JPH0153666B2 - - Google Patents

Description

[Detailed description of the invention] The present invention relates to protease adsorbents.

Conventionally used methods for purifying protease include precipitation using a precipitant such as sulfate, magnesium sulfate, or pentonite, adsorption chromatography using silicic acid, silica gel, hydroxylapatite, etc., and protease purification. Positive or anion exchange chromatography, which utilizes the electrical dissociation properties of chromatography, or a combination thereof are known. In general, these methods can be expected to increase purity as the number of purification steps increases, but on the other hand, they have the drawback of decreasing yield.

Furthermore, since the purification process takes a long time, important problems remain to be solved, such as denaturation and inactivation of the enzyme due to autolysis by the protease itself and digestion by other coexisting proteases.

In recent years, an adsorbent in which a ligand with affinity for protease is bonded to an insoluble carrier has been developed as an excellent purification method for protease, and so-called affinity chromatography, which purifies enzymes using this adsorbent, is highly selective and effective. It has been attracting attention as an excellent method for purifying high-purity proteases because it can obtain the target product in high yield.

To give specific examples, (1) For urokinase, for example, a) a method using lysine or arginine or a derivative thereof as a ligand (Japanese Patent Publication No. 51-44193, Japanese Patent Publication No. 51-20596);
No., JP-A-51-95183, JP-A-51-35481~
No. 35483, b) Method using a urokinase inhibitor contained in placental tissue etc. as a ligand (Tokuko Sho
No. 51-20597, and c) a method using a benzguanidine derivative as a ligand (Japanese Patent Application Laid-open No. 53-88389).

(2) For kallikrein, for example, a method using a kallikrein inhibitor in bovine tissue or human serum as a ligand (Japanese Patent Application Laid-open No. 31017/1983) (3) For enzymes represented by trypsin, a) AP using arginine as a ligand; - Method using Sepharose (Japanese Unexamined Patent Publication No. 48-82083), b) Method using para-aminophenylguanidine, para-aminobenzamidine, or meta-aminobenzamidine as a ligand [Archive Biochemistry.
and Biophysics, Volume 154, Page 501 (1973)].

However, in conventional affinity chromatography, the affinity between the adsorbent and protease is weak, and for example, the adsorption capacity is significantly reduced by protease solutions containing high concentrations of salts, making it impossible to obtain the expected yield. .

In addition, attempts have been made to use protease inhibitors present in animal tissues as ligands, but it is difficult to obtain large quantities of these ligands, and this is far from practical.

Therefore, the present inventors synthesized and investigated various ligands in order to develop protease adsorbents using ligands that are available in large quantities. As a result, the following general formula () derived from L-leupeptin It has been found that an argininal derivative represented by the following formula (wherein A represents a phenylglycyl group or a phenylalanyl group) efficiently adsorbs protease and easily desorbs the target protease by adjusting the pH.

The present invention was completed based on the above findings.

Proteases that can be purified using the protease adsorbent of the present invention are not particularly limited, but trypsin, kallikrein, plasmin, thrombin, and the like are preferred.

The water-insoluble carrier used in the present invention is not particularly limited, and includes, for example, acidic ion exchange resins such as methacrylic acid-divinylbenzene copolymer, cellulose derivatives having acidic groups such as carboxymethyl cellulose, cepharose, agarose, dextran, etc. Examples include polymeric polysaccharides with carboxylic acid groups or sulfonic acid groups introduced,
High molecular weight polysaccharides are preferred.

In order to bind these insoluble carriers and L-argininal derivatives, for example, the following general formula () can be used. (In the formula, A represents a phenylglycyl group or a phenylalanyl group, and R represents a lower alkyl group). may be reacted with the above-mentioned activated insoluble carrier, and then the protecting group of the aldehyde group may be removed.

Here, R includes, for example, a butyl group.

There are known methods for activating the acidic groups in the insoluble carrier, such as a method in which cyanogen bromide is applied to agarose, a method in which water-soluble carbodiimide is applied to agarose having a carboxyalkyl derivative as a spacer, CH-Sepharose (Pharmacia) For example, a method in which a water-soluble carbodiimide is applied to

For example, when a water-soluble carbodiimide is used, the reaction between the L-argininal derivative with a protected aldehyde group and the activated insoluble carrier can be carried out in a solvent at pH 3 to 7, preferably 4 to 6, and at a temperature of 25 to 45
℃, preferably 35 to 40℃ for 10 to 30 hours, preferably 15 to 25 hours. Examples of the solvent used here include a 0 to 50% dimethylformamide solution or a dioxane solution consisting of a salt solution or a buffer solution capable of maintaining a pH of 3 to 7.

To remove the protecting group of the aldehyde group from the obtained reaction product, in a buffer solution having a pH of 1 to 4, preferably 2 to 3, at a temperature of 20 to 50°C, preferably 30 to 45°C, and 24 to 45°C.
Hydrolysis may be carried out for 120 hours, preferably 48 to 96 hours. Buffers used here include 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 sodium,
Examples include those with compositions such as potassium salts.

Various proteases can be obtained using the protease adsorbent obtained by the method described above, for example, in the following manner. Here, a method for purifying urinary kallikrein will be explained.

First, the adsorbent is packed into a column and the pH is adjusted to 5 to 10.
Preferably the pH is 6-8. In this case, the ionic strength is not particularly limited, but it is preferable to equilibrate in advance with a 0.025-0.5M buffer. Examples of the buffer used here include sodium phosphate-phosphoric acid, potassium phosphate-phosphoric acid, sodium acetate-acetic acid, and trishydroxyaminomethane-hydrochloric acid.

Thereafter, a crude urine kallikrein aqueous solution adjusted to pH 5 to 10, preferably 6 to 8, is passed through the adsorbent column to adsorb urine kallikrein onto the adsorbent. Next, the adsorbent adsorbed with urine kallikrein is washed with the above buffer solution, and then adjusted to pH 1 to 4, preferably pH 2 to 3.
A highly pure aqueous solution of urinary kallikrein can be obtained by eluting urinary kallikrein with an acid solution, a water-soluble salt solution, or a buffer solution. Examples of acid solutions used here include aqueous solutions of citric acid, tartaric acid, lactic acid, succinic acid, acetic acid, phosphoric acid, hydrochloric acid, and the like. Examples of water-soluble salt solutions include sodium chloride-hydrochloric acid aqueous solution, potassium chloride-hydrochloric acid aqueous solution, and sodium sulfate/sulfuric acid aqueous solution.
In addition, as buffer solutions, sodium phosphate-phosphoric acid aqueous solution, potassium phosphate-phosphoric acid aqueous solution, citric acid-
Sodium citrate aqueous solution, succinic acid-borax,
Lactic acid - sodium lactate, acetic acid - sodium acetate,
Examples include tartaric acid - sodium tartrate.

In addition to the column method, the method of the present invention may be performed in a batch method.

The aldehyde group-protected L-argininal derivative used in producing the protease adsorbent of the present invention can be produced, for example, by the following method. That is, L-leucyl-L-argininal with a protected aldehyde group is obtained by hydrolyzing an active ester of phenylglycine or phenylalanine with a protected amino group and L-leupeptin with a protected aldehyde group with thermolysin. (Refer to JP-A-55-37185, Example 1) in a solvent, followed by catalytic reduction to remove the protecting group of the amino group. Examples of the N-protecting group used here include protecting groups that can be removed by catalytic reduction, such as benzyloxycarbonyl group and p-methoxybenzyloxycarbonyl group. As the active ester, it is convenient and desirable to use N-hydroxysuccinimide ester, p-nitrophenyl ester, 2,4,5-trifluorophenyl ester, and the like. Further, the solvent used for the condensation may be any commonly used solvent. Further, the catalytic reduction can be carried out in a conventional manner using palladium black or the like in a solvent such as methanol.

The condensation of phenylglycine or phenylalanine with a protected amino group and L-leucyl-L-argininal with a protected aldehyde group can be carried out by using a carbodiimide such as dicyclohexylcarbodiimide or ethyldimethylaminopropylcarbodiimide alone. The method used or N
-method using in combination with hydroxybenztriazole, N-hydroxysuccinimide, N-hydroxy-5-norbornene-2,3-dicarboxamide, etc., or diphenylphosphoryl azide, 1-ethoxycarbonyl-2-ethoxy-1,2- It can also be carried out by a method commonly used for peptide bond formation, such as a method using a condensing agent such as dihydroquinoline.

Next, the present invention will be specifically explained using Examples and Experimental Examples.

Example 1 Preparation of L-phenylalanyl-L-leucyl-L-argininal cephalose (1) 240 mg of N-benzyloxycarbonyl-L-phenylalanine N-hydroxysuccinimide ester and the compound described in JP-A-55-37185 220 mg of L-leucyl-L-argininal dibutyl acetal hydrochloride obtained by the above method was dissolved in 10 ml of N,N'-dimethylformamide under ice cooling, and then 70 ml of triethylamine was added and the mixture was stirred at room temperature for an additional 20 hours. conduct. After distilling off the solvent under reduced pressure, it was subjected to column chromatography using silica gel as a carrier, developed with butanol; butyl acetate: acetic acid: water = 4:2:1:1 (v/v), and Rf0.8 The Sakaguchi reagent positive fraction was concentrated under reduced pressure to obtain 150 mg of N-benzyloxycarbonyl-L-phenylalanyl-L-leucyl-L-argininal dibutyl acetal hydrochloride.

[α] ²⁹ ₅₇₈ = -25.5゜ (C = 0.4ACOH) mp = 88 ~ 91℃ (decomposition) FD-MS; m/z = 683 (M + H) ⁺ (100%,
684 (39.3%) (2) 120 mg of the obtained powder was dissolved in 10 ml of methanol, and catalytic reduction was performed for 2 hours using palladium black. After the reaction was completed, palladium black was removed and the solvent was distilled off to obtain 70 mg of L-phenylalanyl-L-leucyl-L-argininal dibutyl acetal hydrochloride.

Rf; 0.3 (same as developing solvent in (1)) [α] ²⁷ ₅₇₈ = -11.2° (C = 0.7, ACOH) mp = 82 to 84°C FD-MS; m/z = 549 (M + H) ⁺ (100 %),
550 (38.6%), 551 (8.7%) (3) 50 mg of the obtained L-phenylalanyl-L-leucyl-L-argininal dibutyl acetal hydrochloride was added to 0.1 M morpholinoethanesulfonic acid.
Suspend in a mixture of 25 ml and dioxane, adjust the pH to 5, and then add CH-Sepharose 4B.
Add 15ml and stir for 1 hour until the entire amount
Add 500 mg of water-soluble carbodiimide and incubate at 37°C.
Stir for 20 hours. After washing the resin with water, it was immersed in 50 ml of 0.2 M sodium citrate buffer, pH 2.5, at 40°C for 60 hours, the buffer was removed, and washing with water was repeated to obtain 15 ml of the title resin.

Example 2 D,L-phenylglycyl-L-leucyl-L
- Preparation of argininal sepharose 210 mg of N-benzyloxycarbonyl-D,L-phenylglycine N-hydroxysuccinimide ester, 200 mg of L-leucyl-L-argininal dibutyl acetal hydrochloride, and 62μ of triethylamine were prepared as in Example 1. Similarly, react in 10 ml of N,N'-dimethylformamide,
Next, the same treatment as in Example 1 was carried out to obtain N-benzyloxycarbonyl-D,L-phenylglycyl-
130 mg of L-leucyl-L-argininal dibutyl acetal hydrochloride was obtained.

Rf; 0.8 (same solvent as Example 1 (1)) [α] ²⁶ ₅₇₈ = -16.3° (C = 0.5, ACOH) mp = 84 to 87°C FD-MS; m/z = 669 (M + H) ⁺ ( 100%),
670 (48.2%), 671 (12.7%), 595
(15.7%) Dissolve 110mg of the obtained powder in 10ml of methanol,
Catalytic reduction was carried out for 2 hours using palladium black.
After the reaction, palladium black was removed and the solvent was distilled off to give D,L-phenylglycyl-L-leucyl-
L-Argininal dibutyl acetal hydrochloride 60
I got mg.

Rf; 0.3 (same solvent as Example 1 (1)) [α] ²⁶ ₅₇₈ = -17.2° (C = 0.6, ACOH) mp = 70 to 75°C FD-MS; m/z = 535 (M + H) ⁺ ( 100%),
536 (23.9%), 537 (5.6%), 461 (4.8
%) Using 50 mg of the obtained D,L-phenylglycyl-L-leucyl-L-argininal dibutyl acetal hydrochloride, the same operation as in Example 1 was carried out to obtain 15 ml of the title resin.

Example 3 Preparation of D-phenylalanyl-L-leucyl-L-argininalcephalose N-benzyloxycarbonyl-D-phenylalanine N-hydroxysuccinimide ester
218 mg, L-leucyl-L-argininal dibutyl acetal hydrochloride 200 mg, and triethylamine 62μ were reacted in 10 ml of N,N'-dimethylformamide in the same manner as in Example 1,
The same treatment as in Example 1 was carried out to obtain hygroscopic powdery N-
125 mg of benzyloxycarbonyl-D-phenylalanyl-L-leucyl-L-argininal dibutylacetal hydrochloride was obtained.

Rf; 0.8 (same solvent as Example 1 (1)) [α] ²⁶ ₅₇₈ = -17.3° (C = 0.8, ACOH) FD-MS; m/z = 683 (M + H) ⁺ (100%),
684 (40.5%), 685 (10.6%), 609 (5.5
%) 105 mg of the obtained powder was subjected to catalytic reduction in the same manner as in Example 1 to obtain D-phenylalanyl- in the form of a hygroscopic powder.
55 mg of L-leucyl-L-argininal dibutyl acetal hydrochloride was obtained.

Rf; 0.3 (same solvent as Example 1) [α] ²⁶ ₅₇₈ = -34.6° (C = 0.8, ACOH) FD-MS; m/z = 549 (M + H) ⁺ (100%),
550 (28.9%), 551 (6.5%), 447 (3.8
%) Experimental Example 1 Protease adsorbent prepared according to Example 1 10
ml was packed into a column and sufficiently buffered with 50mM trishydroxymethane-hydrochloric acid buffer adjusted to pH 7.5. Crude human urine kallikrein (specific activity) with a titer of 500 kallikrein units was then adjusted to pH 7.5.
A solution containing 1.7 kallikrein units/mg protein was passed through the column, and human urine kallikrein was adsorbed onto the adsorbent. Next, after washing the column with the above buffer solution, human urine kallikrein adsorbed on the column was desorbed using 0.2M citric acid, and the titer was 424.4 kallikrein units (specific activity 13.3 kallikrein units/
mg protein) was obtained. The recovery rate was 85%, and the specific activity increased about 8 times.

Experimental Example 2 A column was filled with 10 ml of the protease adsorbent prepared in Example 2, and the pH was adjusted to 8.2. Sufficient buffering was performed with a 50mM trishydroxymethane-hydrochloric acid buffer (containing 0.02M calcium chloride). Then, the active tribucin content dissolved in 2 ml of the same buffer
10 mg of 75% commercially available trypsin was adsorbed. Next, the column was washed with the above buffer solution, and then trypsin adsorbed on the column was desorbed using 0.1M acetic acid. The activity was measured using benzoyl-L-arginine ethyl ester as a substrate. The purified trypsin had a purity of 95% and a recovery rate of 91%.

Claims (1)

[Claims] 1. General formula (In the formula, A represents a phenylglycyl group or a phenylalanyl group.) A protease adsorbent comprising an argininal derivative represented by the following formula bound to a water-insoluble carrier. 2 General formula (In the formula, A represents a phenylglycyl group or a phenylalanyl group, and R represents a lower alkyl group.) An argininal dialkyl acetal derivative represented by the following.