JPH0153870B2 - - Google Patents

Description

[Detailed Description of the Invention] The present invention is based on the formula [] [In the formula, R represents a lower alkyl group] It relates to an argininal di-lower alkyl acetal represented by the following formula.

The argininal di-lower alkyl acetal of the present invention is useful as a raw material for various protease inhibitors and substrates for affinity chromatography.

Certain actinomycetes (Streptomyces roseus)
leupeptin (acetyl and propionyl-
It has already been revealed by Umezawa, Aoyagi et al. that leucyl-leucyl-argininal) has a strong inhibitory effect on various serine proteases and thiol proteases. [The Journal of Antibiotics Volume 22,
283 pages (1969) and others]. Focusing on the argininal contained in leupeptin, several argininal derivatives have been produced based on its amino acid sequence, and all of them are known to have protease inhibitory effects (Enzyme Inhibitors of Microorganisms). Vial Origin, p. 23, Hamao Umezawa, University of Tokyo Press, 1972), and it is also known that it can be used as a substrate for affinity chromatography (Japanese Patent Application Laid-open No. 37185/1985).

Such argininal derivatives have conventionally been produced by fermentation methods or synthetically using arginine as a raw material.

However, when fermentation is used, it is not possible to obtain an argininal derivative with the desired amino acid sequence. In addition, the method using arginine as a raw material is an extremely complicated method that involves, for example, protecting the carboxylic acid group of arginine and the amino group of the guanidino group, followed by reaction, removal of the protecting group of the carboxylic acid, reduction, and removal of the protecting group of the amino group. be.

However, since the argininal dialkyl acetal of the present invention can be directly reacted with amino acids and peptides using ordinary peptide chemistry techniques as a raw material, it is possible to obtain argininal derivatives with a desired amino acid sequence extremely easily. have

In the present invention, the lower alkyl group is a _C1 - _C6 alkyl group, preferably a _C3 - _C5 alkyl group.

Examples of the compounds of the present invention include di-n-propyl acetal, di-n-butyl acetal, and di-n-pentyl acetal of argininal.

Next, the method for producing the compound of the present invention will be explained.

The compound of the present invention is a mixture of the dialkyl acetal of leupeptin, α-chymotrypsin, and various enzymes, commercially available Pronase E (manufactured by Kaken Pharmaceutical Co., Ltd.).
It can be obtained by allowing it to react for about 60 to 100 hours at around 0.degree. C., adsorbing it on column chromatography using silica gel as a carrier, and then eluting it. It can also be obtained in the same way by reacting exopeptidase such as aminopeptidase M with leucyl argininal dialkyl acetal obtained by reacting thermolysin with dialkyl acetal of leupeptin.

The thus obtained argininal dialkyl acetal itself can be immobilized on a resin having an acidic group and then hydrolyzed to form argininal, thereby becoming an affinity resin such as trypsin. By introducing such as, it is possible to obtain a substrate for affinity chromatography that has a strong affinity for the target protease. For example, when phenylalanine is introduced into the α-amino group of argininal, it becomes a substrate with extremely high affinity for kallikrein.

The advantages of synthesizing peptidic argininal using the argininal dialkyl acetal of the present invention are (i) no need to protect the amino group in the guanidino group, and (ii) mild acid hydrolysis after synthesis. Examples include the ability to convert acetal into an aldehyde group.

To introduce amino acid residues etc. into the argininal dialkyl acetal of the present invention, methods commonly used in peptide chemistry, such as (1) acid halide method (2) N-hydroxysuccinimide, p-nitrophenol , active ester method using , pentachlorophenol, etc. (3) Carbodiimide method using dicyclohexylcarbodiimide, dimethylaminopropylcarbodiimide, etc. (4) Diphenyl phosphoric acid azide, N-ethoxycarbonyl-2-ethoxydihydroquinoline, N-
Ethyl-5-phenyl isoxazolium-
Methods using condensing agents such as 3'-sulfonate 6-chloro-1-p-chlorobenzenesulfonyloxybenzotriazole (5) Mixed acid anhydride method using ethyl chloroformate, isobutyl chloroformate, etc. (6) Methods such as azide method You can use .

Purification of compounds into which residues such as amino acids have been introduced by the methods described above is simple and efficient, as compounds containing guanidide groups are difficult to crystallize. It is true. Furthermore, if necessary, the benzyloxycarbonyl group protecting the amino group may be removed by catalytic reduction using palladium black or the like in a solvent such as methanol. In addition, acetal can be converted into an aldehyde group by hydrolysis, but if it is insoluble in water, methanol,
Using water-miscible solvents such as ethanol, acetone, acetonitrile, and dimethylformamide,
The reaction may be carried out using a mineral acid such as hydrochloric acid or sulfuric acid at 10 to 60°C, preferably 25 to 40°C for several hours. The concentration of the acid used in this case is not particularly limited, but is from 0.3 to
It is preferable to adjust it to the 0.5 standard. After the disappearance of the raw material is confirmed by thin-layer chromatography using silica gel as a carrier, the excess acid is removed using a weakly basic ion exchange resin, such as Dowex WGR, and the salt of the target compound is obtained by freeze-drying. be able to.

Various protease inhibitors can be obtained by hydrolyzing the acetal of the compound thus obtained to form an aldehyde.

This hydrolysis is performed at a pH of 1 to 4, preferably 2 to 4.
Using the acid or buffer solution in step 3, at 20-50℃, preferably at 30℃.
The reaction may be carried out at ~45°C for 24 hours to 120 hours, preferably 48 to 96 hours. There are no particular restrictions on the buffering agent used, and examples include those with a composition consisting of mineral acids such as hydrochloric acid and phosphoric acid, organic acids such as tartaric acid, citric acid, lactic acid, succinic acid, and acetic acid, and their sodium and potassium salts. .

Examples of the compounds obtained in this manner include phenylalanyl argininal and N-benzyloxycarbonyl-prolyl-argininal.

On the other hand, in order to bond an acetalized argininal derivative to a resin, the protecting group of the amino group is removed by a method commonly used in peptide chemistry, and then various acidic groups are added by using the amino group as a functional group. What is necessary is to bind it to a water-insoluble carrier having the following. The water-insoluble carrier used in the present invention is not particularly limited, and includes, for example, ion exchange resins having acidic groups such as methacrylic acid-divinylbenzene copolymer, cellulose derivatives having carboxymethyl groups, and furthermore, cepharose, agarose,
Examples include polymeric polysaccharides such as dextran with acidic groups such as carboxylic acid or sulfonic acid introduced therein.

As a method for bonding these water-insoluble carriers and acetalized argininal derivatives, there are methods generally used when creating affinity resins, such as applying water-soluble carbodiimide to agarose having a carboxyalkyl derivative as a spacer. It is effective to use CH-cephalose (manufactured by Pharmacia) with a water-soluble carbodiimide, or commercially available activated CH-cephalose (manufactured by Pharmacia).

The obtained compound can be easily converted into argininal by hydrolyzing the acetal moiety by the method described above.

Examples of products obtained in this manner include phenylalanyl-argininal cephalose.

The present invention will be specifically described below with reference to Examples.

In addition, thin layer chromatography in the examples
All Rf values are Merck thin-layer plates, silica gel
Using 60F254 plate 0.25 mm, developing solvent n-butanol; butyl acetate; acetic acid; water = 4:2:1:
1 and measured.

The light intensity was measured using a mercury lamp at 578 nm.

The compounds were also confirmed by field desorption mass spectrometry.

Example 1 Preparation of D,L-argininal dibutyl acetal hydrochloride 25 g of leupeptin dibutyl acetal hydrochloride obtained by the method described in JP-A-55-37185 was mixed with 0.02 g of leupeptin dibutyl acetal hydrochloride.
The suspension was suspended in a 0.1 molar N-ethylmorpholine-hydrochloric acid buffer (PH8.0, 1000 ml) containing molar calcium chloride and 0.02 molar manganese chloride, and then 1.25 g of pronase E was added and stirred at 37°C for 72 hours. The reaction solution was passed through HP20 (500 ml), washed with water, eluted with 50% ethanol, and the Sakaguchi reagent positive fraction was collected to obtain 4.8 g of argininal dibutyl acetal hydrochloride.

[α] ²⁵ ₅₇₈ +0.5° (C=0, 8, acetic acid) mp164-166°C FD-MS; 289 (M+H) ⁺ Example 2 Preparation of D,L-argininal dibutyl acetal hydrochloride Example 1 Similarly, 500 mg of leupeptine dibutyl acetal hydrochloride was suspended in 0.1 molar N-ethylmorpholine-hydrochloric acid buffer (PH 9.0, 20 ml) containing 0.02 molar calcium chloride and 0.02 molar manganese chloride, and then Streptomyces Griseus・
25 mg of protease A was added and stirred at 37°C for 96 hours. The reaction solution was heated to HP20 (10
ml) treatment to obtain 47 mg of the title powder.

Example 3 L obtained by the method described in JP-A-55-37185
- Leucyl-D,L-argininal dibutyl acetal hydrochloride 500 mg in the same buffer as in Example 1.
After adding 25 mg of pronase E and stirring at 37°C for 72 hours, HP20 (10 ml) was treated in the same manner as in Example 1 to obtain 125 mg of the title powder.
I got it.

Reference Example 1 (1) Production of N-benzyloxycarbonyl-L-phenylalanyl-D,L-argininal dibutyl acetal hydrochloride 480 mg of N-benzyloxycarbonyl-L-phenylalanine N-hydroxysuccinimide ester and Example 1 D, L- obtained in
Argininal dibutyl acetal hydrochloride 288
mg was dissolved in 10 ml of 50% N,N'-dimethylformamide aqueous solution under ice-cooling, and then 70 μm of triethylamine was added and further stirred at room temperature for 8 hours. After the solvent was distilled off under reduced pressure, it was subjected to column chromatography using silica gel as a carrier to obtain a mixture of butanol; butyl acetate; acetic acid; water = 4:2:
After development at a ratio of 1:1 (v/v), the Sakaguchi reagent positive fraction with Rf0.7 was concentrated under reduced pressure to obtain 300 mg of the title compound.

[α] ²⁹ ₅₇₈ −8.3° (C = 0.4, AcOH) FD-MS; m/z = 570 (M + H) ⁺ (100%),
571 (32.8%), 572 (5.6%), 573 (1.6%), 462
(4.9%), 463 (1.2%) (2) Production of N-benzyloxycarbonyl-L-phenylalanyl-D,L-argininal hydrochloride Obtained N-benzyloxycarbonyl-L
- Phenylalanyl-D,L-argininal dibutyl acetal hydrochloride (100 mg) was mixed with a mixture of 1N hydrochloric acid (1 volume) and acetonitrile (2 volumes) to 37°C.
Hydrolysis was carried out at ℃ for 2 hours, 20 ml of water was added, and the mixture was neutralized with a weakly basic ion exchange resin Dowex WGR (OH type). The aqueous solution was freeze-dried to obtain 72 mg of the title powder.

Rf; 0.43 to 0.65 ^* (Development conditions described above) [α] ²⁶ ₅₇₈ +9.0 (C=0.5, MeOH) FD-MS; m/z440 (M+H) ⁺ (100%), 441
(43.1%), 442 (4.9%) *Reference Natural leupeptin Rf: 0.41-0.28 Leupeptin gives 3 spots under the above thin layer chromatography conditions.

(3) Preparation of L-phenylalanyl-D,L-argininal sepharose 130 mg of N-benzyloxycarbonyl-L-phenylalanyl-D,L-argininal dibutyl acetal hydrochloride obtained in (1) was dissolved in 10 ml of methanol. Catalytic reduction was performed for 4 hours using palladium black. After the reaction was completed, the palladium black was removed and the solvent was distilled off under reduced pressure to obtain 97 mg of powder of L-phenylalanyl-D,L-argininal dibutyl acetal hydrochloride. The obtained powder was suspended in a mixed solution of 30 ml of 0.1 mol-morpholinoethanesulfonic acid and 30 ml of dioxane, and then 20 ml of CH-Sephalose 4B (manufactured by Pharmacia) was added to adjust the pH to 5. Next, a total of 1 g of water-soluble carbodiimide was added over 1 hour while stirring on a 37°C water bath.
Stirred for 20 hours. The resin was transferred to a glass filter, thoroughly washed with water, and then immersed in 100 ml of 0.2M sodium citrate buffer, pH 2.5, at 40°C for 60 hours. Next, the buffer solution was removed and washing was performed with water to obtain 20 ml of the title resin.

Reference Example 2 Production of N-benzyloxycarbonyl-L-prolyl-L-argininal hydrochloride 415 mg of N-benzyloxycarbonyl-L-proline N-hydroxysuccinimide ester, 324 mg of D,L-argininal dibutyl acetal hydrochloride, and triethylamine 140μ was reacted in 10 ml of 50% N,N'-dimethylformamide aqueous solution in the same manner as in Example 2, and treated in the same manner as in Reference Example 1 (1) to obtain 145 mg of powder of the title compound.

Rf: 0.8 (same solvent as Example 2) [α] ²⁴ ₅₇₈ −60.3° (C = 0.3, MeOH) 100 mg of the obtained powder was treated in the same manner as in Reference Example 1 (2) to obtain 73 mg of the title powder. .

Rf; 0.53 to 0.41 [α] ²⁶ ₅₇₈ −77.7゜ (C=0.5, MeOH) FD-MS; m/z=390 (M+H) ⁺ (100%), 391
(26.7%), 372 (21.6%) Experimental Example 1 Protease adsorbent 10 prepared according to Reference Example 1
ml was packed into a column and sufficiently buffered with a 0.1M trishydroxymethane-hydrochloric acid buffer containing 0.1M sodium chloride adjusted to pH 7.5. Thereafter, a solution containing crude human urine kallikrein (specific activity 2.4 kallikrein units/mg protein) with a titer of 782 kallikrein units and adjusted to pH 7.5 was passed through the column, and human urine kallikrein was adsorbed onto the adsorbent. Next, after washing the column with the above buffer, the human urine kallikrein adsorbed on the column was desorbed using the above buffer containing 10mM leupeptin, and then the eluate was
Add 20mM equivalent of sodium borohydride,
The activity was measured and a titer of 564 kallikrein units (specific activity 54.2 kallikrein units/mg protein) was obtained. The recovery rate was 72%, and the specific activity increased about 23 times.

Claims (1)

[Claims] 1 formula [In the formula, R represents a lower alkyl group] Argininal di-lower alkyl acetal.