Method for producing polyethylene glycol derivative having carboxyl group at terminal

Abstract

To provide a method for producing a PEG derivative by removing a reduced form of an N-oxyl compound in a terminal carboxyl group-containing polyethylene glycol (hereinafter, PEG) derivative to obtain a PEG derivative having little residual amount of a reduced form of an N-oxyl compound.SOLUTION: There is provided a method for producing a PEG derivative having a carboxyl group at the terminal, comprising the following steps. Step 1: a PEG compound is subjected to an oxidation reaction using an N-oxyl compound to obtain a PEG derivative having a carboxyl group at the terminal, followed by adding a reducing agent to quench the oxidation reaction. Step 2: a mixed aqueous solution adjusted to a pH of 3.0 or less and an organic solvent selected from aromatic hydrocarbons or the like are mixed under the condition of 0°C or higher, followed by separating into an organic layer and an aqueous layer. Step 3: the organic layer obtained in Step 2 and an aqueous solution adjusted to a pH of 3.0 or less are mixed under the condition of 0°C or higher to separate into an organic layer and an aqueous layer. Step 4: the organic layer obtained in Step 3 is concentrated to recover a PEG derivative having a carboxyl group.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for producing a polyethylene glycol derivative having a terminal carboxyl group. [Background technology]

[0002] When drugs using bio-related substances such as hormones, cytokines, and enzymes are administered to the body, they are usually quickly excreted from the body through glomerular filtration in the kidneys and uptake by macrophages in the liver and spleen. As a result, their half-life in the blood is short, and in some cases they are unable to achieve sufficient pharmacological effects. To solve this problem, drugs have been developed in which bio-related substances are chemically modified with water-soluble polymers such as polyethylene glycol or albumin. Chemically modified bio-related substances can have their half-life in the blood extended by increasing their molecular weight or forming a hydration layer.

[0003] In particular, polyethylene glycol derivatives having a carboxyl group at the end are particularly important materials because they can be used as raw materials for converting to activated esters that form stable amide bonds with lysine residues or primary amino groups at the N-terminus of proteins, or other active groups. PEGylated preparations using polyethylene glycol derivatives having a carboxyl group as raw materials are already on the market, such as SOMAVERT (registered trademark): PEGVISOMANT (Pfizer), Mircera (registered trademark): Methoxy Polyethylene glycol-epoetin beta (F Hoffmann-La Roche), and Macugen (registered trademark): Pegaptanib (Eyetech). In addition, clinical trials of many other PEGylated preparations have been conducted, proving their usefulness.

[0004] Examples of methods for producing a polyethylene glycol derivative having a terminal carboxyl group include the following methods. A method in which the terminal hydroxyl group of a polyethylene glycol compound is bonded to an acrylic ester compound by the Michael reaction, and then the ester is hydrolyzed. A method in which an orthoester compound having a terminal hydroxyl group of polyethylene glycol and a leaving group are bonded by the Williamson reaction, followed by hydrolysis. A method for oxidizing the terminal hydroxyl groups of polyethylene glycol using an N-oxyl compound or its derivative

[0005] By the oxidation reaction of the terminal hydroxyl group of polyethylene glycol using this N-oxyl compound, a polyethylene glycol derivative with high carboxylic acid purity is obtained, but after quenching the reaction, the reduced form of the N-oxyl compound is generated as an impurity. From the viewpoint of the performance and safety of pharmaceuticals, polyethylene glycol derivatives used as pharmaceutical raw materials or pharmaceutical additives are required to have a low impurity content. Impurities derived from low molecular weight compounds used to derivatize the polyethylene glycol terminals cause unexpected side reactions when chemically modifying biologically relevant substances, resulting in the generation of pharmaceutical impurities and reducing the purity of the pharmaceutical. For this reason, it is desirable to have a low content of the reduced form of the above-mentioned N-oxyl compound.

[0006] In this regard, there are few reports on the removal of the reductant of N-oxyl compounds in the synthesis of polyethylene glycol derivatives having a carboxyl group by utilizing an oxidation reaction using an N-oxyl compound.

[0007] For example, Patent Document 1 reports that a terminal hydroxyl group of a polyethylene glycol compound is converted to a carboxylic acid in an aqueous solution using TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl), and then ethanol is added to quench the reaction. The aqueous layer is then washed with ethyl acetate, resulting in the migration of colored impurities, which are assumed to be TEMPO reductants, into the ethyl acetate layer.

[0008] Furthermore, Non-Patent Document 1 reports that after the terminal hydroxyl group of polyethylene glycol is converted to a carboxylic acid in an aqueous solution using TEMPO, sodium sulfite is added to quench the reaction, and the aqueous layer is washed with hexane to remove TEMPO residues.

[0009] Furthermore, Non-Patent Document 2 reports that the terminal hydroxyl group of polyethylene glycol is converted to a carboxylic acid in an aqueous solution using TEMPO, and then low molecular weight impurities used in the reaction are removed by dialysis.

[0010] In addition, Patent Document 2 reports that low molecular weight impurities are removed by adding hexane, a poor solvent, to a polyethylene glycol derivative dissolved in heated ethyl acetate and reprecipitation. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] JP 2022-162784 A [Patent Document 2] JP 2004-197077 A [Non-patent literature]

[0012] [Non-Patent Document 1] Synlett 2018, 29(5), 556-559 [Non-Patent Document 2] ACS Macro Letters 2019, 8(7), 826-834 Summary of the Invention [Problem to be solved by the invention]

[0013] Patent Document 1 and Non-Patent Document 1 do not disclose the remaining amount or removal efficiency of TEMPO residue, and it is unclear whether the TEMPO reduced product can be removed by washing with an organic solvent or by the crystallization described in Patent Document 2. In addition, in Non-Patent Document 1, purification is finally performed by silica gel column chromatography, but this requires the use of a large amount of solvent and a large amount of silica gel becomes waste, making this a production method that is problematic for industrial use. Furthermore, the dialysis described in Non-Patent Document 2 takes a long time of two days and requires freeze-drying after dialysis, making the work complicated, making this a production method that is problematic for industrial use.

[0014] Thus, although the oxidation reaction using an N-oxyl compound is useful for obtaining a polyethylene glycol derivative having a carboxyl group at its terminal with a high carboxylation rate, a simple method for separating the reduced product of the N-oxyl compound produced by quenching after the reaction from the polyethylene glycol derivative without the need for purification using a column or dialysis, which is relatively complicated from an industrial standpoint, has not yet been established, and thus there remains a problem.

[0015] In view of the above problems, the present invention has an object to provide a production method capable of removing the reduced form of an N-oxyl compound contained in a polyethylene glycol derivative having a terminal carboxyl group by an industrially simple method, thereby obtaining a polyethylene glycol derivative having a small amount of the reduced form of an N-oxyl compound remaining. [Means for solving the problem]

[0016] As a result of intensive research by the present inventors to solve the above problems, they have found that the reduced form of the N-oxyl compound can be removed by adjusting the pH of a mixed aqueous solution of a polyethylene glycol derivative having a carboxyl group at its terminal and a reduced form of an N-oxyl compound to a specific value or lower, extracting the mixed aqueous solution with an aromatic hydrocarbon, an alkyl carboxylate or a halogenated hydrocarbon, and then washing the organic layer containing the mixture of a polyethylene glycol derivative having a carboxyl group at its terminal and a reduced form of the N-oxyl compound with an aqueous solution adjusted to a specific value or lower, thereby obtaining a polyethylene glycol derivative having a low remaining amount of the reduced form of the N-oxyl compound.

[0017] That is, the present invention is as follows. [1] A method for producing a polyethylene glycol derivative having a carboxyl group at its terminal, comprising the following steps 1, 2, 3 and 4: Step 1: A process of subjecting a polyethylene glycol compound having a molecular weight of 1,000 to 80,000 and having a hydroxyl group or an aldehyde group at its terminal to an oxidation reaction using an N-oxyl compound to obtain a polyethylene glycol derivative having a carboxyl group at its terminal, and then quenching the oxidation reaction by adding a reducing agent to obtain a mixed aqueous solution containing the reduced product of the N-oxyl compound and the polyethylene glycol derivative having a carboxyl group at its terminal. Step 2: An extraction step in which the mixed aqueous solution adjusted to a pH of 3.0 or less is mixed with an organic solvent containing at least one selected from the group consisting of aromatic hydrocarbons, carboxylic acid alkyl esters, and halogenated hydrocarbons at a temperature of 0° C. or higher, and the solution is separated into an organic layer and an aqueous layer. Step 3: A washing step in which the organic layer obtained in step 2 is mixed with an aqueous solution adjusted to a pH of 3.0 or less at a temperature of 0° C. or higher to separate the organic layer from the aqueous layer. Step 4: A recovery step of recovering a polyethylene glycol derivative having a carboxyl group at its terminal by concentrating the organic layer obtained in the step 3.

[0018] [2] The method according to [1], wherein the polyethylene glycol derivative having a carboxyl group at its terminal is any one of linear polyethylene glycol, branched polyethylene glycol, and multi-arm polyethylene glycol.

[0019] [3] The method according to [1] or [2], characterized in that the average molecular weight of the polyethylene glycol derivative having a carboxyl group at its terminal is 1,000 to 80,000.

[0020] [4] The method according to any one of [1] to [3], wherein the acid used to adjust the pH of the mixed aqueous solution in step 2 and the aqueous solution in step 3 is an acid having an octanol / water partition coefficient of 0.5 or less and a pKa of 6 or less. Effect of the Invention

[0021] According to the present invention, it is possible to remove the reduced form of N-oxyl compounds contained in a polyethylene glycol derivative having a carboxyl group at its terminal, thereby obtaining a polyethylene glycol derivative having a low content of reduced form of N-oxyl compounds. This production method makes it possible to obtain a polyethylene glycol derivative having a carboxyl group at its terminal and having a low content of reduced form of N-oxyl compounds, without carrying out purification such as silica gel column purification or dialysis, which are unsuitable for industrial production. [Brief description of the drawings]

[0022] [Figure 1] NMR chart obtained before purification in Example 1 is shown. [Diagram 2] NMR chart obtained after purification in Example 1 is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The present invention relates to a method for producing a polyethylene glycol derivative having a terminal carboxyl group, comprising the above-mentioned steps 1, 2, 3 and 4.

[0024] (Raw material: polyethylene glycol compound having a terminal hydroxyl group or aldehyde group) The polyethylene glycol compound is preferably represented by formula (1), formula (2) or formula (3).

[0025] [ka]

[0026] Here, POLY represents a linear polyethylene glycol moiety. 2 CH 2 O-(CH 2 CH 2 O) n -CH 2 CH 2 -It is expressed by the formula: F 1 represents a hydroxyl group or an aldehyde group.

[0027] F 2 represents an alkyl group having 1 to 24 carbon atoms (preferably 1 to 4 carbon atoms), an amino group, a protecting group for an amino group, a hydroxyl group or a protecting group for a hydroxyl group, or an aldehyde group. Examples of the protecting group for an amino group include a t-butoxycarbonyl protecting group, a 9-fluorenylmethyloxycarbonyl protecting group, an allyloxycarbonyl protecting group, a benzyloxycarbonyl protecting group, a 2,2,2-trichloroethoxycarbonyl protecting group, a mesyl protecting group, a tosyl protecting group, a nosyl protecting group, a triflyl protecting group, a trimethylsilyl protecting group, a triethylsilyl protecting group, a triisopropylsilyl protecting group, a t-butyldimethylsilyl protecting group, a t-butyldiphenylsilyl protecting group, an acetyl protecting group, a benzoyl protecting group, a pivaloyl protecting group, a trityl protecting group, a monomethyltrityl protecting group, and a dimethyltrityl protecting group. Examples of the protecting group for a hydroxyl group include a methyl protecting group, a t-butyl protecting group, a benzyl protecting group, an alkoxymethyl protecting group, a 2-tetrahydropyranyl protecting group, a benzyloxymethyl protecting group, a mesyl protecting group, a tosyl protecting group, a nosyl protecting group, a triflyl protecting group, a trimethylsilyl protecting group, a triethylsilyl protecting group, a triisopropylsilyl protecting group, a t-butyldimethylsilyl protecting group, a t-butyldiphenylsilyl protecting group, an acetyl protecting group, a benzoyl protecting group, a pivaloyl protecting group, a trityl protecting group, a monomethyltrityl protecting group, and a dimethyltrityl protecting group.

[0028] L 1 and L 4 each independently represents an alkyl group having 1 to 10 carbon atoms, provided that F 2 If is an alkyl group, L 4 represents a single bond. L 2 and L 3 respectively represent an ether bond, an ester bond, an amide bond, a urethane bond or a carbonate bond, provided that F 2 If is an alkyl group, L 3 represents an ether bond.

[0029] [ka]

[0030] In formula (2), POLY 1 represents a linear polyethylene glycol portion, specifically, -CH 2 CH 2 O-(CH 2 CH 2 O) n -CH 2 CH 2 -It is expressed by the formula: POLY 2 represents a similar linear polyethylene glycol portion or a single bond, provided that the average molecular weights of both linear polyethylene glycols may be the same or different.

[0031] E is L1 , L 5 and L 9 is a methine group or a nitrogen atom bonded to L 1 , L 4 , L 5 , L 9 and L 12 each independently represents an alkyl group having 1 to 10 carbon atoms or a single bond, provided that POLY 2 When is a single bond, L 9 and L 12 represents an alkyl group having 1 to 10 carbon atoms or a single bond. L 2 , L 3 , L 6 , L 10 and L 11 each independently represents an ether bond, an ester bond, an amide bond, a urethane bond or a carbonate bond, provided that POLY 2 When is a single bond, L 10 is an ether bond, ester bond, amide bond, urethane bond, carbonate bond or single bond, L 11 is a single bond.

[0032] F 1 represents an alkyl group having 1 to 24 carbon atoms (preferably 1 to 4 carbon atoms), an amino group, a protecting group for an amino group, a hydroxyl group or a protecting group for a hydroxyl group, or an aldehyde group. Examples of the protecting group for an amino group include a t-butoxycarbonyl protecting group, a 9-fluorenylmethyloxycarbonyl protecting group, an allyloxycarbonyl protecting group, a benzyloxycarbonyl protecting group, a 2,2,2-trichloroethoxycarbonyl protecting group, a mesyl protecting group, a tosyl protecting group, a nosyl protecting group, a triflyl protecting group, a trimethylsilyl protecting group, a triethylsilyl protecting group, a triisopropylsilyl protecting group, a t-butyldimethylsilyl protecting group, a t-butyldiphenylsilyl protecting group, an acetyl protecting group, a benzoyl protecting group, a pivaloyl protecting group, a trityl protecting group, a monomethyltrityl protecting group, and a dimethyltrityl protecting group. Examples of the hydroxyl-protecting group include a methyl protecting group, a t-butyl protecting group, a benzyl protecting group, an alkoxymethyl protecting group, a 2-tetrahydropyranyl protecting group, a benzyloxymethyl protecting group, a mesyl protecting group, a tosyl protecting group, a nosyl protecting group, a triflyl protecting group, a trimethylsilyl protecting group, a triethylsilyl protecting group, a triisopropylsilyl protecting group, a t-butyldimethylsilyl protecting group, a t-butyldiphenylsilyl protecting group, an acetyl protecting group, a benzoyl protecting group, a pivaloyl protecting group, a trityl protecting group, a monomethyltrityl protecting group, and a dimethyltrityl protecting group. F 2 represents a hydroxyl group or an aldehyde group.

[0033] [ka]

[0034] In formula (3), X represents a residue obtained by removing a hydroxyl group from a compound selected from the group consisting of glycerol, trimethylolpropane, pentaerythritol, xylitol, dipentaerythritol, hexaglycerol, dicylitol, disucrose, and tripentaerythritol. POLY represents a linear polyethylene glycol portion, specifically, -CH 2 CH 2 O-(CH 2 CH 2 O) n -CH 2 CH 2 -It is expressed by the formula: L1 represents an ether bond. L 2 represents an ether bond, an ester bond, an amide bond, a urethane bond or a carbonate bond. L 3 represents an alkyl group having 1 to 10 carbon atoms (preferably 2 to 6 carbon atoms). F represents a hydroxyl group or an aldehyde group; m is 3 to 8 depending on the structure of X.

[0035] In a particularly preferred embodiment, F in formula (1) 1 is a hydroxyl group, and L 1 is an alkyl group having 2 carbon atoms, and L 2 is an ether bond, F 2 , L 3 , L 4 is a methyl group, an ether bond, a single bond, or a hydroxyl group, an ether bond, or an alkyl group having 2 carbon atoms. In a particularly preferred embodiment, E in formula (2) is a methine group, and L 1 is a methylene group having one carbon atom, and L 2 is an ether bond, and L 3 is an ether bond, and L 4 is a single bond, F 1 is a methyl group, and L 5 is a single bond, L 6 is an ether bond, and L 9 is a methylene group having one carbon atom, and L 10 , POLY 2 , L 11 and L 12 is a single bond, F 2 In a particularly preferred embodiment, X in formula (3) is a pentaerythritol residue, and L 2 is an ether bond, and L 3 is an alkyl group having two carbon atoms, and F is a hydroxyl group.

[0036] (The polyethylene glycol derivative having a carboxyl group at its terminal) The polyethylene glycol derivative having a carboxyl group at its terminal is preferably any one of linear polyethylene glycol, branched polyethylene glycol, and multi-arm polyethylene glycol represented by formula (4), formula (5), or formula (6).

[0037] [ka]

[0038] In formula (4), POLY represents a linear polyethylene glycol portion, specifically, -CH 2 CH 2 O-(CH 2 CH 2 O) n -CH 2 CH 2 -It is expressed by the formula: F 1 represents a carboxyl group. F 2represents an alkyl group having 1 to 24 carbon atoms (preferably 1 to 4 carbon atoms), a carboxyl group, an amino group, a protecting group for an amino group, or a protecting group for a hydroxyl group. Preferred examples of the protecting group for an amino group include a t-butoxycarbonyl protecting group, a 9-fluorenylmethyloxycarbonyl protecting group, an allyloxycarbonyl protecting group, a benzyloxycarbonyl protecting group, a 2,2,2-trichloroethoxycarbonyl protecting group, a mesyl protecting group, a tosyl protecting group, a nosyl protecting group, a triflyl protecting group, a trimethylsilyl protecting group, a triethylsilyl protecting group, a triisopropylsilyl protecting group, a t-butyldimethylsilyl protecting group, a t-butyldiphenylsilyl protecting group, an acetyl protecting group, a benzoyl protecting group, a pivaloyl protecting group, a trityl protecting group, a monomethyltrityl protecting group, and a dimethyltrityl protecting group. As the protecting group for a hydroxyl group, a methyl protecting group, a t-butyl protecting group, a benzyl protecting group, an alkoxymethyl protecting group, a 2-tetrahydropyranyl protecting group, a benzyloxymethyl protecting group, a mesyl protecting group, a tosyl protecting group, a nosyl protecting group, a triflyl protecting group, a trimethylsilyl protecting group, a triethylsilyl protecting group, a triisopropylsilyl protecting group, a t-butyldimethylsilyl protecting group, a t-butyldiphenylsilyl protecting group, an acetyl protecting group, a benzoyl protecting group, a pivaloyl protecting group, a trityl protecting group, a monomethyltrityl protecting group, or a dimethyltrityl protecting group is preferred.

[0039] L 1 and L 4 each independently represents an alkyl group having 1 to 10 carbon atoms, provided that F 2 If is an alkyl group, L 4 represents a single bond. L 2 and L 3 each independently represents an ether bond, an ester bond, an amide bond, a urethane bond, or a carbonate bond, provided that F 2 If is an alkyl group, L 3 is an ether bond.

[0040] [ka]

[0041] In formula (2), POLY 1 represents a linear polyethylene glycol portion, specifically, -CH 2 CH 2 O-(CH 2 CH 2 O) n -CH 2 CH 2 -It is expressed by the formula: POLY 2 represents the same linear polyethylene glycol moiety or a single bond, provided that the average molecular weights of both linear polyethylene glycols may be the same or different.

[0042] E is L 1 , L 5 and L 9 is a methine group or a nitrogen atom bonded to

[0043] L 1 , L 4 , L 5 , L 9 and L 12 each independently represents an alkyl group having 1 to 10 carbon atoms or a single bond (preferably L 1 , L 5 , L 9 is a carbon number of 1 to 4 or a single bond, L 4 and L 12 (has 1 to 5 carbon atoms or a single bond), but POLY 2 When is a single bond, L 9 and L 12 each independently represents an alkyl group having 1 to 10 carbon atoms or a single bond. L 2 , L 3 , L 6 , L 10 and L 11 each independently represents an ether bond, an ester bond, an amide bond, a urethane bond or a carbonate bond, provided that when POLY2 is a single bond, L 10 represents an ether bond, an ester bond, an amide bond, a urethane bond, a carbonate bond or a single bond, and L 11 represents a single bond.

[0044] F 1 represents an alkyl group having 1 to 24 carbon atoms (preferably 1 to 4 carbon atoms), a carboxyl group, an amino group, a protecting group for an amino group, or a protecting group for a hydroxyl group. Examples of the protecting group for an amino group include a t-butoxycarbonyl protecting group, a 9-fluorenylmethyloxycarbonyl protecting group, an allyloxycarbonyl protecting group, a benzyloxycarbonyl protecting group, a 2,2,2-trichloroethoxycarbonyl protecting group, a mesyl protecting group, a tosyl protecting group, a nosyl protecting group, a triflyl protecting group, a trimethylsilyl protecting group, a triethylsilyl protecting group, a triisopropylsilyl protecting group, a t-butyldimethylsilyl protecting group, a t-butyldiphenylsilyl protecting group, an acetyl protecting group, a benzoyl protecting group, a pivaloyl protecting group, a trityl protecting group, a monomethyltrityl protecting group, and a dimethyltrityl protecting group. Examples of the protecting group for a hydroxyl group include a methyl protecting group, a t-butyl protecting group, a benzyl protecting group, an alkoxymethyl protecting group, a 2-tetrahydropyranyl protecting group, a benzyloxymethyl protecting group, a mesyl protecting group, a tosyl protecting group, a nosyl protecting group, a triflyl protecting group, a trimethylsilyl protecting group, a triethylsilyl protecting group, a triisopropylsilyl protecting group, a t-butyldimethylsilyl protecting group, a t-butyldiphenylsilyl protecting group, an acetyl protecting group, a benzoyl protecting group, a pivaloyl protecting group, a trityl protecting group, a monomethyltrityl protecting group, and a dimethyltrityl protecting group. F 2 represents a carboxyl group.

[0045] [ka]

[0046] In formula (6), X represents a residue obtained by removing a hydroxyl group from a compound selected from the group consisting of glycerol, trimethylolpropane, pentaerythritol, xylitol, dipentaerythritol, hexaglycerol, dicylitol, disucrose, and tripentaerythritol. POLY represents a linear polyethylene glycol portion, specifically, -CH 2 CH 2O-(CH 2 CH 2 O) n -CH 2 CH 2 -It is expressed by the formula: L 1 represents an ether bond. L 2 represents an ether bond, an ester bond, an amide bond, a urethane bond, or a carbonate bond. L 3 represents an alkyl group having 1 to 10 carbon atoms (preferably 1 to 5 carbon atoms), F represents a carboxyl group; m is 3 to 8 depending on the structure of X.

[0047] In a particularly preferred embodiment, F in formula (4) 1 is a carboxyl group, and L 1 is an alkyl group having one carbon atom, and L 2 is an ether bond, F 2 , L 3 , L 4 is a methyl group, an ether bond, a single bond, or a carboxyl group, an ether bond, or an alkyl group having one carbon atom. In a preferred embodiment, E in formula (2) has a methine group, and L 1 is a methylene group having one carbon atom, and L 2 is an ether bond, and L 3 is an ether bond, and L 4 is a single bond, F 1 is a methyl group, and L 5 is a single bond, L 6 is an ether bond, and L 9 , L 10 , POLY 2 , L 11 , L 12 is a single bond, F 2 In a preferred embodiment, X in formula (3) is a residue obtained by removing a hydroxyl group from pentaerythritol, and L 2 is an ether bond, and L 3 is an alkyl group having one carbon atom, and F is a carboxyl group.

[0048] The weight average molecular weight of the polyethylene glycol derivative having a carboxyl group at its terminal is in the range of 1,000 to 80,000, preferably 1,000 to 40,000, more preferably 2,000 to 40,000.

[0049] (Process 1) In step 1, the polyethylene glycol compound is subjected to an oxidation reaction using an N-oxyl compound to obtain a polyethylene glycol derivative having a terminal carboxyl group, and then a reducing agent is added to quench the oxidation reaction, thereby obtaining a mixed aqueous solution containing the reduced form of the N-oxyl compound and the polyethylene glycol derivative having a terminal carboxyl group.

[0050] The N-oxyl compound may be TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl), a TEMPO derivative, or a combination thereof. The TEMPO derivative may be a TEMPO derivative having a methoxy group, a hydroxy group, a carboxyl group, an oxo group, a cyano group, an acetamide group, a dimethylamino group, a methacryloyloxy group, a bromoacetamide group, an iodoacetamide group, a glycidyloxy group, a phosphonooxy group, a propynyloxy group, an isothiocyanate group, or a benzoyloxy group at the 4th position on the piperidine ring of TEMPO. In a particularly preferred embodiment, TEMPO or a TEMPO derivative having a methoxy group, a hydroxy group, an oxo group, or an acetamide group at the 4th position on the piperidine ring of TEMPO, or a combination thereof, may be selected, and TEMPO is particularly preferred.

[0051] During the oxidation reaction, it is preferable to add a reoxidizing agent to the reaction system. The reoxidizing agent is an oxidizing agent capable of activating an N-oxyl compound, and is selected from any one or combination of hypohalous acid or its salt (hypochlorous acid or its salt, hypobromous acid or its salt, hypoiodous acid or its salt), haloous acid or its salt (chlorous acid or its salt, bromous acid or its salt, iodous acid or its salt), perhalogen acid or its salt (perchloric acid or its salt, periodic acid or its salt), halogen (chlorine, bromine, iodine), halogen oxide (chlorine monoxide, chlorine dioxide, dichlorine hexoxide, bromine dioxide), nitrogen oxide (nitric oxide, nitrogen dioxide, dinitrogen trioxide, etc.), peracid (hydrogen peroxide, peracetic acid, persulfuric acid, perbenzoic acid, etc.). Preferably, it is an alkali metal hypohalous acid salt, more preferably sodium hypochlorite.

[0052] In addition, the oxidation reaction is preferably carried out in a buffer solution. The buffer solution is a neutral to weakly basic buffer solution, and is selected from Good's buffer solutions such as 2-morpholinoethanesulfonic acid buffer solution, as well as any one of phosphate buffer solution, bicarbonate buffer solution, carbonate buffer solution, ammonium buffer solution, Tris buffer solution, citrate buffer solution, borate buffer solution, or a combination thereof, and is preferably a bicarbonate buffer solution.

[0053] Water may be used instead of the buffer solution. In this case, it is preferable to maintain the pH at neutral to weakly basic levels by appropriately adding an inorganic base such as sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium hydrogen carbonate, disodium hydrogen phosphate, trisodium phosphate, or ammonia during the reaction.

[0054] In addition, bromides or iodides may be added as activators for the oxidation reaction, and are selected from any one of ammonium salts (ammonium bromide, ammonium iodide), alkali metal bromides or iodides (bromides such as lithium bromide, potassium bromide, sodium bromide, and iodides such as lithium iodide, potassium iodide, sodium iodide), alkaline earth metal bromides or iodides (calcium bromide, magnesium bromide, strontium bromide, calcium iodide, magnesium iodide, strontium iodide), or combinations thereof.

[0055] The temperature when the oxidation reaction is carried out is 0°C or higher, preferably 0°C to 40°C, more preferably 0°C to 20°C, and particularly preferably 0°C to 10°C.

[0056] The reducing agent is a compound having a reducing effect on the activated N-oxyl compound and the reoxidizing agent, and is selected from any one of a primary alcohol having 1 to 24 carbon atoms and a corresponding aldehyde, a hydrazine compound, citric acid and its salts, succinic acid and its salts, ascorbic acid and its salts, isoascorbic acid and its salts, sulfurous acid and its salts, oxalic acid and its salts, formic acid and its salts, or a combination thereof, and is preferably any one of sodium ascorbate, sodium isoascorbate, and sodium sulfite, and more preferably sodium isoascorbate.

[0057] (Process 2) In step 2, the mixed aqueous solution of step 1 is adjusted to a pH of 3.0 or less, and an organic solvent containing one or more selected from the group consisting of aromatic hydrocarbons, carboxylic acid alkyl esters, and halogenated hydrocarbons is mixed therewith at a temperature of 0° C. or higher, and the solution is separated into an organic layer and an aqueous layer.

[0058] The acid used to adjust the pH of the mixed aqueous solution is preferably an acid having an octanol / water partition coefficient of 0.5 or less and a pKa of 6 or less, or a mixture of two or more of such acids. Particularly preferred is an inorganic acid having an octanol / water partition coefficient of 0.5 or less and a pKa of 6 or less, and more preferably hydrochloric acid, sulfuric acid, or phosphoric acid.

[0059] From the viewpoint of the present invention, the mixed aqueous solution is adjusted to a pH of 3.0 or less, and the pH is preferably 2 or less. From the viewpoint of suppressing deterioration of the polyethylene glycol chain, the mixed aqueous solution is preferably adjusted to a pH of 1 or more.

[0060] The solvent for the organic layer is an organic solvent containing at least one selected from the group consisting of aromatic hydrocarbons, carboxylic acid alkyl esters, and halogenated hydrocarbons. Here, the aromatic hydrocarbon is preferably an aromatic silicon carbide such as toluene or xylene. The carboxylic acid alkyl ester preferably has 1 to 4 carbon atoms, and is preferably an alkyl acetate, with methyl acetate, ethyl acetate, and butyl acetate being particularly preferred. The halogenated hydrocarbon is preferably has 1 to 4 carbon atoms, and is preferably methylene chloride or chloroform.

[0061] The organic solvent may consist of one or more solvents selected from the group consisting of aromatic hydrocarbons, alkyl carboxylates, and halogenated hydrocarbons. Alternatively, it may contain organic solvents other than these solvents, and examples of such organic solvents include aliphatic hydrocarbons such as hexane and heptane, and alcohols such as methanol and ethanol. In addition, when an organic solvent other than aromatic hydrocarbons, alkyl carboxylates, and halogenated hydrocarbons is contained, the ratio of such organic solvents is preferably 5% by mass or less, and more preferably 1% by mass or less.

[0062] The temperature at which step 2 is carried out varies depending on the type and boiling point of the solvent, but is preferably 0° C. to 60° C. In the case where aromatic hydrocarbons are contained, the temperature at which step 2 is carried out is preferably 30° C. or higher, more preferably 45° C. or higher, and particularly preferably 55° C. In the case where halogenated hydrocarbons are contained, the temperature at which step 2 is carried out is 5° C. or higher, preferably 15° C. or higher, and more preferably 25° C. or higher.

[0063] The time required for step 2 is 5 minutes or more, preferably 15 minutes or more, and more preferably 30 minutes or more.

[0064] (Step 3) Step 3 is a water-washing step in which the organic layer obtained in step 2 is mixed with an aqueous solution adjusted to a pH of 3 or less under conditions of 0° C. or higher to separate the organic layer from the aqueous layer.

[0065] In step 3, the acid used to adjust the pH of the aqueous solution is preferably an acid having an octanol / water partition coefficient of 0.5 or less and a pKa of 6 or less, or a mixture of two or more of such acids. Particularly preferred is an inorganic acid having an octanol / water partition coefficient of 0.5 or less and a pKa of 6 or less, and more preferably hydrochloric acid, sulfuric acid, or phosphoric acid.

[0066] From the viewpoint of the present invention, the pH of the aqueous solution is adjusted to 3 or less, and preferably to 2 or less. From the viewpoint of preventing deterioration of the polyethylene glycol chain, the pH of the aqueous solution is preferably 1 or more.

[0067] The temperature at which step 3 is carried out varies depending on the type and boiling point of the solvent, but is preferably 0° C. to 60° C. in general. When aromatic hydrocarbons are contained, the temperature at which step 2 is carried out is preferably 30° C. or higher, more preferably 45° C. or higher, and particularly preferably 55° C. When halogenated hydrocarbons are contained, the temperature at which step 2 is carried out is preferably 5° C. or higher, further preferably 15° C. or higher, and particularly preferably 25° C. or higher.

[0068] The time required for carrying out step 3 is 5 minutes or more, preferably 15 minutes or more, and more preferably 30 minutes or more. From the viewpoint of the present invention, it is preferable to carry out step 3 two or more times. From the viewpoint of productivity and effect, it is preferable to carry out step 3 three or less times.

[0069] (Step 4) In step 4, the organic layer obtained in step 3 is concentrated to recover a polyethylene glycol derivative having a terminal carboxyl group. The method of concentration is not limited, but drying under reduced pressure or in a vacuum is preferred. The crystals obtained by concentration can be further reprecipitated and recovered. Examples of good solvents for reprecipitation include ethyl acetate and toluene, and examples of poor solvents include hexane.

[0070] According to the present invention, it is possible to obtain a composition of a polyethylene glycol derivative having a terminal carboxyl group, in which the content of the reduced form of an N-oxyl compound is 0.1 mol % to 1.0 mol % (more preferably 0.1 mol % to 0.8 mol %). EXAMPLES

[0071] The present invention will be described in more detail below with reference to examples. In the examples, the content of reduced N-oxyl compounds in polyethylene glycol derivatives having a terminal carboxyl group was measured using 1 H-NMR was used. 1 For the H-NMR analysis, JNM-ECP400 and JNM-ECA600 manufactured by JEOL Datum Co., Ltd. were used.

[0072] Example 1 25g (12.5mmol) of linear polyethylene glycol with a molecular weight of 2,000, one end being a hydroxyl group and the other being a methoxyl group, 98mg of TEMPO (Tokyo Chemical Industry Co., Ltd., 0.6mmol), 7g of sodium bicarbonate (Kanto Chemical Co., Ltd.), and 140g of ion-exchanged water were charged into a 500mL four-neck flask, heated under a nitrogen atmosphere, and dissolved at 40°C. 37.2g of sodium hypochlorite solution (Kanto Chemical Co., Ltd., effective chlorine concentration 6.0%) was added dropwise over 30 minutes, and an oxidation reaction was carried out at 40°C for 1 hour and 40 minutes. After the oxidation reaction, 1.24g of sodium ascorbate (Kanto Chemical Co., Ltd.) was charged at 25°C, stirred for 30 minutes, and the oxidation reaction was quenched (step 1).

[0073] After quenching, 6 mol / L hydrochloric acid (Kanto Chemicals) was added to adjust the pH to 2.0, and 125 g of chloroform was added and stirred for 15 minutes at 25° C. After layer separation, the chloroform layer was recovered, and 125 g of chloroform was added again to the aqueous layer, and the same operation was carried out (extraction step 2).

[0074] The obtained chloroform layers were combined and homogenized, and then 125 g of an aqueous hydrochloric acid solution adjusted to pH 2.0 was added and stirred at 15° C. for 15 minutes. After layer separation, the chloroform layer was recovered (water washing step 3). Step 3 was repeated two more times by the same operation.

[0075] Next, chloroform was distilled off from the chloroform layer, and 63 g of ethyl acetate was added and dissolved at 40° C. 2.5 g of magnesium sulfate (Kanto Chemicals) was added and stirred at 40° C. for 30 minutes, after which magnesium sulfate was filtered off. The filtrate was cooled to 20° C., and 150 g of hexane was added to precipitate crystals, which were then filtered off. 100 g of hexane was added to the obtained crystals, and the mixture was stirred, filtered off again, vacuum dried, and the crystals were recovered (recovery process: yield 85%). The changes in the TEMPO reduced product content after step 1, after extraction step 2, and after water washing step 3 are shown in Table 1. FIG. 1 shows an NMR chart of the mixture after step 1 (before purification) in Example 1, and FIG. 2 shows an NMR chart of the purified product after carrying out water washing step 3 three times in Example 1.

[0076] [Table 1]

[0077] Example 2 25g (12.5mmol) of linear polyethylene glycol with a molecular weight of 2,000, one end being a hydroxyl group and the other being a methoxyl group, 98mg (0.6mmol) of TEMPO, 7g of sodium bicarbonate, and 140g of ion-exchanged water were charged into a 500mL four-neck flask, heated under a nitrogen atmosphere, and dissolved at 40°C. 37.2g of sodium hypochlorite solution (effective chlorine concentration 6.0%) was added dropwise over 30 minutes, and an oxidation reaction was carried out at 40°C for 1 hour and 40 minutes. After the oxidation reaction, 1.24g of sodium ascorbate was charged at 25°C, and the mixture was stirred for 30 minutes to quench the oxidation reaction (step 1).

[0078] After quenching, 6 mol / L hydrochloric acid was added to the mixed aqueous solution to adjust the pH to 1.5, 125 g of chloroform was added, and the mixture was stirred at 25° C. for 15 minutes. After separation, the chloroform layer was collected, and 125 g of chloroform was added to the aqueous layer again, and the same operation was carried out. The obtained chloroform layers were combined and homogenized (extraction step 2).

[0079] Next, 125 g of an aqueous hydrochloric acid solution adjusted to pH 1.5 was added to the chloroform layer, and the mixture was stirred at 15° C. for 15 minutes. After layer separation, the chloroform layer was recovered (water washing step 3). The water washing step 3 was repeated two more times in the same manner.

[0080] Next, chloroform was distilled off from the chloroform layer, and 63 g of ethyl acetate was added and dissolved at 40° C. 2.5 g of magnesium sulfate was added and stirred at 40° C. for 30 minutes, after which magnesium sulfate was filtered off. The filtrate was cooled to 20° C., and 150 g of hexane was added to precipitate crystals, which were then filtered off. 100 g of hexane was added to the obtained crystals, and the mixture was stirred, filtered off again, vacuum dried, and the crystals were recovered (recovery step 4: yield 85%). The changes in the TEMPO reduced product content after step 1, after extraction step 2, and after water washing step 3 are shown in Table 2.

[0081] [Table 2]

[0082] Example 3 2.5g (0.06mmol) of glycerol-skeleton branched polyethylene glycol with molecular weight of 40,000, 98mg (0.6mmol) of TEMPO, 0.7g of sodium bicarbonate, and 14g of ion-exchanged water were charged into a 50mL three-neck flask and dissolved at 25°C under a nitrogen atmosphere. After cooling to 6°C, 191mg of sodium hypochlorite solution (effective chlorine concentration 5.9%) was added dropwise and an oxidation reaction was carried out at 6°C for 3 hours. After the oxidation reaction, 14mg of sodium isoascorbate was charged at 5°C and stirred for 10 minutes to quench the oxidation reaction (step 1).

[0083] After quenching, 3.9 g of purified salt was dissolved and 3 mol / L sulfuric acid was added to adjust the pH to 1.0, 13 g of toluene was added, and the mixture was stirred for 30 minutes at 50° C. After layer separation, the toluene layer was collected, and 13 g of toluene was added again to the aqueous layer, and the same operation was carried out (extraction step 2).

[0084] The obtained toluene layers were combined and homogenized, and then 25 g of a 20% aqueous sodium chloride solution adjusted to pH 3.0 with sulfuric acid was added, followed by stirring for 30 minutes at 55° C. (water washing step 3). After layer separation, the toluene layer was recovered, and the water washing step was repeated two more times by the same operation.

[0085] The toluene layer was collected and distilled off, and then 25 g of ethyl acetate was added and dissolved at 40° C. 500 mg of magnesium sulfate was added and stirred at 40° C. for 30 minutes, after which the magnesium sulfate was filtered off. The solution was concentrated and distilled off at 40° C. to collect crystals (recovery step 4: yield 50%). The changes in the TEMPO reduced product content after step 1, after extraction step 2, and after water washing step 3 are shown in Table 3.

[0086] [Table 3]

[0087] Example 4 5g (0.25mmol) of multi-arm polyethylene glycol having a molecular weight of 20,000 and a pentaerythritol skeleton, 8mg (0.05mmol) of TEMPO, 1.4g of sodium bicarbonate, and 28g of ion-exchanged water were charged into a 100mL three-neck flask and dissolved at 25°C under a nitrogen atmosphere. After cooling to 6°C, 3g of sodium hypochlorite solution (effective chlorine concentration 5.9%) was added dropwise, and an oxidation reaction was carried out at 6°C for three and a half hours. After the oxidation reaction, 108mg of sodium isoascorbate was charged at 5°C and stirred for 10 minutes to quench the oxidation reaction (step 1).

[0088] After quenching, 85% phosphoric acid was added to adjust the pH to 3.0, 25 g of chloroform was added, and the mixture was stirred for 30 minutes at 45° C. After layer separation, the chloroform layer was collected, and 13 g of chloroform was added to the aqueous layer again, and the same operation was carried out (extraction step 2).

[0089] The obtained chloroform layers were combined and homogenized, and then 25 g of an aqueous phosphoric acid solution adjusted to pH 1.0 was added thereto, followed by stirring at 45° C. for 30 minutes (water washing step 3).

[0090] The chloroform layer was collected, and the chloroform was distilled off. Then, 25 g of ethyl acetate was added and dissolved at 35° C. 500 mg of magnesium sulfate was added and stirred at 35° C. for 30 minutes, after which the magnesium sulfate was filtered off. The solution was concentrated and distilled off at 40° C. to collect crystals (recovery step 4: yield 91%). The changes in the TEMPO reduced product content after step 1, after extraction step 2, and after water washing step 3 are shown in Table 4.

[0091] [Table 4]

[0092] Example 5 5g (0.25mmol) of multi-arm polyethylene glycol having a molecular weight of 20,000 and a pentaerythritol skeleton, 8mg (0.05mmol) of TEMPO, 1.4g of sodium bicarbonate, and 28g of ion-exchanged water were charged into a 100mL three-neck flask and dissolved at 25°C under a nitrogen atmosphere. After cooling to 6°C, 3g of sodium hypochlorite solution (effective chlorine concentration 5.9%) was added dropwise, and an oxidation reaction was carried out at 6°C for three and a half hours. After the oxidation reaction, 108mg of sodium isoascorbate was charged at 5°C and stirred for 10 minutes to quench the oxidation reaction (step 1).

[0093] After quenching, 8.7 g of purified salt was dissolved, phosphoric acid was added to adjust the pH to 3.0, 25 g of toluene was added, and the mixture was stirred for 30 minutes at 45° C. After layer separation, the toluene layer was collected, and 25 g of toluene was added again to the aqueous layer, and the same operation was carried out (extraction step 2).

[0094] The obtained toluene layers were combined and homogenized, and then 25 g of a 20% aqueous sodium chloride solution adjusted to pH 1.0 with sulfuric acid was added, followed by stirring at 55° C. for 30 minutes (water washing step 3).

[0095] After separation, the toluene layer was collected and the toluene was distilled off, and then 25 g of ethyl acetate was added and dissolved at 40° C. 500 mg of magnesium sulfate was added and stirred at 35° C. for 30 minutes, after which the magnesium sulfate was filtered off. The crystals were collected by concentration and distillation at 40° C. (recovery step 4: yield 85%). Table 5 shows the changes in the TEMPO reduced product content after step 1, after extraction step 2, and after water washing step 3.

[0096] [Table 5]

[0097] Next, the pH of the aqueous layer during extraction and washing was adjusted to about 4, and the removal of the TEMPO-reductant during steps 2 and 3 was evaluated.

[0098] Comparative Example 1 1 g (0.5 mmol) of linear polyethylene glycol with a molecular weight of 2,000, one end of which is a hydroxyl group and the other end of which is a methoxyl group, 4 mg (0.6 mmol) of TEMPO, 0.3 g of sodium bicarbonate, and 5.6 g of ion-exchanged water were charged into a screw-cap bottle, heated under a nitrogen atmosphere, and dissolved at 40° C. 1.5 g of sodium hypochlorite solution (effective chlorine concentration 5.9%) was charged, and an oxidation reaction was carried out at 10° C. for 2 hours. After the oxidation reaction, 54 mg of sodium isoascorbate was charged at 10° C. and stirred for 30 minutes to quench the oxidation reaction (step 1).

[0099] After quenching, 0.1 mol / L hydrochloric acid was added to adjust the pH to 4.1, 5 g of chloroform was added, and the mixture was stirred for 30 minutes at 25° C. After layer separation, the chloroform layer was collected, and 2.5 g of chloroform was added again to the aqueous layer, and the same operation was carried out (extraction step 2).

[0100] The obtained chloroform layers were combined and homogenized, and then 5 g of an aqueous hydrochloric acid solution adjusted to pH 4.2 was added and stirred at 25° C. for 30 minutes. After layer separation, the chloroform layer was collected (water washing step 3). Step 3 was repeated two more times by the same operation.

[0101] The chloroform layer was collected, and the chloroform was concentrated and distilled off to collect crystals (collection step 4). Table 6 shows the changes in the TEMPO reduced product content after step 1, after extraction step 2, and after water washing step 3.

[0102] [Table 6]

[0103] As described above, even when steps 2 and 3 were carried out by adjusting the aqueous layer pH to more than 3, the TEMPO reduced product could not be effectively reduced.

[0104] Next, as described in Patent Document 2, an investigation was conducted as to whether the TEMPO-reduced product could be removed by repeatedly performing reprecipitation by adding hexane, a poor solvent, to a polyethylene glycol derivative dissolved in heated ethyl acetate.

[0105] Comparative Example 2 30 g (15 mmol) of linear polyethylene glycol with a molecular weight of 2,000, one end being a hydroxyl group and the other being a methoxyl group, 118 mg (0.75 mmol) of TEMPO, 8.4 g of sodium bicarbonate, and 168 g of ion-exchanged water were charged into a 500 mL four-neck flask, heated under a nitrogen atmosphere, and dissolved at 40° C. 47 g of sodium hypochlorite solution (effective chlorine concentration 5.9%) was added dropwise over 30 minutes, and an oxidation reaction was carried out at 40° C. for 2 hours. After the oxidation reaction, 1.5 g of sodium ascorbate was charged at 22° C. and stirred for 30 minutes to quench the oxidation reaction (step 1).

[0106] After quenching, 6 mol / L hydrochloric acid was added to adjust the pH to 2.0, 150 g of chloroform was added, and the mixture was stirred for 15 minutes at 22° C. After layer separation, the chloroform layer was recovered, and 150 g of chloroform was added again to the aqueous layer, and the same operation was carried out (extraction step 2).

[0107] After the obtained chloroform layer was homogenized, chloroform was distilled off, 77 g of ethyl acetate was added, and the mixture was dissolved at 40°C. 3.0 g of magnesium sulfate was added, and the mixture was stirred at 40°C for 30 minutes, after which magnesium sulfate was filtered off. The temperature of the filtrate was cooled to 25°C, and 180 g of hexane was added to precipitate crystals, which were then filtered off. 125 g of ethyl acetate was added to the obtained wet crystals, and the crystals were dissolved at 40°C, and the mixture was cooled to 20°C, and 63 g of hexane was added to precipitate crystals, which were then filtered off. The same operation was repeated twice, and 100 g of hexane was added to the obtained wet crystals, and the mixture was stirred, and then filtered off again, vacuum dried, and the crystals were collected (yield 69%). Table 7 shows the change in the content of the TEMPO reduced product upon repeated reprecipitation.

[0108] [Table 7]

[0109] Comparative Example 3 5g (0.25mmol) of multi-arm polyethylene glycol having a molecular weight of 20,000 and a pentaerythritol skeleton, 8mg (0.05mmol) of TEMPO, 1.4g of sodium bicarbonate, and 28g of ion-exchanged water were charged into a 100mL three-neck flask and dissolved at 25°C under a nitrogen atmosphere. After cooling to 6°C, 3g of sodium hypochlorite solution (effective chlorine concentration 5.9%) was added dropwise, and an oxidation reaction was carried out at 6°C for three and a half hours. After the oxidation reaction, 108mg of sodium isoascorbate was charged at 5°C and stirred for 10 minutes to quench the oxidation reaction (step 1).

[0110] After quenching, 85% phosphoric acid was added to adjust the pH to 3.0, 25 g of chloroform was added, and the mixture was stirred for 30 minutes at 45° C. After layer separation, the chloroform layer was collected, and 13 g of chloroform was added to the aqueous layer again, and the same operation was carried out (extraction step 2).

[0111] The obtained chloroform layers were combined and homogenized, and then the chloroform was distilled off. Then, 30 g of ethyl acetate was added and dissolved at 35°C. 500 mg of magnesium sulfate was added and stirred at 35°C for 30 minutes, after which the magnesium sulfate was filtered off. 15 g of hexane was added to the obtained filtrate to precipitate crystals, which were then filtered off. The obtained wet crystals were dissolved in 30 g of ethyl acetate, and crystallization was repeated twice under the same conditions. 20 g of hexane was added to the obtained wet crystals and stirred, then filtered off again, vacuum dried, and the crystals were collected (yield 83%). Table 8 shows the change in the content of the TEMPO reduced product upon repeated reprecipitation.

[0112] [Table 8]

[0113] As described above, even after the third reprecipitation, the reduction in the TEMPO-reduced product had almost plateaued, and the effect was limited.

[0114] Next, as described in Patent Document 1, we investigated whether the TEMPO reduced product could be removed by washing the mixed aqueous solution obtained by the oxidation reaction using TEMPO with an organic solvent (ethyl acetate).

[0115] Comparative Example 4 1 g (0.5 mmol) of linear polyethylene glycol with a molecular weight of 2,000, one end of which is a hydroxyl group and the other end of which is a methoxyl group, 4 mg (0.025 mmol) of TEMPO, 280 mg of sodium bicarbonate, and 6 g of ion-exchanged water were charged, heated under a nitrogen atmosphere, and dissolved at 35°C. 1.5 g of sodium hypochlorite solution (effective chlorine concentration 5.9%) was added dropwise, and an oxidation reaction was carried out at 10°C for 2 hours. After the oxidation reaction, 54 mg of sodium isoascorbate was added at 10°C, and the mixture was stirred for 5 minutes to quench the oxidation reaction (step 1).

[0116] After quenching, 6 mol / L hydrochloric acid was added to adjust the pH to 1.0, and 9 g of ethyl acetate was added and stirred at 25° C. for 30 minutes. Table 9 shows the change in the TEMPO reduced product content before and after washing with ethyl acetate.

[0117] [Table 9]

[0118] In this example, no reduction in the content of the TEMPO reductant was observed.

[0119] As described above, the present invention is a useful purification method by which the amount of reduced N-oxyl compounds contained in polyethylene glycol derivatives having a terminal carboxyl group can be reduced by washing with water, which is preferable for industrial production, and polyethylene glycol derivatives having a low content of reduced N-oxyl compounds can be obtained. [Industrial Applicability]

[0120] The present invention is particularly suitable for medical applications including the modification of polypeptides, enzymes, antibodies and other low-molecular-weight drugs, genes, nucleic acid compounds including oligonucleic acids, nucleic acid medicines and other physiologically active substances, or applications to drug delivery system carriers such as liposomes, polymer micelles and nanoparticles.

Claims

1. A method for producing a polyethylene glycol derivative having a carboxyl group at its terminal, comprising the following steps 1, 2, 3 and 4: Process 1: A process for obtaining a mixed aqueous solution containing a reduced form of the N-oxyl compound and the polyethylene glycol derivative having a carboxyl group at its terminal by subjecting a polyethylene glycol compound having a molecular weight of 1,000 to 80,000 and having a hydroxyl group or an aldehyde group at its terminal to an oxidation reaction using an N-oxyl compound to obtain a polyethylene glycol derivative having a carboxyl group at its terminal, and then quenching the oxidation reaction by adding a reducing agent. Process 2: An extraction step in which the mixed aqueous solution adjusted to a pH of 3.0 or less is mixed with an organic solvent containing at least one selected from the group consisting of aromatic hydrocarbons, carboxylic acid alkyl esters, and halogenated hydrocarbons at a temperature of 0° C. or higher to separate the solution into an organic layer and an aqueous layer. Step 3: A washing step in which the organic layer obtained in the step 2 is mixed with an aqueous solution adjusted to a pH of 3.0 or less at a temperature of 0° C. or higher to separate the organic layer from the aqueous layer. Step 4: A recovery step of recovering a polyethylene glycol derivative having a carboxyl group at its terminal by concentrating the organic layer obtained in the step 3.

2. 2. The method according to claim 1, wherein the polyethylene glycol derivative having a terminal carboxyl group is any one of linear polyethylene glycol, branched polyethylene glycol, and multi-arm polyethylene glycol.

3. 3. The method according to claim 1, wherein the average molecular weight of the polyethylene glycol derivative having a carboxyl group at its terminal is 1,000 to 80,000.

4. 3. The method according to claim 1, wherein the acid used to adjust the pH of the mixed aqueous solution in step 2 and the aqueous solution in step 3 is an acid having an octanol / water partition coefficient of 0.5 or less and a pKa of 6 or less.

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