JPH0160038B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel human limboblastoid interferon C-terminal peptide. In this specification, when amino acids, peptides, protective groups, active groups, etc. are indicated by abbreviations
The IUPAC, IUB regulations or common symbols in the field shall be followed, examples of which are listed below. Furthermore, when an amino acid or the like can have optical isomers, the L-isomer is indicated unless otherwise specified. Ser: Serine Leu: Leucine Thr: Threonine Asn: Asparagine Gln; Glutamine Glu: Glutamic acid Arg: Arginine Lys: Lysine Tyr: Tyrosine OBzl: Benzyloxy group Z: Carbobenzoxy group NHS: N-hydroxysuccinimide group Tos: p -Toluenesulfonyl group Boc: tertiary butoxycarbonyl group Interferon is an antiviral glycoprotein or protein that is produced when living cells are infected with a virus, and its use can be used to prevent or prevent viral diseases. It is thought to be treatable and has been attracting attention in recent years. The currently elucidated human interferons are β-type interferon (Fibro blast interferon), α-type interferon (Leucocyte interferon, Lympho blastoid interferon).
It is classified into γ-type interferon (interferon) and γ-type interferon (immune interferon). However, at present, a technique for purifying these interferons into a single glycoprotein or protein has not yet been established. The present inventors believe that by using an antibody that specifically reacts with human α-type interferon, the antigen-
We believed that human α-type interferon could be purified by antibody reaction, and based on this idea, we selected human α-type interferon with high sensitivity and conducted intensive research to obtain antibodies that showed specific reactivity against the interferon. It's here. In the process, a certain type of peptide having the C-terminal peptide chain of human limphoblastoid interferon, which is a type of human α-type interferon, was synthesized and an antigen was synthesized using this as a hapten. It has been found that desired antibodies having specific reactivity against type interferon can be obtained. The present invention was completed based on this new knowledge. That is, the present invention provides the general formula R-Glu-Ser-Leu-Arg-Ser-Lys-Glu (1) [wherein R is a hydrogen atom, H-Thr-Asn-Leu-
Gln group, H-Ser-Leu-Ser-Thr-Asn-Leu-
Gln group, or H-Tyr-Ser-Leu-Ser-Thr-
Indicates Asn-Leu-Gln group. ] This relates to the C-terminal peptide of human limphoblastoid interferon represented by: The peptide represented by the above general formula (1) of the present invention has not been synthesized to date. Not only can it be easily synthesized by simple operations using readily available commercially available amino acids, but it also has a specific amino acid sequence and can be used to produce large quantities of a certain antigen with a defined recognition site. Moreover, from the antigen obtained using the above-mentioned peptide as a hapten, compared to the case where natural α-type interferon is used as an antigen, it is possible to obtain antibodies with high specificity for human α-interferon in large quantities and constantly stably. Can be done. Therefore, the antibody can be used for the purification of human α-interferon by binding it to, for example, an affinity chromatography carrier and using the chromatography. The peptide represented by the above general formula (1) of the present invention is a peptide corresponding to the C-terminal peptide chain of human limboblastoid interferon. This is a conventional peptide synthesis method, specifically,
The Peptides, Volume 1 (1966)
[Schroder and Luhke, Academic Press,
NewYork, USA] or “Peptide Synthesis”
For example, the azide method, chloride method, acid anhydride method, mixed acid anhydride method, DCC method, active ester method (p-nitrophenyl ester method,
N-hydroxysuccinimide ester method, cyanomethyl ester method, etc.), method using Woodward reagent K, carbodiimidazole method, redox method, DCC/additive (HONB, HOBt,
It can be manufactured by methods such as HoSu). In the above method, both solid phase synthesis method and liquid phase synthesis method can be applied, but liquid phase synthesis method is preferable. Usually, the peptide of the present invention is produced according to the general polypeptide synthesis method described above, for example, by the so-called stepwise method in which amino acids are sequentially condensed one by one to the terminal amino acid, or by a method in which the peptides are divided into several fragments and coupled together. be done. More specifically, the above-mentioned peptide consists of a raw material having a reactive carboxyl group corresponding to one of two types of fragments that are divided into two at an arbitrary position of the bond, and a raw material having a reactive amino group corresponding to the other fragment. If the resulting condensate has a protecting group, it can be produced by removing the protecting group using a conventional method. In many cases, it is usually desirable to protect lysine in the synthesis reaction step of the peptide of the present invention. Further, in the final step of the above reaction, all protecting groups are usually removed from the protected peptide in which at least one of the constituent amino acid residues of the peptide is protected. Furthermore, in the above synthetic reaction step, functional groups that should not participate in the reaction are protected with a conventional protecting group, and the protecting group is removed after the reaction is completed. Furthermore, the functional groups involved in the reaction are usually activated. These reaction methods are known, and the reagents used therein can be appropriately selected from known methods. Examples of protecting groups for amino groups include carbobenzoxy, tert-butyloxycarbonyl, tert-
amyloxycarbonyl, isobornyloxycarbonyl, p-methoxybenzyloxycarbonyl, 2-chloro-benzyloxycarbonyl, adamantyloxycarbonyl, trifluoroacetyl, phthalyl, formyl, o-nitrophenylsulfenyl, diphenylphosphinothioyl Examples include. Examples of protective groups for carboxyl groups include alkyl esters (e.g. alkyl esters such as methyl, ethyl, propyl, butyl, tert-butyl, etc.), benzyl esters, p-nitrobenzyl esters, p-methoxybenzyl esters, and p-chlorobenzyl esters. , benzhydryl ester, carbobenzoxyhydrazide, tert
Examples include groups that can form -butyloxycarbonyl hydrazide, trityl hydrazide, etc. Examples of the guanidino group-protecting group for arginine include nitro, tosyl, p-methoxybenzenesulfonyl, carbobenzoxy, isobornyloxycarbonyl, adamantyloxycarbonyl, and the like. The guanidino group may also be protected in the form of a salt of a suitable acid such as benzenesulfonic acid, toluenesulfonic acid, hydrochloric acid, sulfuric acid, and the like. The hydroxyl groups of threonine and serine can be, but do not necessarily need to be protected, for example by esterification or etherification. Examples of groups suitable for this esterification include lower alkanoyl such as acetyl, aroyl such as benzoyl, and groups derived from carbonic acid such as benzoyloxycarbonyl and ethyloxycarbonyl.
Examples of groups suitable for etherification include benzyl, tetrahydropyranyl, and tert-butyl. Examples of activated carboxyl groups include:
For example, the corresponding acid chlorides, acid anhydrides or mixed acid anhydrides, azides, active esters (methyl alcohol, ethyl alcohol, benzyl alcohol,
Pentachlorophenol, p-nitrophenol, N-hydroxysuccinimide, N-hydroxybenztriazole, N-hydroxy-5-
Examples include esters with norbornene-2,3-dicarboximide, etc. The peptide bond forming reaction may be carried out in the presence of a condensing agent such as a carbodiimide reagent such as dicyclohexylcarbodiimide or carbodiimidazole, or tetraethylpyrophosphite. The peptide of the present invention represented by the above general formula (1) is
Depending on the type of group represented by R, it is more specifically produced according to the methods [] to [] shown below.

【table】

【table】

【table】

[Table] ↓

【table】

[Table] [In the above [] to [], A is a protecting group for an amino group, B is an active group for a hydroxyl group or a carboxyl group, C is a protecting group for a guanidino group of arginine, D
represents a protecting group for the ε-amino group of lysine, E represents a protective group for the γ-carboxyl group of glutamic acid, and F represents a protective group for the carboxyl group. ] In the above, preferable A is:
Boc, Z, p-methoxybenzyloxycarbonyl group, etc., B is preferably an active ester residue such as N-hydroxysuccinimide, p-nitrophenyl ester, or a mixed acid anhydride such as isobutyloxycarbonyl group. residues, azides, etc.
Preferred examples of C include nitro, tosyl, etc., preferred examples of D include tosyl, E.
Preferred examples include benzyloxy, etc.
Preferred examples of F include alkyl ester residues and tert-butoxycarbonyl hydrazide. In the above method [], amino acid (2) and amino acid
The reaction with (3) can be carried out in the presence of a solvent. As the solvent, various solvents known to be usable in the peptide condensation reaction, such as anhydrous or hydrous dimethylformamide, dimethyl sulfoxide, pyridine, chloroform, dioxane,
Dichloromethane, tetrahydrofuran, ethyl acetate, N-methylpyrrolidone, hexamethylphosphoric triamide, or a mixed solvent thereof may be used. The ratio of amino acid (3) and amino acid (2) to be used is not particularly limited and can be appropriately selected within a wide range, but usually the latter is used in an equal amount to 50% of the former.
It is best to use twice the amount, preferably the same amount to 1.5 times the amount. The reaction temperature is within the range known to be usable for peptide bond-forming reactions, usually from about -40 to about 60
℃, preferably selected from the range of about -20 to about 40℃. The reaction time is generally about several minutes to 30 hours. In method [], the reaction between peptide (5) and amino acid (6), the reaction between peptide (8) and amino acid (9), the reaction between peptide (11) and amino acid (12), and the reaction between peptide (14) and amino acid ( Reaction with amino acid (6) and amino acid (4)
The reaction with 5) can be carried out in the same manner as the reaction between amino acid (2) and amino acid (3) above. Also, in method [], the reaction between amino acid (18) and amino acid (19), the reaction between peptide (21) and amino acid (22), the reaction between peptide (24) and amino acid (25), and peptide (27)
Reaction between and peptide (28), reaction between amino acid (19) and amino acid (31) in method [], peptide (33)
Reaction of and amino acid (6) and reaction of peptide (35) and peptide (36), and reaction of peptide (39) and amino acid (40) and peptide (42) in method []
The reaction between peptide (36) and peptide (36) can also be carried out in the same manner as above. Peptides (4), (7), obtained by each of the above reactions
(10), (13), (16), (20), (23), (26), (29), (32), (3
The elimination reaction of protecting group A in 4), (37) and (43) is as follows:
It is carried out in the usual manner. Such methods include, for example, reductive methods (e.g. hydrogenation using catalysts such as palladium or palladium black, reduction with metallic sodium in liquid ammonia), acidolysis (e.g. trifluoroacetic acid, hydrogen fluoride, methanesulfonic acid, hydrogen bromide, etc.). Acidolysis with strong acids such as acids), etc. Hydrogenation using the above catalyst can be carried out, for example, at a hydrogen pressure of 1
It can be carried out at atmospheric pressure and 0 to 40°C. The amount of catalyst used is usually about 100 mg to 1 g, and generally 1 to 48 g.
The reaction completes in about an hour. In addition, the above acidolysis is carried out without a solvent, usually at about 0 to 30°C, preferably at 0.
This can be carried out at ~20°C for about 15 minutes to 1 hour. The amount of acid used is usually about 5 to 10 times the amount of the raw material compound. Furthermore, the above reduction with metallic sodium in liquid ammonia is carried out using such amount of metallic sodium that the reaction solution is colored permanent blue for about 30 seconds to 10 minutes, and is usually carried out at -40 to -
It can be carried out at around 70℃. Also, protecting group C of peptide (10) and peptide (16),
Protecting group D of (29), (37) and (45), peptide (4) and (
6)
protecting group E and peptides (26), (34), (43) and (4
The protecting group F in 6) can be prepared by the above-mentioned reductive method, respectively.
It can be deprotected similarly. Amino acids (2), (3), (6), (9), (15), (18), (19), (22), (25) used in the above methods () to ()
，
(31), (40) and (45) may be known commercial products, and peptides (12), (27), (35) and (42) may be known commercial products or mixed acid anhydride method, azide It is possible to use those obtained by chemical methods. The above mixed acid anhydride method is carried out using an alkylhalocarboxylic acid such as methyl chloroformate, methyl bromoformate, ethyl chloroformate, ethyl bromoformate, isobutyl chloroformate, etc. in the presence of a basic compound in a suitable solvent. . Examples of basic compounds include triethylamine, trimethylamine, pyridine, dimethylaniline, N-methylmorpholine, 1,5-diazabicyclo[4,3,
0] nonene-5 (DBN), 1,5-diazabicyclo[5,4,0] undecene-5 (DBU), 1,
Organic bases such as 4-diazabicyclo[2,2,2]octane (DABCO) and inorganic bases such as potassium carbonate, sodium carbonate, potassium bicarbonate, and sodium bicarbonate can be used. Examples of solvents include various solvents commonly used in the mixed acid anhydride method, specifically halogenated hydrocarbons such as methylene chloride, chloroform, and dichloroethane, aromatic hydrocarbons such as benzene, toluene, and xylene, diethyl ether, Ethers such as tetrahydrofuran and dimethoxyethane, esters such as methyl acetate and ethyl acetate, aprotic polar solvents such as N,N-dimethylformamide, dimethyl sulfoxide, and hexamethylphosphoric triamide, etc. can be used.
The reaction is usually carried out at -20 to 100°C, preferably -20 to 50°C, and the reaction time is generally 5 minutes to 10 hours, preferably 5 minutes to 2 hours. The azidation method is carried out by first reacting an activated carboxyl group with an alcohol such as methyl alcohol, ethyl alcohol, or benzyl alcohol with hydrazine hydrate in an appropriate solvent. It will be done. As the solvent, for example, dioxane, dimethylformamide, dimethyl sulfoxide, or a mixed solvent thereof can be used. The amount of hydrazine hydrate to be used is usually 5 to 20 times, preferably 5 to 10 times, by molar amount relative to the activated carboxyl group. The reaction is usually carried out at 50°C or lower, preferably -
It is carried out at 20-30°C. In this way, a compound (hydrazine derivative) in which the carboxyl group of the terminal amino acid is substituted with hydrazine can be produced. A compound in which the carboxyl group of the terminal amino acid is substituted with azide is produced by reacting the hydrazine derivative obtained above with a nitrite compound in a suitable solvent in the presence of an acid. Hydrochloric acid is usually used as the acid. As the solvent, for example, dioxane, dimethylformamide, dimethyl sulfoxide, or a mixed solvent thereof can be used. Further, as the nitrite compound, for example, sodium nitrite, isoamyl nitrite, nitrosyl chloride, etc. can be used. The nitrite compound is generally used in an equimolar to 2-fold molar amount, preferably in an equimolar to 1.5-fold molar amount, relative to the hydrazine derivative. The reaction is usually carried out at -20 to 0°C, preferably -20 to -10°C, and is generally completed in about 5 to 10 minutes. The peptide (28) in the above method [] can be obtained by removing the protecting groups A and E of the peptide (16), and the peptide (36) in the above method []
can be obtained by removing the protecting group A of peptide (29). Such deprotecting group reactions may be carried out in accordance with the method described above. The peptide of general formula (1) produced as described above is isolated and purified from the reaction mixture by peptide separation means such as extraction, partitioning, column chromatography, etc. In this way, the synthetic peptide of the present invention represented by general formula (1), ie, the C-terminal peptide of human lymphoblastoid interferon, is obtained. The synthetic peptide thus obtained is
By introducing radioactive iodine such as I ¹²⁵ or I ¹³¹ , it can be used as a labeled peptide, which is a raw material for producing labeled antigens used in radioim assay (RIA method). The above radioactive iodine can be introduced by a conventional iodination method, for example, an oxidative iodination method using chloramine T [WMHunter and FC
Greenwood; Nature, 194 , P495 (1962),
Biochem. J. 89 , P114 (1963)]. Specifically, for example, a suitable solvent such as
This is carried out in a solvent such as 0.2M phosphate buffer (PH=7.4) in the presence of chloramine T at around room temperature for about 10 to 30 seconds. The ratio of peptide, radioactive iodine, and chloramine T to be used is, for example, when introducing one radioactive iodine per tyrosine, approximately 1 millicury of radioactive iodine and 10 millicuries of chloramine T per nanomole of tyrosine molecules contained in the peptide. ~
It is best to use about 100 nanomoles of radioactive iodine, and when introducing two radioactive iodines per tyrosine, use about 2 millicuries of radioactive iodine and about 10 to 100 nanomoles of chloramine T per 1 nanomol of tyrosine molecules contained in the peptide. Good to use.
The radioactive iodine-labeled peptide thus produced is isolated and purified by conventional separation means such as extraction, partitioning, column chromatography, dialysis, etc. The peptides thus obtained can be stored by lyophilization, if necessary. Furthermore, the synthetic peptide of the present invention represented by the above general formula (1) can be used as a hapten for producing human α-type interferon antigen. The method for producing the human α-type interferon antigen will be described in detail below. The human α-type interferon antigen is produced by using at least one peptide of the present invention as a hapten and reacting this with a suitable carrier in the presence of a hapten-carrier binding reagent. As the carrier bound to the hapten in the above method, a wide range of high-molecular natural or synthetic proteins commonly used in the preparation of antigens can be used. Examples of the carrier include animal serum albumins such as horse serum albumin, bovine serum albumin, rabbit serum albumin, human serum albumin, and sheep serum albumin, horse serum globulin, bovine serum globulin, rabbit serum globulin, human serum globulin,
Animal serum globulin such as sheep serum globulin, horse thyroglobulin, bovine thyroglobulin, rabbit thyroglobulin, human thyroglobulin, animal thyroglobulin such as ovine thyroglobulin, horse hemoglobulin, bovine hemoglobulin, rabbit hemoglobulin, Animal hemoglobulins such as human hemoglobulin and sheep hemoglobulin, animal hemocyanins, and proteins extracted from roundworms (Ascaris extract, Japanese Patent Application Laid-Open No. 16414/1983, J.
Immun., 111 , 260-268 (1973), J.Immun.
122, 302-308 (1979), J.Immun., 98 , 893-900
(1967) and Am.J.Physiol., 199 , 575-578
(1960) or further purified versions of these), polylysine, polyglutamic acid,
Examples include lysine-glutamic acid copolymers, copolymers containing lysine or ornithine, and the like. As the hapten-carrier binding reagent, a wide variety of those commonly used in the production of antigens can be used. Specifically, crosslinking between amino groups, aliphatic dialdehydes such as glyoxal, malondialdehyde, glutaraldehyde, succinaldehyde, and adipaldehyde, and crosslinking between thiol groups. For example, dimaleimide compounds such as N,N'-o-phenylene dimaleimide and N,N'-m-phenylene dimaleimide, for example, metamaleimide benzoyl-N-hydroxy for crosslinking amino groups and thiol groups. Succinimide ester, 4
-(maleimidomethyl)-cyclohexane-1-
Maleimide carboxyl-N-hydroxysuccinimide ester compounds such as carboxyl-N'-hydroxysuccinimide ester, reagents used in the usual peptide bond-forming reaction to form an amide bond between an amino group and a carboxyl group, such as N,N-dicyclohexylcarbodiimide, N ―
Ethyl-N'-dimethylaminocarbodiimide,
Examples include dehydration condensation agents such as carbodiimides such as 1-ethyl-3-diisopropylaminocarbodiimide and 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbosiimide. In addition, as the above hapten-carrier binding reagent,
It is also possible to use a combination of a diazonium arylcarboxylic acid such as p-diazonium phenylacetic acid and a conventional peptide bond-forming reaction reagent, such as the above dehydration condensation agent. The above antigen production reaction can be carried out, for example, in an aqueous solution or
The reaction is carried out in a normal buffer solution having a pH of 7 to 10, preferably a buffer solution having a pH of 8 to 9, at a temperature of 0 to 40°C, preferably around room temperature. The reaction is usually carried out for about 1 to 24 hours, preferably 3
Completed in ~5 hours. Typical buffer solutions used in the above may include the following. 0.2N sodium hydroxide - 0.2M boric acid - 0.2M potassium chloride buffer, 0.2M sodium carbonate - 0.2M boric acid - 0.2M potassium chloride buffer, 0.05M sodium tetraborate - 0.2M boric acid -
0.05M sodium chloride buffer, 0.1M potassium dihydrogen phosphate-0.05M sodium tetraborate buffer In the above, the proportions of hapten, hapten-carrier binding reagent, and carrier can be determined as appropriate;
Generally, it is preferable to use the carrier in an amount of 2 to 6 times the weight of the hapten, preferably 3 to 5 times the weight, and the hapten-carrier binding reagent to be used in an amount of about 5 to 10 times the weight. Through the above reaction, a human α-type interferon antigen consisting of a peptide-carrier complex in which a carrier and a hapten are bound is obtained through the mediation of a hapten-carrier binding reagent. The antigen obtained after completion of the reaction can be easily isolated and purified by conventional methods such as dialysis, gel filtration, and fractional precipitation. Moreover, the antigen can be preserved by conventional freeze-drying methods. In this way, a human α-interferon antigen consisting of a complex of at least one C-terminal peptide of human α-interferon of the present invention and a carrier is obtained. The antigen usually has an average of 5 to 20 moles of peptide bound to 1 mole of protein, and all of these antigens are highly reproducible and enable the production of highly specific antibodies against human α-type interferon. . Particularly preferred is one in which the binding molar ratio of the peptide to the above-mentioned protein is 1:8 to 15 because it has even higher specificity and can produce antibodies with high titer and high sensitivity. Preparation of antibodies using the antigen obtained above is carried out as follows. That is, it is carried out by administering the above-mentioned antigen to a mammal and collecting antibodies produced in the living body. There are no particular restrictions on the mammal used for antibody production, but it is usually desirable to use rabbits or guinea pigs. For antibody production, a predetermined amount of the antigen obtained above is diluted with physiological saline to an appropriate concentration and mixed with Complete Freubd's Adjuvant to prepare a suspension. may be administered to a mammal. For example, the above suspension is injected intradermally (antigen amount: 0.5-5 mg/time) into rabbits, and then every two weeks,
Immunization may be carried out by administering for 10 months, preferably 4 to 6 months. Antibodies are collected by collecting blood from the immunized animal at a time when large amounts of antibodies are produced after the final administration of the suspension, usually 1 to 2 weeks after the final administration, and centrifuging the blood, followed by separating and collecting the serum. It is done by doing. According to the above, based on the specificity of the antigen used, it is possible to obtain antibodies with excellent specificity for human α-type interferon, high titer, and high sensitivity. The thus obtained human α-interferon antibody can be used, for example, in the RIA method to quantify human α-interferon with high accuracy. Further, the antibody can be used in enzyme immunoassay (EIA) method, florescence immunoassay (FIA) method, etc. by labeling it with an enzyme or a fluorescent substance. Furthermore, the antibody can be made into an insolubilized antibody by reacting with a known insolubilizing substance. In order to explain the present invention in more detail, examples of the production of the peptide of the present invention, an antigen using the same, and an antibody from the antigen will be given below, but the present invention is not limited thereto. The Rf value in each production example was measured by thin layer chromatography on silica gel using the following mixed solvent. Rf〓...1-butanol-acetic acid-water (4:1:
5) Rf =...1 Butanol-pyridine-acetic acid-water (15:10:3:12) <Synthesis of peptide> Production example 1 1 Production of Z-Lys (Tos)-Glu (OBzl)-OBzl H-Glu (OBzl) - Dissolve 4.18 g of OBzl.Tos in 30 ml of dimethylformamide (DMF), add 1.21 ml of triethylamine (TEA), and cool while stirring. Separately, 4.35 g of Z-Lys(Tos)-OH was dissolved in 30 ml of tetrahydrofuran (THF), 0.98 ml of N-methylmorpholine was added, the mixture was cooled to -15°C, and 1.27 ml of isobutyl chloroformate was added dropwise with stirring. dripping
After 30 seconds, add the cold DMF solution prepared above to the solution,
The mixture was stirred at 0°C for 5 minutes, then in a 40°C water bath for 1 minute, and then at 15°C for 30 minutes. THF and DMF are distilled off from the reaction solution under reduced pressure, and the residue is extracted with ethyl acetate. The extract is washed successively with 1N citric acid, saturated brine, saturated aqueous sodium bicarbonate, and saturated brine, dried over anhydrous sodium sulfate, and then ethyl acetate is distilled off. Ethyl ether is added to the obtained oily residue to solidify it, and this is reprecipitated from ethyl acetate-ether to obtain 5.37 g of the desired product. Rf = 0.96 Rf = 0.90 Elemental analysis value (as C ₄₀ H ₄₅ N ₃ O ₉ S) Calculated value (%) C64.59 H6.10 N5.65 Actual value (%) C64.13 H5.95 N5. 63 2(a) Production of H-Lys(Tos)-Glu-OH Dissolve 5.21 g of Z-Lys(Tos)-Glu(OBzl)-OBzl in a mixture of 80 ml of methanol and 20 ml of 10% acetic acid, and add a small amount of palladium black. and stir overnight under H ₂ gas introduction. After the reaction is completed, the catalyst is removed by suction, the liquid is distilled under reduced pressure, and the residue is poured into water and freeze-dried to obtain the desired product. Rf=0.29 Rf=0.52 2(b) Production of Z-Ser-Lys(Tos)-Glu-OH Dissolve 2.13 g of Z-Ser-NHNH ₂ in 20 ml of DMF, add 4.20 ml of 6N hydrochloric acid/dioxane, and - Cool to 15°C and add 1.13 ml of isoamyl nitrite while stirring.
After the reaction solution becomes hydrazide test negative
Neutralize by dropping a small amount of a solution of 3.53 ml of TEA and 1.20 ml of cold DMF. This azide-containing solution was mixed with H-Lys(Tos)-Glu-OH obtained in (a) above and 1.96 ml of TEA.
The mixture was stirred at -10 to -15°C for 2 hours and then at 4°C for 20 hours. DMF was distilled off under reduced pressure, the residue was extracted with ethyl acetate, the ethyl acetate layer was washed with 1N citric acid and saturated brine,
After drying over anhydrous sodium sulfate, ethyl acetate was distilled off under reduced pressure. Ethyl ether is added to the resulting residue to solidify it, and reprecipitation is performed from ethyl acetate-ether to obtain 4.14 g of the desired product. Rf=0.64 Rf=0.65 Elemental analysis value (as C ₂₉ H ₃₈ N ₄ O ₁₁ S・1/2H ₂ O) Calculated value (%) C52.80 H5.96 N8.49 Actual value (%) C52. 87 H5.69 N8.46 3(a) Production of H-Ser-Lys (Tos)-Glu-OH Mix 4.03 g of Z-Ser-Lys (Tos)-Glu-OH with 60 ml of methanol and 40 ml of 10% acetic acid. Add a small amount of palladium black and stir overnight while introducing H ₂ gas. After the reaction is completed, the catalyst is removed by suction, the liquid is distilled under reduced pressure, and the residue is poured into water and freeze-dried to obtain the desired product. Rf=0.23 Rf=0.48 3(b) Z―Arg(NO ₂ )―Ser―Lys(Tos)―Glu
-Production of OH H-Ser-Lys (Tos)-Glu-OH obtained in (a) above
Dissolve in 20 ml of DMF, add 1.74 ml of TEA, and cool while stirring. On the other hand, Z―Arg(NO ₂ )―OH2.41g
Dissolved in THF20ml, N-methylmorpholine 0.70
ml and cooled to -15°C, and 0.86 ml of isobutyl chloroformate was added dropwise while stirring. After 30 seconds of dropping, the cold DMF solution prepared above was added to the solution, and the mixture was reduced to 0.
Stir at 15°C for 5 minutes, then in a 40°C water bath for 1 minute, and then at 15°C for 30 minutes. THF and DMF are distilled off from the reaction solution under reduced pressure, and the residue is extracted with 2% acetic acid-saturated butanol. The extract is washed five times with 2% acetic acid saturated with n-butanol, distilled under reduced pressure, replaced with water to remove acetic acid, and further replaced with methanol to remove water. Ethyl ether is added to the resulting oily residue to solidify it, and this is reprecipitated from ethyl acetate-methanol to obtain 4.09 g of the desired product. Rf=0.42 Rf=0.65 Elemental analysis value (as C ₃₅ H ₄₉ N ₉ O ₁₄ S・1/2H ₂ O) Calculated value (%) C48.83 H5.85 N14.64 Actual value (%) C49. 22 H6.05 N14.11 4 Production of Z-Ser-Leu-NHNH ₂ Dissolve 2.54 g of Z-Ser-NHNH ₂ in 20 ml of DMF, add 5.00 ml of 6N hydrochloric acid/dioxane, cool to -15°C, and stir. Add 1.34 ml of isoamyl nitrite. After the reaction solution becomes hydrazide test negative
Drop a small amount of a solution of 4.20 ml of TEA and 1.40 ml of cold DMF to neutralize. This azide-containing solution was mixed with H-Leu
-Cold DMF with 1.96 g of OC ₂ H ₅ HCl and 1.40 ml of TEA
Add to 15 ml of solution and stir the mixture at -10 to -15°C for 2 hours, then at 4°C for 20 hours. DMF was distilled off under reduced pressure, the residue was extracted with ethyl acetate, the ethyl acetate layer was washed successively with 1N citric acid, saturated brine, saturated aqueous sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, and then washed with ethyl acetate. is distilled off under reduced pressure. Petroleum ether is added to the resulting residue and washed by decantation. Dry the oily substance under reduced pressure in a desiccator, and add 50ml of methanol to the dried substance.
Dissolve 100% hydrazine monohydrate 2.50% under ice cooling.
ml and left at room temperature for 20 hours, then methanol was distilled off under reduced pressure. Ethyl ether was added to the residue to solidify it, which was dried in a desiccator, washed with water to remove excess hydrazine monohydrate, and reprecipitated from methanol-ethyl acetate to obtain the desired product 2.68.
get g. Rf = 0.73 Rf = 0.76 Elemental analysis value (as C ₁₇ H ₂₆ N ₄ O ₅ ) Calculated value (%) C55.73 H7.15 N15.29 Actual value (%) C55.72 H7.01 N15.42 5(a) Production of H-Arg-Ser-Lys(Tos)-Glu-OH Z-Arg(NO ₂ )-Ser-Lys(Tos)-Glu-
Suspend 2.00 g of OH in a mixture of 30 ml of methanol and 30 ml of 50% acetic acid, and add a small amount of palladium black.
Stir for 36 hours under _H2 gas introduction. After the reaction is complete, the catalyst is removed by suction, the liquid is distilled under reduced pressure, the residue is poured into water and freeze-dried, and after 18 hours it is dissolved in water again and freeze-dried to obtain the desired product. Rf = 0.05 Rf = 0.41 5(b) Z―Ser―Leu―Arg―Ser―Lys (Tos)―
Production of Glu-OH Dissolve 1.03 g of Z-Ser-Leu-NHNH ₂ in 15 ml of DMF, add 1.41 ml of 6N hydrochloric acid/dioxane, and -15
Cool to ℃ and add 0.38 ml of isoamyl nitrite while stirring. After the reaction solution becomes hydrazide test negative
Drop a small amount of a solution of 1.18 ml of TEA and 0.40 ml of cold DMF to neutralize. This azide-containing solution was mixed with the H-Arg-Ser-Lys(Tos)-Glu-OH obtained in (a) above and
Add 0.66 ml of TEA to 10 ml of cold DMF solution and stir the mixture for 2 hours at -10 to -15°C and then for 20 hours at 4°C. DMF was distilled off under reduced pressure, and the residue was converted into a water-saturated n-
Extract with butanol, wash the butanol layer five times with n-butanol saturated water, and evaporate under reduced pressure. Add ethyl ether to the resulting residue to solidify it, and reprecipitate from methanol-ethyl acetate to obtain 1.92 g of the desired product. Rf=0.33 Rf=0.69 Elemental analysis value (as C ₄₄ H ₆₆ N ₁₀ O ₁₅ S・2H ₂ O) Calculated value (%) C50.66 H6.76 N13.43 Actual value (%) C50.57 H6 .51 N15.34 6(a) H―Ser―Leu―Arg―Ser―Lys(Tos)―
Production of Glu-OH Z-Ser-Leu-Ser-Lys(Tos)-Glu-
Suspend 1.84 g of OH in a mixture of 30 ml of methanol and 30 ml of 10% acetic acid, and add a small amount of palladium black.
Stir for 13 hours under _H2 gas introduction. After the reaction is completed, the catalyst is removed by suction, the liquid is distilled under reduced pressure, and the residue is poured into water and freeze-dried to obtain the desired product. Rf = 0.09 Rf = 0.53 6(b) Boc―Glu(OBzl)―Ser―Leu―Arg―Ser
-Lys(Tos)-Glu-OH production H-Ser-Leu-Arg-Ser-Lys obtained in (a) above
Dissolve (Tos)-Glu-OH in 20ml of DMF,
Add 0.51ml of TEA under ice-cooling and add Boc-Glu.
(OBzl) - Add 0.95g of ONHS and stir the mixture at room temperature.
Stir for 24 hours. DMF is distilled off under reduced pressure, the residue is extracted with water-saturated n-butanol, and the butanol layer is washed five times with n-butanol-saturated water. The butanol layer was distilled off under reduced pressure, and ethyl ether was added to the resulting residue to solidify it, followed by reprecipitation from methanol-ethyl acetate to obtain 1.70 g of the desired product. Rf=0.42 Rf=0.60 Elemental analysis value (as C ₅₃ H ₈₁ N ₁₁ O ₁₈ S・2H ₂ O) Calculated value (%) C51.82 H6.97 N12.55 Actual value (%) C51.27 H6 .58 N15.47 7(a) Boc―Glu―Ser―Leu―Arg―Ser―Lys
Production of (Tos)-Glu-OH Boc-Glu(OBzl)-Ser-Leu-Arg-Ser-
Lys(Tos)-Glu-OH150mg with methanol 30ml
Dissolve in a mixture with 30 ml of 10% acetic acid, add a small amount of palladium black, and stir for 18 hours while introducing H ₂ gas.
After the reaction is completed, the catalyst is removed by suction, the liquid is distilled under reduced pressure, and the residue is poured with water and freeze-dried to obtain the desired product. 7(b) H-Glu-Ser-Leu-Arg-Ser-Lys
Production of (Tos)-Glu-OH Boc-Glu-Ser-Leu-Arg- obtained in (a) above
Dissolve Ser―Lys(Tos)―Glu―OH in THF,
Leave at room temperature for 15 minutes. Add about 30 ml of anhydrous ether, filter the precipitate, wash with anhydrous ether, and dry under reduced pressure in a desiccator containing potassium hydroxide-phosphorus pentoxide to obtain the desired product. 7(c) H-Glu-Ser-Leu-Arg-Ser-Lys-
Production of Glu-OH H-Glu-Ser-Leu-Arg-Ser obtained in (b) above
-Lys(Tos)-Glu-OH is dissolved in liquid ammonia previously dried with sodium metal and small pieces of sodium metal are added under stirring until the solution remains blue for 30 seconds to 1 minute. Further, crystalline NH ₄ Cl was added to neutralize excess sodium, and ammonia was completely evaporated and distilled at room temperature. The target product ( 58 mg of the peptide of the present invention is obtained. This is called "peptide A". Rf=0.04 Rf=0.34 Elemental analysis value (as C ₃₄ H ₆₁ N ₁₁ O ₁₄・C ₂ H ₄ O ₂・3H ₂ O) Calculated value (%) C44.95 H7.44 N16.02 Actual value ( %) C45.05 H7.11 N15.84 8(a) Production of H-Gln-NHNHBoc 16.00 g of Z-Gln-NHNHBoc in 100 g of methanol
ml, add a small amount of palladium black and add _H2
Stir for 18 hours under gas introduction. After the reaction is completed, the catalyst is removed by suction, the liquid is distilled under reduced pressure, and the residue is dried under reduced pressure in a desiccator to obtain the desired product. Rf = 0.37 Rf = 0.58 8(b) Production of Z-Leu-Gln-NHNHBoc H-Gln-NHNHBoc obtained in (a) above is added to THF50
ml, add 5.51 g of Z-Leu-ONHS under ice cooling, and stir at room temperature for 18 hours. THF was distilled off under reduced pressure,
The residue was extracted with ethyl acetate, and the ethyl acetate layer was diluted with 1N
The mixture is washed successively with citric acid, saturated brine, saturated aqueous sodium bicarbonate, and saturated brine, dried over anhydrous sodium sulfate, and then ethyl acetate is distilled off. Ethyl ether is added to the obtained oily residue to solidify it, and this is reprecipitated from methanol-ethyl ether to obtain 6.04 g of the desired product. Rf = 0.81 Rf = 0.86 Elemental analysis value (as C ₂₄ H ₃₇ N ₅ O ₇ ) Calculated value (%) C56.79 H7.35 N13.80 Actual value (%) C56.67 H7.15 N13.75 9(a) Production of H-Leu-Glu-NHNHBoc Suspend 2.79 g of Z-Leu-Gln-NHNHBoc in 80 ml of methanol and add a small amount of palladium black.
Stir for 32 hours under _H2 gas introduction. After the reaction is completed, the catalyst is removed by suction, the liquid is distilled off under reduced pressure, and the residue is dried under reduced pressure in a desiccator to obtain the desired product. Rf = 0.33 Rf = 0.66 9(b) Production of Z-Asn-Leu-Gln-NHNHBoc H-Leu-Gln-NHNHBoc obtained in (a) above
Dissolve in 30 ml of DMF and cool while stirring. On the other hand, Z-
Dissolve 1.61 g of Asn-OH in 30 ml of THF, add 0.62 ml of N-methylmorpholine, cool to -15°C, and dropwise add 0.80 ml of isobutyl chloroformate while stirring. dripping
After 30 seconds, add the cold DMF solution prepared above to the solution,
The mixture was stirred at 0°C for 5 minutes, then in a 40°C water bath for 1 minute, and then at 15°C for 30 minutes. THF and DMF are distilled off from the reaction solution under reduced pressure, and the residue is extracted with ethyl acetate. The extract is washed successively with 1N citric acid, saturated brine, saturated aqueous sodium bicarbonate, and saturated brine, dried over anhydrous sodium sulfate, and then ethyl acetate is distilled off. Ethyl ether was added to the resulting oily residue to solidify it, and methanol-
Reprecipitate with ethyl acetate to obtain 2.55 g of the desired product. Rf = 0.63 Rf = 0.77 Elemental analysis value (as C ₂₈ H ₄₃ N ₇ O ₉ ) Calculated value (%) C53.10 H6.97 N15.77 Actual value (%) C53.67 H6.63 N15.68 10(a) Production of Z-Thr-ONHS Dissolve 1.28 g of Z-Thr-OH in 30 ml of THF, add 0.58 g of NHS, cool on ice, and then dissolve in N, N
-Dicyclohexylcarbodiimide (DCC) 1.05
Add g. This mixture was stirred at 4°C for 24 hours,
The precipitate was removed, the liquid was distilled off under reduced pressure, ethyl ether was added to the residue, and the residue was washed by decantation.
The obtained oily substance is dried under reduced pressure in a desiccator to obtain the desired product. 10(b) Production of H-Asn-Leu-Gln-NHNHBoc 2.43 g of Z-Asn-Leu-Gln-NHNHBoc is suspended in 80 ml of methanol, a small amount of palladium black is added, and the suspension is stirred for 18 hours while introducing H ₂ gas. After the reaction is completed, the catalyst is removed by suction, the liquid is distilled off under reduced pressure, and the product is dried under reduced pressure in a desiccator to obtain the desired product. Rf = 0.31 Rf = 0.64 10(c) Z―Thr―Asn―Leu―Gln―NHNHBoc
Production of H-Asn-Leu-Gln- obtained in (b) above
NHNHBoc is dissolved in 30 ml of DMF, and to this solution is added the 20 ml DMF solution of Z-Thr-ONHS obtained in (a) above under ice cooling, and the mixture is stirred at room temperature for 18 hours.
1N citric acid is added to the residue obtained by distilling off DMF under reduced pressure to solidify it, and reprecipitation is performed from methanol-ethyl acetate to obtain 2.12 g of the desired product. Rf = 0.82 Rf = 0.79 Elemental analysis value (as C ₃₂ H ₅₀ N ₈ O ₁₁ ) Calculated value (%) C53.18 H6.97 N15.50 Actual value (%) C52.85 H6.95 N15.28 11(a) Production of Z―Thr―Asn―Leu―Gln―NHNH ₂ Z―Thr―Asn―Leu―Gln―NHNHBoc0.91
Dissolve g in 8 ml of TFA and leave at room temperature for 15 minutes.
Add 80 ml of anhydrous ether to quickly filter out the precipitate, wash with anhydrous ether, and dry under reduced pressure in a desiccator containing potassium hydroxide-phosphorus pentoxide to obtain the desired product. Rf = 0.34 11(b) H-Glu(OBzl)-Ser-Leu-Arg-Ser
-Lys(Tos)-Glu-OH production Boc-Glu(OBzl)-Ser-Leu-Arg-Ser-
Dissolve 1.00g of Lys(Tos)-Glu-OH in 8ml of TFA and leave at room temperature for 15 minutes. Add 80 ml of anhydrous ether to quickly filter out the precipitate, wash with anhydrous ether, and dry under reduced pressure in a desiccator containing potassium hydroxide-phosphorus pentoxide to obtain the desired product. Rf = 0.24 Rf = 0.44 11(c) Z―Thr―Asn―Leu―Gln―Glu (OBzl)
―Ser―Leu―Arg―Ser―Lys(Tos)―Glu―
Production of OH Z-Thr-Asn-Leu-Gln- obtained in (a) above
Dissolve NHNH ₂ in 10 ml of DMF, add 0.63 ml of 6N hydrochloric acid/dioxane, cool to -15°C, and add 0.17 ml of isoamyl nitrite while stirring. After the reaction solution becomes negative for the hydrazide test, cool 0.53 ml of TEA.
Add a small amount of DMF 0.40ml solution dropwise to neutralize. This azide-containing solution was mixed with the H-Glu obtained in (b) above.
(OBzl)-Ser-Leu-Arg-Ser-Lys(Tos)-
Add 0.24 ml of Glu-OH and TEA to 10 ml of cold DMF solution and heat the mixture at -10 to -15°C for 2 hours, then at 4°C.
Stir and react for 18 hours. Furthermore, in the same way as in (a) above, Z―Thr―Asn―Leu―Gln―
A reaction product obtained by treating 1.16 g of NNHHBoc with TFA is added to the above reaction mixture, and the mixture is stirred and reacted at the same temperature for 24 hours. DMF is distilled off under reduced pressure, and the residue is extracted with water-saturated n-butanol, washed five times with n-butanol-saturated water, and distilled under reduced pressure. Add ethyl ether to the residue to solidify it, reprecipitate from methanol-ethyl acetate, wash with hot methanol,
get the object. 11(d) H―Thr―Asn―Leu―Gln―Glu―Ser―
Production of Leu-Arg-Ser-Lys(Tos)-Glu-OH Z-Thr-Asn-Leu-Gln-Glu obtained in (c) above
(OBzl)-Ser-Leu-Arg-Ser-Lys(Tos)-
Glu-OH is suspended in a mixture of 50 ml of methanol and 50 ml of 30% acetic acid, a small amount of palladium black is added, and the mixture is stirred for 18 hours while introducing H ₂ gas. After the reaction was completed, the catalyst was removed by suction, the liquid was concentrated under reduced pressure, and methanol was completely distilled off.
25, collect the desired fractions, and freeze-dry to obtain 740 mg of the desired product. Rf=0.17 Rf=0.35 Elemental analysis value (as C ₆₆ H ₉₉ N ₁₇ O ₂₃・C ₂ H ₄ O ₂・2H ₂ O) Calculated value (%) C48.91 H7.08 N15.64 Actual value ( %) C48.82 H6.63 N15.74 12 H―Thr―Asn―Gln―Glu―Ser―Leu―
Production of Arg-Ser-Lys-Glu-OH H-Thr-Asn-Gln-Glu-Ser-Leu-Arg
Dissolve 51.0 mg of -Ser-Lys(Tos)-Glu-OH in liquid ammonia previously dried with sodium metal and add small pieces of sodium metal under stirring until the solution maintains a blue color for 30 seconds to 1 minute. More crystals
NH ₄ Cl was added to neutralize excess sodium, and ammonia was completely distilled off at room temperature.
Collect the desired fraction by gel filtration with Sephadex G-25 gel using % acetic acid.
After concentrating this, water is added and freeze-dried to obtain 32 mg of the target product (peptide of the present invention). This is called "peptide B". Rf=0.01 Rf=0.37 Elemental analysis value (as C ₅₃ H ₉₃ N ₁₇ O ₂₁・C ₂ H ₄ O・3H ₂ O) Calculated value (%) C46.57 H7.32 N16.79 Actual value (% ) C46.10 H6.98 N16.82 13 Production of Z-Leu-Ser-OCH ₃ Dissolve 1.81 _g of H-Ser-OCH 3.HCl in 25 ml of DMF, add 1.62 ml of TEA, and cool on ice to -10°C. While stirring, add 4.21 g of Z-Leu-ONHS and continue stirring at room temperature for 18 hours. DMF was distilled off under reduced pressure, the residue was extracted with ethyl acetate, the ethyl acetate layer was washed with water, dried over anhydrous sodium sulfate, and then ethyl acetate was distilled off under reduced pressure. Ethyl ether is added to the resulting residue to solidify it, and reprecipitated from ethyl acetate-ether to obtain 2.68 g of the desired product. Rf = 0.81 Rf = 0.82 Elemental analysis value (as C ₁₈ H ₂₆ N ₂ O ₆ ) Calculated value (%) C59.00 H7.15 N7.65 Actual value (%) C58.62 H7.03 N7.65 14(a) Production of H-Leu-Ser-OCH ₃・HCl Add 4.20 g of Z-Leu-Ser-OCH ₃ to 40 ml of methanol.
and 11.46 ml of 1N hydrochloric acid, add a small amount of palladium black, and stir for 18 hours while introducing H ₂ gas. After the reaction is completed, the catalyst is removed by suction, the liquid is distilled under reduced pressure, and water is further added and distilled under reduced pressure, which is repeated three times. The residue is dried under reduced pressure in a desiccator containing phosphorus pentoxide to obtain the desired product. Rf=0.38 14(b) Production of Z-Ser-Leu-Ser-OCH ₃ Dissolve 3.19 g of Z-Ser-NHNH ₂ in 25 ml of DMF, add 6.30 ml of 6N HCl/dioxane, and heat at -15°C.
Cool and add 1.69 ml of isoamyl nitrite while stirring. After the reaction solution becomes hydrazide test negative
Add a small amount of a solution of 5.29 ml of TEA and 1.76 ml of cold DMF to neutralize. Add this azide-containing solution to the H-Leu-Ser-OCH _3.HCl obtained in (a) above and 1.60 ml of TEA.
Add the mixture to 20 ml of cold DMF solution at -10 to -15
Stir at 4°C for 2 hours, then at 4°C for 18 hours.
DMF was distilled off under reduced pressure, water was added to the residue to solidify it,
Reprecipitation from methanol-ethyl acetate yields 4.11 g of the desired product. Rf = 0.76 Rf = 0.79 Elemental analysis value (as C ₂₁ H ₃₁ N ₃ O ₈ ) Calculated value (%) C55.62 H6.89 N9.27 Actual value (%) C55.48 H6.92 N9.18 15 Production of Z-Ser-Leu-Ser-NHNH ₂ Dissolve 2.00 g of Z-Ser-Leu-Ser-OCH ₃ in 40 ml of methanol and add 100% NH ₂ NH ₂ under ice cooling.
Add 1.10 ml of H ₂ O and leave at room temperature for 18 hours. After the reaction was completed, the solvent was distilled off under reduced pressure, ether was added to solidify, excess NH ₂ NH ₂ .H ₂ O was removed by adding water, and reprecipitation was performed from methanol-ethyl acetate to obtain 1.91 g of the target product. obtain. Rf = 0.43 Rf = 0.73 Elemental analysis value (as C ₂₀ H ₃₁ N ₅ O ₇ ) Calculated value (%) C52.97 H6.89 N15.44 Actual value (%) C52.85 H6.70 N15.44 16(a) Z―Ser―Leu―Ser―Thr―Asn―Leu―
Gln-Glu-Ser-Leu-Arg-Ser-Lys (Tos)
-Production of Glu-OH Z-Ser-Leu-Ser-NHNH ₂ 70.42mg in DMF5
ml, add 0.78 ml of a 10-fold dilution of 6N hydrochloric acid/dioxane in DMF, cool to -15°C, add 0.20 ml of a 10-fold dilution of isoamyl nitrite in DMF with stirring,
After the reaction solution becomes negative for hydrazide test,
Drop a small amount of 0.65 ml of TEA 10 times diluted in DMF to neutralize. This azide-containing solution was added to the H-Thr-Asn-Leu-Gln-Glu-Ser-Leu obtained in 11(d) above.
-Arg-Ser-Lys(Tos)-Glu-OH151mg
Add 0.29 ml of a 10-fold dilution of TEA in DMF to 5 ml of cold DMF, and stir the mixture at -10 to -15°C for 2 hours, then at 4°C for 18 hours. Furthermore, Z-Ser-Leu-Ser
- Add 117.36 mg of NHNH ₂ and react for 24 hours. DMF was distilled off under reduced pressure, and the residue was extracted with water-saturated n-butanol.
Washed twice with 2% acetic acid saturated with n-butanol five times, concentrated under reduced pressure, added water and further concentrated under reduced pressure,
After completely distilling off n-butanol, it is freeze-dried.
This is reprecipitated with ethyl acetate-ether to obtain the desired product. 16(b) H―Ser―Leu―Ser―Thr―Asn―Leu―
Gln―Glu―Ser―Leu―Arg―Ser―Lys―Glu
-Manufacture of OH Z-Ser-Leu-Ser-Thr-Asn-Leu-Gln-
Glu―Ser―Leu―Arg―Ser―Lys(Tos)―Glu―
Dissolve 150 mg of OH in liquid ammonia previously dried with sodium metal and add small pieces of sodium metal under stirring until the solution maintains a blue color for 30 seconds to 1 minute.
Further, crystalline NH ₄ Cl was added to neutralize excess sodium, and after completely evaporating ammonia at room temperature, the eluate was filtered through Sephadex G-25 gel using 50% acetic acid, and the fractions were collected. After concentration, water is added and lyophilized to obtain 94 mg of the target product (peptide of the present invention). This is called "peptide C". Rf=0.02 Rf=0.39 Elemental analysis value (as C ₆₅ H ₁₂₀ N ₂₀ O ₂₆・C ₂ H ₄ O ₂・H ₂ O) Calculated value (%) C48.19 H7.24 N16.78 Actual value ( %) C47.93 H6.93 N16.49 17(a) Production of Z-Tyr-Osu Dissolve 1.04g of Z-Tyr-OH in 30ml of THF,
- Add 0.38g of hydroxysuccinimide, cool on ice, add 0.68g of DCC under ice-cooling, and cool the mixture at 4℃.
Stir for 18 hours. The precipitate is removed by suction filtration, the liquid is concentrated under reduced pressure, and ethyl ether and petroleum ether are added to the residue, followed by decantation and drying to obtain the desired product. 17(b) Production of H-Ser-Leu-Ser-OCH ₃ Z-Ser-Leu-Ser- obtained in Reference Example 14(b)
Suspend 1.00 g of OCH ₃ in 20 ml of methanol and 20 ml of 10% acetic acid, add a small amount of palladium black, and stir for 14 hours while introducing H ₂ gas. After the reaction is completed, the catalyst is removed by suction, and the liquid is concentrated under reduced pressure, then water is added and freeze-dried to obtain the desired product. Rf = 0.35 Rf = 0.65 17(c) Production of Z-Tyr-Ser-Leu-Ser-OCH ₃ H-Ser-Leu-Ser-OCH ₃ obtained in (b) above
Dissolved in 10ml of DMF, added 0.31ml of TEA under ice cooling, and added the cold Z-Tyr-OSu obtained in (a) above to this solution.
Add DMF solution under stirring. Store the mixture at room temperature for 18
After stirring for an hour, DMF was distilled off under reduced pressure, water was added to the residue to solidify it, and reprecipitation was performed from methanol-ether and then methanol-ethyl acetate to obtain the desired product (1.08 g).
get g. Rf = 0.78 Rf = 0.82 Elemental analysis value (as C ₃₀ H ₄₀ N ₄ O ₁₀ ) Calculated value (%) C58.43 H6.54 N9.09 Actual value (%) C58.14 H6.58 N9.16 17(d) Production of Z-Tyr-Ser-Leu-NHNH ₂ Dissolve 1.00 g of Z-Tyr-Ser-Leu-Ser-OCH ₃ in methanol and add 100% NH ₂ NH ₂ under ice cooling.
Add 0.82 ml of H ₂ O and leave at room temperature for 18 hours. Methanol was distilled off under reduced pressure, ethyl ether was added to the residue to solidify it, and excess NH ₂ NH ₂ was removed by washing with water.
After removing H ₂ O and reprecipitating with methanol-ether, the product was washed with hot methanol to obtain 0.81 g of the desired product. Rf = 0.45 Rf = 0.76 Elemental analysis value (as C ₂₉ H ₄₀ N ₆ O ₉ ) Calculated value (%) C56.48 H6.54 N13.63 Actual value (%) C56.12 H6.57 N13.58 18(a) Z―Tyr―Ser―Leu―Thr―Asn―Leu―
Gln-Glu-Ser-Leu-Arg-Ser-Lys (Tos)
-Production of Glu-OH Z-Tyr-Ser-Leu-Ser-NHNH ₂ 42.3mg
Dissolved in 4ml of DMF and 6N hydrochloric acid/dioxane.
Add 0.34ml of DMF 10 times diluted solution, cool to -15℃,
10 times diluted solution of isoamyl nitrite in DMF 0.09 under stirring
After the reaction solution becomes negative in the hydrazine test, add 0.29 ml of a 10-fold dilution of TEA in DMF little by little to neutralize it. The solution containing this azide was
―Thr―Asn―Leu―Gln―Glu―Ser―Leu―Arg
-Ser-Lys(Tos)-Glu-OH50.0mg and TEA
Add to 4ml of cold DMF solution of 10x DMF dilution 0.10,
The mixture was heated at -10 to -15℃ for 2 hours, then at 4℃ for 18 hours.
Stir for an hour. Furthermore, Z-Tyr-ser-Leu-Ser-
Add 42.3 mg of NHNH ₂ and react for 24 hours. DMF
was distilled off under reduced pressure, and the residue was dissolved in water-saturated n-butanol 30
ml, and dilute the extract with n-butanol saturated water for 10 min.
twice and then five times with 2% acetic acid saturated with n-butanol. The organic layers are collected and concentrated under reduced pressure, and after distilling off n-butanol, they are freeze-dried and reprecipitated with ethyl acetate-ether to obtain the desired product. 18(b) H―Tyr―Ser―Leu―Ser―Thr―Asn―
Leu―Gln―Glu―Ser―Leu―Arg―Ser―Lys
-Production of Glu-OH Z-Tyr-Ser-Leu-Ser-Thr obtained in (a) above
―Asn―Leu―Gln―Glu―Ser―Leu―Arg―Ser
-Lys(Tos)-Glu-OH is dissolved in liquid ammonia previously dried with sodium metal and a small piece of sodium metal is added under stirring until the solution remains blue for 30 seconds to 1 minute. Further, crystalline NH ₄ Cl was added to neutralize excess sodium, and after completely evaporating ammonia at room temperature, the eluate was filtered through Sephadex G-25 gel using 50% acetic acid, and the fractions were collected to obtain the desired product. (Peptide of the present invention) 33 mg is obtained. This is called "peptide D". Rf=0.02 Rf=0.35 Elemental analysis value (as C ₇₄ H ₁₂₃ N ₂₁ O ₂₈・C ₂ H ₄ O ₂・4H ₂ O) Calculated value (%) C47.71 H7.30 N15.79 Actual value ( %) C47.32 H7.24 N15.82 <Production of antigen> Production example 1 Peptide synthesis 5 mg of peptide A obtained in Production Example 7(c) and 15 mg of bovine serum albumin (hereinafter abbreviated as "BSA") Dissolve in 2 ml of ammonia acetate buffer (0.1 mol, pH 7.0). Add 0.11 ml of 0.1 molar glutaraldehyde solution to this solution and stir at room temperature for 5 hours. The reaction mixture was then stirred for 48 h, 4
Dialyze against 1 ml of water at °C. Change the water 5 times during dialysis. Thereafter, the solution containing the peptide-protein complex is freeze-dried to obtain 15 mg of human α-type interferon antigen (hereinafter referred to as "antigen"). This antigen has peptide A for 1 mole of BSA.
is combined with an average of 10 moles. The binding rate of this peptide A and BSA can be determined by further adding the obtained antigen to Cephadex G-50 (eluent: physiological saline,
Detection: 0D280nm, flow rate: 3ml/hour, fractional amount: 1ml each)
And since the presence of peptide A is not recognized,
Peptide A bound to BSA by the gel filtration
The fraction of peptide A and the fraction of other products (dimer of peptide A) are separated, a calibration curve of the standard concentration of peptide dimer is created, the amount of the dimer is determined, and this is used as the starting material. Peptide A used as
It was calculated assuming that the value subtracted from the amount is all bound to BSA. The same applies to each antigen obtained in the following antigen production examples. Production Example 2 5 mg of peptide B obtained in Peptide Synthesis Production Example 12
and 5 mg of BSA in ammonium acetate buffer (0.1
Mol, PH7.0) Dissolve to 2 ml. Add 0.11 ml of 0.1 molar glutaraldehyde solution to this solution and stir at room temperature for 5 hours. The reaction mixture was then heated for 48 hours.
Dialyze against 1 part water at 4°C. Change the water 5 times during dialysis. Thereafter, the solution containing the peptide-protein complex is freeze-dried to obtain 9 mg of human α-type interferon antigen (hereinafter referred to as "antigen"). The obtained antigen has an average of 9 moles of peptide B bound to 1 mole of BSA. Production Example 3 Peptide C 5 obtained in Peptide Synthesis Production Example 16(b)
mg and 25 mg of BSA are dissolved in 4 ml of water. Add 200% dicyclohexyl carbodiimide (DCC) to this solution.
mg and stirred at room temperature for 5 hours. The reaction mixture is then dialyzed against water 2 at 4° C. for 48 hours. Change the water 5 times during dialysis. Then the peptide-
Human alpha interferon antigen (hereinafter referred to as "antigen") is obtained by freeze-drying the solution containing the protein complex.
Get 28mg. The obtained antigen has an average of 12 moles of peptide C bound to 1 mole of BSA. Production Example 4 Peptide D-4 obtained in Peptide Synthesis Production Example 18(b)
mg and 20 mg of BSA are dissolved in 2 ml of ammonium acetate buffer (0.1 molar, PH 7.0). 0.1 in this solution
Add 0.11 ml of molar glutaraldehyde solution,
Stir at room temperature for 5 hours. The reaction mixture is then dialyzed against 1 part water at 4°C for 48 hours. Change the water 5 times during dialysis. Thereafter, the solution containing the peptide-protein complex is freeze-dried to obtain 22 mg of human α-type interferon antigen (hereinafter referred to as "antigen"). The obtained antigen has an average of 9 moles of peptide D bound to 1 mole of BSA. <Production of antibody> 100 μg of the antigen obtained in Antigen production example 3 was added to 1.5 ml.
After dissolving in physiological saline, add Freund's auxiliary solution to
The suspension prepared by adding 1.5 ml to 7 rabbits (2.5
~3.0Kg), and the same amount is administered 6 times every 2 weeks. Thereafter, the same amount as the first dose was administered three times every month. Seven days after the final administration, blood is collected from the test animal and centrifuged to collect antiserum to obtain human α-type interferon antibody (hereinafter referred to as "antigen"). Production Examples 2 to 4 Using the antigens obtained in Antigen Production Examples 1, 2, and 4, human α-interferon antibodies (antibodies, and) were prepared in the same manner as in Production Example 1 above, respectively.
are obtained respectively.・ Production of labeled peptide H-Tyr-Ser-Leu-Ser-Thr-Ans-Leu
―Gln―Glu―Ser―Leu―Arg―Ser―Lys―Glu
-OH, ie, peptide D, is labeled using chloramine T as follows. That is, 5 μg of the above peptide was added to 20 μg of 0.5M phosphate buffer (PH7.0) with Na[ ¹²⁵ ] (carrier free
NEN) Add 1 millicurie of 0.5 molar phosphate buffer, then add 20 μ of 0.5 molar phosphate buffer of 70 mg chloramine T/ml. The reaction is terminated by stirring for 30 seconds at room temperature and adding 50 μ of 60 mg/ml sodium metabisulfite (Na ₂ S ₂ O ₅ ) in 0.5 M phosphate buffer. Then add 1% to the reaction solution.
100μ of a cold sodium iodide aqueous solution was added, and the reaction mixture was transferred to a Sephadex G-25 column (1.0×
30cm) (eluent 0.25% BSA, 10mM
0.05 molar phosphate buffer containing EDTA and 0.02% _NaN3 , PH 7.4) to obtain ¹²⁵ -labeled peptide D. - Measurement of titer Measure the titer of the antibody obtained above as follows. That is, the antibody was dissolved in physiological saline at 10, 10 ² ,
10 ³ , 10 ⁴ , 10 ₅ ... dilute (initial) twice,
To 100μ of each of these, add 0.1ml of ^125- labeled peptide (the labeled peptide obtained above was diluted to about 9500cpm) and 0.05M phosphate buffer (PH = 7.4) [0.25% BSA, 10mM EDTA.
and 0.02% NaN ₃ ] and incubated at 4℃ for 24 hours.
After incubation for hours, the resulting conjugate of antibody and ^125- labeled antigen is separated from unreacted (unbound) ^125- labeled peptide by the dextran-activated charcoal method and centrifugation method (4°C, 30 minutes, 3000 rpm), and the Count the binding rate (%) of the antibody to the ^125- labeled peptide at each dilution concentration.
Measure. The binding rate (%) of the antibody to the ^125- labeled peptide is plotted on the vertical axis, and the dilution factor (initial concentration) of the antibody is plotted on the horizontal axis, and the binding rate is plotted at each concentration. Determine the antibody dilution ratio at which the binding rate is 50%, that is, the antibody titer. As a result, the antibody titer was 50,000.・Human α-type interferon specificity test of antibodies Human β-type interferon at various concentrations (manufactured by Tokyo Metropolitan Clinical Research Institute, specific activity 3×) was used as a test sample.
10 ⁶ U/mg protein), peptide synthesis production example 16
Peptide C obtained in (b), ie, the peptide chain of human α-type interferon, and human α-type interferon (manufactured by Hayashibara Institute, Limphoblastoid Interferon) are used. Also as a standard diluent 0.25
A 0.05 molar phosphate buffer (PH 7.4) containing %BSA, 5mM EDTA and 0.02% _NaN3 is used. Add 0.2 ml of standard diluent and sample to each test tube.
0.1 ml, 0.1 ml of the antibody obtained in antibody production example 3, and
¹²⁵ labeled peptide (labeled peptide obtained above diluted to approximately 2800 cpm) 0.1
ml and incubated at 4℃ for 72 hours.
Normal porcine serum
Add 0.1 ml, then add 0.5 ml of a suspension of activated carbon coated with dextran, and leave at 4°C for 30 minutes.
Next, centrifugation was performed for 30 minutes at 4°C and 3000 rpm to separate the conjugate between the antibody and the ^125- labeled peptide and the unreacted (unbound) ^125- labeled peptide, and the radiation was counted. Assuming that the binding rate (Bo) corresponding to the titer is 100%, determine the percentage of the conjugate (B) between the antibody and the ^125- labeled peptide at the concentration and dilution rate of the test sample. The obtained results show that the antibody is clearly differentiated in its reactivity to human α-type interferon and reactivity to human β-type interferon, and this indicates that it is a highly specific antibody with low cross-reactivity to β-type interferon. I understand that. Similar tests were also conducted on the antibody and, as a result, it was confirmed that it was highly specific for human α-type interferon, almost the same as the antibody.

Claims (1)

[Claims] 1 General formula R-Glu-Ser-Leu-Arg-Ser-Lys-Glu-
OH [In the formula, R is a hydrogen atom, H-Thr-Asn-Leu-
Gln group, H-Ser-Leu-Ser-Thr-Asn-Leu-
Gln group or H-Tyr-Ser-Leu-Ser-Thr-Asn
-Leu-Gln group] C-terminal peptide of human limphoblastoid interferon.