JP2629375B2 - Method for producing amino-protected dopa or dopa derivative - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel dopa having a protected amino group which enables the chemical synthesis of a 3,4-dihydroxyphenylalanine (hereinafter referred to as "dopa")-containing peptide. And dopa derivatives.

[Conventional technology]

Adhesive proteins secreted by shellfish such as mussels and barnacles have strong adhesion to low energy surfaces such as polytetrafluoroethylene. One of the constituent amino acids abundantly contained in this adhesive protein is dopa,
It is said that dopa is 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 for chemically synthesizing a peptide containing dopa in the same manner as a peptide containing ordinary amino acids has been strongly desired in developing new adhesives and pharmaceuticals.

When a peptide is synthesized by a chemical synthesis method using an amino acid as a raw material, various protections for the side chain amino group, amino group, carboxyl group and hydroxy group other than the amino group and carboxyl group of the amino acid involved in the dehydration condensation reaction are provided. It is necessary to protect with a group and, if necessary, remove the protecting group from the intermediate product (deprotection) so that it can participate in the subsequent reaction.

Methods for protecting the amino group in the main chain include a method using a benzyloxycarbonyl group, a method using a t-butyloxycarbonyl group, a method using a 9-fluorenylmethoxycarbonyl group (Fmoc group), and the like. A method using an Fmoc group, which can be deprotected under conditions and enables chemical synthesis of a peptide by a solid phase method, has recently attracted attention.

Protection of the amino group by the Fmoc group of ordinary amino acids was developed by LA Carpino et al. (J. Org. Che.
m., 1972, 37 , 3404-3409), Maienhofer (J. Mei
enhofer) et al. (Int. J. Protein R)
es., 1979, 13 , 35-42). Thereafter, C. Chang et al. (Int. J. Protein Res., 1980, 15 , 59-66); I. Schn et al. (Synthesis, 1986, 4 , 303-305); (Int. J. Protein Res., 1987, 3
0, the 431-432), various improvements have been made. According to these, when an Fmoc group is introduced into the amino group of an amino acid, the amino acid is reacted with 9-fluorenylmethoxycarbonyl chloride in the presence of sodium carbonate, or the amino acid is reacted with 9-fluorene in the presence of sodium hydrogen carbonate. Nylmethylpentafluorophenyl carbonate or reacting the amino acid with 9
-Fluorenylmethyl-N-succinimidyl carbonate.

When protecting the amino group of an amino acid having a functional group such as a hydroxyl group, an amino group or a carbonyl group in the side chain with an Fmoc group, the functional group in the side chain must be previously protected with a t-butyl group, a t-butyloxycarbonyl group, a benzyl group. It is better to protect with a group or a p-toluenesulfonyl group. For example, if tyrosine protects the side chain hydroxy group with dichlorobenzyl group, the yield of amino acid protected with Fmoc group is 85%.
The unprotected yield is better than 70% in the literature by RC Milton et al., Supra.

On the other hand, it is known that the stability of dopa becomes extremely high even in neutral or alkaline conditions when a dopa is reacted with boric acid or a borate in an aqueous solution to form a boric acid complex (JP-A-48-26745). Gazette).

[Problems to be solved by the invention]

When the amino acid is dopa, due to the effects of basic conditions such as sodium carbonate and sodium bicarbonate,
None of the conventional methods can produce a dopa in which an amino group is protected with an Fmoc group. Therefore, peptides containing dopa in the molecule could not be produced either.

The present invention provides a novel method for producing dopa and a dopa derivative in which an amino group is protected by an Fmoc group, which enables chemical synthesis of a dopa-containing peptide.

[Means for solving the problem]

The present invention relates to a compound of the general formula [I] (Wherein R ¹ represents hydrogen, a lower alkyl group or an aryl group), after reacting a dopa or a dopa derivative with a borate or a phosphate, the following general formula [II] (Where R ² is Or halogen. Wherein a 9-fluorenylmethoxycarbonyl compound represented by the following general formula [III] is reacted: (In the formula, R ¹ represents hydrogen, a lower alkyl group or an aryl group.) The present invention relates to a method for producing a dopa or a dopa derivative in which an amino group represented by the formula (I) is protected.

Any of the L-form, D-form and DL-form may be used as the compound represented by the general formula [I], and R ¹ is H (dopa)
Can easily obtain a reagent grade commercial product.

Specific examples of the lower alkyl group of the group R ¹ in the compound of the general formula [I] include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl and the like. , With the corresponding anhydrous alkyl alcohol. Specific examples of the aryl group of the group R ¹ include phenyl, pentachlorophenyl, pentafluorophenyl, benzyl, p-nitrophenyl, When R ¹ is phenyl, it is reacted with anhydrous phenol under acidic conditions, and R ¹ is pentachlorophenyl, pentafluorophenyl, p-nitrophenyl or In the case of the above, the amino group and the hydroxyl group of dopa are protected in advance by a suitable ordinarily used protecting group, and then reacted with the corresponding phenol derivative, followed by removing the protecting groups for the amino group and the hydroxyl group.

As for the compound represented by the general formula [II], commercially available reagent grade products can be easily obtained.

Specific examples of the halogen in the compound of the general formula [II] include chlorine, bromine and iodine.

The reaction between the compound represented by the general formula [I] and the borate or the phosphate, and the reaction between the reaction product and the compound represented by the general formula [II] are performed in a solvent. ,
For example, a mixed solvent of water and one or more organic solvents selected from acetonitrile, diethyl ether, dioxane, acetone and the like can be used.

The borate used in the present invention includes tetraborate, metaborate and the like, and particularly, sodium salt and potassium salt are easily used. As the phosphate, trisodium phosphate,
Disodium phosphate, monosodium phosphate, and those obtained by replacing the sodium salt thereof with a potassium salt may be mentioned. In the present invention, at least one of these compounds is used. The amount of the borate or phosphate is an amount sufficient for the compound represented by the general formula [I] and boron or phosphorus to form a chelate compound.

The reaction temperature and reaction time of the compound represented by the general formula [I] and the borate or phosphate may be set so as to select the conditions under which the above chelate compound is formed.
Stir at 100 ° C. for 3 minutes to 24 hours.

After reacting the compound represented by the general formula (I) with a borate or a phosphate, the reaction temperature and reaction time when reacting the compound represented by the general formula (II) are as follows. The condition may be appropriately selected so as to occur, for example, 0 to 100
Stir at 1 ° C. for 1-6 hours. At this time, the pH of the reaction solution was set to 8
If it is adjusted to 1212 and further stirred for 1 to 10 minutes, the Fmoc-forming reaction can be sufficiently performed. As the pH adjuster, sodium carbonate or sodium hydrogencarbonate whose pH does not change rapidly is preferable.

Although the atmosphere of the above reaction may be in the air, it is preferable to carry out the reaction in an atmosphere of an inert gas such as nitrogen or argon in order to suppress side reactions as much as possible.

The thus-obtained compound represented by the general formula [III] is prepared by acidifying the reaction solution with an acid such as hydrochloric acid or sulfuric acid (for example, at pH 1).
-6), extraction with an organic solvent such as ether, ethyl acetate, tetrahydrofuran, dichloromethane, chloroform, etc., removal of the organic solvent by an appropriate method such as concentration under reduced pressure, and a mixed solvent such as dichloromethane / hexane, methanol / ether, etc. Or purified by column chromatography or the like if necessary to obtain a purified product.

Starting from the compound represented by the general formula [III] obtained by the present invention and a normal amino acid in which an amino group and / or a carboxyl group and / or a functional group of a side chain is protected with a suitable protecting group, A peptide containing dopa therein is chemically synthesized.

〔Example〕

(Example 1) A mixed solution of 20 ml of acetonitrile and 10 ml of acetone was added to 593 mg (3 mmol) of 3,4-dihydroxyphenyl-L-alanine (manufactured by Sigma), and a 0.01 M sodium tetraborate buffer (pH 9. 18) Add 30 ml, stir at room temperature for 15 minutes, add 1015 mg (3 mmol) of 9-fluorenylmethyl N-succinimidyl carbonate (manufactured by Cambridge Research Biochemicals) and add 12 ml at room temperature.
Stirred for hours. 788 mg of sodium carbonate in the reaction mixture
(7.4 mmol) was added little by little, and the mixture was further stirred for 5 minutes. The unreacted raw material was extracted three times with 15 ml of ether, and the aqueous layer was adjusted to pH 2 with concentrated hydrochloric acid. The reaction product was extracted from the aqueous layer three times with 20 ml of ethyl acetate, and the ethyl acetate layer was washed once with 20 ml of a 0.1 N hydrochloric acid aqueous solution. After drying the ethyl acetate solution with anhydrous magnesium sulfate, the ethyl acetate was distilled off, and the residue was recrystallized from dichloromethane / hexane to obtain 271 mg (yield 67%) of a reaction product.

 The analysis results of this reaction product are as follows.

Melting point: 159 ° C. ¹ H nuclear magnetic resonance spectrum: Table 1 ¹³ C nuclear magnetic resonance spectrum: Table 2 High-performance liquid chromatogram: FIG. 1 Infrared absorption spectrum: FIG. 2 In FIG. 1, the reaction product has a retention time of 3.4. In minutes. Under the same analytical conditions, the starting material 3,4-dihydroquinphenyl-L-alanine appears at a retention time of 1.2 minutes, and the other starting material 9-fluorenylmethyl N-succinimidyl carbonate appears at a retention time of 8.9 minutes. Has been confirmed separately.

From the above results, it was confirmed that the reaction product was a compound represented by the formula [IV].

The melting point was measured by a differential scanning calorimeter (PerkinElmer, D
SC-7 type) using a heating rate 10 ° C. / min, measured at a sample amount 1.8 mg, measured by ¹ H and ¹³ C nuclear magnetic resonance spectra nuclear magnetic resonance apparatus (manufactured by Bruker, AC-250 type) The high-performance liquid chromatograph (body: Waters, 600 series) uses micro-bonder spheres (μBondas
phere) 5 μC18-100Å (3.9 mm × 15 cm, manufactured by Waters) was used, and the developing solvent was 0.1% trifluoroacetic acid-water.
A 50:50 mixture of 0.1% trifluoroacetic acid-acetonitrile,
The measurement was performed at a flow rate of 1.0 ml / min and a detection wavelength of 220 nm, and the infrared absorption spectrum was measured by a KBr tablet method using an infrared analyzer (Hitachi model 270-50).

Example 2 591 mg of 3,4-dihydroxyphenyl-L-alanine (3
Mmol) and sodium tetraborate decahydrate 1145mg
(3 mmol) was added with 40 ml of water, stirred at room temperature for about 60 minutes, and 1018 mg (3 mmol) of 9-fluorenylmethyl N-succinimidyl carbonate was added to acetonitrile 10
It was dissolved in ml and added, followed by stirring at room temperature for 17 hours. 5.0 g of sodium carbonate was added little by little to the reaction mixture, and the mixture was stirred at room temperature for about 10 minutes. The unreacted raw material was extracted three times with 20 ml of ether.
The aqueous layer was adjusted to pH 2 by adding concentrated hydrochloric acid. The reaction product was extracted from the aqueous layer three times with 20 ml of ether, the extract was dried over anhydrous magnesium sulfate, the ether was distilled off, the residue was recrystallized from methanol / ether, and the reaction product was recovered.
00 mg (87% yield) was obtained.

As a result of analyzing the reaction product, the melting point, ¹ H nuclear magnetic resonance spectrum, ¹³ C nuclear magnetic resonance spectrum, high performance liquid chromatogram, and infrared absorption spectrum were the same as those in Example 1, and the reaction product was represented by the formula [IV ] Was confirmed.

(Example 3) 591 mg of 3,4-dihydroxyphenyl-L-alanine (3
Mmol) and 524 mg (3 mmol) of potassium monohydrogen phosphate, 40 ml of water were added, and the mixture was stirred at room temperature for about 60 minutes.
1017 mg (3 mmol) of fluorenylmethyl N-succinimidyl carbonate dissolved in 10 ml of acetonitrile was added, and the mixture was stirred at room temperature for 17 hours. 5.0 g of sodium carbonate was added little by little to the reaction mixture, and the mixture was stirred at room temperature for about 10 minutes.
The unreacted raw material was extracted three times with 20 ml of ether, and the aqueous layer was adjusted to pH 2 by adding concentrated hydrochloric acid. The reaction product was extracted from the aqueous layer three times with 20 ml of ether, the extract was dried over anhydrous magnesium sulfate, the ether was distilled off, and the residue was recrystallized from methanol / ether to give 867 mg of the reaction product ( Yield 68
%).

As a result of analyzing the reaction product, the melting point, ¹ H nuclear magnetic resonance spectrum, ¹³ C nuclear magnetic resonance spectrum, high performance liquid chromatogram, and infrared absorption spectrum were the same as those in Example 1, and the reaction product was represented by the formula [IV ] Was confirmed.

〔The invention's effect〕

The present invention provides a novel dopa having an amino group protected by an Fmoc group and a derivative thereof, and enables chemical synthesis of a dopa-containing peptide which has not been achieved before.

[Brief description of the drawings]

FIG. 1 is a high performance liquid chromatogram of the compound of the present invention, and FIG. 2 is an infrared absorption spectrum of the compound of the present invention.

Claims (1)

(57) [Claims] 1. A compound of the formula [I] (In the formula, R ¹ represents hydrogen, a lower alkyl group or an aryl group) represented by the formula (4), wherein a borate or a phosphate salt is formed with a 3,4-dihydroxyphenylalanine or a 3,4-dihydroxyphenylalanine derivative. After reacting, the following general formula [II] (Where R ² is Or a halogen) represented by the following general formula [III]: (Wherein R ¹ represents hydrogen, a lower alkyl group or an aryl group). A method for producing a 3,4-dihydroxyphenylalanine or a 3,4-dihydroxyphenylalanine derivative in which an amino group represented by the following formula is protected.