JPS6238368B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a highly reactive polymer having a large number of carboxyl groups (or salts thereof) in the side chain and an active group containing a thiol group or an active disulfide bond at one end of the main chain, and a method for producing the same. Regarding. The purpose of the present invention is to manufacture target-directed anti-cancer drugs (anti-tumor drugs) by combining anti-tumor antibodies, etc. that have the ability to bind to targets such as tumor cells, and cytotoxic substances, such as anti-cancer drugs. The object of the present invention is to provide a polymer that can be used to effectively and efficiently combine the two. BACKGROUND ART Conventionally, it has been known to use a reactive polymer as a mediator in order to efficiently bind an antitumor antibody and a cytotoxin to produce a target-directed anticancer agent. For example, in JP-A No. 51-126281, an antitumor immunoglobulin and a polymer carrier having 5 to 500 anticancer drug molecules covalently bonded to each molecule, such as polyglutamic acid, are bonded through an amide bond to form an antitumor drug. It is disclosed that it has been obtained. The antitumor agent obtained by this method selectively binds to tumor cells,
It is a drug of great interest as it is expected to exert toxicity on tumor cells. However, a drawback of this known antitumor agent is that the antitumor antibody and the toxic moiety (polymer carrier bound to the antitumor agent) are bound to each other through an amide bond, that is, via a free amino group or carboxyl group in the antitumor antibody. The point is that this is done through a combination of Immunoglobulins also have many amino groups and carboxyl groups in their antigen recognition sites. Therefore, when a cytotoxic substance or toxic moiety is bound to an anti-tumor immunoglobulin through an amide bond, the cytotoxic substance will also bind to the antigen recognition site of the anti-tumor immunoglobulin, and as a result, the resulting Antitumor drugs no longer have the ability to bind to tumor cells at all, or
Otherwise, a problem arises in that it is lowered. In addition, in the method described in JP-A-51-126281, within the antibody molecule and within the polyglutamic acid molecule,
Alternatively, amide bonds are formed between molecules of the same type. And the antitumor agents obtained as a result of the formation of these undesirable amide bonds have reduced performance,
Furthermore, there is the problem that they contain high molecular weight substances that are unsuitable for use in tumor treatment. The present inventors have conducted extensive research to solve the drawbacks of the prior art, and have found that it is possible to bind cytotoxins or toxic moieties using disulfide bonds that exist in limited positions in immunoglobulins. It was discovered that an antitumor agent free from the above-mentioned drawbacks can be obtained. The present invention provides a reactive polymer that can be optimally used in the production of such antitumor agents or other target-directed drugs. That is, in the present invention, 60 mol% or more of the structural units consist of structural units represented by the formula [], In [Formula [], Z represents a hydrogen atom or a monovalent cation. m represents an integer from 1 to 4. ] When the constituent units of formula [] are not 100 mol%,
The remaining structural units are expressed by the formula [′] [In formula [′], R is -H, -CH ₃ ,

It is a group represented by [Formula] or -CH ₂ OH. ], and has an active group represented by the formula [] at the carboxyl terminal of the main chain, In [formula], W represents an alkylene group having 1 to 4 carbon atoms, and X is a hydrogen atom or a 2-pyridylthio group, a 4-pyridylthio group, a 3-carboxy-
4-nitrophenylthio group, 4-carboxy-2
-pyridylthio group, N-oxy-2-pyridylthio group, 2-nitrophenylthio group, 4-nitro-
Forms an active disulfide bond with an adjacent sulfur atom selected from the group consisting of 2-pyridylthio group, 2-benzothiazoylthio group, 2-benzimidazoylthio group, and N-phenylamino-N'-phenyliminomethylthio group. It is a group that can be used. R ₁
represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ] It is a reactive polymer with a degree of polymerization of 5 to 3000. The reactive polymer of the present invention can bind cytotoxic substances such as anticancer drugs to it by utilizing the large number of carboxyl groups (or salts thereof) present in the side chain, and can also bind cytotoxic substances such as anticancer drugs to the reactive polymer present at one end of the main chain. It is possible to bind immunoglobulins such as anti-tumor antibodies to this by utilizing the active group. In formula [], Z is a hydrogen atom or a monovalent cation, such as Na ⁺ , K ⁺ , NH ₄ ⁺ . m is 1
It represents an integer of ~4, but m is preferably 1 or 2. In addition, in the reactive polymer of the present invention, among the structural units represented by the formula [], for example, those in which m=1 and those in which m=2 may be mixed. These total 60 of the total constituent units.
It is sufficient if it is at least 80 mol%, preferably at least 80 mol%. In the reactive polymer of the present invention, 40
Constituent units other than those represented by the formula [] may be contained within a range of less than mol%. Examples of these include α-amino acids, such as glycine, alanine, phenylalanine, and serine, which do not have a carboxyl group (or a salt thereof) in the α-position side chain. Such a structural unit consisting of an α-amino acid does not participate in any way in binding with a cytotoxic substance, but it regulates the water solubility of the reactive polymer and the lipid solubility and water solubility of the polymer obtained by binding the cytotoxic substance. It may be helpful. Therefore, if there is no particular need to adjust fat solubility or water solubility, it is practically advantageous to use a product that does not contain such a constituent unit consisting of an α-amino acid. In formula [], X represents a hydrogen atom or a group capable of forming an active disulfide bond with the adjacent sulfur atom, and the latter is, for example, a 2-pyridylthio group.

[Formula] 4-pyridylthio group

[Formula] 3-carboxy-4-nitro phenylthio group

[Formula] 4-carboxy-2-pyridylthio group

[Formula] N-oxy-2-pyri dilthio group

[Formula] 2-nitrophenyl Thio group

[Formula] 4-nitro-2-pyri dilthio group

[Formula] 2-benzothi Azoylthio group

[Formula] 2-ben Zoimidazoylthio group

[Formula] and N-phenylamino-N'-phenyliminomethylthio group

There is a [formula]. In formula [], W represents a divalent organic group,
It is not particularly limited as long as it is an inert group that does not participate in any reaction during the process of obtaining the reactive polymer of the present invention and the subsequent reaction process. These groups include, for example, 2-aminoethanethiol residue (-
linear or cysteine benzyl ester residues such as CH ₂ CH ₂ −)

[Formula] and homocysteine benzyl ester residue

Alkylene group having a side chain such as [Formula], 4-aminothiophenol residue

Examples include phenylene groups with no substituents or with substituents as shown in [Formula],
Particularly preferred is an alkylene group having 1 to 4 carbon atoms.
R ₁ is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, preferably a hydrogen atom. Among the reactive polymers of the present invention, those in which X is a hydrogen atom, that is, the carboxyl terminal of the main chain has the following formula [-a] [In formula [-a], the definitions of W and R ₁ are the same as in formula []. ] A method for producing a reactive polymer having an active group represented by the following will be explained. In this method, 60 mol% or more of the structural units consist of structural units represented by the above formula [], and the following formula [] is present in the main chain or at the carboxyl terminal of the main chain. In [formula [], the definitions of W and R ₁ are the same as in formula []. R ₂ represents an alkyl group, an aralkyl group, or an aryl group when the group represented by the formula [] is a terminal group of the main chain, and when the group represented by the formula [] is present in the main chain, teeth,

It is a divalent group represented by the formula: However, W' is a divalent organic group that is the same as or different from W, and is bonded to S in the formula []. R ₁ ' is the same as or different from R ₁ and represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ] This is a method of reductively cleaving disulfide bonds in the polymer by reacting a hydrophilic polymer having a disulfide bond-containing group represented by the following with a thiol compound or a borohydride compound. The reaction between a hydrophilic polymer and a thiol compound is usually carried out in a homogeneous reaction system using water or an organic solvent such as dimethylformamide or dimethyl sulfoxide as a reaction solvent. Suitable thiol compounds include, for example, dithiothreitol and 2-mercaptoethanol. The thiol compound is used in a molar amount of 1 to 100 times the amount of disulfide bonds in the polymer. The reaction temperature is preferably -5° to 70°C, and the reaction time is preferably 5 minutes to 10 days. When using a borohydride compound, such as sodium borohydride or potassium borohydride, the reaction with the polymer is usually carried out in an aqueous solution. The method for producing the hydrophilic polymer having a disulfide bond-containing group represented by the above formula [] will be described in detail in Reference Examples below, but the outline thereof is as follows. For example, glutamic acid benzyl ester (γ-benzyl-L-glutamic acid) is reacted with phosgene to obtain γ-benzyl-L-glutamate N-carboxylic acid anhydride, which is converted into, for example, n-propyl 2-aminoethyl disulfide. (CH ₃ CH ₂ CH ₂ S-SCH ₂ CH ₂ NH ₂ ) to form a polymer, and when this polymer is decomposed with an acid or alkali, a hydrophilic polymer having the group of the above formula [] at the end of the molecule is obtained. is obtained. Anhydride is polymerized using, for example, cystamine (H ₂ NCH ₂ CH ₂ SSCH ₂ CH ₂ NH ₂ ) to form a polymer, and when this polymer is decomposed with acid or alkali, the group of the above formula [] is added to the main chain. A hydrophilic polymer having the following properties is obtained. Among the reactive polymers of the present invention, those in which X is a group capable of forming an active disulfide bond with the adjacent sulfur atom, that is, the carboxyl terminal of the main chain has the following formula [-b] [In formula [-b], the definitions of W and R ₁ are the same as in formula []. X' represents a group capable of forming an active disulfide bond with the adjacent sulfur atom. ] A method for producing a reactive polymer having an active group represented by the following will be explained. The method involves reacting a polymer containing an active group represented by the formula [-a] at the carboxyl terminal of the molecule obtained as described above, that is, a polymer containing a thiol group, with an active disulfide compound. This is the way to do it. As the active disulfide compound, for example, 2-pyridyl disulfide

[Formula] 4-pyridyldisul Fido

[Formula] 5,5'-dithiobis(2-nitrobenzoic acid)

[Formula] 4-carboxy-2-pyridyl disulfide N-oxy-2-pyridyl disulfide

[Formula] 2-nitrophenyldi sulfide

[Formula] 4-nitro-2-pyridyl disulfide

[Formula] 2-benzothiazoyl disulfide

[Formula] 2-benzimidazoyl disulfide

[Synthesis of γ-benzyl-L-glutamate N-carboxylic acid anhydride]

A dispersion was prepared by adding 10.0 g of γ-benzyl-L-glutamic acid to 120 ml of anhydrous tetrahydrofuran. Separately, under a nitrogen atmosphere, add 20 ml of trichloromethyl chloroformate to 10.0 g of carbon black.
It was gradually added dropwise over 70 minutes to generate phosgene. The generated phosgene is the γ-benzyl-L
- The dispersion of glutamic acid was inhaled under a nitrogen atmosphere. After 70 minutes, the dispersion became a pale yellow transparent liquid, so the phosgene was stopped, and then nitrogen was sucked in for 1.5 hours to remove unreacted phosgene. The solvent was distilled off from the resulting transparent liquid under reduced pressure under a nitrogen stream (140 mmHg, 27°C). 150 ml of anhydrous n-hexane was added to the residue to dissolve it, and the mixture was stirred on an ice bath for 5 minutes to precipitate a white solid. This solid was purified by reprecipitation twice in a system of ethyl acetate-n-hexane (anhydrous) under a nitrogen atmosphere, sucked off, and dried under reduced pressure to produce γ-benzyl-L-glutamate N-carboxylic anhydride (described below). 7.75 g (having the structural formula) were obtained as a white solid. The melting point of this product was 94.0-94.5°C (decomposition), and the yield was 69.8%. Reference Example 2 7.75 g of γ-benzyl-L-glutamate N-carboxylic acid anhydride obtained in Reference Example 1 was dried 1,4
-Dissolved in 185 ml of dioxane under nitrogen atmosphere with stirring. To the solution thus obtained, a solution obtained by dissolving 95 mg of cystamine (H ₂ NCH ₂ CH ₂ SSCH ₂ CH ₂ NH ₂ ) in 10 ml of dry dioxane was added and mixed, and the mixture was incubated at room temperature under nitrogen atmosphere for 24 hours. The mixture was stirred to carry out a polymerization reaction. After the reaction, the reaction mixture was added to the isopropyl ether of 4 with stirring to precipitate the produced polymer. When the precipitated white polymer was taken and dried under reduced pressure, the yield was 6.19
The yield was 95.9%. The average molecular weight of the obtained polymer was determined by the viscosity method (dichloroacetic acid, 25.0°C) and was found to be 47,300 (P. Doty et al., J. Am. Chem Soc., Vol. 78, 947).
(see p. 1956). The obtained polymer has the following formula poly-γ from the raw materials, initiator, and reaction mechanism used.
It was reasonably estimated that the substance was mainly composed of -benzyl-L-glutamate, and was also confirmed by infrared absorption spectrum. 3.11 g of the poly-γ-benzyl-L-glutamate obtained above was dissolved in a mixed solution of 25.0 ml of trifluoroacetic acid and 4.5 ml of anisole. To the thus obtained solution, 25.0 ml of methanesulfonic acid was added, and the mixture was stirred for 20 minutes on an ice bath under a nitrogen atmosphere, and then stirred for 30 minutes at room temperature to carry out an acid decomposition reaction of the γ-benzyl ester. After the reaction, the reaction mixture is
The polymer was precipitated by stirring into 450 ml of isopropyl ether. The precipitated white polymer was suctioned off and suspended in 50 ml of water. About 60 ml of saturated water was added and mixed to this, and the mixture was stirred at room temperature for 30 minutes to carry out a carboxyl group neutralization reaction.
Thereafter, the resulting reaction solution was dialyzed against pure water at 4° C. for 3 days using a cellulose tube, and then freeze-dried to obtain 1.91 g of a white solid. When the obtained solid was examined by infrared absorption spectrum, it was confirmed that the absorption of benzyl ester had disappeared and that the carboxyl group had become a sodium salt. The yield of poly-L-glutamate as sodium salt was 89.3%. In addition, viscosity method (salt-phosphoric acid buffer mixture,
The average molecular weight determined using ionic strengths of 0.11 and 1.01 (25.5°C) was 29.200 (RBHawkins et al.
Macromolecules, vol. 5, p. 294, 1972).
The obtained polymer is mainly composed of a sodium salt of poly-L-glutamate of the following formula. Reference Example 3 5.50 g of γ-benzyl-L-glutamate N-carboxylic acid anhydride obtained in Reference Example 1 was dried 1,4
-Dissolved in 150 ml of dioxane under nitrogen atmosphere with stirring. A solution obtained by dissolving 142 mg of n-propyl 2-aminoethyl disulfide (CH ₃ CH ₂ CH ₂ SSCH ₂ CH ₂ NH ₂ ) in 10 ml of dry dioxane was added to the solution obtained in this manner, and the mixture was stirred under a nitrogen atmosphere. The mixture was stirred at room temperature for 40 hours to carry out a polymerization reaction. After the reaction, the reaction mixture was added to the isopropyl ether of 4 with stirring to precipitate the produced polymer. When the precipitate was collected and dried under reduced pressure, the yield was 4.41 g, with a yield of 96.3%. Next, 4.00 g of the obtained polymer was dissolved in a mixed solution of 35 ml of trifluoroacetic acid and 5.0 ml of anisole, and 35 ml of methanesulfonic acid was added, and the mixture was incubated for 30 minutes under ice-cooling under a nitrogen atmosphere and for 30 minutes at room temperature. By stirring, the γ-benzyl ester was acid-decomposed. After the reaction was completed, the reaction mixture was added to 540 ml of isopropyl ether with stirring, and the polymer was precipitated and collected. This was dissolved in 100 ml of 5.0% heavy sour water to carry out a carboxyl group neutralization reaction, and then the reaction solution was dialyzed against pure water at 4° C. for 3 days using a cellulose tube. The resulting solution was freeze-dried to yield 2.33 g (77.4% yield) of sodium poly-L-glutamate as a hygroscopic flocculent solid. No benzyl ester absorption was observed in the infrared absorption spectrum of the product, confirming that the carboxyl group was converted into a sodium salt. The average molecular weight was determined to be 16,700 by the same method as above. The obtained polymer is
It is mainly composed of sodium salt of poly-L-glutamate of the following formula. Reference Example 4 10.0 g of γ-benzyl-L-glutamate N-carboxylic acid anhydride obtained in Reference Example 1 and 0.23 g of L-alanine N-carboxylic acid anhydride were dried 1,4-
The mixture was added to 280 ml of dioxane under a nitrogen atmosphere and dissolved with stirring. To the solution thus obtained, a solution obtained by dissolving 198 mg of 4-aminophenyl disulfide in 10 ml of dry dioxane was added and mixed.
The polymerization reaction was carried out by stirring at room temperature under nitrogen atmosphere for 24 hours. After the reaction, the reaction mixture was added to the isopropyl ether in step 4 with stirring, and the resulting polymer was precipitated. The polymer precipitate was collected and dried under reduced pressure to obtain 8.21 g. The yield was 97%. The obtained polymer is reasonably estimated to be the following copolymer of γ-benzyl-L-glutamate and L-alanine from the raw materials, initiator, and reaction mechanism used, and the infrared absorption spectrum It was also confirmed by. 4.0 g of the thus obtained copolymer was dissolved in a mixed solution of 30 ml of trifluoroacetic acid and 5.0 ml of anisole. Add 30 ml of methanesulfonic acid to the resulting solution and stir for 20 minutes on an ice bath under nitrogen atmosphere.
Thereafter, the mixture was stirred at room temperature for 30 minutes to carry out an acid decomposition reaction of the γ-benzyl ester. After the reaction, the reaction mixture was added to 600 ml of isopropyl ether with stirring to precipitate the polymer. The precipitated white polymer was suctioned off and suspended in 65 ml of water. Add about 80 ml of saturated heavy soybean water to this, mix and keep at room temperature for 30 minutes.
The mixture was stirred for a minute to perform a neutralization reaction of carboxyl groups, thereby forming a homogeneous solution. The obtained solution was mixed with pure water using a cellulose tube at 4°C for 3 hours.
Dialysis for 1 day followed by lyophilization yielded 2.45 g of white solid. When the obtained solid was examined by infrared absorption spectrum, it was confirmed that the absorption of benzyl ester had disappeared and that the carboxyl group had become a sodium salt. The yield as sodium salt was 88%. The obtained polymer is mainly composed of a sodium salt of a polymer of L-glutamate and L-alanine represented by the following formula. Example 1 (1) Production of a reactive polymer having a thiol group at the molecular end: Sodium salt of poly-L-glutamate having a disulfide bond in the main chain obtained in Reference Example 2
292mg (10μ mole) in 0.1M Tris-HCl - 1m
M was dissolved in 10 ml of EDTA solution (PH8.50), 78 mg (1 mmole) of 2-mercaptoethanol was added thereto, and the resulting solution was heated and stirred at 50°C for 3 hours under a nitrogen atmosphere (disulfide bonds were cleaved). ). After the reaction was cooled on ice, 1N hydrochloric acid was added to adjust the pH of the solution to 2.0, and the resulting precipitate was centrifuged. The obtained precipitate was dissolved in approximately 25 ml of 0.1N caustic soda solution, and 1N hydrochloric acid was added to this to adjust the pH.
It was set to 7.0. The wet volume of the solution thus obtained is
A dispersion of 30 ml of activated thiopropyl sepharose 6B resin dispersed in 40 ml of 0.1 M sodium phosphate-1 m M EDTA solution (PH7.0) was added, stirred gently for 12 hours under a nitrogen atmosphere, and SH was added to the molecular end. The polymer containing the groups was adsorbed onto the resin. Then, separate the resin and add 300ml of 0.01M sodium phosphate.
Washed with 1mM EDTA solution (PH7.0). Next, the resin was mixed with 0.1M Tris/hydrochloric acid - 1mM
Disperse in 100ml of EDTA solution (PH8.5) and add 2
- 1.4 g of mercaptoethanol was added and slowly stirred for 12 hours under a nitrogen atmosphere to regenerate the polymer having an SH group at the end of the molecule. Thereafter, the resin was separated and washed with 150 ml of 0.01M Tris/hydrochloric acid-1mM EDTA solution (PH8.0). Combine the liquid and washing liquid, add 1N hydrochloric acid to this under ice cooling, and adjust the pH to 2.
The resulting precipitate was isolated by centrifugation. The obtained precipitate is a reactive polymer having a thiol group at the end of the molecule (Z=
Na, m=2; W=- in formula [-a]
(CH ₂ ) ₂ −, R ₁ =H). (2) Production of a reactive polymer having an active disulfide bond at the end of the molecule: Dissolve the precipitate obtained in (1) above in approximately 25 ml of 0.1N caustic soda solution, and add 1N hydrochloric acid to the PH.
was set to 8.0. Then, 79 mg of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) was added to this solution.
0.1M sodium phosphate and 1mM EDTA solution (PH
8.0) A solution obtained by dissolving in 5 ml was added and stirred for 30 minutes (the SH group at the end of the molecule forms an active disulfide bond). The resulting reaction solution was placed in a cellophane dialysis tube and dialyzed against a 0.9% saline solution for 24 hours and then against pure water for 24 hours at 4°C. Thereafter, by freeze-drying the dialysis fluid, the target molecule terminal is TNB (3-carboxy-4-nitrophenylthio group).

181 mg of a flocculent solid (reactive polymer) of the sodium salt of polyglutamic acid activated with [formula]) was obtained. The yield was 62%. (3) Measurement of the molecular weight of a reactive polymer having an active disulfide bond at the molecular end: Accurately weigh 10.02 mg of the reactive polymer obtained in (2) above, and add it to a 0.1 M Tris/hydrochloric acid-1 m M EDTA solution ( PH
8.0) Approximately 0.1 mg of dithiothreitol solid was added to the solution and stirred. After 10 minutes, the amount of terminal groups in the reactive polymer was determined by measuring the absorption intensity at 412 nm of the released TNB anion, and it was found to be 0.794 μmole. Therefore,
The molecular weight of the obtained reactive polymer was 10.02×10 ^−
3 /0.794×10 ⁻⁶ ≒12600, and the number of constituting glutamic acid units is 12600/151≒83. Example 2 (1) Production of a reactive polymer having a thiol group at the end of the molecule: 100 mg of the sodium salt of poly-L-glutamate having a disulfide bond-containing group at the carboxyl end of the main chain obtained in Reference Example 3 ( 6.00μ
mole), 0.1M Tris-HCl - 1mM EDTA
It was dissolved in 5 ml of solution (PH8.0), 9.24 mg (60 μmole) of dithiothreitol was added thereto, and the resulting solution was heated and stirred at 50° C. for 3 hours under a nitrogen atmosphere. Next, while cooling the reaction solution on ice, 1N hydrochloric acid was added to adjust the pH of the solution to 2.0, and the resulting precipitate was isolated by centrifugation. Approximately 10 ml of the obtained precipitate
It was dissolved in 0.1N sodium carbonate solution, and 1N hydrochloric acid was added thereto to adjust the pH to 7.0. To the solution thus obtained, a wet volume of 9 ml of activated thiopropyl sepharose 6B resin was added to 0.1 M sodium phosphate - 1 m M
A dispersion in 12 ml of EDTA solution (PH7.0) was added and stirred for 12 hours under a nitrogen atmosphere. Then separate the resin and add 200ml of 0.01M sodium phosphate.
Washed with 1mM EDTA solution (PH7.0). Next, the resin was mixed with 0.1M Tris/HCl - 1mM EDTA.
The mixture was dispersed in 50 ml of a solution (PH8.5), 139 mg of dithiothreitol was added thereto, and the mixture was slowly stirred for 12 hours under a nitrogen atmosphere. After that, separate the resin and 0.01M
Tris/hydrochloric acid - 1mM EDTA solution (PH8.0) 70
Washed with ml. The solution and washing solution were combined, 1N hydrochloric acid was added to the solution under ice cooling to adjust the pH to 2, and the resulting precipitate was isolated by centrifugation. The obtained precipitate is a reactive polymer having a thiol group at the end of the molecule. (2) Production of a reactive polymer having an active disulfide bond at the end of the molecule: Dissolve the precipitate obtained in (1) above in 10 ml of 0.1N caustic soda solution, and add 1N hydrochloric acid to bring the pH to 7.0.
And so. Next, 26.4 mg of 2-pyridyl disulfide dissolved in 4 ml of ethanol was added to this solution, and the mixture was stirred for 30 minutes. The resulting reaction solution was placed in a cellophane dialysis tube and dialyzed against 30% ethanol for 24 hours and then against pure water for 24 hours at 4°C. Then, by freeze-drying the dialysis fluid,
The target molecule end is a 2-pyridylthio group

A flocculent solid (reactive polymer) of the sodium salt of polyglutamic acid activated with the formula 56
mg was obtained. The yield was 56%. (3) Measurement of the molecular weight of a reactive polymer having an active disulfide bond at the molecular end: Accurately weigh 9.53 mg of the reactive polymer obtained in (2) above, and add it to a 0.1 M sodium phosphate-1 m M EDTA solution ( About 0.1 mg of solid dithiothreitol was added to the solution and stirred. The amount of terminal groups in the reactive polymer was determined by measuring the absorption intensity at 343 nm of the 2-pyridylthio anion liberated after 10 minutes, and it was found to be 0.561 μmole. Therefore, the molecular weight of the obtained reactive polymer is
9.53×10 ⁻³ /0.561×10 ⁻⁶ ≒17000, and the number of constituting glutamic acid units is 17000/151≒113. Example 3 (1) Production of a reactive polymer having a thiol group at the end of the molecule: The hydrophilic polymer having a disulfide bond in the main chain obtained in Reference Example 4 (L-glutamic acid and L-glutamic acid
- Sodium salt of copolymer of alanine) 100mg
0.1M Tris-HCl - 1mM EDTA solution (PH
8.5) Dissolve in 5 ml and add dithiothreitol to this.
Add 10.0 mg and the resulting solution was heated under nitrogen atmosphere for 50
The mixture was heated and stirred at ℃ for 3 hours. Next, while cooling the reaction solution on ice, 1N hydrochloric acid was added to adjust the pH of the solution to 2.0, and the resulting precipitate was isolated by centrifugation. The obtained precipitate was dissolved in 10.0 ml of 0.1N sodium carbonate solution, and 1N hydrochloric acid was added thereto to adjust the pH to 7.0. To the solution thus obtained was added a wet volume of 10 ml of a dispersion of activated thiopropyl sepharose 6B resin in 13 ml of 0.1 M sodium phosphate-1 m M EDTA solution (PH 7.0) and incubated under nitrogen atmosphere for 12 hours. Stirred. The resin was then separated and washed with 200 ml of 0.01M sodium phosphate-1mM EDTA solution (PH7.0). Next, the resin was mixed with 0.1M Tris/HCl - 1mM EDTA.
It was dispersed in 50ml of solution (PH8.5), 150mg of dithiothreitol was added thereto, and slowly stirred for 12 hours under a nitrogen atmosphere.Then, the resin was separated and 0.01M
Tris/hydrochloric acid-1mM EDTA solution (PH8.0) 100
Washed with ml. The solution and washing solution were combined, 1N hydrochloric acid was added to the solution under ice cooling to adjust the pH to 2, and the resulting precipitate was isolated by centrifugation. The obtained precipitate is a reactive polymer having a thiol group at the end of the molecule (Z=
Na, m=2; in formula [-a]

[Formula] Ri=H). (2) Production of a reactive polymer having an active disulfide bond at the end of the molecule: The precipitate obtained in (1) above was dissolved in 10 ml of 0.1N caustic soda solution, and 1N hydrochloric acid was added thereto to adjust the pH to 7.7.
And so. Next, 17 mg of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) was added to 2.0 ml of this solution.
A solution obtained by dissolving in acetone was added thereto, and the mixture was stirred for 30 minutes. The resulting reaction solution was placed in a cellophane dialysis tube and diluted against 0.9% saline solution at 4°C.
Dialysis was performed for 24 hours and then for another 24 hours against pure water. Thereafter, by freeze-drying the dialysis fluid, 51 mg of a flocculent solid (reactive polymer) of the sodium salt of a copolymer of L-glutamic acid and L-alanine, in which the target molecular terminal was activated with TNB, was obtained. was gotten.
The yield was 51%. (3) Measurement of the molecular weight of a reactive polymer having an active disulfide bond at the molecular end: Accurately weigh 10.16 mg of the reactive polymer obtained in (2) above, and add it to a 0.1 M sodium phosphate-1 m M EDTA solution ( About 0.1 mg of solid dithiothreitol was added to the solution and stirred. The amount of terminal groups in the reactive polymer was determined to be 1026 μmole by measuring the absorption intensity at 412 nm of the TNB anion released after 10 minutes. Therefore, the molecular weight of the obtained reactive polymer was 10.16×10
³ /1.026×10 ⁶ ≒9900, and the ratio of the constituent glutamic acid units and alanine units is 95:5, so the average number of each constituent unit is 9900/151×0.95+71
×0.05×0.95= 64 and 9900/151×0.95+71×0.05×
0.05=3.4.

Claims (1)

[Claims] 1. 60 mol% or more of the structural units consist of the structural units represented by the formula [], In [Formula [], Z represents a hydrogen atom or a monovalent cation. m represents an integer from 1 to 4. ] When the constituent units of formula [] are not 100 mol%,
The remaining structural units are expressed by the formula [′] [In formula [′], R is -H, -CH ₃ ,
It is a group represented by [Formula] or -CH ₂ OH. ], and has an active group represented by the formula [] at the carboxyl end of the main chain, In [formula], W represents an alkylene group having 1 to 4 carbon atoms, and X is a hydrogen atom or a 2-pyridylthio group, a 4-pyridylthio group, a 3-carboxy-4-nitrophenylthio group, a 4-carboxy -2-pyridylthio group, N-oxy-2-pyridylthio group, 2-nitrophenylthio group, 4-nitro-2-pyridylthio group, 2-benzothiazoylthio group, 2-benzimidazoylthio group and N-
It is a group that can form an active disulfide bond with an adjacent sulfur atom selected from the group consisting of phenylamino-N'-phenyliminomethylthio groups.
R ₁ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ] A reaction polymer with a degree of polymerization of 5 to 3000. 2 60 mol% or more of the structural units consist of the structural units represented by the formula [], In [Formula [], Z represents a hydrogen atom or a monovalent ion. m represents an integer from 1 to 4. ] When the structural unit of formula [] is not 100 mol%,
The remaining structural units are expressed by the formula [′] [In formula [′], R is -H, -CH ₃ ,
It is a group represented by [Formula] or -CH ₂ OH. ], and has a disulfide bond-containing group represented by the formula [] in the main chain or at the carboxyl terminal of the main chain, In [Formula [], W represents an alkylene group having 1 to 4 carbon atoms, and R ₁ is a hydrogen atom or an alkylene group having 1 to 4 carbon atoms.
~4 alkyl group. R ₂ represents an alkyl group, an aralkyl group, or an aryl group when the group represented by the formula [] is a terminal group of the main chain, and when the group represented by the formula [] is present in the main chain, , is a divalent group represented by [Formula]. However, W′ is an alkylene group having 1 to 4 carbon atoms that is the same as or different from W,
It is bonded to S in formula []. R ₁ ' is the same as or different from R ₁ and represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ] Characterized by reductive cleavage of disulfide bonds in a hydrophilic polymer, 60 mol% or more of the constituent units are constituent units represented by the formula [], and the constituent units of the formula [] are not 100 mol% When, the remaining structural units consist of the structural unit represented by [′], and the carboxyl terminal of the main chain has the formula [−
has an active group represented by a], [In formula [-a], the definitions of W and R ₁ are the same as in the above formula []. ] A method for producing a reactive polymer having a degree of polymerization of 5 to 3000. 3 60 mol% or more of the structural units consist of the structural units represented by the formula [], In [Formula [], Z represents a hydrogen atom or a monovalent cation. m represents an integer from 1 to 4. ] When the constituent units of formula [] are not 100 mol%,
The remaining structural units are expressed by the formula [′] [In formula [′], R is -H, -CH ₃ ,
It is a group represented by [Formula] or -CH ₂ OH. ] Consisting of a structural unit represented by the following, and having an active group represented by the formula [-a] at the carboxyl terminal of the main chain, [In formula [-a], W represents an alkylene group having 1 to 4 carbon atoms, and R ₁ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ] 60 mol% of the structural unit characterized by reacting a hydrophilic polymer with an active disulfide compound
The above consists of the constituent units expressed by the formula [],
When the constituent units of the formula [] are not 100 mol%, the remaining constituent units consist of the constituent units represented by the formula ['], and have an active group represented by the formula [-b] at the carboxyl terminal of the main chain. ing, In [formula [-b], the definitions of W and R ₁ are the same as in the above formula [-a]. X' is 2-pyridylthio group, 4-pyridylthio group, 3-carboxy-4-nitrophenylthio group, 4-carboxy-2-pyridylthio group, N-oxy-2-pyridylthio group, 2-nitrophenylthio group group, 4-nitro-2-pyridylthio group, 2-benzothiazoylthio group, 2-benzimidazoylthio group and N-
It is a group that can form an active disulfide bond with an adjacent sulfur atom selected from the group consisting of phenylamino-N'-phenyliminomethylthio groups. ] A method for producing a reactive polymer having a degree of polymerization of 5 to 3000.