JPS59159828A - Reactive polymer and its production - Google Patents

Abstract

PURPOSE:A reactive polymer having numerous carboxyl groups on the side chains and a specified active group on one end of the main chain, which can be used most suitably in the production of a target-directing medicine such as an antineoplastic agent. CONSTITUTION:A reactive polymer with a degree of polymerization of 5-3, comprising at least 60mol%, based on the total structural units, structural unit of formula I (wherein Z is H or a monovalent cation, and m is 1-4) and having an active group of formula II, wherein X is H or a group which, together with the adjacent S atom, can form an active disulfide bond, e.g., a group capable of forming 2-pyridylthio or the like; and W is a bivalent organic group, e.g., 1-4C alkylene. This is a reactive polymer having numerous carboxyl groups on the side chains and having a thiol group or an active disulfide bond-containing active group on one end of the main chain. In the production of a target-directing carcinostatic agent, etc., by binding an antineoplastic antibody capable of binding with a target such as tumor cells with a cytotoxic substance such as a carcinostatic agent, the polymer can be used to bind both components effectively and efficiently.

Description

Detailed description of the invention a. Industrial application field
It also relates to a highly reactive monomer having 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. The purpose of the present invention is to create a target-directed anti-cancer drug (anti-tumor drug) by combining an anti-tumor antibody that has the ability to bind to a target such as a tumor cell with a cytotoxic substance such as an anti-cancer drug. The object of the present invention is to provide a polymer that can be used to effectively and efficiently bind the two during production.

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, a complex in which immunoglobulin KP-bis(2-chloroethyl)amino-L-phenylalanine, etc. is bound (-JP-A-51-61640), a complex in which immunoglobulin is bound to methotrexate, etc. (JP-A-56-1988) -65829
No.), a complex in which chlorambuncle, etc. are bound to immunoglobulin (Japanese Unexamined Patent Publication No. 56-65829).

A complex in which mitomycin-C, etc. is bound to immunoglobulin (Japanese Patent Application Laid-Open No. 55-92325), a complex in which taunomycin is bound to immunoglobulin (Japanese Patent Application Laid-open No. 1447-1987)
No. 23) etc. are publicly known.

Furthermore, JP-A-51-126281 discloses a polymer carrier (e.g., polyglutamic acid) that covalently shares 5 to 500 molecules of an anticancer drug with an antitumor immunoglobulin per molecule.
It has been shown that an antitumor agent was obtained by combining the following with an amide bond.Di].

The cytotoxic complexes obtained by these methods are expected to selectively bind to tumor cells and exert harsh effects on tumor X cells, making them 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, so in order to avoid such difficulties, It has no choice but 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 nest point can be improved. However, lF! In the method described in 1981-126281, 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. Immunoglobulin binds to the polymer carrier, which causes problems such as the resulting complex not only cannot be homogeneous, but also contains polymeric substances that are unsuitable for use as therapeutic agents. be.

C1 Purpose of the Invention The present inventors have conducted extensive research to solve the drawbacks of the prior art, and as a result, the present inventors have discovered a method that contains only one reactive group for the binding reaction with immunoglobulin and is different from that. ,
A polymer carrier containing a large number of reactive groups is prepared for binding substances to cells, and after first binding a large number of cytotoxic substances to the polymer carrier through the multiple reactive groups, the reaction with immunoglobulin is performed. According to K, if the procedure of combining with immunoglobulin is performed, immunoglobulin which does not contain polymeric substances unsuitable for use as a therapeutic agent, but which binds a large number of cytotoxic substances. We have discovered a compound capable of producing cytosomatic material complexes. The present invention also provides a reactive polymer that can be optimally used in the production of such antitumor agents or other target-directed drugs.

d1 Structure and operation of the invention The present invention consists of 60 moles or more of the structural units represented by the formula Cl), and has an active group represented by the formula [■] at the ζ terminal of the main chain. 1 x-s-w-c-...[■] A reactive polymer having a degree of polymerization of 5 to 3000 and a method for producing the same.

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. By utilizing the existing active groups, immunoglobulins such as anti-tumor antibodies can be bound to this.

In formula [1), Z is a hydrogen atom or a monovalent cation,
For example, Na'', K'', and NH4+. m is 1 to 4
m represents an integer of 1 or 2, but preferably m is 1 or 2. In addition, in the reactive polymer of the present invention, formula CI)
Among the structural units represented by , for example, those with m = 1 and those with m = 2 may be mixed. The total number of these components should be 60 moles or more, preferably 80 moles or more of the total composition.

The reactive polymer of the present invention may contain structural units other than the structural units represented by formula [■] within a range of less than 40 moles of all structural units.

Examples of these include glycine that does not have a carboxyl group (or a salt thereof) in the α-position side chain.

There are α-amino acids such as alanine, phenylalanine, and serine.

Although such a composition consisting of α-amino acids does not appear to be involved in binding with cytotoxic substances, it does affect the water solubility of reactive polymers and the lipid solubility and water solubility of polymers obtained by binding cytotoxic substances. It may help you adjust. Therefore, for station buildings that do not particularly require adjustment of fat solubility or water solubility, it is practically advantageous to use a product that does not contain such a constituent unit consisting of α-amino acids.

In formula [■], X represents a hydrogen atom or a group capable of forming an active disulfide bond with an adjacent sulfur atom,
As the latter, for example, 2-piphenylthio group, dilthio group (HOOC [3-s->, N-oxy-2 formula [1-cho], W represents a divalent arithmetic group, and the reactive polymer of the present invention is obtained. Examples of these groups include straight-chain groups such as 2-aminopropionic acid residues (-C112CH2-), which are not particularly limited as long as they are inert groups that do not participate in any reactions during the process and subsequent reaction processes. Alternatively, examples include N-pene (S2fl) lene group, phenylene group having no substituent such as 4-mercaptobenzoic acid residue (-C>->, or having a substituent, but alkylene group having 1 to 4 carbon atoms) Particularly preferred are the groups l, l-I.

The reactivity of the present invention (among the combinations, the formula ([X in 3 is a hydrogen atom, that is, the amino terminal of the main chain has the following formula C
r1-a) +1 H3-W-C-・・・・・・・・・・・・・・・[:ll
A method for producing a reactive polymer having an active group represented by [-1] will be explained.

In this method, 60 mol% of the constituent nails are based on the above formula [1]
Consisting of structural units represented by, the degree of polymerization is 5 to 3000
The hydrophilic polymer of formula [! ■] R1S -W-Co-Horse......CilD (represents.
The amino terminus of the polymer is acylated by reacting with an acylating agent containing a protected thiol group represented by
This is a method for producing the desired reactive polymer by removing the protective group of the thiol group.

+7'7 g member 6, which is the raw material used in this method.
A hydrophilic polymer in which 0 mole or more consists of 4G55i units represented by the formula [■] and has a degree of polymerization of 5 to 3000 is, for example, a polyasparagine polymer corresponding to the case where m = 1 in the formula [■] ``=2'', a copolymer of aspartic acid and glutamic acid, and a unit structure that cannot be expressed by the formula [■'', for example, 40 monomers with the structure of alanine. Aspartic acid-alanine copolymer containing up to 1.

It is a hydrophilic polymer such as glutamine butyro-alanine co-pentamer. These polymers can be prepared by a conventional method, for example, by a method in which N-carboxydiV'-hydrate of AMITSUBA Q' is used without involving the polymerization reaction. To give a specific example, poly-L-glutamic acid can be easily obtained by introducing γ-benzyl-L-glutamate into N-monthly peroxide hydrate, subjecting it to a polymerization reaction, and then removing the benzyl group. I can do seven things.

Another raw material used in this method, formula [1■]
In the acylating agent containing a protected thiol group represented by, for example, the group may be a group that forms a (C disulfide) with an adjacent sulfur atom. For example, a methylthio group (C13S-), a

Chilthio group (CI,, CI(2CH2CH7S-)
Alkylthio-substituted alkylthio groups such as 2-bi1j di, ・[
・Groups that can be recognized as forming a 1,000-group ruphid can be listed. R, also represents an acyl group such as an acetyl group ("H3C) + nolobionyl group (CM3CM2+! -), or a group in which m- in a pendiacylating agent can activate the adjacent carbonyl group, such as succinimidyl group. Gishi group () Carboxylic acid residue of acid anhydride, azide group [-N,
] etc. are preferably used groups. In the acylating agent represented by formula (II), W Id is the same as the explanation for formula [■]. Formula C! Specific examples of acylating agents containing a protected thiol group represented by [:] include succinimidyl 3, and R1, which represents an N-acetyl homocysteine protecting group, is an activating group for a carbonyl group. Compounds in which P is incorporated into the molecule (ift) can also be used in the Bokuto method.

For the hydrophilic polymers used in the reaction of this method, the protective thiol-containing agent is 1 to 1
oo times equivalent, preferably 5 times to 100 times equivalent fc
J4: Yes. The solvents used are buffer (pH 5-8), DM
Solvents that form a homogeneous solution, such as F, are preferred. The reaction can also proceed more advantageously in the presence of a base such as triethylamine or sodium carbonate. The reaction temperature was 0°C to 50°C.

Preferably it is 4°C to 30°C. Reaction time is 1 hour to 3
It is day.

After the reaction is complete, add hydrochloric acid if the reaction solution is a buffer solution.

By adding an acid such as sulfuric acid to reduce the reaction solution pu to 4 or less, causing a precipitate of a hydrophilic polymer in which an acyl group having a protected thiol group is bonded to the amine terminal, and then pouring this into a door, Purify the product. If the reaction solution uses a solvent other than Mercury, add a buffer solution and
It can be purified by a similar method. It can also be purified by KX dialysis or gel filtration.

Thus, the tl obtained is a new aqueous polymer in which an acyl group having a protected thiol group is bonded to the amine end. A method for obtaining a reactive polymer having an active group represented by the formula [:IT-1] (C will be described below.
i! ! ! The method for removing the group varies depending on the type of protecting group. For example, formula C! When R7 forms a disulfide with the adjacent sulfur atom in TD (d,
A method of selectively cleaving disulfide groups 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, the hydrophilic polymer is converted into a reactive polymer having a thiol-containing group at the amine end. I can do it.

The amount of reducing agent used is preferably 1 to 100 equivalents.

The reaction temperature is preferably θ°C to 50°C, and the reaction time is preferably 1 minute to 3 hours. In formula C1[], when R1 is an acyl group, it is eliminated by hydrolysis.

The reaction is carried out in an aqueous solution using an alkali such as hydroxylamine, sodium carbonate, or sodium hydroxide as a catalyst.
Perform using 0 equivalent. The reaction temperature is preferably 0 to 50°C and the reaction time is preferably 10 minutes to 11) hours. When R1 is a benzyl group in formula C1If), the hydrophilic polymer is treated with excess trifluoroacetic acid, methanesulfonic acid, or a mixture thereof. The reaction temperature is θ°C to 30°C, and the reaction time is 30 minutes to 10 hours. It should be noted that the protected area represented by the formula [town 1'1. For example, when N-acetylhomocystine thiolactone is used as the acylating agent containing a thiol group, the deprotection reaction proceeds simultaneously with the unarylation reaction, so there is no need to carry out the deprotection reaction multiple times.

The amine end of the main chain thus obtained has the formula ('[I-a
) contains a low molecular weight substance d: does not contain gold as an impurity, but
The unreacted -
It is possible that it contains 1.

Next, this unreacted polymer substance is removed by ζ,
Method 1c for purifying the product by counter-tanning is described below. The purification is carried out by chromatography in a buffer using a resin that binds a compound having a negative thiol group, such as, for example, 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. Ru. A buffer 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 can be acidified to precipitate the polymer, or it can be dialyzed to remove low-molecular substances.The target product can also be obtained by freeze-drying.

Next, in the uninvented reactive compound, if X in the formula CII, ] disturbs the ability to form an active disulfide viscosity with the neighboring 9 host atoms, i.e., at the amino terminal of the main chain. The following formula cm-b] 1 x'-s-w-c-・・・・・・・・・・・・ [几-b
](represents.) A method for producing a reactive polymer having an active group represented by () will be described.

The method is to react a polymer containing an active group represented by the formula Cll-, ) at the amine end, that is, a polymer containing a thiol group, with an active disulfide compound. Examples of active disulfide compounds include:
2-pyridyl disulfide (○-8-s-(1'3)
, 4-pyridyl disulfide (N○-8-8CN
), 5.5'-dithiobis(2-nitrobenzoic acid) -2-pyridyl disulfide 4-nitro-2-pyridyl disulfide benzimidazoyl disulfide.

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 normal polymer. Reaction temperature: 1°C to 70°C9 Reaction time: 1 minute to 24°C
The time is appropriate.

Purification after reaction 1d1 A method in which a buffer is added to the reaction i1M, and then the polymer is precipitated with acidity.

This can be easily carried out by dialysis of the reaction solution and then freeze-drying.

Next, the formula [■-b
] A reactive polymer having an active group represented by the formula [!] is attached to the amine end of the main chain. A method for converting into a reactive polymer having an active group represented by [T-a] 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 borohydride. The reaction solvent is preferably water or dimethylformamide. The amount of reducing agent used is preferably 1 to 100 equivalents. Reaction temperature i-1: 0-5
0℃.

A suitable reaction time is 1 minute to 3 hours. Purification is carried out by conventional acid precipitation methods, gel filtration methods, dialysis methods, etc.

The reactive polymer of the present invention has a large number of highly reactive carboxyl groups (or salts thereof) in the side chain, and a highly reactive thiol group or an active disulfide bond at the molecular end. By using the reactivity of these groups, immunoglobulins and cytotoxins can be effectively and efficiently bound, for example, by the method described below, and the resulting conjugate can be used as a target-directed drug. It can be used as

As the cellular substance introduced into the side chain, for example, an anticancer agent containing an amine 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
The reaction is 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, the carboxyl groups in the polymer may be activated with, for example, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride or dicyclohexylcarbodiimide, or the carboxyl groups may be activated in the form of a mixed acid anhydride. It may be activated. The reaction temperature is 0℃
The suitable reaction time for one reaction at ~100°C is 40 minutes to 10 days.

e0 Effects of the Invention The thus obtained reactive polymer in which a cytotoxic substance is bonded to the side chain still has a highly reactive thiol group or active disulfide bond at the end of the molecule, so that the reaction of such a group or bond is prevented. The reactive polymer of the present invention is made 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 having the toxic portion as the toxic moiety. Specific manufacturing methods include, for example, the following methods.

■ Treat anti-tumor immunoglobulin with pepsin.

A dimer of Feb' is obtained, and the disulfide bond at the hinge portion is cleaved reductively using, for example, a thiol reagent to obtain Fab' having a thiol group in the molecule. Then, Fab' bearing a thiol group is reacted with a polymer in which a cytotoxic substance is further bound to the reactive polymer of the present invention having an active disulfide bond to obtain the desired antitumor complex.

■ A polymer in which a cytotoxic substance is further bonded to the reactive polymer of the present invention having a thiol group is added to an antitumor immunoglobulin into which a maleimide group has been introduced by, for example, acting on bucunimidyl m-maleimidobenzoate. The reaction is performed to obtain the desired antitumor complex.

The antitumor agents thus replaced are expected to selectively bind to tumor cells and exert toxicity to them.

Hereinafter, the present invention will be described in detail with reference to Examples. Example 1 l-1) Production of poly-L-glutamic acid into which an acyl group containing a protected thiol group was introduced at the amine end (hereinafter abbreviated as N-terminus) .

Sodium salt of poly-L-glutamic acid (average molecule i2
1,000) in a solution of 226.5 mg in 0.1 M sodium phosphate buffer (pH 7, s) (15 mJ),
A solution of 6s, sq of N-succiimidyl 3-(2-pyridyldithio)propionate (hereinafter abbreviated as 5PDP) in ethanol (6ml) was added in two portions under stirring.
The reaction was allowed to proceed for 1.5 hours at room temperature. The reaction solution was put into a cellophane dialysis tube and diluted with 0.01 M') acid buffer (p
When low-molecular substances are removed by dialysis against H7,s) at 4°C for 2 days, a 3-(2-pyridyldithio)propionyl group is introduced to the amino terminal of the poly-L-glutamic acid molecule through an amide bond, forming a A solution joml containing poly-L-glutamic acid having a disulfide bond was obtained.

1-2) Po17L- having a thiol group at the N-terminus
Production of glutamic acid.

7 mg of dithiothreitol was added to 5 ml of a solution containing poly-L-glutamic acid having a disulfide bond and having a 3-(2-pyridyldithio)propionyl group introduced into the amino terminus obtained by CO2N a l-1). 0.
1 M sodium IJ phosphate (pH 6,0) buffer2.
A solution in Oml 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. The obtained poly-L-glutamic acid precipitate (including poly-L-glutamic acid having a thiol group at the end and poly-L-glutamic acid not having a thiol group at the end) was diluted with 0.01 NHCl.
Washed with.

On the other hand, thiopropyl sepharose 6B gel
(manufactured by Pharmacia) 15-0.1M sodium phosphate (pH 6,0)-1mM ethylenediaminetetraacetic acid (hereinafter abbreviated as ED TA) (pH 6,0)
) Dispersed in 40 trl of buffer solution, and in the resulting dispersion bath solution,
The poly-L-curtamic acid obtained above was added to the same buffer solution 1.
Add the solution obtained by dissolving it in 0 ml of water, and stir gently for 11 minutes at room temperature in a nitrogen atmosphere. This binds poly-L-glutamic acid having a thiol group at the end to the resin. Next, the diluted fat was separated and moxibusted with 0.01M phosphoric acid at pH 7.5, followed by washing for several minutes.

The resin thus obtained was diluted with 0°IM to IJ-hydrochloric acid.
Dispersed in 50ml of 1mM MEDEA (pH 8,5) buffer and 1.17ml of 2-mercaptoethanol. was added thereto, 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) lithium-hydrochloric acid-0.1M
Wash well with MEDTA (pH 8,5) buffer, then adjust the filtrate and washings to pH 1,8 with hydrochloric acid while cooling with water.
The resulting precipitate of boron-L-glutamic acid having a thiol group at its terminal was centrifuged.

1-3) Production of IJI,-glutamic acid with an active disulfide group at the N-terminus.

1-2) The poly-
I,C,S of L-glutamic acid was again dissolved in 0.4 M sodium phosphate-im biEDT'A (pH 7,5
) 23 n
A solution of Bi in ethanol (4 ml) was added to 0.1 M sodium phosphate-1 m MEDTA (pH 6,0) 10- to the resulting solution, and after reacting at room temperature T for 30 minutes, (poly-L-glutamic acid (the end of which becomes an active disulfide group) Put the reaction solution into a cellophane tube, and
, dialyzed against oiM sodium phosphate (PH 7,5) buffer for 6 hours and against pure for 1 day. The recovered liquid was reduced to 30 m under reduced pressure and freeze-dried to obtain 35.474 g of a flocculent solid of poly-11-glutamic acid (sodium salt) with a 2-pyridyldithio group introduced at its terminal (ga
yield 15.6%). From the results of Example 1-4), A4
The resulting polymer contains a 2-pyridyldithio group at the terminal and has a molecular weight of 17,800. 1-4) A polymer having a thiol group at the N-terminus.

- Production of glutamic acid. Accurately weigh the sample of the actual plate (1.895 ml).

3.00 mg of 0.1 M sodium phosphate buffer (pH
7,2) Dissolve K, add a small piece of dithiothreitol, and remove the absorption derived from liberated 2-mercaptopyridine (3
43 fat m, molecular extinction coefficient ε = soso) was measured (A
=0.286). The amount of terminal groups in the precisely weighed sample is 0.
．． 1062 μmole, therefore, the molecular weight of the poly-L-glutamic acid molecule containing a terminal active disulfide of the same Zamble A is 17,800, and the glutamic acid (
The number of units of sodium salt) was calculated to be 118 since the mass number of 1 unit was 151.

The reaction solution was transferred to Sephadex G-25 (1, OX40 cm
, 0.01 M phosphate buffer - 1 m MEDTA (
When low-molecular substances are removed by passing the solution to pH 6,0>), N
- A solution of poly-L-glutamic acid having a thiol group at the terminal was obtained.

Example 2 2-1) Production of a copolymer of glutamic acid and alanine containing an acyl group containing a protected thiol group at the N-terminus - Sodium (average molecular weight) of a copolymer of L-glutamic acid and L-alanine 4i17,200. Glutamic acid-Annine constituent equivalent ratio 30.3:1).

l 72fi (g) to Tris-hydrochloric acid (pH; 3.
2) Add N-boo 1! to the solution in (10fnl). Naruhomofufufinthiolactone 48 IJFJ ethanol (4
g) The solution was added in two portions and stirred at room temperature for 10 hours. Put the reaction solution into a cellophane dialysis tube and
MEDTA-0.01M phosphate buffer (pH 6.0
) at 4°C for 2 days to remove borrowed molecular substances.
16 ml of a solution containing a thiol group-containing polymer produced by introducing N-acetylhomocysteine into the amino terminal of a copolymer of L-glutamic acid and L-alanine through an amide bond was obtained.

The same method as shown in Example 1-2) was used, that is, the polymer into which thiol groups had been introduced was once bonded to the thiopropyl Sepharose 6B gel, and no thiol groups were introduced.
A purified product of a polymer having a thiol group (precipitate) was obtained by removing the unreacted polymer and then eluting the polymer with an enlarged thiol group from the gel.

2-2) Production of a copolymer of L-glutamic acid and L-alanine having an active disulfide group at the N-terminus.

L- having a thiol group at the end obtained in 2-1)
To the precipitate of the copolymer of glutamine 9 and L-alanine, add 0.4MIJ acid buffer and 1mM EDTA (pH 7) again.
, 5) Add and dissolve 2 ml f, 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 having a 2-hyridyldithio group introduced at its terminal. A flocculent solid of 22.4T9 (weight yield: 13φ) of a copolymer of L-alanine and L-alanine (sodium 1M salt) was obtained. From the results of Examples 2 to 3), the obtained polymer contained all 2-pyridyldithio groups at the terminals and had 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.

The polymer 1.154+I containing 2-pyridyldithio base at the end returned in 2-2) was added (weighed in foil, Example 1-4).
) was reacted with cinaothreitol. The molecular weight of the terminal active group-mercap-2-mercap in the same sample was calculated by measuring the twinning derived from mercap and tobiridine.

Terminal active group amount 0.083/Jmole, , molecular weight 1
3,900.

The reaction solution was diluted with 0.01 M phosphate buffer - 1 mrt (EDT
'A (pH 6, 0) to remove low molecular weight substances, L-glutamic acid with a thiol group at the N-terminus and L-glutamic acid with a thiol group at the N-terminus.
- A solution of a copolymer of alanine was obtained.

Treatment Example 3 3-1. ) Bodies containing all thiono L groups at the N-terminus 1,1 1
．． - Production of glutamic acid.

Sodium salt of poly-L-glutamic acid (average molecular weight 1
7,000 ) 170 ■ with 0.1 M phosphoric acid) I
Dissolve in 5 ml of Jum buffer (pH 7,3) and add S
- A solution of 346Tq of acetylmercaptoconophosphoric anhydride dissolved in 2.0- dimethylformamide was added in two portions, and the mixture was stirred at room temperature for 2 hours while maintaining the pH between 7°θ and 7.5. . The reaction solution was then placed in a cellophane dialysis tube and diluted with 0.0'IM phosphate buffer (pH 7,
5) was sufficiently dialyzed at 4°C. Recovery liquid 8, 5 +
7Ie.

Add 2.5 ml of 0.5 M hydroxylamine solution and
The reaction was carried out at 0° C. for 30 minutes to remove the acetyl group protecting the thiol group.

1 NHCl was added to the reaction solution at 0°C to adjust the pH to 2.
After allowing the mixture to stand for 2 hours, the resulting precipitate was centrifuged to obtain a precipitate of poly-L-glutamic acid having a thiol group introduced into its N-terminus.

Furthermore, using the same method as shown in 1-2), that is, the polymer into which thiol groups have been introduced is once bonded to the thiopropyl Sepharose 6B gel, the unreacted polymer into which thiol groups have not been introduced is removed, and then the thiol groups are A purified product of a polymer having a thiol group was obtained by eluting the introduced polymer from the gel, and acidified with hydrochloric acid to obtain a precipitate.

3-2) Polycarbonate having an active disulfide group at the N-terminus
Production of L-glutamic acid.

3-1) The poly-
The precipitate of L-glutamic acid was dissolved again in 0.4 M sodium phosphate-1 m MEDTA buffer (PH 7,6), and 39.6 μ of 5,5'-dithiobis(2-nitrobenzoic acid) was added thereto. The mixture was dissolved and reacted for 30 minutes. The reaction solution was placed in a cellophane tube and thoroughly dialyzed against water at 4°C. When the recovered solution is freeze-dried, the target product, 29.6F#, a flocculent solid of poly-L-glutamic acid (sodium salt) containing a 4-nitro-3-carboxyphenyldithio group in the main chain and at the N-terminus, is obtained. Obtained (
weight yield 17.4%). From the results of Example 3-3), the obtained polymer contains a 4-nitro-3-carboxyphenyldithio group at the terminal, and the molecular weight is 14.90.
It is 0.

3-3) Bo IJ L- having a thiol group at the N-terminus
Manufacture of glutamine liquor.

3-2) The Tatsumi combination 2.20 containing a 4-nitro-3-carboxyphenyldithio group at the end
Weigh 8■ accurately and get 0. I MIJ acid balance liquid 3.00mg
It was dissolved in 7. After adding 10rnR42-mercaptoethanol 0°107 and leaving it for 30 minutes, the released 5-
412 nm derived from mercapto-2-nitrobenhazoic acid
The absorption at K (s = 12,000) was measured, and the molecular weight of the polymer was determined by determining the terminal active groups i and M. Terminus 1 active group amount 0.148 μmole, molecular weight 14.9000 reaction solution 0.01 M phosphoric acid buffer solution-1
After dialysis against mMEDTA (pH 5,8) to remove low-molecular substances, a solution of L-glutamic acid having a thiol group at the N-terminus was obtained.

Claims (1)

[Scope of Claims] 1. 60 or more moles of the structural units must be a structural unit represented by the formula [■] ooz, and the amino terminal of the inventor has the formula [■] 1 x-s-w-c-... ......A reactive polymer having an active group represented by [■] and having a degree of polymerization of 5 to 3,000. 2. The reactive polymer according to claim 1, wherein W in formula [11] is a substituted or unsubstituted alkylene group having 1 to 4 carbon atoms. 3 In formula CrD, X is a 2-pyridylthio group. 4-pyridylthio group, 3-carboxy-4-nitrophenylthio group, 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
3. A reactive polymer according to claim 1 or 2, comprising a group capable of forming an active disulfide bond carried from the group consisting of -phenylamino-N'-phenyliminomethylthio groups. 4. A hydrophilic polymer in which 60 moles or more of the structural units are composed of the structural units represented by the formula CI) and the degree of polymerization is 5 to 3000,
60 mol of a structural unit characterized by reacting with an acylating agent containing a protected thiol group, represented by Cll0, and then removing the protecting group of the thiol group. The polymerization degree is 75 or more, consisting of a structural unit represented by the formula [■], and having an active group represented by the formula CTI-a] at the amino terminal of the main chain;
Method for producing 000 reactive polymers. 5. More than 60 molar units of the constituent units are composed of constituent units represented by the formula CI), υ, the amino terminal of the main chain has the formula (na) MS-W- and -, ,, i, , -0, -9[IT-a
], the degree of polymerization is 5 to 3.
000 reactive polymer and an active disulfide compound, more than 60 moles of the structural units are composed of the structural unit represented by the formula [■], and the amino terminal of the main chain has the formula CI [- b'll x'-s-w-μm ・・・・・・・・・・・・ [■-
b] has an active group, and has a degree of polymerization of 5 to 3.
Method for producing 000 reactive polymers. 6. 60 moles or more of the constituent units are constituent units represented by the formula [■] υ, and the amino terminal of the main chain has the formula CII-b
:] 1 x'-s-w-c-...[-5
91 Formula 1: H-b), the definition of W is to reductively cleave the disulfide bond of a reactive polymer having an active group represented by the above formula) and having a degree of polymerization of 5 to 3000. Characteristic, more than 60 molti of the constituent units are of the formula [■]
A method for producing a reactive polymer having a degree of polymerization of 5 to 3,000, comprising a structural unit represented by the formula (II-a) and having an active group represented by the formula (II-a) at the amino terminal of the main chain.