JP2019065124A - Monodispersed polyethylene glycol mono(meth)acrylate polymer and manufacturing method therefor - Google Patents

Abstract

To provide a comb-type polymer capable of making analysis easy when purity of a pharmaceutical by binding a biofunctional compound and a polyethylene glycol mono(meth)acrylate polymer and a purified article after separation purification is confirmed.SOLUTION: There is provided a single dispersed polyethylene glycol mono(meth)acrylate polymer represented by the formula (1), in which a plurality of main peaks are observed per molecular weight of constitutional units derived from the single dispersed polyethylene glycol mono(meth)acrylate polymer in a matrix support laser elimination ionization time-of-flight type mass spectroscopy.(M)AO-(CHCHO)a-H (1), where (M)A represents a (meth)acryloyl group and a represents an integer of 6 to 40.SELECTED DRAWING: Figure 5

Description

  The present invention relates to monodispersed polyethylene glycol mono (meth) acrylate polymers. More specifically, modification of biofunctional compounds (protein drugs, polypeptides, enzymes, antibodies, antibody drugs, nucleic acid compounds including genes, oligonucleic acids, etc., nucleic acid drugs, other drugs such as anticancer drugs, small molecule drugs, etc.) The present invention relates to a monodispersed polyethylene glycol mono (meth) acrylate polymer applicable as an agent, a polymer structure, in particular, a hydrogel for cell scaffolds, and an element constituting a reactive polymer fine particle.

  In recent years, pharmaceuticals in which polyethylene glycol is linked (modified) to a biofunctional compound have been put to practical use. This is to modify the polyethylene glycol to reduce the antigenicity and immunogenicity of the biofunctional compound, and the apparent size is larger than the glomerular filtration limit, thereby avoiding renal clearance or avoiding cell clearance mechanism. By doing this, a remarkable prolonging effect of circulating half-life in vivo can be expected.

  As polyethylene glycol modifiers, linear polyethylene glycols having a molecular weight of 5,000 to 40,000 and having a methoxy group at the end are generally used. However, when using a high molecular weight polyethylene glycol modifier, drug administration may become difficult because the viscosity of the drug after modification is increased. In order to solve this problem, high molecular weight branched polyethylene glycol is used as a modifier instead of a polyethylene glycol modifier, and a comb polymer obtained by polymerizing polyethylene glycol monomethacrylate having a methoxy group at the end is used. Has been reported.

  For example, Non-Patent Document 1 discloses a comb polymer obtained by polymerizing polyethylene glycol monomethacrylate having a methoxy group at an end having an average molecular weight of 460 or 2000. It is reported that this comb-poly conjugated interferon maintains a low viscosity as compared to the linear polyethylene glycol modifier and exhibits a long circulating half-life in vivo.

  On the other hand, in a pharmaceutical product in which such a polyethylene glycol modifying agent and a biofunctional compound are combined, the number, the number of the polyethylene glycol modifying agent bonded to the biofunctional compound, the efficacy, stability, solubility and toxicity of the modified drug It is known that the drug retention time changes (Non-patent Document 2), and it is necessary to measure the number of bonds and to separate and purify. Methods of measurement include electrophoresis, hydrophobic column chromatography, size exclusion chromatography, etc. Recently, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) is known from the viewpoint of accurate molecular weight. ) Has attracted attention (Non-Patent Document 3).

  In Non-Patent Documents 4 and 5, repeated administration to the same individual of liposome or nanoparticle modified with polyethylene glycol having a methoxy group at the end reduces the half life in blood after the second administration as compared with the first administration. It has been reported that an accelerated blood clearance (ABC) phenomenon may occur. As a solution to this, Non-Patent Document 6 and Patent Document 1 report a polyethylene glycol modifier having a hydroxyl group at the end.

  In a method of producing polyethylene glycol mono (meth) acrylate having a hydroxyl group at an end, ethylene oxide is generally polymerized using 2-hydroxyethyl methacrylate as a raw material. However, in this case, polyethylene glycol mono (meth) acrylate consisting of a single ethylene glycol repeat number can not be obtained, and a crosslinkable compound having a (meth) acryloyl group at both ends is by-produced. If polymerization is carried out in a state in which several% of the crosslinkable compound is contained, crosslinking and gelation occur, and there is a problem that the desired linear polymer can not be obtained.

  As another production method, there is known a method of reacting (meth) acrylic acid under acidic conditions using polyethylene glycol having hydroxyl groups at both ends as a raw material, and a method of reacting an acid anhydride and an acid chloride. In general, polyethylene glycol having hydroxyl groups at both ends of ethylene glycol having a repeat number of 5 or less can be purified by distillation, so that it is possible to synthesize monodispersed polyethylene glycol mono (meth) acrylate by obtaining high purity raw materials . However, when the ethylene glycol repeat number is 6 or more, monodispersed polyethylene glycol mono (meth) acrylate can not be synthesized because polyethylene glycol having a molecular weight distribution produced by polymerization is used as a raw material. Furthermore, since the above-mentioned crosslinkable compound is by-produced because polyethylene glycol having hydroxyl groups at both ends is used as a raw material, problems occur during polymerization.

  For example, Non-Patent Document 7 reports an example in which polyethylene glycol having a number average molecular weight of 1000 and acrylic anhydride are reacted, and it can be seen that 30% of a crosslinkable compound is generated. Although separation purification using an aqueous solution of sodium chloride is performed to obtain polyethylene glycol monoacrylate having a hydroxyl group at an end, the yield is low, and the obtained target contains compounds having different ethylene glycol repeat numbers.

A. godwin, Bioconjugate chem., 2015, 26, p452 A. P. Chapman, Advanced Drug Delivery Review, 2002, 54, p 531 F. M. Veronese, Biomaterials, 2001, 22, p 405 T. Ishida, H. Kiwada, et al., J. control. Release, 2007, 122, p 349 W. Jiskoot, R. M. F. van Schie, et al., Pharmaceutical Research, 2009, 26, 6, p1303 Mark G. P. Saifer, Bioconjugate chem. , 2012, 23, p485 Jia-Heng Lei et al. , J Surf Deterg, 2012, 15, p 117

JP 2012-214747 A

As described above, since the comb polymer is synthesized using polyethylene glycol mono (meth) acrylate containing a compound having different ethylene glycol repeat number as a monomer, in addition to the molecular weight distribution of the polymer main chain, the polymer side Distribution also occurs in the molecular weight of the chain. As a result, when measured by MALDI-TOF MS, m / z = 44 (CH 2 CH 2 O) per molecule ion peak of the observed number of measurement results analysis complicated, and the disadvantage that it is difficult. Furthermore, there is a disadvantage that analysis becomes difficult similarly in purity confirmation after separation and purification.
As a result, when the number of bonded comb polymers can not be accurately measured in a pharmaceutical product in which a biofunctional compound and the comb polymer are bonded, variations in drug efficacy, stability, solubility, toxicity, etc. occur. Quality control may be difficult.

  From the above, it has been desired to develop a comb polymer that can be easily analyzed in the case of checking the purity of a pharmaceutical product in which a biofunctional compound and a polyethylene glycol mono (meth) acrylate polymer are bonded.

  An object of the present invention is to provide a pharmaceutical product in which a biofunctional compound and a polyethylene glycol mono (meth) acrylate polymer are combined, and a comb polymer which can easily be analyzed in the purity confirmation of a purified product after separation and purification. It is to be.

  Another object of the present invention is to provide a process which can minimize by-products in polymerizing polyethylene glycol mono (meth) acrylate to obtain a polymer.

  As a result of intensive studies to achieve the above object, the present inventors have found that matrix-assisted laser desorption / ionization time-of-flight mass spectrometry shows that a plurality of main peaks are structural units derived from polyethylene glycol mono (meth) acrylate. It has been found that monodispersed polyethylene glycol mono (meth) acrylate polymers as observed for each molecular weight solve the above problems.

  Furthermore, in the production of the monodispersed polyethylene glycol mono (meth) acrylate, a crosslinkable compound is not by-produced by combining synthesis steps in a specific order, using a suitable protecting group, and column chromatography etc. The inventors have found that high purity compounds can be obtained only by simple liquid separation extraction without using a purification method, and the present invention has been completed.

That is, the present invention provides the following [1] to [6].
[1] A polymer of monodispersed polyethylene glycol mono (meth) acrylate represented by the formula (1), which is
A plurality of main peaks are observed at intervals of molecular weights of constitutional units derived from the monodispersed polyethylene glycol mono (meth) acrylate by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry. Dispersed polyethylene glycol mono (meth) acrylate polymer.

_{(M) AO- (CH 2 CH} 2 O) a-H ··· (1)

[In the formula (1),
(M) A is a (meth) acryloyl group,
a represents an integer of 6 to 40; ]

[2] A monodispersed polyethylene glycol mono (meth) acrylate polymer comprising the following step [A], step [B], step [C], step [D] and step [E] in this order Manufacturing method.

Step [A]: The compound represented by Formula (4) is subjected to a nucleophilic substitution reaction so as to satisfy Formula (F1), and the compound represented by Formula (4) Step [B]: The compound represented by Formula (4) is (meth) acrylated with (meth) acrylic anhydride or (meth) acrylic acid chloride to obtain a compound represented by Formula (5) Step [C]: a step of deprotecting the compound represented by the formula (5) to obtain a mixture containing monodispersed polyethylene glycol mono (meth) acrylate represented by the formula (1): Step [D]: the above Step of purifying the mixture obtained in Step [C] Step [E]: Step of polymerizing monodispersed polyethylene glycol mono (meth) acrylate represented by Formula (1) obtained in Step [D].

_{HO- (CH 2 CH 2 O)} b-H ··· (2)
[In Formula (2), b shows the integer of 3-37. ]

LO-(CH ₂ CH ₂ O) c-R (3)
[In the formula (3),
L represents a leaving group,
R represents a hydroxyl protecting group which can be deprotected under acidic conditions;
c shows the integer of 3-37. ]

6 ≦ b + c ≦ 40 (F1)
[In the formula (F1),
b is an integer of 3 to 37,
c shows the integer of 3-37. ]

_{HO- (CH 2 CH 2 O)} a-R ··· (4)
[In the formula (4),
R represents a hydroxyl protecting group which can be deprotected under acidic conditions;
a represents an integer of 6 to 40; ]

_{(M) AO- (CH 2 CH} 2 O) a-R ··· (5)
[In the formula (5),
R represents a hydroxyl protecting group which can be deprotected under acidic conditions;
a represents an integer of 6 to 40; ]

[3] The method is characterized in that in the step [D], one or more solvents selected from the group consisting of dichloromethane and chloroform are used as an organic phase, and liquid purification is performed by liquid separation at 0 to 20 ° C. , [2] way.

[4] Monodispersed polyethylene glycol mono (meth) acrylate represented by the formula (1).

_{(M) AO- (CH 2 CH} 2 O) a-H ··· (1)
[In the formula (1),
(M) A is a (meth) acryloyl group,
a represents an integer of 6 to 40; ]

[5] A method for producing monodispersed polyethylene glycol mono (meth) acrylate comprising performing the following step [A], step [B], step [C] and step [D] in this order.

Step [A]: A compound represented by Formula (2) is subjected to a nucleophilic substitution reaction so as to satisfy Formula (F1), and a compound represented by Formula (4) is converted into a compound represented by Formula (4) Step [B]: A step of obtaining a compound represented by Formula (5) by (meth) acrylation of a compound represented by Formula (4) with (meth) acrylic acid anhydride or (meth) acrylic acid chloride Step [C]: A step of deprotecting a compound represented by the formula (5) to obtain a mixture containing monodispersed polyethylene glycol mono (meth) acrylate represented by the formula (1) Step [D]: the above step A step of purifying said mixture obtained in [C]

_{HO- (CH 2 CH 2 O)} b-H ··· (2)
[In Formula (2), b shows the integer of 3-37. ]

LO-(CH ₂ CH ₂ O) c-R (3)
[In the formula (3),
L represents a leaving group,
R represents a hydroxyl protecting group which can be deprotected under acidic conditions;
c shows the integer of 3-37. ]

6 ≦ b + c ≦ 40 (F1)
[In the formula (F1),
b is an integer of 3 to 37,
c shows the integer of 3-37.

_{HO- (CH 2 CH 2 O)} a-R ··· (4)
[In the formula (4),
R represents a hydroxyl protecting group which can be deprotected under acidic conditions;
a represents an integer of 6 to 40; ]

_{(M) AO- (CH 2 CH} 2 O) a-R ··· (5)
[In the formula (5),
R represents a hydroxyl protecting group which can be deprotected under acidic conditions;
a represents an integer of 6 to 40; ]

[6] The method is characterized in that in the step [D], one or more solvents selected from the group consisting of dichloromethane and chloroform are used as an organic phase, and liquid purification is performed by liquid separation at 0 to 20 ° C. , [5] way.

  According to the present invention, there is provided a comb polymer which can be easily analyzed when checking the purity of a pharmaceutical product obtained by combining a biofunctional compound and a polyethylene glycol mono (meth) acrylate polymer and the purified product after separation and purification. it can.

  Furthermore, according to the present invention, monodispersed polyethylene glycol mono (meth) acrylate, which is a raw material of the monodispersed polyethylene glycol mono (meth) acrylate polymer, and an easy manufacturing method without by-production of a crosslinkable compound. It becomes possible to offer.

It is a reverse phase chromatography measurement result of the compound 5 measured in Example 2-1. It is a result of reverse phase chromatography measurement of polyethylene glycol monomethacrylate (manufactured by Aldrich, 409529, average molecular weight 500) having a hydroxyl group at a commercial end measured in Comparative Example 2-3. It is a reverse phase chromatography measurement result of polyethyleneglycol monomethacrylate (The product made by Aldrich, 447943, average molecular weight 465) which has the methoxy group in the commercial end measured by Comparative Example 2-4. It is a gel permeation chromatography measurement result of the polymer 1 measured in Example 3. FIG. It is a MALDI-TOF MS measurement result of the polymer 1 measured in Example 4. FIG. It is a gel permeation chromatography measurement result of the comparative polymer 1 measured by Comparative Example 3-1. It is a gel permeation chromatography measurement result of the comparative polymer 2 measured by Comparative Example 3-2. It is a MALDI-TOF MS measurement result of the comparative polymer 2 measured by the comparative example 4. FIG.

Hereinafter, preferred embodiments of the present invention will be described in detail.
Monodispersed polyethylene glycol mono (meth) acrylate in the present invention is a compound having a hydroxyl group and a (meth) acryloyl group at the end of monodispersed polyethylene glycol. In addition, monodispersed polyethylene glycol is a compound having a purity of 90% or more (hereinafter referred to as chain length purity) of a compound consisting of a specific ethylene glycol repeat number.

This monodispersed polyethylene glycol mono (meth) acrylate is represented by Formula (1).
_{(M) AO- (CH 2 CH} 2 O) a-H ··· (1)

  In said Formula (1), (M) A shows a (meth) acryloyl group, a is an integer of 6-40 which represents the repeating number of monodispersed polyethylene glycol. When it is used as a drug bound to a biofunctional compound, a is preferably 8 to 24 from the viewpoint of suppressing the aggregation of the hydrophobic biofunctional compound.

  The chain purity of the compound represented by the formula (1) is measured by reverse phase chromatography. In reverse phase chromatography, after identifying each peak using a mass spectrometer for the detector, measure and calculate by changing the detector to a suggested refractometer on the same column, developing solvent conditions Purity is determined from the area value of each peak. The measurement conditions are not particularly limited as long as each peak is detected separately, but when a mass spectrometer is used as a detector, for example, measurement is performed under the following conditions.

Equipment: Waters Co., Ltd. Alliance 2695
Detector (mass spectrometer): Quattro micro tandem mass spectrometer manufactured by Waters Co., Ltd.
Column: TSKgel ODS-80Ts manufactured by Tosoh Corporation
(Particle diameter 5 μm, column size 4.6 mm × 25 cm)
Developing solvent: 5 mM ammonium acetate methanol / distilled water = V1 / V2
V1 and V2 represent the volume ratio of methanol and distilled water. V1 and V2 are suitably selected by the repeating number of monodispersed polyethylene glycol of the compound to be measured.

When a suggested refractometer is used for the detector, for example, measurement is performed under the following conditions.
Detector (differential refractometer): RI-8020 manufactured by Tosoh Corp.
Column: TSKgel ODS-80Ts manufactured by Tosoh Corporation
(Particle diameter 5 μm, column size 4.6 mm × 25 cm)
Developing solvent: 5 mM ammonium acetate methanol / distilled water = V1 / V2
V1 and V2 represent the volume ratio of methanol and distilled water, and are synonymous with V1 and V2 used for measurement conditions when a mass spectrometer is used as a detector.

<Method of producing monodispersed polyethylene glycol mono (meth) acrylate>
The monodispersed polyethylene glycol mono (meth) acrylate of the present invention can be produced by a production method comprising at least the following [Step A], [Step B], [Step C], and [Step D] in this order: .

(Step [A])
The step [A] according to the present invention is a compound HO- (CH ₂ CH ₂ O) b-H represented by the following formula (2) (2)
A compound represented by the following formula _{(3) LO- (CH 2 CH} 2 O) c-R ··· (3)
And the condition 6 ≦ b + c ≦ 40 represented by the following formula (F1) (F1)
By nucleophilic substitution reaction to satisfy the compounds represented by the following formula _{(4) HO- (CH 2 CH} 2 O) a-R ··· (4)
Is a process of obtaining

  In the above formula (2), b represents an integer of 3 to 37, in the above formula (3), L represents a leaving group, R represents a protecting group for a hydroxyl group which can be deprotected under acidic conditions, c Represents an integer of 3 to 37. Moreover, b in Formula (2) and c in Formula (3) are b + c = 6-40, and satisfy | fill the conditions represented by said Formula (F1).

  The compound represented by the said Formula (2) and the compound represented by Formula (3) can use a commercial item, and can be obtained by a well-known synthetic | combination method. Moreover, as a compound represented by said Formula (3), the compound which added the leaving group to the compound represented by said Formula (4) obtained by this process [A] can be utilized. In addition, it is possible to synthesize | combine the compound with many ethylene glycol repeating numbers by repeating a process [A] using the compound which added the leaving group to the compound represented by said Formula (4).

Reaction product containing the compound represented by the formula (4) by subjecting the compound represented by the formula (2) and the compound represented by the formula (3) to nucleophilic substitution reaction in the presence of a base You can get In said Formula (4), R is a protecting group of the hydroxyl group which can be deprotected under acidic conditions, and a shows the integer of 6-40. Further, R in the formula (4) is derived from R in the formula (3). In the reaction product, the following formula (6)
_{RO- (CH 2 CH 2 O)} d-R ··· (6)
The compound represented by is contained as an impurity. In following formula (6), R shows the protective group of the hydroxyl group which can be deprotected under acidic conditions, d shows the integer of 8-80. Further, R in the following formula (6) is derived from R in the above formula (3).

  In the above formulas (3) and (4), R may be a protecting group of a hydroxyl group which can be deprotected under acidic conditions, and may be a protecting group which does not decompose the (meth) acrylic ester upon deprotection in the subsequent step [C]. There is no particular limitation. As the protective group, those which can be deprotected under acidic conditions, preferably trityl (Trt) group, p-methoxybenzyl (PMB) group, tetrahydropyranyl (THP) group, bis (2-chloroethoxy) methyl (BOM) group Α-naphthyldiphenylmethyl group, p-methoxyphenyldiphenylmethyl group, t-butyldimethylsilyl (TBS) group, isopropyldimethylsilyl group, tribenzylsilyl group, triisopropylsilyl (TIPS) group, more preferably trityl (Trt) And p-methoxybenzyl (PMB) and t-butyldimethylsilyl (TBS).

  In the above formula (3), L is a leaving group and is not particularly limited as long as it produces an ether bond by the nucleophilic substitution reaction, and tosyl group, mesyl group, trifluoromethanesulfonyl group, iodine atom, bromine atom And are selected from chlorine atoms.

  The nucleophilic substitution reaction can be carried out in a solvent. The solvent is not particularly limited as long as the solvent does not react with the compound represented by the formula (2) and the compound represented by the formula (3), for example, tetrahydrofuran, acetonitrile, dimethylformamide, dichloromethane, chloroform And aprotic polar solvents, and mixtures thereof. The amount of the solvent used is usually 1 to 100 times, preferably 2 to 50 times, and most preferably 3 to 30 times the weight ratio of the compound represented by the formula (3). When the amount of the solvent used is less than the lower limit, the compound represented by the formula (3) is bonded to both ends of the compound represented by the formula (2). If the amount exceeds the upper limit, the progress of the nucleophilic substitution reaction tends to be delayed.

  In the nucleophilic substitution reaction, the amount of the compound represented by the formula (2) is usually 1.1 to 50 times, preferably at a molar ratio to the compound represented by the formula (3). Is 1.5 to 30 times, more preferably 2.0 to 20 times. When the amount of the compound represented by the formula (2) is less than the lower limit, the compound represented by the formula (3) is bonded to both ends of the compound represented by the formula (2) The production amount of the compound represented by the formula (6) tends to be large, while when it exceeds the upper limit, the progress of the nucleophilic substitution reaction tends to be delayed.

  As the base used for the nucleophilic substitution reaction, there is no problem if the reaction proceeds, and examples thereof include metallic sodium, sodium hydride and potassium tert-butoxide. The amount of the base used is usually 1.1 to 10 times, preferably 1.2 to 5 times the molar ratio of the compound represented by the formula (3).

  The reaction temperature of the nucleophilic substitution reaction varies depending on the solvent used and the like, but is usually 0 to 100 ° C. If the reaction temperature is below the lower limit, the reaction may be delayed, whereas if the temperature is above the upper limit, the side reaction may proceed due to an excessive temperature. Moreover, as reaction time of the said nucleophilic substitution reaction, although it changes with conditions, such as said reaction temperature, about 0.2 to 48 hours are preferable normally.

  In the step [A], a reaction product containing the compound represented by the formula (4) and the compound represented by the formula (6) can be obtained by such a nucleophilic substitution reaction. The reaction product may be used as it is unrefined and used in the next step [B], and the compound represented by the formula (4) is purified by silica gel column chromatography, separation extraction treatment, adsorbent treatment, etc. Although the compound represented by the formula (6) is not reactive in the reaction in the step [B] described later, and may be purified in the step described later, it is unrefined. It can be used.

(Step [B])
Process [B] which concerns on this invention (meth) acrylates the compound represented by said Formula (4), and following formula (5)
_{(M) AO- (CH 2 CH} 2 O) a-R ··· (5)
Is a process of obtaining In said Formula (5), (M) A shows a (meth) acryloyl group, R shows the protective group of the hydroxyl group which can be deprotected by acid condition conditioning, and a shows the integer of 6-40.

  In the step [B], the reaction product obtained in the step [A] is reacted with either (meth) acrylic anhydride or (meth) acrylic acid chloride in the presence of a base to obtain the above formula A reaction product containing the compound represented by (5) and the compound represented by the formula (6) can be obtained.

  The reaction in step [B] can be carried out in a solvent. Examples of the solvent include toluene, tetrahydrofuran, acetonitrile, dimethylformamide, dichloromethane, chloroform, and mixtures thereof. The amount of the solvent used is usually 1 to 100 times, preferably 2 to 50 times, and most preferably 3 to 30 times the mass ratio of the compound represented by the formula (4). When the amount of the solvent used is less than the lower limit, it may be difficult to control the reaction due to heat generation. On the other hand, when the amount exceeds the upper limit, the progress of the reaction tends to be delayed.

  As a base used at a process [B], when (meth) acryl chloride is used as a (meth) acrylating reagent, a triethylamine, a diisopropyl ethylamine, a pyridine is mentioned, for example. The amount of the base used is usually 1.1 to 10 times, preferably 2 to 5 times the molar ratio of the compound represented by the formula (4).

  When (meth) acrylic anhydride is used as the (meth) acrylate reagent as the base used in step [B], for example, dimethylaminopyridine is added as a base catalyst in addition to bases such as triethylamine, diisopropylethylamine, and pyridine be able to. The amount of the base used is usually 1.1 to 10 times, preferably 2 to 5 times the molar ratio of the compound represented by the formula (4). The amount of the organic catalyst used is usually 0.01 to 1.0 times by mole, preferably 0.05 to 0.5 times the molar ratio of the compound represented by the formula (4) It is.

  The reaction temperature of step [B] varies depending on the solvent used and the like, but is usually -20 to 40 ° C. If the reaction temperature is less than the lower limit, the progress of the reaction may be delayed. If the upper limit is exceeded, the generated compound represented by the formula (5) may cause a polymerization reaction. There is. Moreover, as reaction time of the said reaction, although it changes with conditions, such as reaction temperature, about 0.2 to 48 hours are preferable normally.

  In addition, in the step [B], excess (meth) acrylic acid anhydride and (meth) acrylic acid chloride can be removed after completion of the reaction. As the method, an alcohol can be added to quench excess (meth) acrylic acid anhydride and (meth) acrylic acid chloride, and remove at least under reduced pressure. As the alcohol, there is no problem as long as the (meth) acrylic acid ester produced by reacting with excess (meth) acrylic acid anhydride and (meth) acrylic acid chloride can be concentrated under reduced pressure, for example, having 4 or less carbon atoms Alcohols are mentioned, preferably methanol, ethanol, isopropyl alcohol, most preferably methanol. When an alcohol having a large number of carbon atoms is used, it is necessary to heat the (meth) acrylic acid ester to be formed under reduced pressure, which may cause a polymerization reaction of the compound represented by the formula (5). is there.

  The amount of the alcohol used is usually 0.01 to 2 times by weight, preferably 0.05 to 0.5 times by weight the amount of (meth) acrylic acid anhydride and (meth) acrylic acid chloride. . When the amount used is less than the lower limit, (meth) acrylic acid anhydride and (meth) acrylic acid chloride remain, and in step [C] and later, (meth) acrylic acid anhydride and (meth) acrylic acid chloride are A reacted by-product may be generated, and on the other hand, when the amount exceeds the upper limit, unreacted alcohol remains, and when the liquid separation extraction process is performed, the layer separation may be hindered. The temperature at which the alcohol is added is usually 30 ° C. or less, preferably 20 ° C. or less. When the said temperature exceeds an upper limit, there exists a possibility that the compound represented by the said Formula (5) may raise | generate a polymerization reaction.

  As a pressure reduction degree in vacuum concentration, 50 kPa or less is preferable, 40 kPa or less is more preferable, and 30 kPa or less is more preferable. If the degree of vacuum is equal to or higher than the above-mentioned value, the (meth) acrylic acid ester produced can not be distilled off, or it may take time to evaporate.

  The vacuum concentration can be performed while stirring to prevent bumping, and can also be performed while bubbling air, since the compound represented by the formula (5) suppresses polymerization.

  The temperature in vacuum concentration is usually 60 ° C. or less, preferably 40 ° C. or less. When the said temperature exceeds an upper limit, there exists a possibility that the compound represented by the said Formula (5) may raise | generate a polymerization reaction.

  Since the excess (meth) acrylic acid anhydride and (meth) acrylic acid chloride can be removed by the above operation, (meth) acrylic acid anhydride and (meth) acrylic acid in the reaction after [Step C] It is no longer possible to produce by-products in which chloride is reacted.

  In the step [B], a reaction product containing the compound represented by the formula (5) and the compound represented by the formula (6) can be obtained by such a reaction. The reaction product may be used as it is unrefined and used in the next [Step C], and the compound represented by the formula (5) is purified by silica gel column chromatography, separation extraction treatment, adsorbent treatment, etc. Although it may be used afterward, in the present invention, it can be used unrefined because purification is possible in the steps described later.

(Step [C])
Process [C] which concerns on this invention is a process of deprotecting the compound represented by said Formula (5), and obtaining the mixture containing the compound represented by said Formula (1). In the reaction product, the compound represented by the formula (6) is deprotected, the following formula (7)
_{HO- (CH 2 CH 2 O)} d-H ··· (7)
The compound represented by is contained as an impurity. In said Formula (7), d shows the integer of 8-80.

  As a method of deprotecting the protecting group of a hydroxyl group which can be deprotected under the above acidic conditions, known methods can be used, and for example, the method described in Protective Groups in Organic Sysnthesis by GREEN WUTS is effective. is there. Moreover, when R in said Formula (5) and (6) is Trt group, TBS group, and PMB group, it is possible to make it deprotect by the conversion reaction under acidic conditions. As the conversion reaction under the above acidic conditions, for example, the reaction is carried out at 60 ° C. in 1 M hydrochloric acid, or the reaction is carried out by adding a catalytic amount of p-toluenesulfonic acid monohydrate in methanol. There is a method such as

  In the step [C], a reaction product containing the compound represented by the formula (1) and the compound represented by the formula (7) can be obtained by such a deprotection reaction. The reaction product may be used as it is in the crude process of the next step [D], and the compound represented by the formula (1) is purified by silica gel column chromatography, separation extraction treatment, adsorbent treatment, etc. Although it may be used afterward, in the present invention, it can be used unrefined because purification is possible in the steps described later.

(Step [D])
In the step [D] according to the present invention, the monodispersed polyethylene glycol mono (meth) acrylate is purified by purifying a reaction product containing the compound represented by the formula (1) obtained in the step [C]. It is a process to obtain.

  The compound represented by the formula (7) generated as an impurity in the step [C] is a compound contained as compared to monodispersed polyethylene glycol mono (meth) acrylate since both terminals are hydroxyl groups. Has a large difference in polarity. Therefore, they can be easily separated and removed by purification operations such as silica gel column chromatography, liquid separation extraction treatment, adsorbent treatment and the like. In particular, according to the production method of the present invention, it is possible to obtain the monodispersed polyethylene glycol mono (meth) acrylate of the present invention of high purity only by a simple liquid separation extraction treatment without purification by silica gel column chromatography. it can.

  Examples of the liquid separation and extraction treatment include a method including a washing step of liquid separation washing with an aqueous solution at 25 ° C. or lower after dissolving the reaction product obtained in the step [C] in an organic solvent. Examples of the organic solvent include toluene, chloroform and dichloromethane. Among these, chloroform and dichloromethane are preferable from the viewpoint of the solubility of the compound represented by the formula (1). The amount of the organic solvent used is usually 2 to 30 times the mass ratio of the reaction product containing the compound represented by the formula (1) and the compound represented by the formula (7) , Preferably 3 to 20 times. When the amount of the organic solvent used is less than the lower limit, the compound represented by the formula (1) may be dissolved in the aqueous layer, and when it exceeds the upper limit, the formula (7) may be used. The compound represented by may be dissolved in the organic layer.

  In the washing step, the aqueous solution is not particularly limited as long as it can dissolve the compound represented by the formula (7), and examples thereof include aqueous solutions of ion exchanged water, sodium chloride, and potassium chloride. The amount of the aqueous solution to be used is usually 2 to 30 times, preferably, the mass ratio of the reaction product containing the compound represented by the formula (1) and the compound represented by the formula (7). Is 3 to 20 times. When the amount of the aqueous solution used is less than the lower limit, the washing efficiency of the compound represented by the formula (7) decreases, and when it exceeds the upper limit, it is represented by the formula (1) Compounds may dissolve into the water layer.

  In the washing step, as the ratio of the organic solvent to the aqueous solution, the value of the organic solvent / aqueous solution is usually 0.2 to 3.0 and 0.5 to 2.0 in mass ratio. Is preferred.

  As a temperature of the said washing | cleaning process, it is preferable that it is 0-25 degreeC, and it is still more preferable that it is 5-20 degreeC. When the temperature exceeds the upper limit, the compound represented by the formula (7) tends to be difficult to dissolve and remove in the organic layer. The number of times of the liquid separation washing is not particularly limited, and the compound represented by the formula (7) contained in the organic solvent by thin layer chromatography (TLC), mass spectrometry (MS) measurement, etc. It is preferable to carry out several times while checking.

  In the step [D], monodispersed polyethylene glycol mono (meth) acrylate of the present invention containing the compound represented by the formula (1) in high purity can be easily obtained by such separation extraction treatment. it can. According to the present invention, since it is possible to remove the impurities generated in the steps [A] to [C] by such simple purification, purification by silica gel column chromatography or the like is unnecessary in each step. It is. The monodispersed polyethylene glycol mono (meth) acrylate containing the compound represented by the above-mentioned formula (1) is used as it is for the production of the polymer of monodispersed polyethylene glycol mono (meth) acrylate of the present invention. Although it is possible, it may be further purified by treatment such as crystallization, adsorbent treatment, silica gel column chromatography and the like.

(Step [E])
Process [E] concerning this invention superposes | polymerizes the monodispersed polyethyleneglycol mono (meth) acrylate represented by Formula (1) obtained by said process [D], and is monodispersed polyethyleneglycol mono (meth) acrylate Is a process of obtaining

  The molecular weight of the polymer obtained by radical polymerization of monodispersed polyethylene glycol mono (meth) acrylate represented by the above formula (1) is not particularly limited, and polymerization is performed so that the performance required in each application can be exhibited. Although conditions and the like can be prepared and appropriately determined, usually, when it is used as a medicine having a weight average molecular weight of about 1000 to 1000000 and the polymer is bound to a biofunctional compound, 2000 to 40,000 is preferable.

  The monodispersed polyethylene glycol mono (meth) acrylate represented by the formula (1) polymerizes a mixture of a compound represented by the formula (1) and another monomer capable of copolymerizing with the compound represented by the formula (1). be able to.

  The other monomer can be appropriately selected depending on the application, and, for example, diethylaminoethyl (meth) acrylate, polyethylene glycol mono (meth) acrylate, glycerol (meth) acrylate, (meth) acryloyloxyethyl phosphate, (meth) Acryloyloxyethyl phosphorylcholine, N-methylcarboxybetaine (meth) acrylate, N-methylsulfobetaine (meth) acrylate, methyl (meth) acrylate, glycidyl (meth) acrylate, dimethylaminoethyl (meth) acrylate, 2-hydroxyethyl methacrylate , Various (meth) acrylic acid esters such as 2-methoxyethyl (meth) acrylate; various vinyl ethers such as methyl vinyl ether; others, acrylamide, N, N'- Dimethyl acrylamide, N-isopropyl (meth) acrylamide, (meth) acrylic acid, allyl alcohol, acrylonitrile, acrolein, vinyl acetate, sodium vinyl sulfonate, styrene, chlorostyrene, vinyl phenol, vinyl cinnamate, vinyl chloride, vinyl bromide, Various radically polymerizable monomers such as butadiene, vinylene carbonate, itaconic acid, itaconic acid ester, fumaric acid, fumaric acid ester, maleic acid and maleic acid ester may be mentioned. When using the said polymer as a pharmaceutical couple | bonded with the biofunctional compound, a homopolymer is desirable.

  The compound represented by the formula (1) may be used for polymerization in the bulk state as it is, or a solution may be added for polymerization. Moreover, if a compound represented by said Formula (1) melt | dissolves as a solvent, it will not be specifically limited, A common solvent can be used. For example, polar aprotic solvents such as acetone, dioxane, dimethylformamide (DMF), dimethylsulfoxide (DMSO), tetrahydrofuran (THF), polar protic solvents such as methanol, and nonpolar solvents such as dichloromethane and toluene can be used.

  The radical polymerization of the compound represented by the formula (1) can be carried out by thermal polymerization or photopolymerization. The thermal polymerization can be performed using a thermal polymerization initiator. As a thermal polymerization initiator, for example, peroxide type radical initiators (benzoyl peroxide, ammonium persulfate, etc.) or azo type radical initiators (azobisisobutyronitrile (AIBN), 2,2'-azobis-dimethyl) Valeronitrile (ADVN, etc.), 2,2'-azobiscyanovaleric acid (ACVA), water-soluble or oil-soluble redox radical initiators (consisting of dimethyl aniline and benzoyl peroxide) can be used.

  The photopolymerization can be performed, for example, by ultraviolet light (UV) with a wavelength of 254 nm or electron beam (EB) irradiation with an acceleration voltage of 150 to 300 kV. Under the present circumstances, although use of a photoinitiator is arbitrary, it is preferable to use from the point of reaction time. Examples of the photopolymerization initiator include 2-hydroxy-2-methyl-1-phenyl-1-propanone, 1-hydroxy-cyclohexyl phenyl ketone and the like, but from the viewpoint of solubility etc. 2-hydroxy-2-methyl-1 Preferred is -phenyl-1-propanone.

  Radical polymerization of the compound represented by the above formula (1) can also be carried out by living radical polymerization, and more specifically, atom transfer radical polymerization (ATRP method), reversible addition fragmentation chain transfer polymerization ( The RAFT polymerization method and the nitroxide-mediated polymerization method (NMP method) can be used. In particular, in modification applications of biofunctional compounds, the reversible addition fragmentation chain transfer polymerization method (RAFT polymerization method) is preferable from the viewpoint of ease of introduction of reactive functional groups to the polymer main chain and no metal. Known methods can be used as the method of the RAFT polymerization, and the methods described in, for example, WO 99/31144, WO 98/01478 and US Pat. No. 6,153,705 are effective.

  When the monodispersed polyethylene glycol mono (meth) acrylate polymer of the present invention produced by the steps [A] to [E] is measured by MALDI-TOF MS, the main peak is represented by the formula (1) It is observed for each molecular weight of the structural unit derived from monodispersed polyethylene glycol mono (meth) acrylate. In other words, the spacing between adjacent main peaks corresponds to the molecular weight of the structural unit derived from monodispersed polyethylene glycol mono (meth) acrylate.

In the present invention, the measurement conditions of the MALDI-TOF MS are the following conditions.
Instrument: AB SCIEX TOF / TOF 5800 SYSTEM
Matrix: α-cyano-4-hydroxycinnamic acid (5 mg / ml solution, water / acetonitrile = 5/5 v / v 0.1% trifluoroacetic acid)
Sample concentration: 0.4 mg / ml
Calibration sample: Standard reagent (molecular weight 1000 to 16000) manufactured by BRUKER
Measurement method: Linear method

Here, the main peak is a monoisotopic ion peak excluding a background peak, an isotope ion peak, a peak derived from a matrix or an impurity, and is derived from monodispersed polyethylene glycol mono (meth) acrylate It is the peak with the highest intensity observed in the width of the building block.
More specifically, in the chart obtained by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry, when the height of the highest peak is 100, the peak is 70 to 100 in height. It is referred to as the main peak. In the present invention, a plurality of main peaks are observed at intervals corresponding to the molecular weight M of the structural unit derived from monodispersed polyethylene glycol mono (meth) acrylate.
At the same time, in the range from (molecular weight of each main peak-M / 2) to (molecular weight of each main peak + M / 2), the height of any peak other than the main peak is 60% or less Do. Such peaks not observed at intervals corresponding to the molecular weight M of the constitutional unit derived from monodispersed polyethylene glycol mono (meth) acrylate are peaks derived from fluctuation, impurities, and background.
The number of main peaks observed at intervals corresponding to the molecular weight M of the structural unit derived from monodispersed polyethylene glycol mono (meth) acrylate is preferably 3 or more, more preferably 4 or more, and particularly preferably 5 or more. The upper limit of the number of main peaks observed at intervals corresponding to the molecular weight M is not particularly limited, but may be 15 or less or 10 or less from the viewpoint of the actual polymerization reaction.

Hereinafter, the present invention will be more specifically described based on examples, but the present invention is not limited to the following examples. In each synthesis example, JMTC-400 manufactured by Nippon Denshi Co., Ltd. is used for nuclear magnetic resonance ( ¹ H-NMR) measurement, and for mass spectrometry (ESI-MS) measurement, Quattro micro tandem type mass manufactured by Waters Corporation. An analyzer was used.

（式中、Ｔｒｔはトリチル基を表す） Example 1-1
Synthesis Example I-1:
Synthesis of Compound 2 in which a in Formula (3) is 4, R is a Trityl Group, and L is a Mesyl Group)

(Wherein, Trt represents a trityl group)

First, Compound 1 was synthesized as shown in the above formula. To a two-necked round-bottomed flask was added tetraethylene glycol (417 g, 2.12 mol), 2,6-di-t-butyl-p-hydroxytoluene (417 mg, 1.89 mmol) and toluene (375 ml). A Dean-Stark tube and a condenser were attached and heated to reflux to remove water azeotropically (45 ml of toluene was collected). Purge the inside of the eggplant flask with nitrogen, add triethylamine (52.5 g, 0.52 mol), trityl chloride (120 g, 0.43 mol) and dimethylaminopyridine (5.25 g, 0.042 mol), and then at room temperature Stir for 4 hours. After 4 hours, the disappearance of trityl chloride was confirmed using thin layer chromatography (hexane: ethyl acetate = 1: 1 by vol), and 200 ml of 1 M aqueous hydrochloric acid solution was added. After adding 20 ml of toluene to the mixture, the solution was separated. The organic phase was washed once with 100 ml of 1 M aqueous hydrochloric acid, once with 100 ml of saturated aqueous sodium bicarbonate and twice with 100 ml of saturated brine. Sodium sulfate was added to the organic phase, dried and filtered. The filtrate was concentrated under reduced pressure to obtain pale yellow clear liquid compound 1.
Yield 164g
MS (ESI +): Compound 1 454.5 [M ⁺ NH ₄ ] ⁺

（式中、Ｍｓはメシル基を表す）

(Wherein, Ms represents a mesyl group)

Next, Compound 2 was synthesized as shown in the above formula. Compound 1 in an eggplant flask (164
800 ml of toluene was added to g, <0.38 mol). The inside of the eggplant flask was purged with nitrogen, triethylamine (62.4 ml, 0.45 mol) and mesyl chloride (31.9 ml, 0.41 mol) were added, and the mixture was stirred at room temperature for 2 hours. After 2 hours, the disappearance of Compound 1 was confirmed by ESI-MS, and 300 ml of 1 M HCl aq was added to separate the layers. The organic phase was washed once with 300 ml of 1 M aqueous hydrochloric acid, twice with 300 ml of saturated aqueous sodium bicarbonate and once with 300 ml of saturated brine. Sodium sulfate was added to the organic phase, dried and filtered. The filtrate was concentrated under reduced pressure to obtain pale yellow clear liquid compound 2.
Yield 189g
MS (ESI +): Compound 2 532.2 [M ⁺ NH ₄ ] ⁺

(Step [A])
Synthesis Example I-2:
Synthesis of Compound 3 in which a in Formula (4) is 8 and R is Trityl Group)

Next, Compound 3 was synthesized as shown in the above formula. Sodium hydride (20.8 g) was placed in a two-necked eggplant flask and purged with nitrogen. It was washed twice with dehydrated hexane (50 ml × 2 times), and 500 ml of acetonitrile was added and cooled to 0 °. Acetonitrile 100 ml was mixed with tetraethylene glycol (579 g, 2.94 mol) azeotropically dehydrated three times with 50 ml of toluene, added to a dropping funnel, and added dropwise over 30 minutes. After completion of the dropwise addition, 100 ml of acetonitrile was mixed with Compound 2 (189 g, <0.37 mol) azeotropically dehydrated three times with 50 ml of toluene, and the mixture was added to the same dropping funnel and dropped over 5 minutes. After completion of the dropwise addition, the reaction mixture was heated to 80 ° C. and stirred for 4 hours. After 4 hours, it was confirmed by ¹ H-NMR spectrum (CDCl ₃ ) that the compound 2 disappeared, and the mixture was allowed to cool to room temperature. The reaction mixture was concentrated under reduced pressure, and 200 ml of toluene was added to the residue. The toluene solution was washed twice with 200 ml of saturated aqueous ammonium chloride solution and three times with 200 ml of saturated brine. Sodium sulfate was added to the organic phase, dried and filtered. The filtrate was concentrated under reduced pressure to obtain pale yellow clear liquid compound 3.
Yield 212g
MS (ESI +): Compound 3 630.8 [M ⁺ NH ₄ ] ⁺

(Step [B])
(Synthesis example I-3:
Synthesis of Compound 4 in which a in Formula (5) is 8 and R is Trityl Group)

Next, Compound 4 was synthesized as shown in the above formula. Compound 3 (34.7 g, 56.6 mmol), triethylamine (31.3 ml, 226 mmol), and methacrylic anhydride (17.4 g, 113) azeotropically dehydrated three times with toluene (20 ml) in a two-necked eggplant flask The mmol) was added and dissolved in toluene (375 ml). After cooling to 0 ° C., dimethylaminopyridine (2.77 g, 22.7 mmol) was added and stirred at 0 ° C. for 2 hours. After 2 hours, the disappearance of compound 3 was confirmed using ESI-MS. Methanol (1 ml) was added to the reaction mixture and stirred at room temperature for 1 hour to consume excess methacrylic anhydride. Saturated aqueous ammonium chloride solution (10 ml) was added to the reaction solution and concentrated under reduced pressure. Toluene (100 ml) was added to the obtained residue. The organic phase was washed twice with saturated aqueous ammonium chloride solution (100 ml), twice with saturated aqueous sodium hydrogen carbonate solution (100 ml), and twice with saturated brine (100 ml). Sodium sulfate was added to the organic phase, dried and filtered. The filtrate was concentrated under reduced pressure to give pale yellow liquid compound 4.
Yield 32.0 g
MS (ESI +): compound 4 698.4 [M ⁺ NH ₄ ] ⁺

(Step [C], [D])
(Synthesis example I-4:
Synthesis of Compound 5 wherein a in Formula (1) is 8)

Next, Compound 5 was synthesized as shown in the above formula. Compound 4 (32.0 g, 47.0 mmol), toluenesulfonic acid monohydrate (4.48 g, 23.6 mmol) and methanol (120 ml) and hexane (100 ml) were added to an eggplant flask. The extraction of hexane (100 ml) was repeated once every hour, and the addition of fresh hexane (100 ml) was repeated, and after stirring for 4 hours at room temperature, disappearance of compound 4 was confirmed using ESI-MS. To the mixture was added saturated aqueous sodium hydrogen carbonate solution (30 ml) to stop the reaction, and the solvent was evaporated under reduced pressure. Dichloromethane (150 ml) was added and the organic phase was washed three times with 100 ml of ion-exchanged water at 20 ° C. and once with 150 ml of saturated brine. Sodium sulfate was added to the organic phase, dried and filtered. The filtrate was concentrated under reduced pressure to obtain pale yellow clear liquid compound 5. The molar yield based on compound 2 was 53.9%.
Yield 14.2g
MS (ESI +): Compound 4 456.3 [M ⁺ NH ₄ ] ^+
1 H-NMR (CD ₃ OD, 400 MHz):
7.28 (s, 1 H), 6. 13 (s, 1 H), 5. 57 (t, 2 H)
3.64 (m, 30 H), 2.62 (b, 1 H)

（式中、ＰＭＢはｐ−メトキシベンジル基を表す） (Example 1-2)
(Step [A])
Synthesis Example II-1:
Synthesis of Compound 7 in which a in Formula (5) is 12 and R is a PMB Group)

(Wherein PMB represents p-methoxybenzyl group)

Next, Compound 7 was synthesized as shown in the above formula. Sodium hydride (11.4 g) was placed in a two-necked eggplant flask and purged with nitrogen. It was washed twice with dehydrated hexane (50 ml × 2 times), and 500 ml of acetonitrile was added and cooled to 0 °. Acetonitrile (100 ml) was mixed with octaethylene glycol (1087 g, 2.94 mol) azeotropically dehydrated with 50 ml of toluene three times, added to a dropping funnel, and added dropwise over 30 minutes. After completion of the dropwise addition, 100 ml of acetonitrile was mixed with compound 6 (290 g, <0.37 mol) azeotropically dehydrated with 50 ml of toluene three times, added to the same dropping funnel and dropped over 5 minutes. After completion of the dropwise addition, the reaction mixture was heated to 80 ° C. and stirred for 4 hours. After 4 hours, it was confirmed by ¹ H-NMR spectrum (CDCl ₃ ) that the compound 6 disappeared and was allowed to cool to room temperature. The reaction mixture was concentrated under reduced pressure, and 200 ml of toluene was added to the residue. The toluene solution was washed twice with 200 ml of saturated aqueous ammonium chloride solution and three times with 200 ml of saturated brine. Sodium sulfate was added to the organic phase, dried and filtered. The filtrate was concentrated under reduced pressure to obtain pale yellow clear liquid compound 7.
Yield 220g

(Step [B])
Synthesis Example II-2:
Synthesis of Compound 8 in which a in Formula (5) is 12 and R is a PMB Group)

Next, Compound 8 was synthesized as shown in the above formula. Compound 7 (36.0 g 56.6 mmol) which was azeotropically dehydrated three times with toluene (50 ml) in a two-necked eggplant flask, triethylamine (31.3 ml, 226 mmol), methacrylic acid chloride (11.8 g, 113 mmol) Was dissolved in toluene (375 ml). After stirring at 0 ° C. for 2 hours, disappearance of compound 7 was confirmed using ESI-MS. Saturated aqueous ammonium chloride solution (10 ml) was added to the reaction solution and concentrated under reduced pressure. Toluene (100 ml) was added to the obtained residue. The organic phase was washed twice with saturated aqueous ammonium chloride solution (100 ml), twice with saturated aqueous sodium hydrogen carbonate solution (100 ml), and twice with saturated brine (100 ml). Sodium sulfate was added to the organic phase, dried and filtered. The filtrate was concentrated under reduced pressure to give pale yellow liquid compound 8.
Yield 33.1 g

(Step [C], [D])
Synthesis Example II-3:
Synthesis of Compound 9 wherein a in Formula (1) is 12)

Next, compound 9 was synthesized as shown in the above formula. Compound 8 (33.1 g, 47.0 mmol), toluenesulfonic acid monohydrate (4.48 g, 23.6 mmol) and methanol (120 ml) and hexane (100 ml) were added to an eggplant flask. The extraction of hexane (100 ml) was repeated once every hour, and the addition of fresh hexane (100 ml) was repeated, and after stirring for 4 hours at room temperature, disappearance of compound 8 was confirmed using ESI-MS. To the mixture was added saturated aqueous sodium hydrogen carbonate solution (30 ml) to stop the reaction, and the solvent was evaporated under reduced pressure. Chloroform (150 ml) was added and the organic phase was washed three times with 100 ml of ion-exchanged water at 10 ° C. and once with 150 ml of saturated brine. Sodium sulfate was added to the organic phase, dried and filtered. The filtrate was concentrated under reduced pressure, and silica gel chromatography (developing solvent: ethyl acetate, methanol) was performed to obtain a transparent liquid compound 8. The molar yield based on compound 6 was 44.8%.
Yield 16.6g

（式中、ＴＢＳはｔ−ブチルジメチルシリル基を表す） (Example 1-3)
(Step [A])
Synthesis Example III-1:
Synthesis of Compound 10 in which a in Formula (5) is 24 and R is a TBS Group)

(Wherein, TBS represents a t-butyldimethylsilyl group)

Next, compound 11 was synthesized as shown in the above formula. Sodium hydride (11.4 g) was placed in a two-necked eggplant flask and purged with nitrogen. It was washed twice with dehydrated hexane (50 ml × 2 times), and 500 ml of acetonitrile was added and cooled to 0 °. Acetonitrile (100 ml) was mixed with dodecaethylene glycol (1604 g, 2.94 mol) azeotropically dehydrated three times with 50 ml of toluene, added to a dropping funnel, and added dropwise over 30 minutes. After completion of the dropwise addition, 100 ml of acetonitrile was mixed with Compound 10 (367 g, <0.37 mol) azeotropically dehydrated three times with 50 ml of toluene, and the mixture was added to the same dropping funnel and dropped over 5 minutes. After completion of the dropwise addition, the reaction mixture was heated to 80 ° C. and stirred for 4 hours. After 4 hours, it was confirmed by ¹ H-NMR spectrum (CDCl ₃ ) that the compound 10 disappeared and was allowed to cool to room temperature. The reaction mixture was concentrated under reduced pressure, and 200 ml of toluene was added to the residue. The toluene solution was washed twice with 200 ml of saturated aqueous ammonium chloride solution and three times with 200 ml of saturated brine. Sodium sulfate was added to the organic phase, dried and filtered. The filtrate was concentrated under reduced pressure to obtain pale yellow clear liquid compound 11.
Yield 411g

(Step [B])
Synthesis Example III-2:
Synthesis of Compound 12 wherein a in Formula (5) is 24 and R is a TBS group)

Next, Compound 12 was synthesized as shown in the above formula. Compound 11 (67.3 g, 56.6 mmol), triethyl amine (22.9 ml, 226 mmol), and methacrylic anhydride (17.4 g, 113) azeotropically dehydrated with toluene (20 ml) three times in a two-necked eggplant flask The mmol) was added and dissolved in toluene (375 ml). After cooling to 0 ° C., dimethylaminopyridine (2.77 g, 22.7 mmol) was added and stirred at 0 ° C. for 2 hours. After 2 hours, disappearance of compound 11 was confirmed using ESI-MS. Methanol (1 ml) was added to the reaction mixture and stirred at room temperature for 1 hour to consume excess methacrylic anhydride. Saturated aqueous ammonium chloride solution (10 ml) was added to the reaction solution and concentrated under reduced pressure. Toluene (100 ml) was added to the obtained residue. The organic phase was washed twice with saturated aqueous ammonium chloride solution (100 ml), twice with saturated aqueous sodium hydrogen carbonate solution (100 ml), and twice with saturated brine (100 ml). Sodium sulfate was added to the organic phase, dried and filtered. The filtrate was concentrated under reduced pressure to give pale yellow liquid compound 12.
Yield 59.1g

(Step [C], [D])
(Synthesis example III-3:
Synthesis of Compound 13 wherein a in Formula (1) is 24)

Next, compound 13 was synthesized as shown in the above formula. Compound 12 (59.1 g, 47.0 mmol), toluenesulfonic acid monohydrate (4.48 g, 23.6 mmol) and methanol (120 ml) and hexane (100 ml) were added to an eggplant flask. Hexane (100 ml) was withdrawn once every hour, and addition of fresh hexane (100 ml) was repeated, and after stirring for 4 hours at room temperature, disappearance of compound 12 was confirmed using ESI-MS. To the mixture was added saturated aqueous sodium hydrogen carbonate solution (30 ml) to stop the reaction, and the solvent was evaporated under reduced pressure. Chloroform (75 ml) dichloromethane (75 ml) was added and the organic phase was washed three times with 100 ml of ion-exchanged water at 5 ° C. and once with 150 ml of saturated brine. Sodium sulfate was added to the organic phase, dried and filtered. The filtrate was concentrated under reduced pressure, and crystallization was performed with ethyl acetate and hexane to obtain a transparent liquid compound 13. The molar yield based on compound 10 was 46.4%.
Yield 32.4g

(Measurement of chain length purity)
(Example 2-1)
The chain length purity of the compound 5 synthesized in Example 1-1 first identifies each peak by reverse phase chromatography using a mass spectrometer as a detector, and then uses a differential refractometer as a detector. Then, reverse phase chromatography measurement was performed using the same column and developing solvent, and determined from the calculated area value of each peak. When using a mass spectrometer for the detector, use the instrument as an Alliance 2695 manufactured by Waters Co., Ltd., the detector (mass spectrometer) as a Quattro micro tandem mass spectrometer manufactured by Waters Co., Ltd., and as a column to Tosoh Corp. Manufactured by TSKgel ODS-80Ts (particle diameter 5 μm, column size 4.6 mm × 25 cm), using 5 mM ammonium acetate methanol / distilled water = 55/45 (volume ratio) as a developing solvent, and a flow rate of 0.6 mL / mL The measurement was performed under the conditions of min, column temperature 40 ° C., sample concentration 0.01 mg / g, and injection volume 5 μL.

When a differential refractometer is used for the detector, the instrument is a build GPC system HLC-8220 manufactured by Tosoh Corp., a detector (differential refractometer) RI-8020 manufactured by Tosoh Corp., and a column Tosoh Corp. Co., Ltd. TSKgel ODS-80Ts (particle diameter 5 μm, column size 4.6 mm × 25 cm) was used as a developing solvent, 5 mM ammonium acetate methanol / distilled water = 55/45 (volume ratio), respectively. The test was performed under the conditions of 6 mL / min, column temperature 40 ° C., sample concentration 0.2 mg / mL, and injection volume 40 μL.
FIG. 1 shows the results of reverse phase chromatography measurement using a differential refractometer as a detector.

  As a result of measurement using a mass spectrometer, the peak having a retention time of 4 to 7 minutes is a peak derived from a solvent, and the peak detected at 11.0 to 11.8 minutes is a single peak of 7 in formula (1). The dispersed polyethylene glycol monomethacrylate was detected, and the peaks detected at 11.8 to 13.3 minutes were monodispersed polyethylene glycol monomethacrylates in which a is 8 and 9 in the formula (1). As described above, the detected peak is only the monodispersed polyethylene glycol monomethacrylate having a value of 7 to 9 in the formula (1), and according to the measurement result using the differential refractometer, the a in the formula (1) is The chain purity of monodispersed polyethylene glycol monomethacrylate of 8 was 96.3%.

(Example 2-2)
The chain purity of the compound 9 synthesized in Example 1-2 was the same as in Example 2-1 except that the developing solvent was changed to 5 mM ammonium acetate methanol / distilled water = 60/40 (volume ratio). Calculated. As a result, the detected peak is only monodispersed polyethylene glycol monomethacrylate in which a is different in Formula (1), and the chain length purity of the monodispersed polyethylene glycol monomethacrylate in which a is 12 in Formula (1) is 95. It was 4%.

(Example 2-3)
The purity of the compound 13 synthesized in Example 1-3 was changed to Example 2 except that the developing solvent was changed to 5 mM ammonium acetate methanol / distilled water = 65/35 (volume ratio) and the column temperature was changed to 45 ° C. It calculated by the method similar to 1. As a result, the detected peak is only monodispersed polyethylene glycol monomethacrylate in which a is different in Formula (1), and the chain length purity of the monodispersed polyethylene glycol monomethacrylate in which a is 24 in Formula (1) is 94. It was 6%.

(Comparative Example 1-1)
(Synthesis of Comparative Compound 1)
Compound 3 (34.7 g 56.6 mmol) obtained in Synthesis Example I-2 azeotropically dehydrated with toluene (50 ml) three times in a two-necked eggplant flask, toluenesulfonic acid (10.7 g, 56.4 mmol) ), Methacrylic acid (9.7 g, 113 mmol) were added and dissolved in toluene (375 ml). After stirring at 90 ° C. for 6 hours, disappearance of compound 3 was confirmed using ESI-MS. The reaction mixture was washed twice with saturated aqueous sodium hydrogen carbonate solution (100 ml) and twice with saturated brine (100 ml). Sodium sulfate was added to the organic phase, dried and filtered, and the filtrate was concentrated under reduced pressure. To the residue was added methanol (100 ml) and washed 5 times with hexane (100 ml). The methanol phase was concentrated under reduced pressure to obtain pale yellow liquid comparative compound 1.
30g yield

(Comparative example 1-2)
(Synthesis of Comparative Compound 2)
A reaction was performed in the same manner as in Synthesis Example I-4 except that separation of the dichloromethane-water system in Synthesis Example I-4 was not performed, and Comparative compound 2 was obtained.

(Comparative example 2-1)
The chain purity of the comparative compound 1 synthesized in Comparative Example 1-1 was measured by the same method as Example 2-1. As a result, according to the measurement using a mass spectrometer, a compound in which a methacryl group is introduced at both ends of polyethylene glycol or a compound in which a is 4 to 12 in the formula (6) in which both ends are a hydroxyl group Was found to be mixed as an impurity. From the measurement results using a differential refractometer, using the area value of monodispersed polyethylene glycol monomethacrylate alone, the chain length purity of the monodispersed polyethylene glycol monomethacrylate having a of 8 in Formula (1) is 95%. However, the purity including the area value of other impurities was 70.2%.

(Comparative Example 2-2)
The chain purity of Comparative Compound 2 synthesized in Comparative Example 1-2 was measured in the same manner as Example 2-1. As a result, it was found by measurement using a mass spectrometer that the compound represented by a of 4 to 12 is mixed as an impurity in the formula (6) in which both ends are hydroxyl groups. From the measurement results using a differential refractometer, using the area value of monodispersed polyethylene glycol monomethacrylate alone, the chain length purity of the monodispersed polyethylene glycol monomethacrylate having a of 8 in Formula (1) is 95% However, the purity including the area value of other impurities was 88.3%.

(Comparative example 2-3)
The purity of commercially available polyethylene glycol monomethacrylate having a hydroxyl group at a terminal (manufactured by Aldrich, product number 409529, average molecular weight 500) was measured in the same manner as in Example 2-1. FIG. 2 shows the results of reverse phase chromatography measurement using a differential refractometer as a detector.

  A retention time of 4 to 7 minutes is a peak derived from a solvent, a peak of 7 to 11 minutes is polyethylene glycol, and 11 to 35 minutes includes polyethylene glycol monomethacrylates having different polyethylene glycol chain lengths, and 25 to 25 minutes. By 60 minutes, polyethylene glycol dimethacrylates of different polyethylene glycol chain lengths were contained. Since it is a mixture having a distribution as polyethylene glycol monomethacrylate having a hydroxyl group at an end, and impurities are also present, calculation of chain purity and purity is difficult.

(Comparative example 2-4)
The purity of commercially available polyethylene glycol monomethacrylate (manufactured by Aldrich, product number 447943, average molecular weight 465) having a methoxy group at an end was measured by the same method as in Example 2-1.
FIG. 3 shows the results of reverse phase chromatography measurement using a differential refractometer as a detector.

  As a result of measurement using a mass spectrometer, the peak having a retention time of 4 to 7 minutes is a peak derived from a solvent, the peak of 10 to 12 minutes is polyethylene glycol methyl ether, and the peaks of 12 to 35 minutes are each polyethylene glycol It was a polyethylene glycol monomethacrylate having a methoxy group at the end of repeating number 4 to 17. Since it is a mixture having distribution as polyethylene glycol monomethacrylate having a methoxy group at the end, and impurities are mixed, calculation of chain length purity and purity is difficult.

(Example 3)
(RAFT polymerization of Compound 5)
Next, the compound 5 synthesized in Example 1-1 was subjected to radical polymerization to synthesize a polymer 1. Compound 5 (1.44 g, 3.13 mmol), 4-cyanopentanoic acid dithiobenzoate (37.2 mg, 0.133 mmol), 4-4 'azobis (4-cyanovaleric acid) (37) in an eggplant flask .3 mg, 0.133 mmol) was dissolved in DMF (10 ml). After bubbling nitrogen for 15 minutes, the temperature was raised to 70 ° C. to initiate polymerization. After 4 hours, the reaction solution was reprecipitated with 90 ml of an acetone / hexane mixed solvent (1/4 v / v) to obtain Polymer 1.

The number average molecular weight and the molecular weight distribution (weight average molecular weight / number average molecular weight) of the obtained polymer 1 were determined by gel permeation chromatography using a differential refractometer as a detector. Instrument: Tosoh Corp. Build GPC System HLC-8220, Detector: Differential Refractometer RISK: Tosoh Corp. RI-8020, Column: Agilent Mixed-D (particle diameter 5 μm, column Size 4.6 mm × 25 cm) are linked in two, and 11.5 mM lithium bromide-containing dimethylformamide is used as a developing solvent, flow rate 0.6 mL / min, column temperature 40 ° C., sample concentration 0.2 mg / mL, injection volume It performed on the conditions of 70 microliters. Methyl methacrylate was used as a standard.
FIG. 4 shows the results of gel permeation chromatography measurement.

  As a result of gel permeation chromatography measurement, the number average molecular weight of the polymer 1 was 12400, and the molecular weight distribution was 1.09.

(Example 4)
(MALDI-TOF MS measurement)
The molecular weight of Polymer 1 synthesized in Example 3 was measured by matrix-assisted laser desorption / ionization (MALDI-TOF MS). MALDI-TOF MS measurement uses AB SCIEX TOF / TOF 5800 SYSTEM, and α-cyano-4-hydroxycinnamic acid (5 mg / ml solution, water / acetonitrile = 5/5 v / v 0.1% trifluoro) as a matrix Using acetic acid), the sample was mixed with the matrix at 1/2 (v / v) to prepare a solution with a final concentration of 0.4 mg / ml, of which 1 μl was loaded on the plate. The calibration was performed using a BRUKER standard reagent (molecular weight: 1000 to 16000), and the measurement was performed by the linear method.
As a result of the measurement, as shown in FIG. 5, the main peak was detected for each molecular weight (438) of the structural unit derived from the monomer (compound 5).

  This makes it easy to analyze the number of polymer 1 bound to the drug when it is used as a drug bonded to a biofunctional compound.

(Comparative Example 3-1)
(RAFT Polymerization of Polyethylene Glycol Monomethacrylate Having a Hydroxyl Group at the Commercially Available Terminal)
Comparative Polymer 1 was synthesized by radically polymerizing commercially available polyethylene glycol monomethacrylate having a hydroxyl group at a terminal (manufactured by Aldrich, product number 409529, average molecular weight 500). Polyethylene glycol monomethacrylate (2.16 g, 4.65 mmol), 4-cyanopentanoic acid dithiobenzoate (55.9 mg, 0.020 mmol), 4-4 'azobis (4-cyanovaleric acid) in an eggplant flask (28.0 mg, 0.010 mmol) was dissolved in DMF (15 ml). After bubbling nitrogen for 15 minutes, the temperature was raised to 70 ° C. to initiate polymerization. After 4 hours, the reaction solution was reprecipitated with 90 ml of an acetone / hexane mixed solvent (1/4 v / v) to obtain Comparative Polymer 1.

  The number average molecular weight of the obtained comparative polymer 1 was measured in the same manner as in Example 3. The gel permeation chromatography measurement result is shown in FIG. As a result of gel permeation chromatography measurement, in Comparative Polymer 1, a part of the polymer is crosslinked by the polyethylene glycol dimethacrylate having a methacryl group at both ends contained as an impurity in the monomer, and a plurality of peaks are confirmed, and the number average It was difficult to calculate molecular weight and molecular weight distribution.

(Comparative example 3-2)
(Raft polymerization of polyethylene glycol monomethacrylate having a methoxy group at a commercially available end)
Comparative Polymer 2 was synthesized by radical polymerization of commercially available polyethylene glycol monomethacrylate (manufactured by Aldrich, 447943-100 ML, average molecular weight 465) having a methoxy group at an end. Polyethylene glycol monomethacrylate (2.16 g, 4.65 mmol), 4-cyanopentanoic acid dithiobenzoate (55.9 mg, 0.020 mmol), 4-4 'azobis (4-cyanovaleric acid) in an eggplant flask (28.0 mg, 0.010 mmol) was dissolved in DMF (15 ml). After bubbling nitrogen for 15 minutes, the temperature was raised to 70 ° C. to initiate polymerization. After 4 hours, the reaction solution was reprecipitated with 90 ml of an acetone / hexane mixed solvent (1/4 v / v) to obtain Comparative Polymer 2.

  The number average molecular weight of the obtained comparative polymer 2 was measured in the same manner as in Example 3. FIG. 7 shows the results of gel permeation chromatography measurement. As a result of gel permeation chromatography measurement, the number average molecular weight of the comparative polymer 2 was 9000, and the molecular weight distribution was 1.11.

(Comparative example 4)
(MALDI-TOF MS measurement of comparative polymer 2)
The molecular weight of the comparative polymer 2 synthesized in Comparative Example 3-2 was subjected to MALDI-TOF MS measurement in the same manner as in Example 4. As a result of the measurement, many peaks were observed as shown in FIG. This is because the molecular ion peak is separated and detected for each ethylene glycol repeating number of m / z = 44 (CH ₂ CH ₂ O), and it is observed that it is an ethylene glycol unit, m / z = 44 (CH ₂₎ This is because each of CH ₂ O) was observed, and when it is used as a drug bound to a biofunctional compound, analysis of the number of bound comparative polymer 2 to the drug becomes complicated.

  As described above, according to the present invention, monodispersed polyethylene glycol mono (meth) acrylate containing a hydroxyl group at the end and containing, as a main component, a compound consisting of a single ethylene glycol repeating number, in particular with high purity. It is possible to provide As described above, the monodispersed polyethylene glycol mono (meth) acrylate of the present invention can reduce the complexity at the time of analysis due to the small number of compounds different in ethylene glycol repetition number. In addition, since it does not contain a crosslinkable compound, it is possible to obtain a target linear polymer without causing crosslinking or gelation at the time of radical polymerization.

Furthermore, the method for producing monodispersed polyethylene glycol mono (meth) acrylate of the present invention can be carried out using a suitable protecting group and combining the synthesis steps in a specific order without generating a crosslinkable compound by-product and also using column chromatography. Since the above-mentioned high purity monodispersed polyethylene glycol mono (meth) acrylate can be produced only by liquid separation extraction without using a purification method such as chromatography, it is a production method particularly suitable for industrialization.

Claims (6)

A polymer of monodispersed polyethylene glycol mono (meth) acrylate represented by the formula (1), wherein
A plurality of main peaks are observed at intervals corresponding to the molecular weight of the constitutional unit derived from the monodispersed polyethylene glycol mono (meth) acrylate by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry. , Monodispersed polyethylene glycol mono (meth) acrylate polymers.

_{(M) AO- (CH 2 CH} 2 O) a-H ··· (1)
[In the formula (1),
(M) A is a (meth) acryloyl group,
a represents an integer of 6 to 40; ] A method for producing a monodispersed polyethylene glycol mono (meth) acrylate polymer, comprising performing the following step [A], step [B], step [C], step [D] and step [E] in this order .

Step [A]: The compound represented by Formula (4) is subjected to a nucleophilic substitution reaction so as to satisfy Formula (F1), and the compound represented by Formula (4) Step [B]: The compound represented by Formula (4) is (meth) acrylated with (meth) acrylic anhydride or (meth) acrylic acid chloride to obtain a compound represented by Formula (5) Step [C]: a step of deprotecting the compound represented by the formula (5) to obtain a mixture containing monodispersed polyethylene glycol mono (meth) acrylate represented by the formula (1): Step [D]: the above A step of purifying said mixture obtained in step [C]

Step [E]: A step of polymerizing the monodispersed polyethylene glycol mono (meth) acrylate represented by the formula (1) obtained in the step [D].

_{HO- (CH 2 CH 2 O)} b-H ··· (2)
[In Formula (2), b shows the integer of 3-37. ]

LO-(CH ₂ CH ₂ O) c-R (3)
[In the formula (3),
L represents a leaving group,
R represents a hydroxyl protecting group which can be deprotected under acidic conditions;
c shows the integer of 3-37. ]

6 ≦ b + c ≦ 40 (F1)
[In the formula (F1),
b is an integer of 3 to 37,
c shows the integer of 3-37. ]

_{HO- (CH 2 CH 2 O)} a-R ··· (4)
[In the formula (4),
R represents a hydroxyl protecting group which can be deprotected under acidic conditions;
a represents an integer of 6 to 40; ]

_{(M) AO- (CH 2 CH} 2 O) a-R ··· (5)
[In the formula (5),
R represents a hydroxyl protecting group which can be deprotected under acidic conditions;
a represents an integer of 6 to 40; ]
The method is characterized in that in the step [D], one or more solvents selected from the group consisting of dichloromethane and chloroform are used as the organic phase, and liquid purification is performed by liquid separation at 0 to 20 ° C. for purification. Method 2 described.
Monodispersed polyethylene glycol mono (meth) acrylate represented by Formula (1).

_{(M) AO- (CH 2 CH} 2 O) a-H ··· (1)

[In the formula (1),
(M) A is a (meth) acryloyl group,
a represents an integer of 6 to 40; ]
A method for producing monodispersed polyethylene glycol mono (meth) acrylate comprising performing the following step [A], step [B], step [C] and step [D] in this order.

Step [A]: A compound represented by Formula (2) is subjected to a nucleophilic substitution reaction so as to satisfy Formula (F1), and a compound represented by Formula (4) is converted into a compound represented by Formula (4) Step [B]: A step of obtaining a compound represented by Formula (5) by (meth) acrylation of a compound represented by Formula (4) with (meth) acrylic acid anhydride or (meth) acrylic acid chloride Step [C]: A step of deprotecting a compound represented by the formula (5) to obtain a mixture containing monodispersed polyethylene glycol mono (meth) acrylate represented by the formula (1) Step [D]: the above step A step of purifying said mixture obtained in [C]

_{HO- (CH 2 CH 2 O)} b-H ··· (2)
[In Formula (2), b shows the integer of 3-37. ]

LO-(CH ₂ CH ₂ O) c-R (3)
[In the formula (3),
L represents a leaving group,
R represents a hydroxyl protecting group which can be deprotected under acidic conditions;
c shows the integer of 3-37. ]

6 ≦ b + c ≦ 40 (F1)
[In the formula (F1),
b is an integer of 3 to 37,
c shows the integer of 3-37. ]

_{HO- (CH 2 CH 2 O)} a-R ··· (4)
[In the formula (4),
R represents a hydroxyl protecting group which can be deprotected under acidic conditions;
a represents an integer of 6 to 40; ]

_{(M) AO- (CH 2 CH} 2 O) a-R ··· (5)
[In the formula (5),
R represents a hydroxyl protecting group which can be deprotected under acidic conditions;
a represents an integer of 6 to 40; ]

  The method is characterized in that in the step [D], one or more solvents selected from the group consisting of dichloromethane and chloroform are used as the organic phase, and liquid purification is performed by liquid separation at 0 to 20 ° C. for purification. Method 5 described.

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