JP3202744B2 - Protein modification reagent containing polyethylene glycol derivative - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a high-purity polyethylene glycol derivative useful as a protein modification reagent or the like, and a novel method for producing a polyethylene glycol derivative.

[Prior art] Since a protein obtained from a microorganism or a heterologous animal and having a different structure from that derived from human is non-self for human,
It produces antibodies when administered to humans. It has been known that repeated administration of this substance causes anaphylactic shock, and seriously threatens life. One of the useful means for solving such a problem is
The following equation [Wherein, R represents an alkyl group, and n represents a positive integer that can be arbitrarily changed. A polyethylene glycol derivative (I) [hereinafter, simply referred to as compound (I)] is reacted with an amino group of a protein to lead to a polyethylene glycol-modified protein,
Methods for reducing the antigenicity of the protein are known.

[Problems to be solved by the invention]

However, when synthesis of compound (I) was attempted by the method described in the following literature, as a result of analysis by high-performance gel filtration chromatography,
The reaction product was found to be a mixture containing compound (I). That is, when trying to obtain a compound (I) having an average molecular weight of 10,000 using polyethylene glycol monoalkyl ether having an average molecular weight of 5,000 and cyanuric chloride,
According to the method described in JP-B-61-42558, 20 g of monomethoxy polyethylene glycol having a molecular weight of 5,000 was dissolved in 100 ml of anhydrous benzene containing 10 g of anhydrous sodium carbonate, and
After refluxing at 30 ° C. for 30 minutes, 2.4.6-trichloro-s-
365 mg of triazine was added and reacted at 80 ° C. for 24 hours under reflux. The reaction residue was removed by filtration, 300 ml of petroleum ether was added to generate a precipitate, and the precipitate was washed several times with petroleum ether, and 730 mg was obtained according to the method described in JP-A-62-115280. Of cyanuric chloride to 40 g of polyethylene glycol monomethyl ether (average molecular weight
5000), to a mixture of 200 ml of benzene, 20 g of anhydrous sodium carbonate and 10 g of molecular sieve 3A,
After reacting for 0 hours, the procedure of forming a precipitate with 400 ml of petroleum ether, dissolving this in benzene and reprecipitating with petroleum ether was repeated three times, and in each case, [Wherein R and n are as defined above], a mixture comprising several compounds having a molecular weight in the range of 5,000 to a high molecular weight region mainly comprising compound (III) (R = CH ₃ ) is obtained. (FIGS. 2 and 3). Also, according to the method described in Chemistry Letters, 773 (1980), 730 mg
Of cyanuric chloride was added to a mixture of 40 g of polyethylene glycol monomethyl ether (average molecular weight 5000), 200 ml of dry benzene, 20 g of anhydrous sodium carbonate and 10 g of molecular sieve 3A, and reacted at 80 ° C. for 44 hours.
The procedure of forming a precipitate with 400 ml of petroleum ether, dissolving this in benzene, and reprecipitating with petroleum ether was performed in 6 steps.
By repeating the process twice, a mixture comprising the compound (III) (R = CH ₃ ), the compound (I) (R = CH ₃ ) and the compound in the high molecular weight region was obtained (FIG. 4). Furthermore, Japanese Journal of Cancer Research, 77 , 1264 (198
According to the method described in 6), 60 g of 1.12 g of cyanuric chloride
Polyethylene glycol monomethyl ether, 200ml
Of anhydrous benzene, 20 g of anhydrous sodium carbonate and 20 g of molecular sieve 4A, reacted at 80 ° C. for 120 hours, distilled off benzene, dissolved in acetone, and then precipitated with petroleum ether three times. By repeating, a mixture containing various compounds mainly composed of compounds in the high molecular weight region was obtained (FIG. 5).

In this document, gel filtration chromatography using Sephadex G-100 as a carrier was carried out to obtain a purified product showing a single peak at a molecular weight of 10,000 in this chromatography, and a uniform compound (I) (R = CH ₃ ) is stated to have been obtained. However, in recent years, high-speed gel filtration chromatography, which has better separation ability than low-speed gel filtration chromatography using Sephadex G-100 or the like as a carrier, has been developed, making it possible to carry out separations that were previously impossible. , 56 , 1481 (1984)). That is, analysis by low-speed gel filtration chromatography using Sephadex G-100 or the like as a carrier is an insufficient method for purity analysis. fact,
According to this document, gel filtration chromatography using Sephadex G-100 as a carrier was performed, and after fractionation at the main peak showing a molecular weight of 10,000 in this document, the portion showing a single peak in this chromatography was subjected to high-speed gel filtration chromatography. As a result, the mixture was a mixture containing various compounds mainly composed of compounds in the high molecular weight region (FIG. 6).

Thus, when a protein is modified using a mixture containing various compounds having such a wide range of molecular weights, the modified protein becomes heterogeneous, and it is extremely difficult to always obtain a protein of constant quality. There are problems such as difficulty.

On the other hand, the target compound (I) can be efficiently purified from a mixture containing various compounds having such a wide range of molecular weights by means of industrial separation and purification (recrystallization, reprecipitation, ultrafiltration, etc.). The fact is that it has not been possible to achieve a high purity.

Therefore, an object of the present invention is to provide a highly pure compound (I).

Another object of the present invention is to provide a novel production method capable of producing compound (I).

[Means for solving the problem]

As a result of intensive studies, the present inventors have succeeded in obtaining compound (I) with extremely high purity, and have further studied to complete the present invention.

That is, the present invention uses the formula [Wherein, R represents an alkyl group, and n represents a positive integer that can be arbitrarily changed. The high purity polyethylene glycol derivative represented by the formula (1), that is, the high purity compound (I) is not provided. The present invention also relates to a compound (I) having a purity of at least 75% by high performance gel filtration chromatography.
Is to be provided. The present invention further relates to a compound (II) represented by the following formula: ROCH ₂ CH _{2 n} OH (II) wherein R and n are as defined above, and cyanuric chloride in the presence of a Group IIB metal compound. And a compound formed by reacting compound (II) with one molecule of each cyanuric chloride. [Wherein, R and n have the same meanings as described above] It is intended to provide a highly pure compound (I) having very few by-products such as the compound (III) represented by the following formula. Still another invention relates to a method for preparing a compound (II) from cyanuric chloride.
II A process for producing compound (I) by reacting in the presence of a Group B metal compound.

In each of the above formulas, the alkyl group represented by R preferably has 1 to 18 carbon atoms, for example, methyl, ethyl, n-
Propyl, isopropyl, n-butyl, isobutyl, se
c-butyl, n-pentyl, isopentyl, n-hexyl, isohexyl, n-octyl, n-nonyl, isononyl, n-decyl, isodecyl, n-undecyl, n-
Dodecyl, n-tridecyl, n-tetradecyl, n-pentadecyl, n-hexadecyl, n-heptadecyl, n
-Octadecyl, etc., among which those having 1 to 4 carbon atoms are particularly preferred. Most preferred is methyl.

In the above formulas, a positive integer represented by n is 10 to 700
Is particularly preferable, and 50 to 350 is particularly preferable.

The high-purity compound (I) can be produced, for example, as follows.

That is, it is produced by reacting compound (II) with cyanuric chloride in the presence of a Group IIB metal compound in a suitable solvent. The reaction time is generally 1-300 hours, and the reaction temperature is generally 50-140 ° C, preferably about 70-110 ° C. The solvent used here may be any solvent as long as it is inert to the reagent used in the present invention, for example, aromatic hydrocarbon solvents such as benzene and toluene and halogenated hydrocarbon solvents such as 1,2-dichloroethane. Is mentioned.

As the group IIB metal compound to be coexisted, a group IIB metal oxide such as zinc oxide, cadmium oxide and mercury oxide is preferably used. It is also desirable to take measures to remove water from the reaction system. For example, molecular sieves are used.

The purity of the high-purity polyethylene glycol derivative of the present invention was measured using high-speed gel filtration chromatography under the following conditions.

(Conditions) Column: TSK-gel G3000SW φ7.5 mm x 60 cm (Tosoh Corporation) Eluent: ethanol / [0.01 M phosphate buffer (pH 7) +
0.2 M saline] = 1/19 Flow rate: 0.7 ml / min Detection wavelength: 254 nm The analysis shown in FIGS.

As shown in FIG. 1, the polyethylene glycol derivative according to the present invention is much higher in purity than any obtained by the prior art (FIGS. 2 to 6), and the purity by high performance gel filtration chromatography is 75%. %.

Here, the purity by high performance gel filtration chromatography is the area percentage of the peak formed by compound (I) with respect to the area formed by the high performance gel filtration chromatography chart. Usually, data processing is performed using a data processor such as, for example, Chromatopack CR-6A (Shimadzu).

Since the reaction product obtained by the above method already contains the compound (I) in high purity, it can be used as it is, for example, as a protein modification reagent, but if it is desired to obtain a compound (I) with higher purity. For example, industrial separation and purification means such as recrystallization, reprecipitation, and ultrafiltration are preferably applied.

A protein such as L
-A modified asparaginase obtained by modifying an amino group in an asparaginase molecule is a conventional asparaginase (JP-B-61-42558).
Bulletin, Chemistry Letters, 773 (1980), Japanese Journal of Cancer Research, 77 , 126
4 (1986)]. Wherein R and n are as defined above, and x is 1 to
Shows 92. ] It is a modified asparaginase containing the modified asparaginase represented by the following formula with high purity.

Modification of a protein with the highly pure compound (I) of the present invention can be performed by a method similar to the conventional method. That is,
The reaction may be performed in a buffer solution of boric acid, phosphoric acid, acetic acid, or the like having a pH of 8 to 10 at 0 ° C. to room temperature for 1 to 72 hours.
Compound (I) is usually prepared by adding 1 to the number of amino groups of the protein.
It is used in an amount of up to 20 times the molar amount, but is changed depending on the desired degree of modification. The reaction solution may be subjected to ultrafiltration, ion exchange chromatography, affinity chromatography, gel filtration chromatography, hydrophobic chromatography, reverse-phase high-performance liquid chromatography, electrophoresis,
Purification by commonly used means such as dialysis can yield the desired modified protein.

〔Example〕

Hereinafter, the present invention will be described more specifically with reference to Examples and Reference Examples, but these do not limit the present invention.

Example 1 Production of 2,4-bis-methoxypolyethylene glycol-6-chloro-s-triazine 110 g of polyethylene glycol monomethyl ether having an average molecular weight of 5000 and 25 g of molecular sieve 4A were refluxed in benzene 0.5 for 6 hours at 80 ° C. . After allowing to cool to room temperature, 50 g of zinc oxide and 1.85 g of cyanuric chloride were added, and the mixture was heated under reflux at 80 ° C. for 53 hours. After cooling to room temperature, benzene
After adding 0.5 and filtering, the filtrate was concentrated to dryness to obtain 108 g of the title compound.

Physical property values ・ High-speed gel filtration chromatography Column: TSK-gel G3000SW φ7.5mm × 60cm (Tosoh Corporation) Eluent: ethanol / [0.01M phosphate buffer (pH7) +
0.2 M saline] = 1/19 Flow rate: 0.7 ml / min Detection wavelength: 254 nm Retention time: 20,967 min Purity: 91.5% (area percentage) Chart: As shown in FIG. 1 ・ Silica gel thin layer chromatography Silica gel: Kieselgel 60 (Merck Development solvent: methylene chloride / methanol = 15/2 Rf value: 0.5 Example 2 Production of 2,4-bis-methoxypolyethylene glycol-6-chloro-s-triazine Polyethylene glycol monomethyl ether having an average molecular weight of 5000 11 g was heated and refluxed at 80 ° C. for 4 hours in 50 ml of benzene, allowed to cool to room temperature, and then molecular sieve 4A2.
5 g was added, and the mixture was heated under reflux at 80 ° C. for 4 hours. After allowing to cool to room temperature, 13.5 g of mercury oxide (yellow) and 184 mg of cyanuric chloride were added, and the mixture was heated under reflux at 80 ° C. for 27 hours. After cooling to room temperature, 50 ml of benzene was added and the mixture was centrifuged. The benzene was distilled off and dried, and 9.79 g of the title compound (purity 78.2% in high-speed gel filtration chromatography using TSKG3000SW)
I got

Example 3 Production of 2,4-bis-methoxypolyethylene glycol-6-chloro-s-triazine 11 g of polyethylene glycol monomethyl ether having an average molecular weight of 5,000 was heated under reflux at 80 ° C. for 4 hours in 50 ml of benzene at room temperature. After cooling down to molecular sieve 4A2.
5 g was added, and the mixture was heated under reflux at 80 ° C. for 4 hours. After allowing to cool to room temperature, 7.8 g of cadmium oxide and 184 mg of cyanuric chloride were added, and the mixture was heated under reflux at 80 ° C. for 34 hours. After cooling to room temperature, 50 ml of benzene was added and the mixture was centrifuged. The benzene was distilled off and dried, and 9.35 g of the title compound (purity 7 in high-speed gel filtration chromatography using TSK gel G3000SW) was added.
8.5%).

Example 4 Gel filtration purification of 2,4-bis-methoxypolyethylene glycol-6-chloro-s-triazine 800 mg of the reaction product obtained in Example 1 was dissolved in 20 ml of water, and TSK gel G3000SW (21.5 mmφ × 600 mm, (Eluted with water) and subjected to gel filtration purification. The fraction containing the target compound was lyophilized, and 324 mg (TSK
gel 93000% in high performance gel filtration chromatography using G3000SW).

Physical properties Melting point: 56-58 ° C Elemental analysis: C 53.65 (54.17), H 8.99 (9.05), N 0.46
(0.41), Cl 0.33 (0.35) The values in parentheses are theoretical values when the molecular weight of polyethylene glycol monomethyl ether is 5004.

¹³ C-NMR (CDCl ₃ , complete decoupling) δ: 58.6, 67.9, 68.2, 69.7, 69.9, 70.1, 70.3, 70.6, 71.5,
171.6, 172.1 Reference Example 1 Production of Polyethylene Glycol-Modified Asparaginase 976 mg of asparaginase was dissolved in 195 ml of a 0.1 M borate buffer solution adjusted to pH 10.0, and the high-purity 2,4 obtained in Example 1 was dissolved.
29.0 g of -bis-methoxypolyethylene glycol-6-chloro-s-triazine were added at 4 ° C. in three portions over 60 minutes. After stirring at 4 ° C. for 22 hours, water was added to adjust to 4.5, neutralized with a 5% acetic acid aqueous solution, and then subjected to ultrafiltration to obtain 6.5 as an aqueous solution having a protein content of 0.14 mg / ml.

Physical property column: High performance gel filtration chromatography (TSK-gel
G4000PW _XL φ7.8mm × 30cm, 2 columns, guard column TSKguard column PW _XL φ6.0mm × 4cm) (manufactured by Tosoh Corporation) Eluent: 0.2 M saline Flow rate: 0.6 ml / min Detection wavelength: 254 nm Retention time: 24.3 min Effect of the Invention] The high-purity compound (I) of the present invention is not only modified for the purpose of decreasing the antigenicity of the protein, but also modified for the purpose of delaying in vivo clearance, improving delivery to tissues, and the like. Useful for modification. By modifying the protein using the high-purity compound (I), a modified protein having satisfactory quality as a pharmaceutical composition, for example, a modified asparaginase can be easily obtained.

Further, according to the production method of the present invention, the compound (I) having a higher purity as described above can be produced as compared with the conventional method.

[Brief description of the drawings]

FIG. 1: High performance gel filtration chromatography of high purity polyethylene glycol derivatives obtained by the method of Example 1. FIG. 2: High-performance gel filtration chromatography of a polyethylene glycol derivative obtained according to the method described in JP-B-61-42558. FIG. 3: High-performance gel filtration chromatography of a polyethylene glycol derivative obtained according to the method described in JP-A-62-115280. Figure 4: High performance gel filtration chromatography of a polyethylene glycol derivative obtained according to the method described in Chemistry Letters, 773 (1980). Figure 5: High performance gel filtration chromatography of polyethylene glycol derivatives obtained according to the method described in Japanese Journal of Cancer Research 77 1264 (1986). FIG. 6: High-speed gel filtration chromatography of a polyethylene glycol derivative obtained by synthesis according to the method described in Japanese Journal of Cancer Research 77 1264 (1986) and then purifying by gel filtration chromatography according to the literature.

──────────────────────────────────────────────────続 き Continuation of the front page (72) Inventor Hiroo Maeda 3-1-198 Kasuganaka, Konohana-ku, Osaka-shi, Sumitomo Pharmaceutical Co., Ltd. (56) References JP-A-62-115280 (JP, A) JP-A-63-126900 (JP, A) JP-A-1-60375 (JP, A) JP-A-3-95200 (JP, A) Jpn. J. Cancer Res. , Vol. 77, p. 1264-1270 (1986) Cancer and Chemotherapy, Vol. 11 (10), p. 227-1235 (1984) (58) Fields investigated (Int. Cl. ⁷ , DB name) C07K 17/08 C08G 65/321 BIOSIS (DIALOG) CA (STN) WPIDS (STN)

Claims (4)

(57) [Claims] (1) Expression [Wherein, R represents an alkyl group having 1 to 18 carbon atoms, and n represents 10
Represents an integer of ~ 700. ] A protein-modifying reagent containing a polyethylene glycol derivative represented by the above formula, wherein the purity of the derivative in the protein-modified reagent by high-performance gel filtration chromatography is 75% or more. 2. The protein-modifying reagent according to claim 1, wherein the purity of the derivative is 91.5% or more. 3. The protein-modifying agent according to claim 1, wherein in the formula (I), n is an integer of 50 to 350. 4. The protein modifying reagent according to claim 1, wherein in the formula (I), R is an alkyl group having 1 to 4 carbon atoms.