Method for producing polyethylene glycol derivative having carboxyl group at terminal

Abstract

A method for producing a polyethylene glycol derivative having a carboxyl group at a terminal has a step 1, a step 2, a step 3, and a step 4. Step 1:　A polyethylene glycol compound is subjected to an oxidation reaction using an N-oxyl compound to obtain a polyethylene glycol derivative having a carboxyl group at a terminal. A reducing agent is then added to quench the oxidation reaction to obtain a mixed aqueous solution containing a reduced form of the N-oxyl compound and a polyethylene glycol derivative having a carboxyl group at a terminal. Step 2:　The mixed aqueous solution which has been adjusted to a pH of 3.0 or less is mixed with an organic solvent containing one or more selected from the group consisting of aromatic hydrocarbons, carboxylic acid alkyl esters, and halogenated hydrocarbons, at 0°C or higher, and the mixture is separated into an organic layer and a water 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 at 0°C or higher, and the mixture is separated into an organic layer and a water layer. Step 4:　A polyethylene glycol derivative having a carboxyl group is recovered by concentrating the organic layer obtained in step 3.

Description

Method for producing polyethylene glycol derivatives having a terminal carboxyl group

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

[0002] When administered into the body, pharmaceuticals that use bio-related substances such as hormones, cytokines, and enzymes are typically rapidly excreted from the body through glomerular filtration in the kidneys or uptake by macrophages in the liver and spleen. This results in a short half-life in the blood, and in some cases, the drug does not achieve sufficient pharmacological effect. To solve this problem, pharmaceuticals 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 blood half-life extended by increasing their molecular weight or forming a hydration layer.

[0003] In particular, polyethylene glycol derivatives having a terminal carboxyl group 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 N-terminal primary amino groups of proteins, or to other active groups. PEGylated preparations using polyethylene glycol derivatives having a carboxyl group as raw materials are already on the market, including SOMAVERT (registered trademark): PEGVISOMANT (Pfizer), Mircera (registered trademark): Methoxy Polyethylene glycol-epoetin beta (F. Hoffmann-La Roche), and Macugen (registered trademark): Pegaptanib (Eyetech). Additionally, clinical trials of many other PEGylated preparations have been conducted, proving their usefulness.

[0004] Examples of methods for producing polyethylene glycol derivatives having a terminal carboxyl group include the following: - A method in which the terminal hydroxyl group of a polyethylene glycol compound is bonded to an acrylic ester compound by a Michael reaction, followed by ester hydrolysis; - A method in which the terminal hydroxyl group of a polyethylene glycol is bonded to an orthoester compound having a leaving group by a Williamson reaction, followed by hydrolysis; - A method in which the terminal hydroxyl group of a polyethylene glycol is oxidized using an N-oxyl compound or a derivative thereof.

[0005] Although the oxidation reaction of the terminal hydroxyl groups of polyethylene glycol using this N-oxyl compound produces polyethylene glycol derivatives with high carboxylic acid purity, the reduced form of the N-oxyl compound is generated as an impurity after quenching the reaction. Polyethylene glycol derivatives used as pharmaceutical raw materials or pharmaceutical additives are required to have low impurity content from the viewpoint of pharmaceutical performance and safety. Impurities derived from low-molecular-weight compounds used to derivatize the polyethylene glycol terminals can cause unexpected side reactions during chemical modification of 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 N-oxyl compound.

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

[0007] For example, Patent Document 1 reports that, after converting the terminal hydroxyl groups of a polyethylene glycol compound to carboxylic acids with TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl) in an aqueous solution, ethanol is added to quench the reaction, and the aqueous layer is washed with ethyl acetate, whereby colored impurities, which are assumed to be TEMPO reductants, migrate to the ethyl acetate layer.

[0008] Furthermore, Non-Patent Document 1 reports that the terminal hydroxyl groups of polyethylene glycol are converted to carboxylic acids using TEMPO in an aqueous solution, 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 groups of polyethylene glycol are converted to carboxylic acids using TEMPO in an aqueous solution, 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 to cause reprecipitation.

[0011] Japanese Patent Publication No. 2022-162784 Japanese Patent Publication No. 2004-197077

[0012] Synlett 2018, 29(5), 556-559ACS Macro Letters 2019, 8(7), 826-834

[0013] Patent Document 1 and Non-Patent Document 1 do not disclose the amount of residual TEMPO residue or its removal efficiency, and it is unclear whether the reduced TEMPO can be removed by washing with an organic solvent or by the crystallization described in Patent Document 2. In addition, Non-Patent Document 1 finally performs purification by silica gel column chromatography, which requires the use of a large amount of solvent and results in a large amount of silica gel being wasted, 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 process complicated and therefore 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 at a high carboxylation rate, there remains a problem in that no easy method has been established for separating the reduced product of the N-oxyl compound produced by quenching after the reaction from the polyethylene glycol derivative, without requiring purification using a column or dialysis, which is relatively complicated from an industrial standpoint.

[0015] In view of the above problems, the present invention aims 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.

[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 less, extracting the mixed aqueous solution with an aromatic hydrocarbon, a carboxylic acid alkyl ester or a halogenated hydrocarbon, and then washing the organic layer containing the mixed solution 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 less, thereby obtaining a polyethylene glycol derivative having a small amount of the reduced form of the N-oxyl compound remaining.

[0017] That is, the present invention provides the following: [1] A method for producing a polyethylene glycol derivative having a terminal carboxyl group, comprising the following steps 1, 2, 3 and 4: Step 1: a step of subjecting a polyethylene glycol compound having a terminal hydroxyl group or aldehyde group and a molecular weight of 1,000 to 80,000 to an oxidation reaction using an N-oxyl compound to obtain a polyethylene glycol derivative having a terminal carboxyl group, and then adding a reducing agent to quench the oxidation reaction to obtain a mixed aqueous solution containing a reduced form of the N-oxyl compound and the polyethylene glycol derivative having a terminal carboxyl group; Step 2: an extraction step of mixing the mixed aqueous solution, adjusted to a pH of 3.0 or less, with an organic solvent containing one or more selected from the group consisting of aromatic hydrocarbons, carboxylic acid alkyl esters, and halogenated hydrocarbons, under conditions of 0°C or higher, to separate the solution into an organic layer and an aqueous layer; Step 3: a water-washing step of mixing the organic layer obtained in Step 2 with an aqueous solution adjusted to a pH of 3.0 or lower, under conditions of 0°C or higher, to separate the solution into an organic layer and an aqueous layer; Step 4: a recovery step of concentrating the organic layer obtained in Step 3 to recover the polyethylene glycol derivative having a terminal carboxyl group.

[0018] [2] The method according to [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.

[0019] [3] The method according to [1] or [2], wherein the average molecular weight of the polyethylene glycol derivative having a terminal carboxyl group 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 the step 2 and the aqueous solution in the step 3 is an acid having an octanol / water partition coefficient of 0.5 or less and a pKa of 6 or less.

[0021] According to the present invention, reduced forms of N-oxyl compounds contained in polyethylene glycol derivatives having a terminal carboxyl group can be removed, thereby obtaining polyethylene glycol derivatives having a low content of reduced N-oxyl compounds. This production method makes it possible to obtain polyethylene glycol derivatives having a terminal carboxyl group and having a low content of reduced N-oxyl compounds without performing purification procedures unsuitable for industrial production, such as silica gel column purification or dialysis.

[0022] 1 shows an NMR chart obtained before purification in Example 1. FIG. 2 shows an NMR chart obtained after purification in Example 1.

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

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

[0025]

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

[0027] F ２represents 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.

[0028] L １ and L ４ each independently represents an alkyl group having 1 to 10 carbon atoms, provided that F ２ When L is an alkyl group, ４ represents a single bond. 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 When L is an alkyl group, ３ represents an ether bond.

[0029]

[0030] In formula (2), POLY １ represents a linear polyethylene glycol moiety, specifically, —CH ２ CH２ O-(CH ２ CH ２ O) ｎ -CH ２ CH ２ - is represented by the formula: ２ represents a similar 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.

[0031] E is L １ , 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. 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 represents an ether bond, an ester bond, an amide bond, a urethane bond, a carbonate bond or a single bond, L １１ is a single bond.

[0032] F １represents 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 group-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. ２ represents a hydroxyl group or an aldehyde group.

[0033]

[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 moiety, specifically, —CH ２ CH ２ O-(CH ２ CH ２ O) ｎ -CH ２ CH ２ - is represented by the formula: L １ represents an ether bond.２ represents an ether bond, an ester bond, an amide bond, a urethane bond or a carbonate bond. 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, and 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, and 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, L is a single bond, F １ is a methyl group, and L ５ is a single bond, and 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, and F ２ is a hydroxyl group. 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] (Polyethylene glycol derivative having a terminal carboxyl group) The polyethylene glycol derivative having a terminal carboxyl group 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]

[0038] In formula (4), POLY represents a linear polyethylene glycol moiety, specifically, —CH ２ CH ２ O-(CH ２ CH ２ O) ｎ -CH ２ CH ２ - is expressed by the formula: F １ represents a carboxyl group. ２ 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. Preferred protecting groups 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. Preferred hydroxyl-protecting groups 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.

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

[0040]

[0041] In formula (2), POLY １ represents a linear polyethylene glycol moiety, specifically, —CH ２ CH ２ O-(CH ２ CH ２ O) ｎ -CH ２ CH ２ - is represented by the formula: ２ represents a similar 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 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 represents a group having 1 to 4 carbon atoms or a single bond; L ４ and L １２ represents a group having 1 to 5 carbon atoms or a single bond), where 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. ２ , L ３ , L ６ , L １０ and L １１each 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, 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 hydroxyl group-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. ２ represents a carboxyl group.

[0045]

[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 moiety, specifically, —CH ２ CH ２ O-(CH ２ CH ２ O) ｎ -CH ２ CH ２ - is represented by the formula: L １ represents an ether bond. ２ represents an ether bond, an ester bond, an amide bond, a urethane bond or a carbonate bond; ３ represents an alkyl group having 1 to 10 carbon atoms (preferably 1 to 5 carbon atoms); F represents a carboxyl group; and 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, and 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, and F １ is a methyl group, and L ５ is a single bond, and L ６ is an ether bond, and L ９ , L １０ , POLY ２ , L １１ , L １２ is a single bond, and F ２In 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 terminal carboxyl group ranges from 1,000 to 80,000, preferably from 1,000 to 40,000, and more preferably from 2,000 to 40,000.

[0049] (Step 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 a reduced form of the N-oxyl compound and a polyethylene glycol derivative having a terminal carboxyl group.

[0050] Examples of the N-oxyl compound include TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl), TEMPO derivatives, and combinations thereof. The TEMPO derivative may be a TEMPO derivative having a methoxy group, hydroxy group, carboxyl group, oxo group, cyano group, acetamido group, dimethylamino group, methacryloyloxy group, bromoacetamido group, iodoacetamido group, glycidyloxy group, phosphonooxy group, propynyloxy group, isothiocyanate group, or benzoyloxy group at the 4-position on the piperidine ring of TEMPO. In a particularly preferred embodiment, TEMPO or a TEMPO derivative having a methoxy group, hydroxy group, oxo group, or acetamido group at the 4-position on the piperidine ring of TEMPO, or a combination thereof, may be selected. 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 the N-oxyl compound, and is selected from hypohalous acids or salts thereof (hypochlorous acid or salts thereof, hypobromous acid or salts thereof, hypoiodous acid or salts thereof), haloous acids or salts thereof (chlorous acid or salts thereof, bromous acid or salts thereof, iodous acid or salts thereof), perhalogen acids or salts thereof (perchloric acid or salts thereof, periodic acid or salts thereof), halogens (chlorine, bromine, iodine), halogen oxides (chlorine monoxide, chlorine dioxide, dichlorine hexoxide, bromine dioxide), nitrogen oxides (nitric oxide, nitrogen dioxide, dinitrogen trioxide, etc.), peracids (hydrogen peroxide, peracetic acid, persulfuric acid, perbenzoic acid, etc.), or combinations thereof. Preferably, it is an alkali metal hypohalous acid, more preferably sodium hypochlorite.

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

[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 hydrogencarbonate, disodium hydrogenphosphate, 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 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, and 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 at which 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 that has a reducing effect on the activated N-oxyl compound and the reoxidizing agent, and is selected from any one of C1 to C24 primary alcohols and their corresponding aldehydes, hydrazine compounds, 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, and 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] (Step 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, thereby separating the solution 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 such acids. Particularly preferred are inorganic acids 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, alkyl carboxylates, and halogenated hydrocarbons. The aromatic hydrocarbon is preferably an aromatic silicon carbide such as toluene or xylene. The alkyl carboxylate 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 preferably has 1 to 4 carbon atoms, and is preferably methylene chloride or chloroform.

[0061] The organic solvent may consist solely of one or more solvents selected from the group consisting of aromatic hydrocarbons, alkyl carboxylic acid esters, and halogenated hydrocarbons. Alternatively, organic solvents other than these solvents may be included. Examples of such organic solvents include aliphatic hydrocarbons such as hexane and heptane, and alcohols such as methanol and ethanol. Furthermore, when organic solvents other than aromatic hydrocarbons, alkyl carboxylic acid esters, and halogenated hydrocarbons are included, the proportion 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 at a temperature of 0°C or higher, thereby separating 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 such acids. Particularly preferred are inorganic acids having an octanol / water partition coefficient of 0.5 or less and a pKa of 6 or less, and more preferred are hydrochloric acid, sulfuric acid, and 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 suppressing 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 may vary 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 carboxyl group at its terminal. The concentration method is not limited, but reduced pressure or vacuum drying 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 a reduced form of an N-oxyl compound is 0.1 mol % to 1.0 mol % (more preferably 0.1 mol % to 0.8 mol %).

[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 25 g (12.5 mmol) of linear polyethylene glycol having a molecular weight of 2,000, one end hydroxyl group and the other end methoxy group, 98 mg of TEMPO (Tokyo Chemical Industry Co., Ltd., 0.6 mmol), 7 g of sodium bicarbonate (Kanto Chemical Co., Ltd.), and 140 g of ion-exchanged water were charged into a 500 mL four-neck flask, heated under a nitrogen atmosphere, and dissolved at 40°C. 37.2 g of sodium hypochlorite solution (Kanto Chemical Co., Ltd., available 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, 1.24 g of sodium ascorbate (Kanto Chemical Co., Ltd.) was charged at 25°C, and the mixture was stirred for 30 minutes to quench the oxidation reaction (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 resulting chloroform layers were combined and homogenized, and then 125 g of a hydrochloric acid solution adjusted to pH 2.0 was added, followed by stirring 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 using the same procedure.

[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 the 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 resulting crystals, and the mixture was stirred. The crystals were then filtered again and vacuum dried to recover the crystals (recovery step: yield 85%). Table 1 shows the changes in the TEMPO reduced product content after step 1, extraction step 2, and water-washing step 3. Furthermore, FIG. 1 shows the NMR chart of the mixture after step 1 (before purification) in Example 1, and FIG. 2 shows the NMR chart of the purified product after water-washing step 3 was carried out three times in Example 1.

[0076]

[0077] Example 2 25 g (12.5 mmol) of linear polyethylene glycol having a molecular weight of 2,000, one end hydroxyl group and the other end methoxy group, 98 mg (0.6 mmol) of TEMPO, 7 g of sodium bicarbonate, and 140 g of ion-exchanged water were charged into a 500 mL four-neck flask, heated under a nitrogen atmosphere, and dissolved at 40° C. 37.2 g of sodium hypochlorite solution (available 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, 1.24 g of sodium ascorbate was charged at 25° C. and 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, and 125 g of chloroform was added and stirred at 25° C. for 15 minutes. After layer separation, the chloroform layer was recovered, and 125 g of chloroform was added again to the aqueous layer, and the same procedure was repeated. The obtained chloroform layers were combined and homogenized (extraction step 2).

[0079] Next, 125 g of an aqueous solution of hydrochloric acid 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 the magnesium sulfate was filtered off. The filtrate was cooled to 20°C, and 150 g of hexane was added to precipitate crystals, which were filtered off. 100 g of hexane was added to the obtained crystals, and the mixture was stirred. The crystals were again filtered off and vacuum dried to recover the crystals (recovery step 4: yield 85%). Table 2 shows the changes in the TEMPO reduced product content after step 1, extraction step 2, and water-washing step 3.

[0081]

[0082] Example 3 2.5 g (0.06 mmol) of glycerol-based branched polyethylene glycol having a molecular weight of 40,000, 98 mg (0.6 mmol) of TEMPO, 0.7 g of sodium bicarbonate, and 14 g of ion-exchanged water were charged into a 50 mL three-neck flask and dissolved at 25° C. under a nitrogen atmosphere. After cooling to 6° C., 191 mg of sodium hypochlorite solution (available chlorine concentration 5.9%) was added dropwise, and an oxidation reaction was carried out at 6° C. for 3 hours. After the oxidation reaction, 14 mg 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, and 13 g of toluene was added and stirred for 30 minutes at 50° C. After layer separation, the toluene layer was recovered, and 13 g of toluene was added again to the aqueous layer, and the same operation was performed (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 using the same procedure.

[0085] The toluene layer was recovered and evaporated, 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 evaporated at 40° C. to recover crystals (recovery step 4: yield 50%). The changes in the TEMPO reduced product content after step 1, extraction step 2, and water washing step 3 are shown in Table 3.

[0086]

[0087] Example 4 5 g (0.25 mmol) of multi-arm polyethylene glycol having a molecular weight of 20,000 and a pentaerythritol skeleton, 8 mg (0.05 mmol) of TEMPO, 1.4 g of sodium bicarbonate, and 28 g of ion-exchanged water were charged into a 100 mL three-neck flask and dissolved at 25° C. under a nitrogen atmosphere. After cooling to 6° C., 3 g of sodium hypochlorite solution (effective chlorine concentration 5.9%) was added dropwise, and an oxidation reaction was carried out at 6° C. for 3.5 hours. After the oxidation reaction, 108 mg 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, and 25 g of chloroform was added and stirred for 30 minutes at 45° C. After layer separation, the chloroform layer was recovered, and 13 g of chloroform was added again to the aqueous layer, 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 solution of phosphoric acid adjusted to pH 1.0 was added, followed by stirring at 45° C. for 30 minutes (water washing step 3).

[0090] The chloroform layer was recovered, and the chloroform was distilled off. 25 g of ethyl acetate was then added and dissolved at 35°C. 500 mg of magnesium sulfate was added, and the mixture was 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 recover crystals (recovery step 4: yield 91%). The changes in the TEMPO reduced product content after step 1, extraction step 2, and water washing step 3 are shown in Table 4.

[0091]

[0092] Example 5 5 g (0.25 mmol) of multi-arm polyethylene glycol having a molecular weight of 20,000 and a pentaerythritol skeleton, 8 mg (0.05 mmol) of TEMPO, 1.4 g of sodium bicarbonate, and 28 g of ion-exchanged water were charged into a 100 mL three-neck flask and dissolved at 25° C. under a nitrogen atmosphere. After cooling to 6° C., 3 g of sodium hypochlorite solution (effective chlorine concentration 5.9%) was added dropwise, and an oxidation reaction was carried out at 6° C. for 3.5 hours. After the oxidation reaction, 108 mg 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, and phosphoric acid was added to adjust the pH to 3.0, and 25 g of toluene was added and stirred for 30 minutes at 45° C. After layer separation, the toluene layer was recovered, 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 layer separation, the toluene layer was recovered and the toluene was distilled off. 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 mixture was concentrated and distilled off at 40° C. to recover crystals (recovery step 4: yield 85%). Table 5 shows the changes in the TEMPO reduced product content after step 1, extraction step 2, and water washing step 3.

[0096]

[0097] Next, the pH of the aqueous layer during extraction and washing was adjusted to about 4, and the removal of TEMPO reduced products when steps 2 and 3 were carried out was evaluated.

[0098] Comparative Example 1: 1 g (0.5 mmol) of linear polyethylene glycol having a molecular weight of 2,000, one end hydroxyl group and the other end methoxy group, 4 mg (0.6 mmol) of TEMPO, 0.3 g of sodium bicarbonate, and 5.6 g of ion-exchanged water were placed in a screw-cap bottle, heated under a nitrogen atmosphere, and dissolved at 40° C. 1.5 g of sodium hypochlorite solution (available chlorine concentration 5.9%) was added, 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 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, and 5 g of chloroform was added and stirred for 30 minutes at 25° C. After layer separation, the chloroform layer was recovered, 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 resulting chloroform layers were combined and homogenized, and then 5 g of an aqueous hydrochloric acid solution adjusted to pH 4.2 was added, followed by stirring at 25° C. for 30 minutes. After layer separation, the chloroform layer was recovered (water washing step 3). Step 3 was repeated two more times using the same procedure.

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

[0102]

[0103] As described above, even when steps 2 and 3 were carried out with the aqueous layer pH adjusted to above 3, the TEMPO reduced products could not be effectively reduced.

[0104] Next, as described in Patent Document 2, it was investigated whether the TEMPO reduced product could be removed by repeatedly 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 having a molecular weight of 2,000, one end hydroxyl group and the other end methoxy 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 (available 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 the mixture was 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, and 150 g of chloroform was added and 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] The resulting chloroform layer was homogenized, and the chloroform was distilled off. 77 g of ethyl acetate was added and 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 the 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 resulting wet crystals, and the crystals were dissolved at 40°C. After cooling to 20°C, 63 g of hexane was added to precipitate crystals, which were then filtered off. The same procedure was repeated twice, and 100 g of hexane was added to the resulting wet crystals, and the mixture was stirred. The crystals were then filtered off again and vacuum dried, and the crystals were recovered (yield 69%). Table 7 shows the change in the TEMPO reduced product content during repeated reprecipitation.

[0108]

[0109] (Comparative Example 3) 5 g (0.25 mmol) of multi-arm polyethylene glycol having a molecular weight of 20,000 and a pentaerythritol skeleton, 8 mg (0.05 mmol) of TEMPO, 1.4 g of sodium bicarbonate, and 28 g of ion-exchanged water were charged into a 100 mL three-neck flask and dissolved at 25° C. under a nitrogen atmosphere. After cooling to 6° C., 3 g of sodium hypochlorite solution (effective chlorine concentration 5.9%) was added dropwise, and an oxidation reaction was carried out at 6° C. for 3.5 hours. After the oxidation reaction, 108 mg 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, and 25 g of chloroform was added and stirred for 30 minutes at 45° C. After layer separation, the chloroform layer was recovered, and 13 g of chloroform was added again to the aqueous layer, and the same operation was carried out (extraction step 2).

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

[0112]

[0113] As described above, even after the third reprecipitation, the reduction in the TEMPO reduced product had almost reached a plateau, 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 having a molecular weight of 2,000, one end being a hydroxyl group and the other being a methoxy 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 (available 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 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, followed by stirring for 30 minutes at 25° C. Table 9 shows the change in the content of reduced TEMPO before and after washing with ethyl acetate.

[0117]

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

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

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

[0121] Although the present invention has been described in detail and with reference to specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the present invention. This application is based on a Japanese patent application (Patent Application No. 2023-164163) filed on September 27, 2023, the contents of which are incorporated herein by reference.

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: Step 1: a step 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 a reduced form of the N-oxyl compound and the polyethylene glycol derivative having a carboxyl group at its terminal. Step 2: an extraction step of mixing the mixed aqueous solution adjusted to a pH of 3.0 or less with an organic solvent containing one or more selected from the group consisting of aromatic hydrocarbons, carboxylic acid alkyl esters and halogenated hydrocarbons under a condition of 0°C or higher to separate the solution into an organic layer and an aqueous layer. Step 3: a water-washing step of mixing the organic layer obtained in the step 2 with an aqueous solution adjusted to a pH of 3.0 or less under a condition of 0°C or higher to separate the solution into an organic layer and an 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. 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. The method according to claim 1 or 2, wherein the average molecular weight of the polyethylene glycol derivative having a carboxyl group at its terminal is 1,000 to 80,000.

4. The method according to claim 1 or 2, 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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