention, an arginine-containing peptide and a peptide containing arginine and tryptophan can be produced in high yield and high purity.

[Brief description of the drawings]

FIG. 1 is a diagram showing analytical HPLC data of a decapeptide (crude peptide) obtained in Example 4.

FIG. 2 is a diagram showing analytical HPLC data of a hexapeptide (crude peptide) obtained in Example 5.

FIG. 3 is a diagram showing analytical HPLC data of a hexapeptide (crude peptide) obtained in Example 6.

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-1-197465 (JP, A) JP-A-63-250360 (JP, A) JP-A-62-207251 (JP, A) Japanese Patent Publication No. 37- 17963 (JP, B1) Special Table 6-506680 (JP, A) (58) Fields investigated (Int. Cl. ⁶ , DB name) C07C 279/16 C07K 1/06 C07K 7/06 CA (STN) CAOLD (STN) REGISTRY (STN)

Claims (7)

(57) [Claims] An arginine derivative represented by the general formula (1). Embedded image (Wherein, R _{1 to} R ₈ each independently represent a hydrogen atom, an alkyl group having 1 to 4 carbon atoms or an alkoxy group having 1 to 4 carbon atoms, and R ₉ or R ₁₀ are both hydrogen atoms, A hydrogen atom and the other is fluorenylmethyloxycarbonyl (F
a moc) group, one being a hydrogen atom and the other being an alkyl group having 1 to 4 carbon atoms, or one being an Fmoc group and the other being having 1 to 4 carbon atoms.
Represents an alkyl group. ) 2. The arginine derivative according to claim 1, wherein 4 to 7 of R _{1 to} R ₈ are hydrogen atoms. 3. The arginine derivative according to claim 1, wherein R _{1 to} R ₈ are all hydrogen atoms. 4. The arginine derivative according to claim 1, wherein one of R _{1 to} R ₈ is a methoxy group and the remaining is a hydrogen atom. 5. The arginine derivative according to claim 1, wherein R ₃ is a methoxy group and the remainder is a hydrogen atom. 6. A hydrochloride of the arginine derivative according to any one of claims 1 to 5. 7. A method for producing an arginine-containing peptide, wherein the arginine derivative according to any one of claims 1 to 5 or the hydrochloride of the arginine derivative according to claim 6 is used. A method for producing an arginine-containing peptide.