JPS6120560B2 - - Google Patents

Description

[Detailed description of the invention]

Glucagon is a hormone that plays an extremely important role in sugar metabolism in animals, and measurement of its blood concentration is therefore extremely important clinically. The present application relates to antigens useful for producing antibodies for immunoassays useful for measuring the blood concentration of this hormone, particularly glucagon derived from the pancreas. Pancreatic glucagon (H-His-Ser-Gln-Gly-
Thr―Phe―Thr―Ser―Asp―Tyr―Ser―Lys―
Tyr―Leu―Asp―Ser―Arg―Arg―Ala―Gln―
Asp―Phe―Val―Gln―Trp―Len―Met―Asn―
Thr-OH) and intestinal glucagon exist, and pancreatic glucagon is said to be the main one that affects blood sugar levels. Therefore, the clinically necessary measurement value is mainly the blood concentration of glucagon derived from the pancreas. It is well known that the production of antibodies with specificity that can be used clinically is extremely rare, and that antibodies that cross-react with both pancreatic glucagon and intestinal glucagon are mainly obtained. The present inventors synthesized various peptides with the aim of producing antigens that can produce antibodies specific to pancreatic glucagon.
As a result of the investigation, we obtained an unexpected new finding that a pentadecapeptide extending from the 15th position of glucagon to the C-terminus is extremely useful for this purpose, and the present invention was completed. The present invention relates to 1 H-Asp-Ser-Arg-Arg-Ala
―Gln―Asp―Phe―Val―Gln―Trp―Leu―Met
This invention relates to a condensation product obtained by condensing a novel peptide (2) represented by -Asn-Thr-OH with peptide (2) and bovine serum albumin with glutaraldehyde. In this specification, when amino acids, peptides, protective groups, active groups, etc. are indicated by abbreviations,
They are IUPAC - IUB Commission on
The abbreviations are based on Biological Nomenelature or common abbreviations in the field, 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. Arg: Arginine Trp: Tributophane Asn: Asparagine Asp: Aspartic acid Thr: Threonine Ser: Serine Glu: Glutamic acid Gln: Glutamine Ala: Alanine Val: Valine Met: Methionine Met (O): Methionine sulfoxide Leu: Leucine Phe: Phenyl Alanine Z: Carbobenzoxy Boc: t-Butyloxycarbonyl OBu ^t : t-Butyl ester OBzl: Benzyl ester ONB: N-Hydroxy-5-norbornene-
2,3-dicarboximide ester MBS: p-methoxybenzenesulfonyl HONB: N-hydroxy-5-merbornene-
2,3-dicarboximide DOC: N,N'-dicyclohexylcarbodiimide DCU: N,N'-dicyclohexylurea DMF: N,N'-dimethylformamide NMP: N-methyl-2-pyrrolidone TFA: Trifluoroacetic acid THF: Tetrahydrofuran TEA: Triethylamine DCHA: Dicyclohexylamine CMC: Carboxymethylcellulose BSA: Bovine serum albumin The peptide () of the present invention can be produced by conventional methods of peptide synthesis. Either a solid phase synthesis method or a liquid phase synthesis method may be used, but the liquid phase synthesis method is often advantageous. Such means of peptide synthesis are described, for example, in “The Peptides”, Volume 1 (1966),
Schroder and Lubke, Academic Press,
New York, USA or “Peptide Synthesis”
Izumiya et al., Maruzen Co., Ltd. (1975). Examples include imidazole method, redox method, and DCC/additive (eg, HONB, HOBt, HoSu) method. The compound () of the present invention has a starting material having a reactive carboxyl group corresponding to one of two fragments divided into two at any position of its peptide bond, and a reactive amino group corresponding to the other fragment. When the raw materials are condensed using a conventional method for peptide synthesis and the resulting condensate has a protecting group, it can be produced by removing the protective group using a conventional method. In the reaction process for producing peptide (), it is often desirable to protect Asp, and as a final step, the protecting group is removed from the protected peptide () in which at least one of the constituent amino acid residues of the peptide () is protected. In many cases, it can be produced by eliminating all of the Protection of functional groups that should not participate in the reaction of raw materials, protective groups, removal of the protective groups, activation of functional groups that participate in the reaction, etc. can also be appropriately selected from known methods or methods. As the protecting group for the amino group of the raw material, for example, carbobenzoxy, t-butyloxycarbonyl,
t-amyloxycarnyl, isobornyloxycarbonyl, p-methoxybenzyloxycarbonyl, 2-chloro-benzyloxycarbonyl,
Examples include adamantyloxycarbonyl, trifluoroacetyl, phthalyl, formyl, O-nitrophenylsulfenyl, diphenylphosphinothioyl, and the like. Examples of protecting groups for carboxyl groups include alkyl esters (e.g., methyl,
ester groups such as ethyl, propyl, butyl, t-butyl), benzyl ester group, p-nitrobenzyl ester group, p-methoxybenzyl ester group, p-chlorobenzyl ester group, benzhydryl ester group, carbobenzoxyhydrazide group , t-butyloxycarbonyl hydrazide group,
Examples include trityl hydrazide group. As a protecting group for the guanidino group of arginine,
Examples include nitro group, tosyl group, p-methoxybenzenesulfonyl group, carbobenzoxy, isobornyloxycarbonyl, and adamantyloxycarbonyl group. The guanidino group may also be protected in the form of an acid (eg, benzenesulfonic acid, toluenesulfonic acid, hydrochloric acid, sulfuric acid) salt. The hydroxyl group of threonine can be protected, for example, by esterification or etherification.
Examples of groups suitable for this esterification include lower alkanoyl groups such as acetyl groups, aroal groups such as benzoyl groups, and groups derived from carbonic acid such as benzyloxycarbonyl groups and ethyloxycarbonyl groups. Further, examples of groups suitable for etherification include benzyl group, tetrahydropyranyl group, and t-butyl group. However, the hydroxyl group of threonine does not necessarily need to be protected. Methionine may be protected in the form of sulfoxide. Raw materials with activated carboxyl groups include corresponding acid anhydrides, azides, active esters (pentachlorophenol, p-nitrophenol, N-hydroxysuccinimide, N-hydroxybenztriazole, N-hydroxy- Examples include esters with 5-norbornene-2,3-dicarboximide, etc. The peptide bond forming reaction may be carried out in the presence of a dehydrating agent (eg, a carbodiimide reagent such as dicyclohexylcarbodiimide, carbodiimidazole, etc.). This peptide condensation reaction can be carried out in the presence of a solvent. The solvent may be appropriately selected from those known to be usable in peptide condensation reactions. Examples include anhydrous or water-containing dimethylformamide, dimethyl sulfoxide, pyridine, chloroform, dioxane, dichloromethane, tetrahydrofuran, ethyl acetate, N-methylpyrrolidone, or a suitable mixture thereof. The reaction temperature is appropriately selected from the range known to be usable for peptide bond forming reactions, usually about -40°C to about 60°C, preferably about -20°C to about 0°C.
It is appropriately selected from the range of °C. After the completion of the condensation reaction, if the product has a protecting group, it can be removed by a conventional method. Such conventional methods include, for example, reductive methods (e.g., hydrogenation using catalysts such as palladium black, reduction with metallic sodium in liquid ammonia), acidolysis (e.g., with strong acids such as triooroacetic acid, hydrogen fluoride, methanesulfonic acid, etc.). acidolysis). The peptide () produced as described above can be collected from the reaction mixture by peptide separation means, extraction, distribution, column chromatography, etc. Since the peptide () has an argine residue,
It can also be extracted as salt. Examples of acids that can form salts include inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, lactic acid, citric acid, oxalic acid, and maleic acid. Acid can be given. The raw material from which the peptide (2) of the present invention can be produced can also be produced by sequentially condensing each amino acid according to its amino acid sequence using the conventional method of peptide synthesis. The condensation of the pentadecapeptide () of the present invention thus obtained with bovine serum albumin can be carried out using the known glutaraldehyde method [e.g., PROCEEDINGS
OF THE SOIETY FOR EXPERIMENTAL BIOLOGY
and Medicine, 128, 347-350 (1968)]. Bovine serum albumin is used as a carrier for antigens, and in some cases it may be replaced with other proteins that can be used for the same purpose. The optimal ratio of pentadecapeptide () to bovine serum albumin is 10 mg to approximately 20 mg, and a reaction pH of around 7.3 often gives good results. Further, the reaction time is often 2 to 6 hours at room temperature, but around 3 hours is often particularly appropriate. The condensation product thus prepared can be stored by dialysis against water at around 4° C. and freeze-drying in a conventional manner. The condensation product of the present invention produced as described above can be administered to various mammals (e.g., rabbits, mice) according to conventional methods to efficiently produce a product that is specific for pancreatic glucagon. Antibodies with good reactivity can be produced. Peptide () is
Any amount effective to produce antibodies may be sufficient; for example, subcutaneous administration of about 2 mg once to rabbits five times at two-week intervals may produce antibodies. In the following examples, thin layer chromatography was carried out using Silica Gel Plate 60F ₂₅₄ manufactured by Merck Co., Ltd. or Cellulose Plate, Avicel SF manufactured by Futokoshi Yakuhin Co., Ltd., and the following developing solvent was used. Rf ¹ : Chloroform: Methanol: Acetic acid = 9:
10.5 Rf ² :Chloroform:methanol:water=7:
3:0.5 Rf ³ :n-butanol:pyridine:acetic acid:water=
30:20:6:24 Example 1 (1) Production method of Boc-Asn-Thr OBzl Dissolve 112g of Boc-Thr OBzl in 300ml of TFA,
After shaking at room temperature for 20 minutes, add 30 ml of concentrated hydrochloric acid,
Concentrate the solution under reduced pressure. Dissolve the residue in 1 THF, cool on ice, and neutralize by adding 50 ml of TEA. To this, Boc―Asn―OH 76.7g, HONB64, 5g,
Add 74.3g of DCC and stir for 15 hours. precipitated
After filtering off the DCU, the solvent is distilled off under reduced pressure and the residue is dissolved in ethyl acetate. This is washed successively with 10% citric acid water (300 ml x 3), saturated sodium bicarbonate water (300 ml x 3), and water (300 ml x 3), and dried over anhydrous sodium sulfate. After the solvent was distilled off under reduced pressure, ether (1) was added to the residue, the powder was collected by filtration, and then recrystallized from acetonitrile. Yield 105.1g (75.2%) mp165-166℃
[α] ²³ _D -13.9℃ (c=0.9, in DMF), Rf ¹ 0.51,
Elemental analysis calculation value as C ₂₀ H ₂₉ O ₇ N ₃ : C56.72;
H6.90; N9.92, Experimental value: C57.01; H6.89; N9.94 (2) Production method of Bocc-Met(O)-Asn-Thr-OBzl 50.0g of Boc-Asn-Thr-OBzl and 170ml of TFA After shaking at room temperature for 30 minutes, concentrate, add 500 ml of ether, filter as a powder, and dry.
Dissolve this in 400ml of THF, cool on ice, add 20ml of TEA, and then dissolve Boc-Met(O)-ONB (Boc-Met
Dissolve 31.3 g of (O).OH and 23.3 g of HONB in 200 ml of THF, cool on ice, add 26.8 g of DCC, and stir for 4 hours to prepare. ) and stir for 15 hours. After the solvent was distilled off under reduced pressure, ethyl acetate (200 ml) and ether (200 ml) were added to the residue, the powder was collected by filtration, and reprecipitated from acetonatrile. yield
48.5g (72.0%), mp145-147℃ [α] ^23D _-6.5
(c=1.1, in DMF, Rf ¹ 0.19, elemental analysis calculation as C ₂₅ H ₃₈ O ₉ N ₄ S: C52.62; H6.71; N9.82;
S5.62, experimental value: C52.44; H6.73; N9.60; S5.15 (3) Boc-Leu-Met(O)-Asn-Thr-Production method of OBzl Boc-Met(O)-Asn- Thr―OBzl15.0g
After adding 45 ml of TFA and shaking at room temperature for 45 minutes,
Concentrate, add 100 ml of ether, filter to obtain a powder, and dry. Dissolve this in 50ml of DMF, cool on ice, add 5.7ml of TEA, and add Boc-Leu-ONB.
(Boc―Leu―OH6.69g, HoNB5.70g, DCC6.56g
(prepared above) and stir for 15 hours. After the solvent was distilled off under reduced pressure, 200 ml of ethyl acetate was added to the residue, the powder was collected by filtration, and reprecipitated from acetonitrile-ethyl acetate. Yield 15.0g (83.4%),
mp134-136℃, [α] ²⁴ _D -15.1゜(c=1.0,
(in DMF), Rf ¹ 0.25, elemental analysis calculation as C ₃₁ H ₄₉ O ₁₀ N ₅ S: C54.45; H7.22; N10.24; S4.69,
Experimental value: C54.62; H7.60; N9.89; S3.95 (4) Production of Z-Gln-Trp-OBzl H-Trp-OBzl/paratoluenesulfonate
Dissolve 50.0g in THF500ml, cool on ice, TEA15.4
ml, Z-Gln-OH28.0g, HONBl9.7g,
Add 22.7g of DCC and stir for 15 hours. After filtering off the precipitated DCU, it is concentrated and the residue is dissolved in 300 ml of ethyl acetate. This was mixed with saturated sodium bicarbonate water (150ml x 2) and 10% citric acid water (150ml x 2).
2) Wash sequentially with water (150ml x 2). After distilling off the solvent under reduced pressure, the residue was dissolved in 300 ml of THF.
Insoluble matter is filtered off. After concentrating this, ether
Add 500ml, filter it as a powder, and recrystallize it from acetonitrile. Yield 46.1g (82.8%),
[α] ²³ _D +5.8° (c=1.0, in DMF), Rf ¹ 0.60,
Elemental analysis calculation value as C ₃₁ H ₃₂ O ₅ N ₄ : C66.89;
H5.80; 10.07, Experimental value: C66.79; H5.71; N10.20 (5) Production of Z-Val-Gln-Trp-OH Add 50.0 g of Z-Gln-Trp-OBzl to 700 ml of methanol
When the solution is dissolved in water and subjected to catalytic reduction (catalyst: palladium black) for 5 hours, crystals precipitate, which are collected by filtration. This is suspended in 300 ml of DMF, 13 ml of TEA is added and dissolved, and the catalyst is filtered off. Z-Val- to this
After adding ONB37.0g and stirring for 10 hours, 1N-
Add 100 ml of hydrochloric acid to neutralize, then add 500 ml of water and filter as a powder. Wash this thoroughly with methanol. Yield 43.0g (84.7%), mp246-247
℃〔α〕 ²³ _D +12.4゜(c=0.9, in DMF),
Elemental analysis calculation as Rf ¹ 0.14, C ₂₉ H ₃₅ O ₇ N ₅ :
C61.58; H6.24; N12.38, Experimental value: C61.86;
H6.30; N12.36 (6) Production of Z-Phe-Val-Gln-Trp-OH Dissolve 5.1 g of Z-Val-Gln-Trp-OH in 100 ml of acetic acid, perform catalytic reduction for 3 hours, and then remove the catalyst by filtration. and concentrate. Suspend this in 200ml of DMF and add
Z- prepared from 2ml of TEA and 2.70g of Z-Phe-OH
Add Phe-ONB and stir for 7 hours. The solvent was distilled off under reduced pressure, and aqueous acetic acid was added to the residue to form a gel, which was collected by filtration. This is reprecipitated from methanol. Yield 5.50g (84.5%), mp240℃〔α〕 ^24D _+4.1゜(
c.
= 1.0, in DMF), Rf ¹ 0.15, C ₃₈ H ₄₄ O ₈ N ₆・1/2
Elemental analysis calculation as H ₂ O: C63.23; H6.28;
N11.64, experimental value: C63.11; H6.29; N11.80 (7) Boc―Asp(OBzl)―Phe―Val―Trp―OH
Production of Z―Phe―Val―Gln―Trp―OH8.5g,
Dissolve in a mixture of 150 ml of DMF and 50 ml of acetic acid and perform catalytic reduction for 5 hours. After filtering off the catalyst, concentrate, add 100 ml of methanol to the residue, and collect by filtration as crystals. Suspend this in 200ml of DMF, 3.0ml of TEA,
Bo―Asp(OBzl)ONB(Boc―Asp(OBzl)―
(prepared from 3.9 g of OH, 2.4 g of HONB, and 2.7 g of DCC) and stir for 10 hours. After evaporating the solvent under reduced pressure, aqueous acetic acid is added to the residue, the gel is collected by filtration, and reprecipitated from DMF and water. Yield 6.3g (58.1
%), mp191-192℃ (decomposition), [α] ²⁴ _D -6.2゜ (c=1.1, in DMF), Rf ¹ 0.11C ₄₅ H ₅₇ O ₁₁ N ₇・3/
Elemental analysis calculated value as 2H ₂ O: C60.64;
H6.63; N10.76, experimental value: C60.30; H6.48;
N11.34 (8) Boc―Gln―Asp(OBzl)―Phe―Val―Gln
-Production of Trp-OH Boc-Asp (OBzl)-Phe-Val-Gln-Trp-
Add 50 ml of TFA to 6.0 g of OH in a nitrogen stream, shake for 10 minutes, concentrate, add ether, and filter to obtain a powder. Dissolve this in 100ml of DMF,
Add 2.0ml of TEA and add Boc-Gln-ONB (Boc-Gln
- Prepared from OH1.76g, HONB1.34g, DCC1.54g)
Add and stir for 15 hours. Aqueous acetic acid is added to this, the powder is collected by filtration, and then reprecipitated from acetonitrile and water. Yield 5.50g (82.6%), m.
p.210-212℃ (decomposition), [α] ²⁴ _D -10.1゜(c=
1.1, in DMF), Rf ¹ 0.09, Elemental analysis calculated value as C ₅₁ H ₆₅ O ₁₃ N ₉ : C60.52; H6.47; N12.46, experimental value: C60.19; H6.37; N12. 23 (9) Production of Z-Arg(MBS)-Ala-OBu ^t Dissolve 31.0 g of Z-Ala-OBu _t in 300 ml of methanol and perform catalytic reduction for 5 hours, then filter off the catalyst and concentrate. On the other hand, Z-Arg(MBS)-OH.
Suspend 53.0 g of DCHA in 500 ml of ethyl acetate, add 200 ml of 10% citric acid, shake well, wash with water, and dry over anhydrous sodium sulfate. this
Dissolve in 500 ml of THF and add the previously prepared H
-Ala-OBu _t , add 14.9g of HONB, cool on ice, add 17.1g of DCC and stir for 10 hours. After filtering off the precipitated DCU and concentrating, 500% ethyl acetate was added.
ml, 10% citric acid water (200ml x 3), saturated sodium bicarbonate water (200ml x 3), water (200ml
Wash with x3) and dry with anhydrous sodium sulfate. After the solvent is distilled off under reduced pressure, 300 ml of ether is added to the residue to form a powder, which is collected by filtration. yield
44.5g (66.8%), mp125-127℃, [α] ^25D _-6.0
゜ (c=1.0, in DMF), Rf ¹ 0.62, elemental analysis calculated value as C ₂₈ H ₃₉ O ₈ N ₅ S: C55.52; H6.49; N11.56;
S5.27, experimental value: C55.71; H6.49; N11.81; S5.29 (10) Z―Arg(MBS)―Arg(MBS)―Ala―
Production of OBu ^t Dissolve 43g of z-Arg(MBS)-Ala-OBu _t in 500ml of methanol, reduce for 5 hours, and then concentrate. Dissolve the residue in 200ml of DMF and add Z-Arg
(MBS)-OH(Z-Aug(MBS)-OH・
Add 14.0 g of DCHA (prepared from 49.7 g of DCHA) and 14.0 g of HONB, cool on ice, add 16.1 g of DCC, and stir for 15 hours.
After filtering off the precipitated DCU, concentrate and dissolve the residue in 500 ml of chloroform. This is washed with 10% citric acid water (300 ml x 3), saturated sodium bicarbonate water (300 ml x 3) and water (300 ml x 3) in this order, and dried over magnesium sulfate. After distilling off the solvent under reduced pressure, 300 ml of methanol is added and the crystals are collected by filtration and recrystallized from methanol. Yield 52.4g
(79.2%), mp116-118℃ [α] ²⁵ _D -8.8゜(c
=
1.0, in DMF), Rf ¹ 0.42, C ₄₁ H ₅₇ O ₁₂ N ₉ S ₂・2H ₂ O
Elemental analysis calculation value as: C50.86; H6.35;
N13.02; S6.62, experimental value: C51.05; H6.08;
N13.11; S6.62 (11) Boc―Ser―Aug(MBS)―Arg(MS)―
Manufacturing of Ala-OBu ^t Z-Arg(MBS)-Arg(MBS)-Ala-
Dissolve 30.0 g of ^OBut in a mixture of 80 ml of DMF and 300 ml of methanol, and catalytically reduce the solution for 7 hours. After filtering the catalyst and distilling off the methanol under reduced pressure, add Boc-
Add Ser-OH7.3g and HONB6.3g, cool on ice,
Add 7.3g of DCC and stir for 10 hours. precipitated
The DCU is filtered off, the solvent is distilled off under reduced pressure and the residue is dissolved in 500 ml of chloroform. This is washed with saturated sodium bicarbonate (300 ml x 2) and water (300 ml x 2), and dried over magnesium sulfate. After distilling off the solvent under reduced pressure, dissolve in 30 ml of loloform and apply to a silica gel column (400 g). Developed with a solvent of chloroform: methanol: acetic acid (9:0.7:0.35), collected fractions 2 from 600 ml,
Concentrate and add ether to a powder. yield
25.5g (79.0%), mp85-88℃, [α] ^26D _- 20.9
° (c=1.0, in methanol), Rf ¹ 0.33,
Elemental analysis calculation values as C ₄₁ H ₆₄ O ₁₄ N ₁₀ S ₂・H ₂ O:
C49.09; H6.63; N13.96; S6.39, experimental value:
C48.96; H6.55; N13.70; S5.84 (12) Boc―Asp(OBzl)―Ser―Arg(MBS)―
Production of Arg(MBS)-Ala-OH Boc-Ser-Arg(MBS)-Arg(MBS)-Ala
- Add 50 ml of TFA to 10.5 g of OBu ^t , shake and mix at room temperature for 60 minutes, concentrate, add 300 ml of ether, and filter to obtain a powder. Dissolve this in 50ml of DMF,
TEA4.1ml, Boc―Asp(OBzl)・ONB(Boc―
Asp (OBzl)・OH3.4g, HONB1.97g, DCC2.27g
(prepared above) and stir for 15 hours. After distilling off the solvent under reduced pressure, aqueous acetic acid is added to the residue, and the powder is collected by filtration and dried. This is reprecipitated from acetonitrile ether. Yield 6.0g (51.5
%), mp126-130℃, [α] ²⁴ _D +3.5゜(c=1.0
，
(in DMF), Rf ¹ 0.17, elemental analysis calculation as C ₄₈ H ₆₇ O ₁₇ N ₁₁ S ₂・2H ₂ O: C49.26; H6.12; N13.17;
S5.48, experimental value: C49.62; H5.84; N13, OO;
S5.03 (13) Boc―Gln―Asp(OBzl)―Phe―Val―
Gln―Trp―Leu―Met(O)―Asn―Thr―
Production of OBzl Boc-Leu-Met(O)-Asn-Thr-OBzl
Add 25 ml of TFA to 3.25 g, shake at room temperature for 15 minutes, concentrate, add 100 ml of ether to the residue, make a powder, filter, and dry. This is NMP10
ml, add 2 ml of TEA, shake well, add 100 ml of ether, and filter again as a powder. Dissolve this in 100ml of DMF and add Boc-
Gln―Asp(OBzl)―Phe―Val―Gln―Trp―
After adding 4.80g of OH and 2.70g of HONB and dissolving it,
Cool on ice, add 1.55 g of DCC, and stir for 58 hours to form a gel-like reaction solution. After the solvent is distilled off under reduced pressure, the residue is thoroughly washed with aqueous acetonitrile. Yield 5.75g (76.8%), mp23.4℃ (decomposition)
[α] ²⁶ _D -15.8° (c=0.4, in acetic acid), Rf ² 0.63,
Elemental analysis calculation as C ₇₇ H ₁₀₄ O ₂₀ N ₁₄ S:
C58.61; H6.64; N12.43; S2.03, experimental value:
C59.13; H6.96; N12.40; S1.70 (14) Boc―Asp(OBzl)―Ser―Arg(MBS)
―Arg(MBS)―Ala―Gln―Asp(OBzl)―
Phe-Val-Gln-Trp-Leu-Met(O)-Asn
―Production of Thr―OBzl Boc―Gln―Asp(OBzl)―Phe―Val―Gln―
Trp―Leu―Met(O)―Asn―Thr―OBzl
Add 1 ml of anisole to 5.20 g, then add 35 ml of TFA in a nitrogen stream, stir at room temperature for 15 minutes, concentrate, add 100 ml of ether to the residue, and collect by filtration as a powder. Dissolve this in 20ml of NMP,
Add 2.7 ml of TEA and shake thoroughly, then add 200 ml of ether and filter as a powder. Dissolve this in 150ml of DMF, Boc―Asp(OBzl)―
Ser―Arg(MBS)―Arg(MBS)―Ala―
Add 3.66g of OH and 2.36g of HONB, add ice and salt to -10
Cool to ℃ and add 1.02 g of DCC. The reaction solution was heated to 0°C.
After stirring for 10 hours at room temperature and 24 hours at room temperature, the precipitated DCU was filtered off and the solvent was distilled off under reduced pressure. Add 50 ml of water to the residue, filter it as a powder, and wash thoroughly with aqueous acetonitrile. Yield 5.3g
(61.1%), mp237℃ (decomposition), [α] ²⁶ _D -7.1゜(c
= 0.9, in acetic acid), Rf ² 0.63, C ₁₂₀ H ₁₆₁ O ₃₄ N ₂₅ S ₃・
Elemental analysis calculation value as 2H ₂ O: C54.82;
H6.32; N13.32; S3.57, experimental value: C54.47;
H6.16; N13.07; S3.47 (15) H―Asp―Ser―Arg―Arg―Ala―Gln―
Asp―Phe―Val―Gln―Trp―Leu―Met(O)
-Production of Asn-Thr-OH Boc-Asp (OBzl)-Ser-Arg (MBS)-
Arg(MBS)-Ala-Gln-Asp(OBzl)-Phe-
Val―Gln―Trp―Leu―Met(O)―Asn―Thr―
Add 0.25 ml of anisole to 400 mg of OBzl, add 5 ml of methanesulfonic acid, shake and mix at room temperature for 60 minutes, and then add 100 ml of ether to precipitate an oily substance. After removing the ether by decanting, the residue was dissolved in 10 ml of water and mixed with Amberlite IRA-410.
(acetic acid type) column (1 x 10 cm), cool the eluate (liquid volume 50 ml) on ice, add 10 ml of 3N ammonia water, stir at 0°C for 30 minutes, and freeze-dry. Dissolve this in 30 ml of water, and add it to a CMC column (2.2
x 18 cm) and eluted using a linear gradient method from water (500 ml) to 0.2 M ammonia acetate (500 ml).
Fractions from 150 ml to 195 ml are collected and lyophilized. Yield 150mg. Next, dissolve this in 20 ml of water, apply it to an Amberlite XAD-2 column (1.6 x 5 cm), and mix water (200 ml) with 80% ethanol (200 ml).
Elute with a linear gradient method up to 25 ml, collect fractions from 180 ml to 25 ml, distill off the ethanol, and freeze-dry. Yield 115 mg, [α] ²⁴ _D -33.6° (c=
0.6, 50% acetic acid in water), Rf ³ 0.54 (Avicel), amino acid analysis (4% thioglycolic acid 5.7N - hydrochloric acid hydrolysis): Arg2.03 (2); Trp0.87 (1);
Asp3.03 (3); Thr0.97 (1); Ser0.73
(1); Glu2.13 (2); Ala1.00 (1); Val1.03
(1);Met1.00(1);Leu1.03(1);
Phe1.03 (1), (peptide content 85.7%) (16) H-Asp-Ser-Arg-Arg-Ala-Gln-
Asp―Phe―Val―Gln―Trp―Leu―Met―Asn
-Production of Thr-OH (glucagon (15-29)) H-Asp-Ser-Arg-Arg-Gln-Asp-Phe
―Val―Gln―Trp―Leu―Met(O)―Asn―Thr
- Dissolve 225mg of OH in 20ml of 5% thioglycolic acid water and leave it at 50℃ for 20 hours to precipitate a gel.
After distilling off the water under reduced pressure, 5 ml of 50% acetic acid was added to the residue.
Sefidex G-25 column (2.3×
118cm). Elute with 50% acetic acid water, from 180ml
Fractions up to 24 ml are collected and lyophilized. Yield 220
mg, [α] ²⁴ _D -30.0° (c = 0.50% acetic acid in water),
Rf ³ 0.59 (Avicel), amino acid analysis (4% thioglycolic acid, 5.7N-hydrochloric acid hydrolysis): Arg2.15
(2); Trp0.91 (1); Asp3.13 (3);
Thr0.99 (1); Ser0.87 (1); Glu2.20
(2);Ala1.05(1);Val0.96(1);Met1.00
(1); Leu1.07 (1); Phe1.07 (1); Peptide content 79.3% Example 2 Production of conjugate of glucagon (15-29) and BSA 10 mg of glucagon (15-29), 20 mg of BSA at 0.2 M
Dissolved in 4 ml of phosphate buffer (PH7.3), added 4 ml of 5% glutaraldehyde aqueous solution, stirred at room temperature for 3 hours, and dialyzed at 4°C (2 x 4 water).
Freeze dry. Yield 38mg. Lyophilized product obtained above [glucagon (15-
The results of amino acid analysis of BSA (1 mg of each were hydrolyzed in 1 ml of 6N HCl at 110°C for 24 hours) are shown in the table below.

[Table] The above amino acid analysis results show that approximately 10 molecules of glucagon (15-29) are condensed to one molecule of BSA. Moreover, this condensate exhibits a decomposition point of about 270-300°C. Reference Example 1 Method for producing antibodies 8 mg of a conjugate of glucagon (15-29) and bovine serum produced in the same manner as in Example 2 (2 mg of glucagon (15-29) conjugated with bovine serum) was added to distilled water. Dissolve in 1 ml and add Freund Complete Ajiyuband to this.
Add 1 ml of adjuvant) and mix well to make an emulsion, which is injected subcutaneously into the rabbit at several locations. The above procedure is performed 5 times at two-week intervals, and 10 days after the final injection, blood is collected and pilot assay is performed. As a result, it is possible to obtain a glucagon antibody that specifically reacts with pancreatic glucagon, does not react with intestinal glucagon, and can be used at a final dilution concentration of 63,000 times. or,
This antibody contains various glucagon fragments (glucagon (15-23) NH ₂ , glucagon (1-14)
OMe, glucagon (25-29)) does not react at all.

Claims (1)

[Claims] 1 H-Asp-Ser-Arg-Arg-Ala-Gln-Asp
-Phe-Vel-Gln-Trp-Leu-Met-Asn-Thr
-Peptide represented by OH. 2 H-Asp-Ser-Arg-Arg-Ala-Gln-Asp
―Phe―VAl―Gln―Trp―leu―Met―Asn―Thr
- A condensation product obtained by condensing a peptide represented by OH and bovine serum albumin with glutaraldehyde.