JPS59161394A - Preparation of bleomycin bonded with protein or polypeptide - Google Patents

Abstract

PURPOSE:To provide the titled complex having the bioactivity of bleomycin and useful as an antigen to form antiserum capable of recognizing bleomycin compound, by condensing a specific bleomycin derivative with a protein or a polypeptide. CONSTITUTION:The objective complex can be prepared by condensing the bleomycin derivative of formula (BX is residue obtained by removing hydroxyl group from the carboxyl group of bleomycinic acid; A is bonding chain; B is phenylene or alkylene; Y is carboxyl group or its reactive derivative; n is 1 or 2) {e.g. 3-[N-methyl-N-(3-amino-propyl)amino]propylbleomycin trihydrochloride, etc.} with a protein (e.g. bovine serum albumin) or a polypeptide.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing protein or polypeptide conjugates of pleomycin. Pleomycin is an anticancer antibiotic discovered in 1966 by one of the inventors, Umezawa et al. (Umezawa et al.: Journal of Yari Antibiotics, 2).
00, 1966) It is an n-based water-soluble glycopeptide that easily chelates monoatomic divalent copper produced by the actinomycete Streptomyus verticillus, and is a glycopeptide containing 16 types of tl! body has been produced and isolated.

(For example, Umezawa et al.: Journal of Antihyotics 19A, p. 210, 1966). Among these pleomycins, Al, A2. A5. B2, demethyl A2, etc. are decopper-depleted compounds (hereinafter referred to as ``pleomycin-complex J''), which are already widely used in the clinical field of cancer treatment, especially for flat cell carcinoma. To,
It has achieved excellent results in treating skin cancer, head (fourth layer), lung cancer, and malignant lymphoma.

Also, U.S. Patent No. 3,922,262 and U.S. Patent No. Re
No. 30'451 discloses various pleomycins.
has been done.

The present inventors investigated a method of binding pleomycin to a protein or polypeptide in view of the need for radioimmunoassay (hereinafter referred to as RIA) and enzyme immunoassay (hereinafter referred to as EIA) for pleomycins and their derivatives.

Three previous examples of conjugation of pleomycins with proteins have been found in the literature. A, Broughton and J, E, St.
long 1. ): The same pleomycin-BSA conjugate was obtained by reacting the same pleomycin-BSA conjugate with bovine serum albumin (hereinafter referred to as BSA) in a phosphate buffer using water-soluble sulfodiimide. It is used for RIA. .

(Cancer Re5earch38 1419-1
421.1976) Also, Fujiwara, M.
, YaSuno, K., Kitagawa (1)
GMB S (N -7-(maleimido-
butyloB)-8uccinimide) tepepromycin is acylated, a maleimide group is introduced, and this is reacted with BSA into which an SH group has been introduced to synthesize a pepromycin-BSA conjugate. At the same time (2) MB
S(N-+n-(maleimidobenzoylo
A maleimide group is introduced by acylating the de-isolated pepromycin with Xysuccinimide, and this is reacted with the SH group of β-galactosigose to synthesize a pepromycin-β-galactosidase conjugate. (Cancer Research
4], 4121-4128゜1981) However, it has not been described that the pleomycin (or its derivative)-protein conjugate produced by any of the above methods has the biological activity of pleomycins;
Since the decoppered compound is acylated or exposed to reaction conditions that generate an amide bond, the resulting conjugate naturally has a primary amine group attached to the diaminopropionic acid amide moiety, which is essential for the expression of pleomycin activity. It is thought that pleomycin is used in the reaction and has lost its biological activity.

In addition, since the terminal amine moieties of pleomycin and pepromycin are free in the conjugate obtained by the above-mentioned binding method, the anti-Jffi serum prepared using these also recognizes the terminal amine of pleomycin and pepromycin. , it is thought to distinguish between the two. Therefore, in the above method, it is considered necessary to prepare separate antisera for each of pleomycin and pepromycin.

Therefore, the inventors investigated various ways to produce pleomycin protein or peptide conjugates that retain the activity of pleomycin and can produce anti-blood 'IN' that recognizes various pleomycins. Pleomycin that destroys amines After protecting the active site of the 1:5 conductor as a copper complex, pleomycin with a group that blocks carboxylic acid or its reactive derivatives introduced into the terminal amine group is condensed with a protein or peptide. It has been found that a pleomycin-protein or peptide conjugate can be obtained by this method.

Furthermore, anti-blood I'h obtained from rabbits administered with the protein or peptide conjugate of pleomycin prepared by the method of the present invention can be used for RIA and EIA of both bleomycin and pepromycin because it recognizes the pleomycin backbone. .

That is, the present invention relates to the following general formula [I] [B X ] JJ) I-A-NH2-rL(CH2-
B-Y) IT [I] In the formula, [BX] is a residue obtained by removing the hydroxyl group from the carboxyl group of pleomycin acid, which can be represented by the following formula , and A is a bonding chain connecting 7 amino groups.

lower alkylene, or alkylene bonded through an N atom, such as -(CH2)m-NCH3-(CH2)m'-, -(C
H2)+n-NH-(CH2)m'-.

(m and m'' are integers of 2 to 6, preferably 3 or 4), B represents an alkylene group or a phenylene group,
For example, o-, m-, p-0684 or -(C,1(2
)n'-(N=1 to 6), n is 1 or 2, and Y
The present invention relates to the production of protein or polypeptide conjugates of pleomycins, which is characterized by bonding a protein or polypeptide to a novel pleomycin derivative represented by a 6 group representing a carboxyl group or a reactive derivative thereof.

In the pleomycin protein or polypeptide conjugate obtained in the present invention, the number of pleomycins bound to one protein molecule varies depending on the reaction method or the type of protein, but in general, the number of pleomycins bound to one protein molecule varies. The range is ten. The pleomycin derivative of general formula [I] used in the present invention can be used in either a copper-containing form or a copper-free form.

For the condensation of the pleomycin derivative of the above general formula [I] with a protein or polypeptide, a method normally used for condensation of peptides can be used. For example, when Y is a carboxyl group, a method usually used can be used. protein or polypeptide in the presence of a condensing agent, or if Y is a reactive derivative, as is, with the addition of a condensing agent if necessary,
It is sufficient if the protein or polypeptide is not cohabited with the protein or polypeptide.

Among the compounds represented by the general formula [I], A is -((H
2) 3-NGI43-(CH2)3-.

Examples include compounds in which -(CI(2)3-N)I-(CH2)4-, B is p-phenylene. The reactive derivative of the carboxyl group represented by Y in the above general formula [I] is not particularly limited as long as it can react with the amine group of a protein or polypeptide; Ester 9 conductors are used.

Among the compounds of general formula [I], representative ones include -
The compounds in the table are mentioned.

Table 1 Compound A B
n X number 1] H3 1-(CH2)3-N-(CH2)3- +
1 -OH2-(CH2)3-N-(CH2)3-
+ 2 = OHH3 9-(CH2)3-NH-(CH2)4- Fragrance 1
-0H10-(CH2)3-NH-(CH2)4-
Incense 2 -OH.

The compound represented by the general formula [II] can be produced as follows.

General formula [II] [BX]-NH-A-NH2[II] [wherein [BX], A is the same as above. The general formula [III] CHO-11 is added to the pleomycin conductor shown by
3-COX [III] [wherein B and X are the same as above.

By reductively condensing the carbonyl compound represented by, the compound of formula -M [II can be obtained as (II).

Examples of the reducing agent used in the condensation include borohydride compounds such as cyanoborohydride and thorium borohydride. Catalytic reduction may also be carried out using a catalyst such as palladium on carbon. When the carbonyl compound is used in an amount of 1 to 1.5 moles, a derivative with n=1 is mainly obtained, and when 3 moles or more is used, a derivative with n=2 is obtained.

The solvent used in the reaction is methanol, water, dimethylforma, acetonitrile, or a combination thereof. The temperature may be 0 to 50'C. More specifically, n
For derivatives of =1, the reaction time from 0°C to 25°C is preferably 3 hours to 70 hours. When using an aldehyde with low solubility, it is effective to lengthen the time.

In order to remove the 6^ conductor that has been polished as described in 2 above, if a boron hydride compound is used, it should be reacted with hydrochloric acid. After adjusting the pH of ν to 1 and stirring at room temperature for 5 to 10 minutes to decompose excess reducing agent, it is neutralized, methanol is distilled off under reduced pressure, and excess aldehyde and ketone are extracted and removed with ether or butanol. Then, the following desalting operation was performed. In other words, the suction resin example is Amberlite ■XAD-2 (
(manufactured by Rohm and Haas) into a column packed with distilled water, and adsorbed with l-1. Horn (
After washing away salts with distilled water, add acidic water-containing methanol, for example, 1150N hydrochloric acid solution in water - methanol (J:4
v/v), collect the blue pleomycin derivative fraction, and use anion exchange resin, TOWEX, if necessary.
44 (OH type: manufactured by The Tau Chemical Company), concentrated under pressure and freeze-dried to obtain a coarse blue powder of the derivative.

To further improve the purity, perform the following operation.

Dissolve the above powder in distilled water and adjust the pH to 4.5 in advance.
It is injected into a column packed with CM Sephadex C-25 (Na type 10, manufactured by Pharmacia, Fine Chemical Co., Ltd.) equilibrated with 1/20 molar vinegar-intoxicated l.lium mild species I solution and adsorbed. Elution is performed by a linear nail gradient method in which the sodium concentration is gradually increased to 10 molar by successively adding sodium chloride to the above buffer. X is OH
In this case, in this chromatography, the 5^ conductor with n = 2 elutes fastest, followed by the derivative with n = 1, and unreacted raw materials elute last, so use an ultraviolet absorption monitor. It is possible to separate by

If fractionation of the target product or contamination with impurities is found, the chromatography described above may be performed again to completely remove it.

The thus obtained fraction of the target product was desalted using the previously used Anne...Lye AAD-2 desalting method, and then
When freeze-dried, the copper-containing pleomycin derivative is obtained as a blue amorphous powder.

When the pleomycin derivative produced by the method described above is subjected to hydrolysis in 6N hydrochloric acid for 105°CCl2O, the decomposition products [L-threonine, β-amine-β-(4 -amino-6-carboxy-5-7thylopyrimidin-2-yl)propionic acid, 4-amino-3-oxy-2-methyl-n-pentanoic acid, β-oxy-L-histidine, β-amino-L −
Alanine, 2'-(2-amine left methyl)-2-4-bithiazyl-4-carboxylic acid] and a carboxyl group corresponding to the raw material pleomycin of the general formula [Technology 1], represented by the general formula [I ■] When the amine NH2-A-NH2-yL(CH2BCOOH)n
[IV] [In the formula, A and B are the same as above.

detected. Also, in metatrilysis using Ann/List 15, the same as pleomycin, growth, 3
Methyl glycoside of -0-carbamoyl-D-mannose was detected by gas chromatography.

The following two facts prove that the pleomycin derivative produced by the method of the present invention has the chemical structure represented by the above formula [I].

In addition, in the case of active ester type derivatives in which X is other than OH in formula 1, after subjecting the compound represented by general formulas [II] and [Engine II] to a condensation reaction, an organic solvent such as acetone is added to the reaction solution. In addition, the target product represented by the general formula [I] (and the raw material [II]) is precipitated, and the crude product obtained by removing the raw material represented by the formula [III], or when the above isolation method is performed. The intermediate crude product may be used for conjugation with proteins or polypeptides.

Furthermore, active ester derivatives in which X is other than OH in the 4Q formula [I] can be produced as follows.

Compounds that are X or OH in general formula [I] and general formula
[VcoHX [V] It can be produced by condensing a compound represented by the formula "wherein X is the same as above" with a condensing agent.

As a condensing agent, dicyclohexylcarbodiimide, 1
-Ethyl-3-(3-dimethylaminepropyl)carbodiimide, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide, diimpropylcarbodiimide, diphenylphosphorazide (DPPA)
, diethylphosphorocyanidate (DEPC), etc.

Compounds represented by the general formula [V] include p-nitrophenol, O+P-dinitrophenol, pentachlorophenol, 2.4.5-trichlorophenol, pentafluorophenol, N-hydroxysuccinimide, N-hydroxy-5- Examples include norbornene-2,3-dicarboximide.

The solvent used in this condensation may be any solvent as long as it does not affect the reaction, but usually -% formula [11 and [V
] Preferred polar solvents that dissolve the raw material compounds represented by are water, dimethylformamide, dimethylacetamide, acetonitrile, and a combination of these four solvents. General formula [I
] Compound (on the other hand, the proportion of the compound of -qt formula [V] and the condensing agent is 0.5 to 20 equivalents, preferably 1 to 10 equivalents).

The condensation product thus obtained is the desired active ester, as shown in the IR absorption spectrum at 1780° and 1740°C.
This is clear from the fact that it shows an absorption band near m', that it forms a bond with protein as described later, and that it easily returns to its original compound with a carboxyl group when hydrolyzed with cold alkaline water.

In the thus obtained compound of general formula [I], X is O
When it is a group other than H, it is simply reacted with a protein or polypeptide in a neutral or slightly alkaline buffer, and in the case of a compound where X is OH, a commonly used condensing agent such as the above-mentioned Condensing agent, preferably 1-ethyl-
A pleomycin-protein or polypeptide conjugate can be obtained by reacting the 3-(3-di7tylaminopropyl)carbodiimide salt butyrate with a protein or polypeptide. The reaction temperature is usually -5° to 60°C, preferably 00 to 30°C.

In the present invention, the ratio of the protein or polypeptide to the compound of general formula [I] is not particularly limited, but it is usually preferable to use an excess of the compound of general formula [I] relative to the protein or polypeptide; [By increasing the amount of compound 11, the amount of pleomycin that binds to the protein or polypeptide can be increased. Examples of the above-mentioned proteins or polypeptides include albumin, proprin-3 enzyme, and polylysine. In order to isolate the target pleomycin-protein or polypeptide conjugate from the above reaction solution, methods commonly used for purifying proteins or polypeptides, such as gel filtration, ultrafiltration, salting out, and dialysis, can be used. All methods are used.

For example, the pleomycin-BSA conjugate prepared using the above-mentioned active ester (copper-containing substance) was prepared by concentrating the reaction solution by ultrafiltration, if necessary, and equilibrating it with a phosphorus buffer at pH 7.5. When injected into a 5ephadeX G50 column and subjected to chromatography, it is separated into two blue fractions. The blue substance with a large molecular weight that is eluted first is the target substance (copper-containing substance), and the blue fraction that is eluted later is
Contains raw active esters and their hydrolysates.

The desired fraction is dialyzed against water and then freeze-dried to obtain a pleomycin-BSA conjugate (copper-containing substance) as an old-colored powder. This product is useful as an antigen for immunization to prepare antiserum for use in EIA and RIA.

The structure of the product is that it is within the molecular weight range of the target product in gel chromatography, that it exhibits a blue color derived from the pleomycin-copper complex, and that it can be separated from normal amino acids derived from proteins or polypeptides by hydrolysis with 6N hydrochloric acid at 110°C for 118 hours. At the same time, β-hydroxyhistidine, which is the above-mentioned specific amino acid derived from pleomycin, is detected, confirming that it has both the structure of a protein or polypeptide and that of pleomycin. Furthermore, it is important to note that biomolecules such as pleomycin A3-5BA conjugate (copper-containing conjugate) influenced the proliferation of HeLa53 cells. Its IC wholesale value was 598 Lg/ml. This fact suggests that if a carrier with specificity for cancer is selected for the protein moiety, pleomycin can be selectively delivered to the cancerous tissue and attacked by cancer cells.

In addition, among the compounds of general formula [I], those that are active esters can bind to proteins under mild conditions of neutrality and below room temperature, so when enzymes are used as proteins, there is less loss of enzyme activity. The pleomycin-enzyme conjugate prepared by the method can be used as a labeled antigen in EIA. The enzyme to be bound may be one that irritates amine groups that are not involved in the activity, such as peroxidase, β-D
- Calactosidase, alkaline phosphatase, glycol oxidase, lysozyme, etc.

Next, the present invention will be specifically explained using examples.

Example 1), Synthesis of compound represented by general formula CI], 3)
-[N-Methyl-N-(3-aminopropyl)aminocopropyl bleomycin 3 salt et (copper-containing body) 1.72
Dissolve g in 115 ml of methanol, add 97+ ng of terephthalaldehyde acid, and then dissolve 45 mg of
of sodium cyanoborohydride was added. room temperature 2
After reacting for 2 hours, the pH of the anti-love liquid was lowered to 1.0 with a 6N aqueous hydrochloric acid solution, and the reaction was stopped by standing for 10 minutes. After neutralizing with lN sodium hydroxide, methanol was distilled off under reduced pressure, and distilled water was added to the residue to make 50+nI.

CM Sephatex ■C-25 (Na+
The mixture was injected into a column (200 ml volume) packed with Pharmacia, Fine, Chemical Co., Ltd., and adsorbed. By continuously adding thorium chloride to the above buffer, the sodium concentration was gradually increased to 1.0 mol. Elution was performed using a linear concentration gradient method, and 120 ml of the blue fraction eluting at around 0.3 mol was collected. The target product was adsorbed by injecting it into a column of Amberlite XAD-2 (manufactured by Rohm & e-Haas) (200 ml volume) that had been filled in advance with distilled water. After cleaning the column with 300ml of distilled water,
0M +j9d aqueous solution-methanol)ly (1:4 v/
v) was eluted. The blue fractions were collected and neutralized with an anion exchange resin, DOWEX ■44 (OH type: manufactured by The Dow Chemical Company), and then concentrated under reduced pressure and freeze-dried to obtain the compounds in Table 1. 1.27 g of blue powder was obtained.

Is the melting point of crystal 20? 'C (decomposition), the ultraviolet absorption maximum measured with distilled water is 292+np, E1$/Icm is IO
I missed it at 7 or 8. The maximum infrared absorption wave number (cm-') measured by the potassium bromide tablet method is 3420.1720.16'40
,1550,1480.1370.11,30,109
There were 0,1050,1010,980,800 te.

Other physical and chemical properties are shown in Table 2.

In the above reaction, 3-[N-methyl-N=(3-aminopropyl)aminocopropylbleomycin 3, hydrochloride (copper-containing substance) 2.35g terephthalaldehydic acid 1, B+j
As a result of using 123 mg of sodium cyanoborohydride, 829 mg of compound 2 (Table 1) was obtained.

The melting point of Motoichi is 215°C (decomposition), the maximum ultraviolet absorption measured with distilled water is 292 m, L, E [/1 cm is 101
．． It was 8. Infrared absorption maximum wave number (cm-') measured by potassium bromide tablet method is 3400.288o, 1720.1
650.1585.1550.1480.138o, 1
300.125o, 11!30.1130.1100.
They were 1060.102o, 890, and 805.

Other physical and chemical properties are shown in Table 2.

Table 2 Compound V multilayer chromatography electrophoresis Rm value number graph (alanine is Rf value tree 1 1) 3ζ
2 1 0.52 1.00 2 0.35 0.91*1
Nijirikageru 130 F 254, Shiranaizugo Q (
Merck & Co.), 20% ammonium acetate-nonol (75:
2? v/v
50 rng of the compound 'Jrta of 1 obtained in step 1) and a) was dissolved in 2 ml of dimethylformamide, 1 g of N-hydroxysuccinimide 60II was added, and then 56 mg of dicyclohexylcarbodiimide was added. After stirring and reacting at room temperature for 20 hours, 20 ml of acetone was added to remove the resulting precipitate. The precipitate was washed with acetone, dried, dissolved in water, insoluble matter was filtered off, and the filtrate was freeze-dried to obtain 5t3mg of blue powder of compound 3 (Table 1). Using the potassium bromide tablet method, A (11 plus Motoichi's maximum infrared absorption wave G (cm-') is 3400.2850.1780
．． 1740.1720.1640.1550.1460
．． 1380.1240.121O11100,1070
, 1050, 1000, 880, 810.

Other physical and chemical properties are shown in Table 3.

By reacting with p-nitrophenol or N-hydroxy 5-norbornene-2,3-dicarfoximide in place of N-hydroxysuccinimide in the above method, compounds 5 and 7 were obtained, respectively. was synthesized.

Their physical and chemical properties are shown in Table 3.

Furthermore, in the above method, compound 2 is used instead of compound 1 to produce tohydroxysuccinimide,
Compounds 4.6.8 were obtained by reacting with P-nitrophenol and N-hydroxy 5-norbornene-2,3-dicarboximide, respectively.

Their physical and chemical properties are shown in Table 3.

c), 3-[N-methyl-N-(3-aminopropyl)
Amine] Propylbleomycin trihydrochloride (copper-containing substance)
300 mg was dissolved in 10 ml of methanol, 110 mg of terephthalaldehyde N-hydroxysuccinimide ester was added, and then 31 mg of sodium cyanoborate was added. After reacting at room temperature for 8 hours, about 100 ml of acetone was added, and the resulting precipitate was collected by filtration. After washing the precipitate with acetone, it is dried to form a blue powder 3.
40+ng was obtained. This powder was subjected to thin layer chromatography under the conditions shown in Table 3 and was a mixture of components.

In this method, terephthalaldehyde buty-p-nitrophenyl ester or terephthalaldehyde N-hydroxy-5-norbornene-2.3
By using the -dicarboxamide ester, a mixture of compound 5.6 and compound 7.8 was obtained, respectively. This mixture can be used as it is for protein binding without isolating each component.

In this method, Compound 11,]2 could be obtained by using pleomycin A5 as the raw material bleomycin. Their Rf values in thin layer chromatography are shown in Table 3.

Table 3 Compound number Thin layer chromatography. - Rf value*1 3 0.72 4 0.39 5 0.30 6 0.01 (0,24)7
' 0.69 8 0.38 11 0, 74 12 0.41 *1 Nijilica gel [(OF254. Silanized (Merck), 20% acetic acid ammonium-methanol (60:
40v/vZ) However, for compound 6), the value for 20% ammonium acetate-nonol (45: 55v/vX) is shown in parentheses.

2) Synthesis of bleomycin and protein conjugate a) Dissolve 20 mg of bovine blood old albumin (BSA) in 1 ml of 0.05 M phosphorus buffer (pH 7,5) to synthesize the compound obtained in 1)-1) above. 15 mg of 3 was added. After the reaction at room temperature overnight, the mixture was injected into a color chamber filled with 150 ml of Sephadex G-50 equilibrated with the above buffer, chromatography was performed using the same buffer, and the eluate was collected in 2.
It was fractionated into 5 ml portions. Blue bands due to copper pleomycin were eluted in fractions 20-28 and 52-70, respectively. Fractions 20-28 are derived from proteins at 280 nm.
The formation of a conjugate was confirmed.

Fractions 22 to 26 were dialyzed overnight against water and then lyophilized.
The blue powder of Qmg bleomycin-BSA conjugate was omitted.

The E1%/1cm of this product at 280nm is 31.
It was 2. BSA, and 280n of pleomycin
When the number of bonds was calculated using the respective E1% values of 8.6 and 105.6 in m, 14 molecules of bleomycin were bound per molecule of B5Al.

In the same manner as in 2)-a), 10 mg of BjS A was reacted with compound 4 for 91 hours, and as a result of purification, 4 mg of the conjugate was obtained. E1%/of this at 280nm
1 cm indicates 21.3, and by calculation similar to a), B
Seven molecules of pleomycin were bound per 5 Al molecules.

c), 60mg of BSA in the same manner as 2)-a)
(0.9 β mol) and compound 3. obtained in 1)-8).
165 mg (approximately 9Q L mol in terms of raw material pleomycin) of the powder of Step 4 was reacted and purified to obtain 43 mg of the conjugate. The E1%/1cm of this product at 280 nm was 408, and 21 molecules of pleomycin were bound to each molecule of BSA.

Similarly, the mixed powder of BSA and active ester was added to BSA.
The reaction and purification were carried out according to 2)-a) at the ratios shown in Table 4 to obtain conjugates with different binding amounts of pleomycin as shown in the table.

Table 4 Pleomycin/Purified Conjugate BSA Molar Ratio E1% Conjugated Bleomycin/B5A 100:1 40.6 21 50:1 30.0 13 25:1 23.3 8 15・1 15.4 4 In addition, BSA As a result of carrying out the reaction using phosphozyme instead, a pleomycin-lysonam conjugate in which 5 molecules of pleomycin were bound per molecule of lysozyme was obtained.

(Reference example) 1) Synthesis of terephthalaldehyde acid P-nitrophenyl ester 65.0 g of terephthalaldehyde fi and 5.58 g of p-nitrophenol were dimethylformamided, and 7.56 g of dicyclohexylcarbodiimide was added under water cooling.
After 0 minutes of extraction, the reaction was further carried out at room temperature for 4 hours. After the reaction, the precipitate precipitated was removed by filtration, and the filtrate was poured into 2 L of ice water and allowed to stand still overnight. Take J% of the resulting silt, dissolve it in a small amount of ethyl acetate, add ether to precipitate, collect by filtration, and dry.
．． 56g was obtained. The melting point of this substance is 134-185°C
, maximum infrared absorption wave lj of quartz crystal measured by potassium bromide tablet method
((cm-') is 3450, 1740, 1705, 16
20, 1600, 1580, 1520, 1490, 13
50, 1270, 1220, 1205, 1080, 8
It was 50,750.

Silica gel 60F254, Silanize (Merck & Co.)
using ethyl acetate-n-hexane (
The Rf value of thin layer chromatography using 1:9) is 0.
．． It was 61.

2), Synthesis of terephthalaldehyde MN-hydroxy-5-norpol 2,no-2,3-dicarboxamitoester, Terephthalaldehyde 5-norbornene-2,3-dicarboxamide 7,16
g, dioxane 75ml and tetrahydrofuran 75ml
Dissolve dicyclohexylcarbodiimide in the foot axilla and cool with water. ．． After adding 56 g of the mixture and stirring for 30 minutes, the reaction was further carried out at room temperature for 4 hours. After the reaction, the precipitate precipitated was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue 7-i was dissolved in 200 ml of ethyl acetate, the insoluble matter 7 was removed by filtration, and the ash was concentrated under pressure. Petroleum ether and diethyl ether were added to the obtained oil to form a powder, which was collected by filtration. Yield 9.48g (91%). The melting point of this substance is 132-133°C, and the maximum infrared absorption wave number (cm =) of the crystal measured by the potassium bromide tablet method is 3450.
1780, 1740, 1710, 1630, 1580,
1505, 1400, 1340, 1240, 1200,
1130, 1100, 1080, 1050, 1040,
1000, 840, 830, 750, 725
There was.

The Rf value of thin-layer chromatography developed with silica gel 80 F 254 and Silanized Q (Merck & Co., Ltd.) ethyl acetate-n-hexane (1:9) was 0.24.

Claims (2)

[Claims] (1) -11ν formula % formula %) [In the formula, [BX] represents the residue obtained by removing the hydroxyl group from the carboxyl group of pleomycin acid, A represents the bonding chain, B represents phenylene or alkylene, and Y represents the carboxyl group. or its reactive 11^ conductor, n is 1 or 2]. or method for producing polypeptide conjugates (2) General formula % formula %) [In the formula, '[BX] represents a residue obtained by removing the hydroxyl group from the carboxyl group of pleomycin acid, A represents a bonding chain, B represents phenylene or alkylene, and Y represents a carboxyl group. or a reactive derivative thereof, n is 1 or 2] Bleomycin derivatives and salts thereof