JP2663747B2 - Method for purifying 4-hydroxyproline or 4-hydroxyproline derivative having an amino group protected - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for purifying 4-hydroxyproline or a 4-hydroxyproline derivative having an amino group protected, which enables the chemical synthesis of a 4-hydroxyproline-containing peptide.

[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 4-hydroxyproline, and it is estimated that 4-hydroxyproline is deeply involved in the adhesion mechanism. Therefore, a peptide containing 4-hydroxyproline is expected to be applied to a new adhesive. 4-Hydroxyproline is also contained in a large amount in peptides having physiological activity on nerves of higher animals, for example, peptides of sea snake neurotoxin. Therefore, a peptide containing 4-hydroxyproline is expected to be applied to pharmaceuticals.
For these reasons, a technique for chemically synthesizing a peptide containing 4-hydroxyproline in the same manner as a peptide containing an ordinary amino acid has been strongly desired.

On the other hand, L. Lapatsani
s) et al. describe a 9-fluorenylmethoxycarbonyl group (Fmo
c) was introduced into the amino group of the main chain of 4-hydroxyproline for the first time (Synthesis, 1983, 671).
-673). In this method, a dimethylformamide solution (or a dioxane solution) of 9-fluorenylmethyl-N-succinimidyl carbonate is added to a mixed solution of an aqueous solution of 4-hydroxyproline and an aqueous solution of sodium carbonate,
After the reaction, unreacted substances and by-products are removed with ethyl acetate, the aqueous layer is adjusted to pH 2 with concentrated hydrochloric acid, and the target substance is extracted with ethyl acetate and recrystallized by adding petroleum ether.

Further, the present inventors have previously reacted 4-hydroxyproline with 9-fluorenylmethylpentafluorophenyl carbonate in the presence of triethylamine, and extracted the reaction product with diethyl ether. By recrystallization, 4-hydroxyproline in which the amino group was protected was obtained (Japanese Patent Application No. 3-774).
No. 94). As described above, in the above two methods, all means for purifying 4-hydroxyproline in which the amino group is protected by the Fmoc group are by recrystallization.

[0005]

However, the recrystallization method requires 9
The method of purifying fluorenylmethoxycarbonyl-4-hydroxyproline requires a long time for crystallization, and the obtained crystals are relatively hard, so that the crystals are dissolved in an organic solvent such as diethyl ether, ethyl acetate, acetonitrile, etc. It takes time to dissolve. The present invention provides a method for purifying an amino-protected 4-hydroxyproline or a 4-hydroxyproline derivative, which can be purified in a short time and the obtained product is easily dissolved in an organic solvent.

[0006]

Means for Solving the Problems In the course of studying the amount of anhydrous magnesium sulfate to be added to the reaction solution for dehydration of the reaction solution, the present inventors have found that the amount of sulfuric anhydride that is at least the minimum amount required for dehydration is greater than that required for dehydration. When magnesium is added, surprisingly, 4-hydroxyproline or 4
The present inventors have found that a hydroxyproline derivative is adsorbed on magnesium sulfate, and completed the present invention.

That is, the present invention provides: Comprising adsorbing on anhydrous magnesium sulfate,

Embedded image (Wherein, R represents hydrogen, a lower alkyl group or an aryl group). The present invention relates to a method for purifying 4-hydroxyproline or a 4-hydroxyproline derivative having an amino group protected.

The magnesium sulfate used in the present invention is preferably anhydrous and does not contain water of crystallization. This anhydrous salt can be easily obtained as a commercial product. The amount of magnesium sulfate used is sufficient to adsorb water contained in the reaction solution and also to adsorb 4-hydroxyproline or a 4-hydroxyproline derivative in which the amino group of the target compound represented by the chemical formula 2 is protected. Amount. The temperature and the treatment time when the target substance is adsorbed on magnesium sulfate vary depending on the amount of magnesium sulfate to be used and the moisture contained in the reaction system, etc., so that the optimal conditions may be selected and set as appropriate. The temperature is 0 to 35 ° C, and the processing time is 1 to 24 hours.

The recovery of the target substance from the magnesium sulfate to which the target substance has been adsorbed may be performed as follows. That is, the magnesium sulfate to which the target substance is adsorbed is collected by a method such as filtration, washed with an appropriate organic solvent such as ether, and then dissolved in a hydrochloric acid aqueous solution having a pH of about 2. The compound represented by Chemical Formula 2 does not dissolve in this operation. The insoluble matter is collected, washed with an organic solvent miscible with water such as acetonitrile, and dried by a method such as drying under reduced pressure to obtain a purified product. A compound in which a carboxyl group and / or a side chain functional group of the compound represented by Chemical formula 2 obtained by the present invention is protected with an appropriate protecting group, and an amino group, a carboxyl group and / or a side chain functional group are A peptide containing a hydroxyproline in the sequence is chemically synthesized from a normal amino acid protected with a protecting group as a raw material.

The ¹ H nuclear magnetic resonance spectrum of the compound obtained in the present invention is shown in FIGS. 1 to 4 (comparative examples in FIGS. 5 to 8) for the portion having absorption, and the infrared absorption spectrum is shown in FIG.
In FIG. 10 (Comparative Example), the results of high performance liquid chromatography are shown in Sample 1 in Table 1 (Comparative Example is Sample 2), and the results of elemental analysis are shown in Sample 2 in Table 2 (Comparative Example is Sample 2). Are shown below. From these results, it was confirmed that the compound obtained in the present invention was N-9-fluorenylmethoxycarbonyl-4-hydroxy-L-proline.

The ¹ H nuclear magnetic resonance spectrum was measured by a nuclear magnetic resonance apparatus (manufactured by Bruker, model AC-250), and high performance liquid chromatography (main unit: Waters, 600 series) was applied to the column by using a microbonder sphere. (ΜBondasphere) 5 μc18-100
Å (3.9 mm × 15 cm, manufactured by Waters), the developing solvent was a 50:50 mixture of water and acetonitrile, the flow rate was 1.0 ml / min, and the detection wavelength was 254 nm.
The infrared absorption spectrum was measured by a KBr tablet method using an infrared analyzer (Hitachi type 270-50).

[Table 1]

[Table 2]

[0012]

EXAMPLE 3.93 g (30 mmol) of trans-4-hydroxy-L-proline (manufactured by Aldrich) was added to 100 m of distilled water.
1 to dissolve and add 9-fluorenylmethyl-N
-A solution prepared by dissolving 11.17 g (33 mmol) of succinimidyl carbonate (manufactured by Cambridge Research Biochemicals) in 150 ml of acetonitrile was added. While cooling this mixed solution with ice water, 15 ml of triethylamine (manufactured by Kanto Chemical Co., Ltd.) was added little by little to 30
After stirring for minutes, the mixture was further stirred at room temperature for 3 hours. 100 ml of distilled water and 15.3 g of sodium chloride were added to the reaction solution, and the mixture was washed three times with 50 ml of diethyl ether, and the pH of the aqueous layer was adjusted to 2 or less with concentrated hydrochloric acid. This solution was extracted three times with 100 ml of diethyl ether, 30 g of anhydrous magnesium sulfate (manufactured by Kanto Kagaku) was added to the ether layer, and the mixture was allowed to stand at room temperature overnight, followed by Millicup filter paper (manufactured by Millipore).
And filtered. The residue on the Millicup filter paper was washed twice with about 1N aqueous hydrochloric acid solution (50 ml), and the water was replaced with acetonitrile while lightly filtering under reduced pressure, followed by drying under reduced pressure.
Reaction product N-9-fluorenylmethoxycarbonyl
6.32 g of 4-hydroxy-L-proline (yield: 6
0%).

Comparative Example 3.93 g (30 mmol) of trans-4-hydroxy-L-proline (manufactured by Aldrich) was added to 100 m of distilled water.
1 to dissolve and add 9-fluorenylmethyl-N
-A solution of 10.12 g (30 mmol) of succinimidyl carbonate (manufactured by Cambridge Research Biochemicals) in 150 ml of acetonitrile was added. While cooling this mixed solution with ice water, 15 ml of triethylamine (manufactured by Kanto Kagaku) was added little by little,
Stirred for minutes. 150 ml of distilled water and 16 g of sodium chloride were added to the reaction mixture, and 50 ml of diethyl ether was added.
After washing three times with 1 l, the aqueous layer was adjusted to pH 2 or less with concentrated hydrochloric acid. This solution was extracted three times with 100 ml of diethyl ether. To the ether layer, 3 g of anhydrous magnesium sulfate (manufactured by Kanto Chemical Co., Ltd.) was added for dehydration, and the mixture was allowed to stand at room temperature overnight. The mixture was concentrated to about 5 ml with a rotary evaporator, crystallized, and the reaction product N-9-fluorenylmethoxycarbonyl-4
6.52 g of -hydroxy-L-proline (yield: 61
%).

[0014]

The present invention provides a method for purifying high-purity 4-hydroxyproline and its derivative in which the amino group is protected with an Fmoc group in a short time. This facilitated the chemical synthesis of the 4-hydroxyproline-containing peptide.

[Brief description of the drawings]

FIG. 1 is a ¹ H nuclear magnetic resonance spectrum of a compound (N-9-fluorenylmethoxycarbonyl-4-hydroxy-L-proline) obtained in an example at a chemical shift of 1.8 to 3.7 ppm.

FIG. 2 is a ¹ H nuclear magnetic resonance spectrum of the compound (N-9-fluorenylmethoxycarbonyl-4-hydroxy-L-proline) obtained in the example at a chemical shift of 4.0 to 5.4 ppm.

FIG. 3 is a ¹ H nuclear magnetic resonance spectrum of the compound (N-9-fluorenylmethoxycarbonyl-4-hydroxy-L-proline) obtained in the example at a chemical shift of 7.0 to 8.1 ppm.

FIG. 4 is a ¹ H nuclear magnetic resonance spectrum of the compound (N-9-fluorenylmethoxycarbonyl-4-hydroxy-L-proline) obtained in the example at a chemical shift of 12.2 to 13.2 ppm.

FIG. 5 is a ¹ H nuclear magnetic resonance spectrum of the compound (N-9-fluorenylmethoxycarbonyl-4-hydroxy-L-proline) obtained in Comparative Example at a chemical shift of 1.8 to 3.7 ppm.

FIG. 6 is a ¹ H nuclear magnetic resonance spectrum of the compound (N-9-fluorenylmethoxycarbonyl-4-hydroxy-L-proline) obtained in Comparative Example at a chemical shift of 4.0 to 5.4 ppm.

FIG. 7 is a ¹ H nuclear magnetic resonance spectrum of the compound (N-9-fluorenylmethoxycarbonyl-4-hydroxy-L-proline) obtained in Comparative Example at a chemical shift of 7.0 to 8.1 ppm.

FIG. 8 is a ¹ H nuclear magnetic resonance spectrum of the compound (N-9-fluorenylmethoxycarbonyl-4-hydroxy-L-proline) obtained in Comparative Example at a chemical shift of 12.2 to 13.2 ppm.

FIG. 9 is an infrared absorption spectrum of the compound (N-9-fluorenylmethoxycarbonyl-4-hydroxy-L-proline) obtained in the example.

FIG. 10: Compound obtained in Comparative Example (N-9-fluorenylmethoxycarbonyl-4-hydroxy-L-proline)
3 is an infrared absorption spectrum.

Claims (1)

(57) [Claims] (1) Comprising a step of adsorbing on anhydrous magnesium sulfate. (Wherein, R represents hydrogen, a lower alkyl group or an aryl group). A method for purifying 4-hydroxyproline or a 4-hydroxyproline derivative having an amino group protected.