JPH0149299B2 - - Google Patents

Description

[Detailed description of the invention]

a Industrial Application Field The present invention is directed to the production of highly reactive polymers that have a large number of carboxyl groups (or salts thereof) in their side chains and an active group containing a thiol group at one end of the main chain. Regarding the law. 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 bind the two. b. Prior Art For the purpose of selectively killing only certain types of cells, attempts have been made to combine various cytotoxic substances with immunoglobulins that can specifically bind to the target cells. For example, p-
Complex containing bis(2-chloroethyl)amino-L-phenylalanine, etc. (Japanese Patent Application Laid-open No. 51-61640
), a complex in which immunoglobulin is bound to methotrexate, etc. (JP-A-56-65829), a complex in which immunoglobulin is bound to chlorambucil, etc. (JP-A-56-65829), immunoglobulin and mitomycin-C, etc. A complex combining (JP-A-55-92325
(No. 1987), a complex in which daunomycin is bound to immunoglobulin (Japanese Patent Application Laid-open No. 144723/1983), and the like are known. Furthermore, JP-A-51-126281 discloses a method in which an antitumor immunoglobulin and a polymer carrier (e.g., polydaltamic acid) to which 5 to 500 anticancer drug molecules are covalently bonded per molecule are bonded through an amide bond. It is disclosed that an antitumor agent was obtained by using The cytotoxic complexes obtained by these methods are expected to exert toxicity on tumor cells to which they have selectively bound, and are thus very interesting drugs. However, when a cytotoxic substance is directly bound to an immunoglobulin, the antigen recognition activity of the immunoglobulin decreases when a large number of cytotoxic substances are bound to the immunoglobulin.
In order to avoid such difficulties, it is necessary to bind only a small number of cytotoxic substances. On the other hand, when a polymer is used as a carrier for a cytotoxic substance, it is thought that the above-mentioned difficulties can be improved. However, in the method described in JP-A-51-126281, the reaction of binding a large number of cytotoxic substances to a polymer carrier and the reaction of binding an immunoglobulin to a polymer-anticancer drug conjugate are the same reaction. of the immunoglobulin binds to the polymeric carrier, leading to problems such as the resulting complex not only being non-uniform but also containing high molecular weight substances that are unsuitable for use as a therapeutic agent. It is. c. Purpose of the Invention As a result of intensive research to solve the drawbacks of the prior art, the present inventors have discovered that a cell that contains only one reactive group for binding reaction with immunoglobulin and that is different from that reactive group. A polymer carrier containing a large number of reactive groups for binding a toxic substance is prepared, and after first binding a large number of cytotoxic substances to the polymer carrier through the multiple reactive groups, the reactive group with the immunoglobulin is bonded to the polymer carrier. Therefore, by following the procedure of binding with immunoglobulin, an immunoglobulin-cytotoxic substance complex that does not contain polymeric substances unsuitable for use as a therapeutic agent and binds a large number of cytotoxic substances can be obtained. discovered that it is possible to manufacture the body. The present invention provides a reactive polymer that can be optimally used in the production of such antitumor agents or other target-directed drugs. d Structure and operation of the invention 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 of 1-4. ] When the constituent units of formula [] are not 100 mol%,
The remaining structural units are 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 amino terminal of the main chain, In [Formula-a], W represents an alkylene group having 1 to 4 carbon atoms. This is a method for producing a reactive polymer having a degree of polymerization of 5 to 3,000. The reactive polymer of the present invention can bind cytotoxic substances such as anticancer drugs to it by utilizing a large number of carboxyl groups (or salts thereof) present in the side chains.
In addition, by utilizing the active group present at one end of the main chain,
Immunoglobulins such as anti-tumor antibodies can be bound to this. 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 an 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-pi Lysylthio group

[Formula] 2-nitrophe Nylthio group

[Formula] 4-nitro-2- Pyridylthio group

[Formula] 2-ben Zothiazoylthio group

[Formula] 2-benzimidazoylthio 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-aminopropionic acid residue (-CH ₂ CH ₂
-) or N-penzoylcysteine residues such as

[Formula] and N-acyl homocysteine residue

【Formula】Yamel Captosuccinic acid residue

Alkylene group having a side chain substituent as shown in [Formula], 4-mercaptobenzoic acid residue

Phenylene groups having no substituents or having substituents as shown in the formula are exemplified, and alkylene groups having 1 to 4 carbon atoms are particularly preferred. Among the reactive polymers of the present invention, those in which X in the formula [] is a hydrogen atom, that is, those having the following formula [-a] at the amino terminal of the main chain [In formula [-a], the definition of W is formula []
Same as in the case of ] A method for producing a reactive polymer having an active group represented by the following will be explained. In this method, a hydrophilic polymer in which 60 mol % or more of the structural units is composed of the structural units represented by the above formula [] and has a degree of polymerization of 5 to 3000 is added with the formula [] R ₁ S-W-CO-R ₂ ...[] [In the formula, the definition of W is the same as in the case of the formula []. R ₁ represents a protecting group for a thiol group. R ₂ represents a group capable of activating an adjacent carbonyl group. ] The desired reactive polymer is produced by reacting with an acylating agent containing a protected thiol group represented by the formula, acylating the amino terminal of the polymer, and then (or simultaneously) removing the protecting group for the thiol group. This is a method of manufacturing. 60 of the constituent units that are the raw materials used in this method
A hydrophilic polymer in which mol% or more is composed of structural units represented by the formula [] and has a degree of polymerization of 5 to 3000 is, for example, polyaspartic acid corresponding to the case where m = 1 in the formula [], m = 2 Polyglutamic acid corresponding to the case of , a copolymer of aspartic acid and glutamic acid, and a component having a unit structure that cannot be expressed by the formula [], such as aspartic acid containing up to 40 mol% of alanine constitutional units. Hydrophilic polymers such as alanine copolymers and glutamic acid-alanine copolymers. These polymers can be prepared by a conventional method, for example, by subjecting N-carboxylic anhydride of an amino acid to a polymerization reaction. To give a specific example, poly-L-glutamic acid can be easily obtained by converting γ-benzyl-L-glutamate into N-carboxyanhydride, subjecting it to a polymerization reaction, and then removing the benzyl group. In the acylating agent containing a protected thiol group represented by the formula [ ], which is another raw material used in the present method, R ₁ can be, for example, a group that forms a disulfide with the adjacent sulfur atom. For example, alkylthio groups such as methylthio group (CH ₃ S-), butylthio group (CH ₃ CH ₂ CH ₂ CH ₂ S-), γ-succinimidyloxycarbonylpropionylthio group

[Formula] Substituted alkylthio group 2-pyridylthio group

[Formula] 3-carboxy-4-nito lophenylthio group

Examples include groups capable of forming an active disulfide such as [Formula]. R ₁ is also an acetyl group

[Formula] Propi onyl group

An acyl group such as [Formula], benzyl group

Also includes [formula]. In the acylating agent represented by the formula [], -R ₂ represents a group capable of activating the adjacent carbonyl group,
For example, succinimidyloxy group

[Formula] Paranitrophenoxy group

Alcohol residue of active ester such as [Formula], t-butylcarboxy group

[Formula] Isobutyloxycarbo xy group

Preferably used groups include carboxylic acid residues of acid anhydrides such as [Formula], azide group [-N ₃ ], and the like. In the acylating agent represented by the formula [], W is the same as described in the formula []. A specific example of an acylating agent containing a protected thiol group represented by the formula [] is succinimidyl 3-(2-pyridyldithio).
Propionate

【formula】

[Formula] W=−CH ₂ CH ₂ −,

[Formula]), dithiobis(succinimidylpropionate)

[Formula] (R ₁ =

[Formula] W=−CH ₂ CH ₂ −,

[Formula], N-benzoylcystine paranitrophenyl ester

【formula】

【formula】

【formula】

For example, N-acetylhomocysteine thiolactone

Compounds such as [Formula] in which R ₁ representing a protecting group for a thiol group and R ₂ representing an activating group for a carbonyl group are incorporated into the molecule can also be used in this method. The acylating agent containing a protected thiol group used for the hydrophilic polymer used in the reaction of this method is used in an amount of 1 to 100 times equivalent, more preferably 5 times to 100 times equivalent. The solvent used is a buffer solution (PH
5 to 8), solvents that form a homogeneous solution, such as DMF, are preferred. The reaction can also proceed more advantageously in the presence of a base such as triethylamine or sodium carbonate. The reaction temperature is 0°C to 50°C, preferably 4°C to 30°C.
It is ℃. Reaction time is 1 hour to 3 days. After the reaction is complete, if the reaction solution is a buffer solution, add hydrochloric acid,
The product is obtained by adding an acid such as sulfuric acid to lower the pH of the reaction solution to 4 or below, causing a precipitate of a hydrophilic polymer in which an acyl group having a protected thiol group is bonded to the amino terminal, and removing the precipitate. refine. If the reaction solution uses a solvent other than water, it can be purified in the same manner after adding a buffer solution. Alternatively, it can be purified by dialysis or gel filtration. The protecting group of the thiol group of the thus obtained hydrophilic polymer in which an acyl group having a protected thiol group is bonded to the amino terminal is removed, and the desired product, the amino terminal of the main chain, is bonded with the formula A method for obtaining a reactive polymer having an active group represented by [-a] will be described. The method for removing the protecting group is
Depends on the type of protecting group. For example, the expression []
When R ₁ forms a disulfide together with the adjacent sulfur atom, a method of reductively cleaving the disulfide group is preferably used. For example, by acting on the hydrophilic polymer in an aqueous solution with a reducing agent such as 2-mercaptoethanol, dithiothreitol, or sodium borohydride, it is converted into a reactive polymer having a thiol-containing group at the amino terminal. can lead. The amount of reducing agent used is preferably 1 to 100 equivalents. Reaction temperature is 0℃~50℃,
The reaction time is preferably 1 minute to 3 hours. In formula [], when R ₁ is an acyl group, it is eliminated by hydrolysis. The reaction is carried out in an aqueous solution using 1 to 100 equivalents of an alkali such as hydroxylamine, sodium carbonate, or sodium hydroxide as a catalyst. Reaction temperature is 0 to 50℃, reaction time is 10 minutes to 10 minutes.
time is preferable. When R ₁ is a benzyl group in formula [], the hydrophilic polymer is treated with excess trifluoroacetic acid, methanesulfonic acid, or a mixture thereof. Reaction temperature is 0℃~30℃, reaction time is
30 minutes to 10 hours. In addition, when N-acetylhomocysteine thiolactone is used as an acylating agent containing a protected thiol group represented by formula [], the deprotecting group reaction proceeds simultaneously with the acylation reaction, so the deprotecting group is overlapped. There is no need to perform a reaction. At the amino terminal of the main chain thus obtained, the formula [
The reactive polymer having an active group represented by -a] does not contain any low-molecular substances as impurities, but it does contain a part of the hydrophilic polymer used as a raw material in an unreacted form. There is a possibility that it is. Next, a method for further purifying the product by removing this unreacted polymeric substance will be described. Its purification is accomplished by performing chromatography in a buffer using a resin that binds a compound containing a thiol group, such as thiopropyl sepharose 6B (manufactured by Pharmacia). . That is, when an unpurified reactive polymer is passed through the column, the reactive polymer containing a thiol-containing group at the amino end binds to the column, and the impurity, a polymer that does not contain a thiol-containing group, is eluted. . A buffered solution such as 2-mercaptoethanol or dithiothreitol is then passed through the column to elute the purified reactive polymer containing thiol-containing groups. The eluate is acidified to precipitate the polymer or dialyzed to remove low-molecular substances.
The desired product can also be obtained by freeze-drying. Next, among the reactive polymers of the present invention, formula []
When X in represents a group capable of forming an active disulfide bond with the adjacent sulfur atom, that is, the following formula [-a] is present at the amino terminal of the main chain: [In formula [-b], the definition of W is the same as in the above formula []. X ¹ 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 is to react a polymer containing an active group represented by the above formula [-a] at the amino terminal, that is, a polymer containing a thiol group, with an active disulfide compound. As the active disulfide compound, for example, 2-pyridyl disulfide

[Formula] 4-pyridyl disulfide

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

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

[Formula] 2-nitropheni Ludisulfide

[Formula] 4-nitro-2-pyridyl disulfide

[Formula] 2-benzothiazoyl disulfide

[Formula] 2-benzimidazoyl disulfide

[Formula] N-phenylamino-N'-phenyliminomethyl disulfide can be mentioned. Both reactions are usually carried out in a homogeneous reaction system using water or an organic solvent such as dimethylformamide or dimethyl sulfoxide as a reaction solvent. Alternatively, the reaction can be carried out using a reaction system in which an active disulfide compound or its acetone solution, dioxane solution, etc. are added to and mixed with an aqueous solution of the polymer. The reaction temperature is -5°C to 70°C, and the reaction time is suitably 1 minute to 24 hours. For purification after the reaction, add a buffer to the reaction solution,
A method for precipitating and removing a polymer with acidity,
This can be easily carried out by dialysis of the reaction solution and then freeze-drying. Next, the reactive polymer having the active group represented by the formula [-b] at the amino end of the main chain is added to the amino end of the main chain, and the reactive polymer having the active group represented by the formula [-a] is added to the amino end of the main chain. A method for converting the compound into a reactive polymer having a group will be described. Preferably, the method is one in which the disulfide group is reductively cleaved. Examples of the reducing agent used include thiols such as 2-mercaptoethanol and dithiothreitol, and borohydride compounds such as sodium borohydride and calcium borohydride. The reaction solvent is preferably water or dimethylformamide.
The amount of reducing agent used is preferably 1 to 100 equivalents. The reaction temperature is preferably 0 to 50°C and the reaction time is preferably 1 minute to 3 hours. Purification is carried out by conventional acid precipitation, gel filtration, dialysis, etc. The reactive polymer of the present invention has a large number of highly reactive carboxyl groups (or salts thereof) in the side chain,
Since it has a highly reactive thiol group or active disulfide bond at the end of the molecule, the reactivity of these groups can be used to effectively and efficiently bind immunoglobulins and cytotoxins, for example, by the method described below. The resulting conjugate can be used as a targeted drug. As the cytotoxic substance introduced into the side chain, for example, an anticancer agent containing an amino group or an imino group in the molecule is used. The reaction between the reactive polymer of the present invention and the cytotoxic substance 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. In the reaction, for example, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride may be used to activate the carboxyl groups in the polymer with dicyclohexylcarbodiimide, or the carboxyl groups may be activated in the form of a mixed acid anhydride. You may also leave it as Appropriate reaction temperature is -40°C to 100°C and reaction time is 10 minutes to 10 days. e Effects of the invention The reactive polymer thus obtained with a cytotoxic substance bound to its side chain still has a highly reactive thiol group or active disulfide bond at the molecular end, so the reaction of such a group or bond is difficult. The reactive polymer of the present invention to which a cytotoxic agent is bound can be obtained by bonding the anti-tumor immunoglobulin or its fragment by utilizing the reactivity of the thiol group, active disulfide group, maleimide group, etc. generated or introduced into the anti-tumor immunoglobulin or its fragment. It is possible to produce a complex having antitumor properties in which the toxic portion is used. Specific manufacturing methods include, for example, the following methods. Antitumor immunoglobulin was treated with pepsin,
A dimer of Fab' is obtained, and the disulfide bond at the hinge portion is reductively cleaved using, for example, a thiol reagent to have a thiol group in the molecule.
Obtain Fab′. and has a thiol group
Fab' is reacted with the reactive polymer of the present invention having an active disulfide bond and a polymer further bound to a cytotoxic substance to obtain the desired antitumor complex. An antitumor immunoglobulin into which a maleimide group has been introduced by, for example, acting with succinimidyl m-maleimidobenzoate is reacted with a polymer in which a cytotoxic substance is further bonded to the reactive polymer of the present invention having a thiol group. to obtain the desired antitumor conjugate. The antitumor agent thus obtained is expected to exert toxicity on tumor cells to which it selectively binds. Hereinafter, the present invention will be explained in detail with reference to Examples. Example 1 1-1 Production of poly-L-glutamic acid into which an acyl group containing a protected thiol group is introduced at the amino terminal (hereinafter abbreviated as N-terminus). To a solution of 226.5 mg of sodium salt of poly-L-glutamic acid (average molecular weight 21000) in 0.1 M sodium phosphate buffer (PH 7.5) (15 ml) was added N-succiimidyl 3-(2-pyridyldithio) propionate (hereinafter referred to as A solution of 68.8 mg (abbreviated as SPDP) in ethanol (6 ml) was added in two portions under stirring, and the mixture was allowed to react at room temperature for 1.5 hours. Place the reaction solution in a cellophane dialysis tube and add 0.01M phosphate buffer (PH7.5).
When low-molecular substances are removed by dialysis at 4°C for 2 days against poly-L-glutamic acid, a 3-(2-pyridyldithio)propionyl group is introduced into the amino terminal of the poly-L-glutamic acid molecule through an amide bond, resulting in a disulfide bond. 50 ml of a solution containing poly-L-glutamic acid were obtained. 1-2 Poly-L having a thiol group at the N-terminus
- Production of glutamic acid. 1-1), 3-
Add 17 mg of dithiothreitol to 50 ml of a solution containing poly-L-glutamic acid with a disulfide bond into which a (2-pyridyldithio)propionyl group has been introduced and a 0.1 M sodium phosphate (PH6.0) buffer solution.
A 2.0 ml solution was added, and the mixture was allowed to react at room temperature for 80 minutes.
The mixture was then acidified with hydrochloric acid, and the resulting precipitate was centrifuged. Precipitate of the obtained poly-L-glutamic acid (contains poly-L-glutamic acid with and without a thiol group at the end)
was washed with 0.01NHCl. Meanwhile, 15 ml of Thiopropyl Sepharose 6B gel (manufactured by Pharmacia) was mixed with 0.1 M sodium phosphate (PH6.0).
1mM ethylenediaminetetraacetic acid (hereinafter EDTA)
(abbreviated as ) (PH6.0) buffer solution, and to the obtained dispersion solution, a solution obtained by dissolving the poly-L-glutamic acid obtained above in 10 ml of the same buffer solution was added, and the solution was heated to room temperature. 1 in a nitrogen atmosphere.
Stirred overnight. This binds poly-L-glutamic acid having a thiol group at the end to the resin. The resin was then filtered and thoroughly washed with 0.01M phosphoric acid buffer pH 7.5. The thus obtained resin was diluted with 0.1M Tris-hydrochloric acid.
The mixture was dispersed in 50 ml of 1m MEDEA (PH8.5) buffer solution, 1.17 g of 2-mercaptoethanol was added, and the mixture was gently stirred at room temperature in a nitrogen atmosphere for 10 hours. As a result, poly-L-glutamic acid having a thiol group at the end is released from the resin. Filter the resin and add 0.01M Tris-HCl-0.1m
Wash thoroughly with MEDTA (PH8.5) buffer, then
The filtrate and washing solution were adjusted to pH 1.8 with hydrochloric acid under ice-cooling, and the resulting precipitate of poly-L-glutamic acid having a thiol group at its terminal was centrifuged. 1-3 Production of poly-L-glutamic acid having an active disulfide group at the N-terminus. The precipitate of poly-L-glutamic acid having a thiol group at the end obtained in step 1-2) was added to the 0.4M sodium phosphate-1mMEDTA (PH7.5) buffer solution 1 again.
A solution of 23 mg of 2-pyridyl disulfide (hereinafter abbreviated as 2-PDS) in ethanol (4 ml) was dissolved in 0.1 M sodium phosphate.
Add to the resulting solution by adding 10 ml of 1m MEDTA (PH6.0) and react for 30 minutes at room temperature.
Put the reaction solution (the terminal of L-glutamic acid becomes an active disulfide group) into a cellophane tube,
Dialysis was performed against 0.01M sodium phosphate (PH7.5) buffer for 6 hours and against pure for 1 day. The recovered liquid was reduced to 30 ml under reduced pressure and freeze-dried to obtain 35.4 mg of a flocculent solid of poly-L-glutamic acid (sodium salt) with a 2-pyridyldithio group introduced at its terminal (weight yield 15.6%). ). From the results of Example 1-4), the obtained polymer contains a 2-pyridyldithio group at the end and has a molecular weight of 17,800. 1-4 Poly-L having a thiol group at the N-terminus
-Process for producing glutamic acid. Accurately weigh a certain amount of sample (1.895mg) and 3.00ml
A small piece of dithiothreitol was added to the solution in 0.1M sodium phosphate buffer (PH7.2), and the absorption (343 nm, molecular extinction coefficient ε = 8080) derived from liberated 2-mercaptopyridine was measured ( A=0.286). The amount of terminal groups in the accurately weighed sample is 0.1062 μmole. Therefore, the molecular weight of the terminal active disulfide-containing poly-L-glutamic acid molecule in the same sample is 17800, and the number of units of glutamic acid (sodium salt) in the same molecule is was calculated to be 118 since the mass number of one unit is 151. After the reaction, transfer to Cephadex G-25 (1.0 x 40 cm,
When low-molecular substances were removed by passing through 0.01M phosphoric acid buffer - 1m MEDTA (PH6.0)), a solution of poly-L-glutamic acid having a thiol group at the N-terminus was obtained. Example 2 2-1 Production of a glutamic acid and alanine copolymer into which an acyl group containing a protected thiol group is introduced at the N-terminus. Sodium copolymer of L-glutamic acid and L-alanine (average molecular weight 17200, glutamic acid-
Alanine constituent equivalent ratio 30.3:1), 172 mg Tris-
A solution of 48 mg of N-acetyl homocysteine thiolactone in ethanol (4 ml) was added in two portions to a solution in hydrochloric acid buffer (PH8.2) (10 ml), and the mixture was heated for 10 min at room temperature.
Stir for hours. Pour the reaction solution into a cellophane dialysis tube and dilute with 1m MEDTA-0.01M phosphate buffer (PH
6.0) at 4°C for 2 days to remove low-molecular substances, N-acetylhomocysteine was introduced into the amino terminal of the copolymer of L-glutamic acid and L-alanine through an amide bond. , 16 ml of a solution containing a polymer having thiol groups was obtained. The same method as shown in Example 1-2) was used, that is, the polymer into which a thiol group had been introduced was once bonded to the thiopropyl Sepharose 6B gel, and the unreacted polymer into which no thiol group had been introduced was removed. Next, a purified product of a polymer having a thiol group was obtained by eluting the polymer having a thiol group from the gel (precipitate). 2-2 Production of a copolymer of L-glutamic acid and L-alanine having an active disulfide group at the N-terminus. To the precipitate of the copolymer of L-glutamic acid and L-alanine having a thiol group at the end obtained in 2-1), add 0.4M phosphoric acid buffer and 1m MEDTA again.
(PH7.5) Add and dissolve 2 ml, and to the resulting solution,
2-PDS was added and reacted in the same manner as in Example 1-2), and further treatment steps of dialysis and freeze-drying were performed to obtain the target product, L-glutamic acid with a 2-pyridyldithio group introduced at the end. 22.4 mg of a flocculent solid of L-alanine copolymer (sodium salt) was obtained (weight yield: 13%). From the results of Example 2-3), the obtained polymer contains a 2-pyridyldithio group at the end and has a molecular weight of 13,900. 2-3 Production of a copolymer of L-glutamic acid and L-alanine having a thiol group at the N-terminus. 1.154 mg of the polymer containing a 2-pyridyldithio group at the end obtained in 2.2) was accurately weighed, and Example 1-
It was reacted with dithiothreitol in the same manner as in 4). By measuring the absorption derived from liberated 2-mercaptopyridine, the amount of terminal active groups in the same sample,
The molecular weight of the polymer was calculated. Terminal active group amount
0.083μmole, molecular weight 13900. The reaction solution was mixed with 0.01M phosphate buffer - 1m MEDTA.
(PH6.0) to remove low molecular weight substances, N-
L-glutamic acid with a thiol group at the end and L
- A solution of a copolymer of alanine was obtained. Example 3 3-1 Poly-L- containing a thiol group at the N-terminus
Production of glutamic acid. 170 mg of sodium salt of poly-L-glutamic acid (average molecular weight 17000) was dissolved in 5 ml of 0.1 M sodium phosphate buffer (PH7.3), and 346 mg of S-acetylmercaptosuccinic anhydride was dissolved in 2.0 ml of dimethylformamide. Add in two times and adjust the pH
While maintaining the temperature between 7.0 and 7.5, the mixture was stirred at room temperature for 2 hours. Next, the reaction solution was placed in a cellophane dialysis tube and diluted with 0.01M phosphate buffer (PH7.5).
It was thoroughly dialyzed at ℃. 8.5ml of recovery liquid. 2.5 ml of 0.5M hydroxylamine solution was added and reacted at 30°C for 30 minutes to remove the acetyl group protecting the thiol group. 1NHCl was added to the reaction solution at 0°C to adjust the pH to 2.0, and after standing for 2 hours, the resulting precipitate was centrifuged to obtain a precipitate of poly-L-glutamic acid with a thiol group introduced at the N-terminus. Ta. Furthermore, using the same method as shown in 1-2), that is, the polymer into which thiol groups have been introduced is bonded to the thiopropyl Sepharose 6B gel, the unreacted polymer into which thiol groups have not been introduced is removed, and then the thiol A purified product of a polymer having a thiol group was obtained by eluting the polymer into which the group had been introduced from the gel, and a precipitate was obtained by acidifying with hydrochloric acid. 3-2 Production of poly-L-glutamic acid having an active disulfide group at the N-terminus. The precipitate of poly-L-glutamic acid having a thiol group at the end obtained in 3-1) was added to 0.4M again.
Sodium phosphate - 1m MEDTA buffer (PH7.6)
Dissolve to 2 ml and add 5,5'-dithiobis(2-
39.6 mg of nitrobenzoic acid) was added and dissolved, and the mixture was allowed to react for 30 minutes. The reaction solution was poured into a cellophane tube and thoroughly dialyzed against water at 4°C. When the recovered liquid is freeze-dried, the target product, 4-nitro-3
-29.6 mg of a flocculent solid of poly-L-glutamic acid (sodium salt) containing a carboxyphenyl dithio group at the N-terminus in the main chain was obtained (weight yield 17.4
%). From the results of Example 3.3), the obtained polymer contains a 4-nitro-3-carboxyphenyldithio group at the end and has a molecular weight of 14,900. 3-3 Poly-L having a thiol group at the N-terminus
- Production of glutamic acid. 3-2), with 4-nitro-3- at the end
2.208 mg of a polymer containing carboxyphenyl dithio groups was accurately weighed and dissolved in 3.00 ml of 0.1M phosphoric acid buffer. 10mM2-Mercaptoethanol 0.10
ml and left for 30 minutes, the absorption at 412 nm (ε = 12000) derived from liberated 5-mercapto-2-nitrobenzoic acid was measured, the amount of terminal active groups was quantified, and the molecular weight of the polymer was calculated. did. Terminal active group amount 0.148μmole, molecular weight 14900. The reaction solution was mixed with 0.01M phosphate buffer - 1m MEDTA.
(PH5.8) to remove low molecular weight substances, N-
A solution of poly-L-glutamic acid having a thiol group at the end was obtained.

Claims (1)

[Scope of Claims] 1 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. ] Consisting of the structural unit represented by
3000 hydrophilic polymer, the formula [] R ₁ S-W-CO-R ₂ ... [] [In the formula, W represents an alkylene group having 1 to 4 carbon atoms. R ₁ represents a protecting group for a thiol group. R ₂ represents a group capable of activating an adjacent carbonyl group. ] 60 of the structural unit characterized by reacting with an acylating agent containing a protected thiol group, and then removing the protecting group of the thiol group.
At least mol% consists of the structural unit 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 amino terminal of the main chain, [In formula [-a], the definition of W is the same as in the above formula []. ] A method for producing a reactive polymer having a degree of polymerization of 5 to 3000. 2 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 of 1-4. ] 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 [-b] at the carboxyl terminal of the main chain, [In formula [-b], W represents an alkylene group having 1 to 4 carbon atoms. X ¹ represents a group capable of forming an active disulfide bond with the adjacent sulfur atom. ] Having an active group represented by
3000 is characterized by reductively cleaving the disulfide bonds of the reactive polymer, in which 60 mol% or more of the structural units are structural units represented by the formula [], and the amino terminal of the main chain has the formula [-a ] A method for producing a reactive polymer having an active group represented by the following and having a degree of polymerization of 5 to 3,000. [In formula [-a], the definition of W is the same as in the above formula [-b]. ]