JP2565034B2 - Novel dopa derivative and its production method - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel dopa derivative capable of chemically synthesizing a β-3,4- (dihydroxyphenyl) alanine (hereinafter referred to as dopa) -containing peptide and a method for producing the same.

[0002]

2. Description of the Related Art Adhesive proteins secreted by shellfish such as mussels and barnacles have strong adhesive strength to low energy surfaces such as polytetrafluoroethylene. One of the constituent amino acids abundantly contained in this adhesive protein is dopa, which is said to be deeply involved in the adhesion mechanism. It is well known that dopa is a precursor of adrenaline and noradrenaline in higher animals, and is already an effective therapeutic agent for Parkinson's disease as a monomer. Therefore, the peptide containing dopa is expected to be applied to pharmaceuticals. As described above, a technique of chemically synthesizing a peptide containing dopa in the same manner as a peptide containing an ordinary amino acid has been strongly desired in developing new adhesives and pharmaceuticals.

When a peptide is synthesized by a chemical synthesis method using amino acid as a raw material, various functional groups such as amino group or carboxyl group of amino acid main chain, amino group, carboxyl group or hydroxy group of side chain are protected by various protecting groups. It is necessary to remove the protecting group from the intermediate product (deprotection) if necessary, and to participate in the subsequent reaction. Known methods for protecting the main chain amino group include a method using a benzyloxycarbonyl group, a method using a t-butyloxycarbonyl group, and a method using a 9-fluorenylmethoxycarbonyl (Fmoc) group. Among them, a method using an Fmoc group, which can be deprotected under mild basic conditions, has recently attracted attention.

[0004] Protection of amino groups by Fmoc groups of some amino acids and application to peptide synthesis have been reported in carpino (LA Ca
rpino) and others (J. Org. Chem., 197).
2, 37 , 3404-3409). Later, J. Meienhofer et al. Applied this to the solid phase (Int.
rotein Res., 1979, 13 , 35-42), C. Chan
g) et al. Fmoc formation of the amino group of an amino acid whose side chain is protected by t-butyl group and the Fmoc compound (the side chain is t
-Amino acid protected with butyl group and amino group with Fmoc group was studied (Int. J. Protein Res., 19
80, 15 , 59-66), and I. Schoen et al. It was reported to be useful (Synthesis, 1986, 4 , 303-305), and Milton (RC Milton) et al.
It has been reported that 9-fluorenylmethyl-N-succinimidyl carbonate is useful for oc formation. (In
t. J. Protein Res., 1987, 30 , 431-432).

The amino group of an amino acid having a functional group such as a hydroxyl group, an amino group and a carboxyl group in its side chain is Fmoc.
And the functional groups of those side chains are t-butyl group, t
-Fm the amino group of amino acid protected by butyloxycarbonyl group, benzyl group or p-toluenesulfonyl group
Comparing the case of oc, when the amino acid is tyrosine, the yield is 7 if the side chain hydroxy group is not protected.
0% (reference value by Schen et al., Supra), whereas the yield of 85% (reference value by Milton et al., Supra) is obtained when the hydroxy group of the side chain is protected by a dichlorobenzyl group. In the case of, the yield is higher. 2 hydroxyl groups on the side chain
Dopa with individual is unstable in neutral or alkaline. However, when it is reacted with boric acid or borate in an aqueous solution to form boric acid complex, the stability is extremely high even in neutral or alkaline. It is known (Japanese Patent Application Laid-Open No. 48-26745). Then, by utilizing this fact, the present inventors synthesized dopa in which the amino group was protected by the Fmoc group (Japanese Patent Laid-Open No. 3-81258).

[0006]

When the amino acid is dopa, chemical synthesis of a dopa-containing peptide is not easy simply by protecting the amino group of the main chain with an Fmoc group, and it is difficult to chemically synthesize the dopa-containing peptide. Stable hydroxyl groups should also be protected. In the present invention, the amino group of the main chain of dopa is Fm.
The purpose of the present invention is to provide a novel dopa derivative protected by an oc group and also protected by two labile hydroxyl groups of the side chain benzene ring, thereby facilitating chemical synthesis of a dopa-containing peptide. .

[0007]

The present invention provides a compound of formula (I)

[Chemical 6] And an N- (9-fluorenylmethoxycarbonyl) -β-3,4- (dihydroxyphenyl) alanine derivative represented by

The compound represented by the formula (I) has the formula (II)

[Chemical 7] After reacting phenylboric acid with the compound represented by the general formula (III)

Embedded image (In the general formula (III), R represents a group represented by the formula (IV), a group represented by the formula (V), or a halogen atom)
It can be produced by reacting an agent.

[Chemical 9]

[Chemical 10]

As the compound represented by the formula (II), any of L-form, D-form and DL-form may be used, and a reagent grade commercial product can be easily obtained. Further, the compound represented by the formula (III) can be easily obtained as a reagent grade commercial product. The above reaction is carried out in a solvent, and as the solvent, for example, a mixed solvent of water and at least one organic solvent selected from acetonitrile, diethyl ether, dioxane, acetone or the like can be used.

When the compound represented by the formula (II) is reacted with phenylboric acid, the amount of phenylboric acid is represented by the formula (II)
The amount of the compound represented by and boron is sufficient to form a chelate compound as shown in formula (I). Expression (I
The reaction temperature and reaction time for reacting the compound represented by I) with phenylboric acid may be appropriately set so that the above chelate compound is produced, and may be, for example, 0 to 100.
Stir for 3 minutes to 24 hours at ℃. After reacting the compound represented by the formula (II) with phenylboric acid, the compound represented by the formula (III)
The reaction temperature and the reaction time when the compound represented by are reacted may be appropriately set so that the Fmoc-forming reaction occurs, and for example, the reaction is performed by stirring at 0 to 100 ° C. for 1 to 6 hours. At this time, the pH of the reaction solution is adjusted to 8 to 12 and further adjusted to 1 to 60.
The Fmoc-forming reaction can be sufficiently carried out by stirring for a minute. As the pH adjuster, sodium carbonate or sodium hydrogen carbonate whose pH does not change rapidly is preferable.

The atmosphere of the above reaction may be atmospheric air, but in order to suppress side reactions as much as possible, it is preferable to carry out under an atmosphere of an inert gas such as nitrogen or argon. The compound represented by the formula (I) thus obtained is acidified with an acid such as hydrochloric acid or sulfuric acid (for example, pH 1 to 6), and then ether, ethyl acetate, tetrahydrofuran, dichloromethane, chloroform, etc. Extract with an organic solvent, remove the organic solvent by an appropriate method such as concentration under reduced pressure, and recrystallize from a mixed solvent such as dichloromethane / hexane or methanol / ether, or if necessary, perform purification operations such as column chromatography. And make it a purified product.

The compound represented by the formula (I) obtained in the present invention may be used as it is or after protecting the carboxyl group with a suitable protecting group, and then the amino group and / or the carboxyl group and / or the side group. A peptide containing dopa in the sequence is chemically synthesized by using a normal amino acid having a chain functional group protected by an appropriate protecting group as a starting material.

[0013]

【Example】

Example 1 β-3,4-dihydroxyphenyl-L-alanine (manufactured by Sigma) was dissolved in 1970 mg (10 mmol) of a mixed solution of 10 ml of acetonitrile and 20 ml of water, and then phenylboric acid (manufactured by Aldrich). 1225
After adding mg (10 mmol) and 1.0 ml of hydrochloric acid and stirring at room temperature for 10 minutes, sodium carbonate was added to adjust the pH.
Was adjusted to 8, 2591 mg (10 mmol) of 9-fluorenylmethoxycarbonyl chloride (manufactured by Tokyo Chemical Industry Co., Ltd.), 10 ml of acetonitrile and 15 ml of water were added, and the mixture was stirred at room temperature for 5 minutes. A sodium hydrogen carbonate solution was added little by little to the reaction mixture solution to adjust the pH of the solution to 8.0.
It was adjusted to 8.5 and stirred for a further 20 minutes. 20 ml of ether was added to the reaction solution, and the mixture was shaken, the aqueous layer was taken, and this extraction operation was repeated twice. Concentrated hydrochloric acid was added to this aqueous layer to adjust the pH.
Was adjusted to about 2, and this was transferred to a separating funnel, 20 ml of benzene was added and shaken, the benzene layer was taken, and this extraction operation was repeated twice more. After sodium sulfate was added to the extract for dehydration, sodium sulfate was removed by filtration,
Benzene was distilled off from the filtrate by concentration under reduced pressure to obtain a reaction product of 3722 mg (yield: 74%).

The analysis results of this reaction product are as follows. Melting point: 142 ° C. ¹ H Nuclear magnetic resonance spectrum: (for part with absorption) FIGS. 1 to 3 ¹³ C Nuclear magnetic resonance spectrum: (for part with absorption) FIGS. 4 and 5 High performance liquid chromatogram: FIG. 6 Infrared Absorption spectrum: FIG. 7 The peak appearing at a retention time of 17.7 minutes in FIG. 6 is the reaction product. The purity of the reaction product calculated from FIG. 6 was 93%. From the results of FIGS. 1 to 7, it was confirmed that the reaction product was the compound represented by the formula (I).

The melting point was measured by using a differential scanning calorimeter (DSC-7 type manufactured by Perkin Elma Co., Ltd.) at a heating rate of 5 ° C./min.
The amount of sample was measured at 2.45 mg, and the ¹ H and ¹³ C nuclear magnetic resonance spectra were measured by a nuclear magnetic resonance apparatus (Bruker AC-2.
50 type) and measured with DMSO-d ₆ solvent containing DCl / D ₂ O, and high performance liquid chromatography (main body: manufactured by Waters Co., 600 series) has 5 μC 18-100Å microbonder sphere (μBondasphere) on the column
(3.9 mm × 15 cm, manufactured by Waters Co.), and the developing solvent was a 10:90 mixed solution of an 80:20 mixed solution of 0.1% trifluoroacetic acid aqueous solution and 0.1% trifluoroacetic acid acetonitrile solution. Change composition linearly in 30 minutes, flow rate is 1.0 ml / min, detection wavelength is 280
nm and the infrared absorption spectrum was measured by the KBr tablet method using an infrared analyzer (Hitachi 270-50 type).

[0016]

INDUSTRIAL APPLICABILITY According to the present invention, the amino group of the main chain of dopa is Fm.
A novel dopa derivative protected by an oc group and having two hydroxyl groups on the side chain benzene ring protected by phenylboron, and a method for producing the same, which facilitates chemical synthesis of a dopa-containing peptide Is.

[Brief description of drawings]

FIG. 1 shows the chemical shift of the compound of the present invention 0.8 to 3.2 pp.
It is a ¹ H nuclear magnetic resonance spectrum in m. FIG. 2 is a ¹ H nuclear magnetic resonance spectrum of the compound of the present invention at a chemical shift of 3.2 to 5.2 ppm. FIG. 3 is a ¹ H nuclear magnetic resonance spectrum of the compound of the present invention at a chemical shift of 6.0 to 8.4 ppm. FIG. 4 is a ¹³ C nuclear magnetic resonance spectrum of the compound of the present invention at a chemical shift of 30 to 70 ppm. FIG. 5 shows the chemical shift 11 of the compound of the present invention.
It is a ¹³ C nuclear magnetic resonance spectrum at 0 to 176 ppm. FIG. 6 is a high performance liquid chromatogram of the compound of the present invention. FIG. 7 is an infrared absorption spectrum of the compound of the present invention.

Claims (2)

(57) [Claims] 1. A compound represented by the formula (I): An N- (9-fluorenylmethoxycarbonyl) -β-3,4- (dihydroxyphenyl) alanine derivative represented by: 2. Formula (II): After reacting phenylboric acid with β-3,4- (dihydroxyphenyl) alanine represented by the general formula (II
I) [Chemical 3] (In the general formula (III), R is a group represented by the following formula (IV),
Represents a group represented by (V) or a halogen atom) Embedded image The method for producing an N- (9-fluorenylmethoxycarbonyl) -β-3,4- (dihydroxyphenyl) alanine derivative according to claim 1, which comprises reacting an Fmoc agent represented by