JPH0680691A - Production of cysteine-containing peptide - Google Patents

Abstract

PURPOSE:To simply produce the subject peptide in high purity by subjecting a cysteine-containing peptide and a 3-nitro-2-pyridylsulfenyl group-protected cysteine-containing protected peptide to formation of a S-S bond, to formation of a peptide bond, and subsequently to removal of the protecting group. CONSTITUTION:A free SH group-having cysteine-containing protected peptide of formula I (X is amino-protecting group; A, B are various protected amino acid residues, protected peptide; (n), (m) are 0, 1) and a 3-nitro-2-pyridylsulfenyl group-protected cysteine-containing protected peptide are subjected to the formation of a S-S bond and subsequently to the formation of a peptide bond to produce a cysteine-containing protected peptide of formula III, which is subjected to a removal of the protecting group by a conventional method to provide the cysteine-containing peptide of formula IV (A', B', C', D' are the respective groups wherein the protecting groups are removed from A, B, C, D, respectively).

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for synthesizing a cystine-containing peptide, more specifically, a cystine-containing peptide, which is expected to have a large effect on the expression of physiological activity and an increase in activity, is bonded to a specific position with an SS bond. The present invention provides a method for synthesizing a compound, which has a high purity and can be easily synthesized.

[0002]

2. Description of the Related Art Conventionally, as a method for synthesizing a cystine-containing peptide, a protected peptide sequence containing a protected cysteine residue is synthesized by a solid phase method or a liquid phase method. To obtain a peptide with an SH group,
A method of forming a disulfide bond using an air oxidation method or an iodine oxidation method is known. Here, the air oxidation method has a problem that it takes a long time to form an S—S bond and cannot be used for a peptide that is poorly soluble in an aqueous solvent. In addition, the iodine oxidation method uses tyrosine during the reaction,
It is known that histidine and tryptophan cause iodine oxidation, and it is important to select a reaction solvent in order to suppress side reactions. In addition, these methods also have many restrictions in terms of their solubility in organic solvents. Recently, as a method for chemically forming a disulfide bond in order to solve these problems, the thallium (III) trifluoroacetate method [J. Chem. Soc. , C
hem. Commun. , 163 (1987)],
S-protected cysteine sulfoxide method [J. Chem. S
oc. Chem. Commun. , 1676 (198
7) description], and the silyl chloride-sulfoxide method [J. Chem. Soc. Chem. Commu
n. , 167 (1991)] is reported as a reaction for forming a disulfide bond at the same time as deprotection.
Further, a 3-nitro-2-pyridylsulfenyl group (Npys group) is used as a protecting group for the thiol group of cysteine,
It has been reported that an intermolecular disulfide bond can be easily formed by reacting with a free SH group [C
hem. Lett. , 921 (1982)].

[0003]

However, the method of synthesizing these cystine-containing peptides is a method of first synthesizing a prescribed protected peptide sequence and then forming an S--S bond, and further synthesizing the protected peptide sequence. This is an improvement only in the subsequent S-S bond formation method, and is not a solution for improving the yield and purity of the protected peptide sequence or suppressing the by-product of intermolecular disulfide bond. Therefore, in these conventional synthetic methods, it is difficult to increase the yield and purity of the protected peptide sequence to a satisfactory level, and it is also difficult to set the reaction conditions for S—S bond formation, resulting in intermolecular S—S. Since the bond formation cannot be suppressed, there is a problem that further reduction in yield and purity in this step cannot be avoided.

[0004]

Means for Solving the Problems The present inventors have used a condensing agent used for ordinary peptide synthesis to convert the SH group to Npys.
When a cysteine or a cysteine-containing protected peptide protected with a group is reacted with a cysteine or a cysteine-containing protected peptide having a free SH group, a one-step reaction,
The S—S bond forming reaction and the peptide bond forming reaction proceed,
It was found that a highly purified cystine-containing protected peptide can be obtained by forming an S—S bond at a specific position, and as a result of further detailed studies, the present invention has been completed.

More specifically, the present invention will be described in more detail. For example, a cysteine-containing protected peptide represented by the following general formula (II) or (V), such as dimethylformamide (D
To the MF) solution, a cysteine-containing protected peptide represented by the following general formula (III) or (VI) is added to first form an S-S bond, and a peptide condensing agent such as diphenylphosphoryl azide (DPPA) / triethylamine (T
EA) is added and the reaction is carried out.
A cystine-containing protected peptide represented by the formula (1) is obtained, and the amino protecting group (X), the carboxyl protecting group (Y), and the side chain protecting group of various amino acids are eliminated by a conventional method to give A method for synthesizing the indicated cystine-containing peptide. In the present invention, X is an amino-protecting group used in ordinary peptide synthesis, and Y is a carboxyl-protecting group. A, B, C and D are various protected amino acid residues or protected peptides.

[0006]

[Chemical 3]

[0007]

[Chemical 4]

[In the formula (II), X represents an amino protecting group, A and B represent various protected amino acid residues or protected peptides, and n and m each represent an integer of 0 or 1. In formula (III), C and D represent various protected amino acid residues or protected peptides, Y represents a carboxyl protecting group, and p and q each represent an integer of 0 or 1. In the formula (IV), X, Y, A, B, C, D, n, m,
Both p and q have the same meanings as above. In formula (I), A ', B', C'and D'represent various unprotected or partially protected amino acid residues or peptides, and n, m, p and q have the same meanings as above. Have. In the formula (V), X, A, B, n and m have the same meanings as described above. In formula (VI), Y, C, D, p and q have the same meanings as described above. Cys indicates a cysteine residue. ]

As an example of the method for producing a cystine-containing peptide of the present invention, a method for synthesizing a tetrapeptide containing cysteine at the 1-position and 4-position will be described below. Of the tetrapeptide synthesis methods, the peptide chain is temporarily extended, and the 1 + 3 condensation method in which the carboxyl group of the cysteine at the 1-position and the amino group of the amino acid at the 2-position are subjected to a condensation reaction, and the carboxyl group of the amino acid at the 2-position The synthetic schemes of the 2 + 2 condensation method in which the condensation reaction is carried out with the amino group of the amino acid at the 3-position are shown in FIGS. 1 and 2.

First, Bo in the 1 + 3 condensation method (FIG. 1)
c-A-B-Cys (Npys) -OBzl (Boc
Is a t-butoxycarbonyl group, Bzl is a benzyl group, and A and B have the same meanings as described above. For stepwise condensation, dimethylphosphinothioyl chloride (Mpt-Cl) is used. Using a mixed acid anhydride method, trifluoroacetic acid (TFA) was used for de-Boc, and the Npys group was methoxybenzyl (MBzl).
Since it can be exchanged with a thiol protecting group including a group, Boc-AB-Cys (MBzl) -OBz
It was synthesized by reacting 1 with Npys-Cl. TFA / H
-AB-Cys (Npys) -OBzl is Boc-
A-B-Cys (Npys) -OBzl is anisole,
2-mercaptoethanol, dimethyl sulfide based T
It can be synthesized by treating with FA. Figure 1
In the above, MA (Mpt-Cl) represents a mixed acid anhydride of dimethylphosphinothioyl chloride, and TFA / MeSM.
e represents trifluoroacetic acid dimethyl sulfide. 1+
Compounds represented by the general formula (IV) according to the 3 condensation method (FIG. 1) are Boc-Cys-OH and TFA.H-A-B-Cy.
It is obtained by reacting s (Npys) -OBzl. As the condensing agent, a condensing agent commonly used in peptide synthesis such as dicyclohexylcarbodiimide (DCC), or a combination of water-soluble carbodiimide (WSC) and 1-hydroxybenzotriazole (HOBt) or N-hydroxysuccinimide (ONSu), or a mixed acid anhydride is used. A combination of a phosphoric acid-based condensing agent diphenylphosphoryl azide (DPPA) or dimethylphosphinothioyl azide (MPTA) and a base can be used. As the base used in the MPTA method, triethylamine (TEA) or N-methylmorpholine (NMM) can be used. In the WSC / HOBt system, the reaction is usually stirred under ice cooling for 1 to 3 hours, and then allowed to react at room temperature for 1 to 30 hours. In the MPTA method, the reaction is usually completed by treatment for 24 hours under ice cooling.

TFA · H in the 2 + 2 condensation method (FIG. 2)
The synthesis of -B-Cys (Npys) -OBzl is Boc
-Amino acid and TFA.H-Cys (Npys) -OB
Zl can be performed by the mixed anhydride method with Mpt-Cl. Boc in 2 + 2 condensation method (Fig. 2)
The synthesis of -Cys-A-OH is carried out using Boc-Cys (Npy
s) -OH and amino acid benzyl ester with Mpt-Cl
Boc-Cys obtained by the mixed acid anhydride method using
(Npys) -A-OBzl was treated with tri-n-butylphosphine (n-Bu ₃ P) in the presence of water or 2-mercaptoethanol under ice cooling for 1 hour to remove the Npys group. It can be carried out by a method of removing the Bzl group by reacting with sodium hydroxide in a methanol-water system at room temperature for 1 to 10 hours without isolation. As the condensing agent, a combination of a condensing agent DCC or WSC and HOBt or ONSu used in ordinary peptide synthesis, or a phosphoric acid-based condensing agent DPPA or MPTA via a mixed acid anhydride and a base can be used. The compound represented by the general formula (IV) according to the 2 + 2 condensation method (FIG. 2) is Boc-Cys-A-O.
It is obtained by reacting H with TFA.H-B-Cys (Npys) -OBzl. 1+ as a condensing agent
All condensing agents used in the 3 condensation method can be used. In the WSC / HOBt system, the reaction is usually under ice cooling, 1
After stirring for ~ 3 hours, the reaction is continued at room temperature for 1-30 hours. In the MPTA method, the reaction is usually completed by treatment under ice cooling for 24 hours. The best yield is obtained when MPTA is used as the condensing agent and triethylamine is used as the base.

The amino-protecting group (X), carboxyl-protecting group (Y) and side chain-protecting groups of various amino acids of the cystine-containing protected peptide (IV) thus obtained are identified by a conventional method to identify The desired cystine-containing peptide (I) represented by the general formula (I) can be obtained in high purity by forming an S—S bond at the position.

[0013]

According to the conventional method for synthesizing a cystine-containing peptide, it is difficult to increase the yield and purity of the protected peptide sequence to a satisfactory level, and moreover, it is difficult to set the reaction conditions for SS bond formation. , It is not possible to suppress the formation of intermolecular S—S bonds, so further yield in this step,
There was a problem of inevitable deterioration of purity. In contrast,
Since the present invention first forms a disulfide bond and then a peptide bond in a single-step reaction, the fragment condensation method, which has been impossible in the past, is utilized to form an S—S bond at a specific position. It is a novel and efficient production method capable of synthesizing a cystine-containing peptide with high purity in a simple manner and with a wide range of fragment selection.

[0014]

EXAMPLES Examples will be shown below for illustrating the present invention in more detail, but they do not limit the present invention. [Example 1]: Synthesis example 1 Boc-Cys (Npys) -OH 37.5 mg
(0.1 mmol) and HCl.H-Cys-OCH ₃ 1
7.2 mg (0.1 mmol) was dissolved in 1 ml of DMF, 0.011 ml of NMM was added under ice cooling, and the mixture was reacted at room temperature for 2 hours, and further, 19 ml of DMF and 27 Mpt-N ₃ 27.
mg and 0.011 ml of NMM were added, and the mixture was reacted for 24 hours under ice cooling. The reaction solution was concentrated, ethyl acetate was added to the residue, the organic layer was washed with purified water, concentrated, and then collected by HPLC to obtain 18.1 mg of the following target product. (Yield 53.9
%) TLC: Rf = 0.6 (chloroform: methanol = 2)
0: 1) MS (FAB): m / z 337 (M + H) ⁺

[0015]

[Chemical 5]

Example 2 Synthesis Example 2 (1 + 3 Condensation Method) Boc-Gly-Phe-Cys (Npys) -OBz
67.0 mg (0.1 mmol) of 1 was dissolved in 0.39 ml of methylene chloride, 0.39 ml of TFA was added under ice cooling, the mixture was reacted at room temperature for 30 minutes, concentrated, and then 1 ml of DMF.
Dissolved in. This solution was mixed with TEA 0.01 while stirring with ice cooling.
4 ml, Boc-Cys-OH 22.1 mg (0.1
Mmol), and reacted at room temperature for 2 hours.
_{19ml, Mpt-N 3 27.0mg,} TEA 0.
014 ml was added under ice cooling and reacted for 24 hours. The reaction solution was concentrated, ethyl acetate was added to the residue, the organic layer was washed with purified water, concentrated, and then subjected to HPLC fractionation.
2 mg was obtained. (Yield 74.9%) HPLC content: 99.4% MS (FAB): m / z 617 (M + H) ⁺

[0017]

[Chemical 6]

[Examples 3 to 6]: Synthetic Examples 3 to 6 Table 1 is the same as Synthetic Example 2 except that glycine and phenylalanine in Synthetic Example 2 are replaced with A and B in Table 1 below.
The compound shown in was obtained.

[0019]

[Table 1]

[Examples 7 to 11]: Synthesis Examples 7 to 11 Mpt-N ₃ , as a condensing agent, was prepared in the same manner as in Example 2.
Instead of TEA, HOBt 20.3mg, WSC ・
HCl 23.0 mg and TEA 0.017 ml were added under ice cooling and reacted for 3 hours and room temperature for 21 hours to obtain the compounds shown in Table 2 below.

[0021]

[Table 2]

Example 12: Synthesis example 12 (2 + 2 condensation method) Boc-Phe-Cys (Npys) -OBzl 6
1.3 mg (0.1 mmol) of methylene chloride 0.39
Dissolve in 3 ml, add 0.39 ml of TFA under ice cooling, and add 3
After stirring for 0 minutes, react at room temperature for 30 minutes, concentrate, and then DMF
Dissolved in 1 ml. This solution was stirred under ice-cooling with TEA.
0.014 ml, Boc-Cys-Gly-OH 2
7.8 mg (0.1 mmol) was added, and the mixture was reacted at room temperature for 2 hours, further DMF 19 ml, Mpt-N ₃ 27.0.
mg and TEA 0.014 ml were added under ice-cooling and reacted for 24 hours. The reaction solution was concentrated, ethyl acetate was added to the residue, the organic layer was washed with purified water, concentrated, and then subjected to HPLC fractionation to obtain 50.4 mg of the target product below. (Yield 81.8
%) HPLC content: 99.6% MS (FAB): m / z 617 (M + H) ⁺

[0023]

[Chemical 7]

Examples 13 to 16: Synthesis Examples 13 to 16 Glycine and phenylalanine in Synthesis Example 12 were replaced by
Compounds shown in Table 3 below were obtained in the same manner as in Synthesis Example 12 except that A and B in Table 3 below were substituted.

[0025]

[Table 3]

[Examples 17 to 21]: Synthesis Examples 17 to 21 Mpt- was used as a condensing agent in the same manner as in Example 12.
Instead of N ₃ and TEA, HOBt 20.3mg, W
SC ・ HCl 23.0mg, TEA 0.017ml
Was added under ice cooling and reacted for 3 hours and room temperature for 21 hours to obtain the compounds shown in Table 4 below.

[0027]

[Table 4]

[0028]

[Brief description of drawings]

FIG. 1 is a synthetic scheme of the 1 + 3 condensation method.

FIG. 2 is a synthetic scheme of the 2 + 2 condensation method.

Claims (2)

[Claims] 1. A cysteine-containing protected peptide represented by the following general formula (II) and a 3-cysteine represented by the following general formula (III):
From the cysteine-containing protected peptide protected with a nitro-2-pyridylsulfenyl group, first, an S—S bond and then a peptide bond are formed to obtain a cystine-containing protected peptide represented by the following general formula (IV). The method for producing a cystine-containing peptide represented by the following general formula (I), which comprises deprotecting [Chemical 1] [In the formula (II), X represents an amino protecting group, A and B represent various protected amino acid residues or protected peptides, and n and m each represent an integer of 0 or 1. In formula (III), C and D are various protected amino acid residues or protected peptides, Y is a carboxyl protecting group, p and q each represent an integer of 0 or 1, and Npy
s represents a 3-nitro-2-pyridylsulfenyl group.
In the formula (IV), X, Y, A, B, C, D, n, m, p and q all have the above meanings. In the formula (I), A ′,
B ', C'and D'represent various unprotected or partially protected amino acid residues or peptides, n, m, p
And q have the meanings given above. Cys represents a cysteine residue. ] 2. A cysteine-containing protected peptide protected by a 3-nitro-2-pyridylsulfenyl group, represented by the following general formula (V):
The method for producing a cystine-containing peptide represented by the general formula (I) according to claim 1, wherein I) is a cysteine-containing protected peptide represented by the following general formula (VI). [Chemical 2] [In the formula (V), X is an amino protecting group, A and B are various protected amino acid residues or protected peptides, n and m are each an integer of 0 or 1, and Npys is 3-nitro-2-pyridylsulfenyl. Indicates a group. In formula (VI), C and D represent various protected amino acid residues or protected peptides, Y represents a carboxyl protecting group, and p and q each represent an integer of 0 or 1.
Cys represents a cysteine residue. ]