JPH0123059B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to enzyme immunoassay ( ^EIA ₎ of human parathyroid hormone (human PTH or its C-terminal fragment) in a test solution. Ser−Gln−Arg−Pro ⁵¹ −Arg
−Lys ⁵³ −Lys−Glu ⁵⁵ −Asp−Asn−Val−
Leu−Val ⁶⁰ −Glu−Ser−His−Glu−Lys ⁶⁵
−Ser−Leu−Gly−Glu−Ala ⁷⁰ −Asp−Lys
−Ala−Asp−Val ⁷⁵ −Asp−Val−Leu−
Thr−Lys ⁸⁰ −Ala−Lys−Ser−Gln ⁸⁴ −OH
[] (In the formula, R ₂ is H or H-R ₃ - group, R ₃
The present invention relates to EIA of human PTH or its C-terminal fragment using an antibody obtained using a peptide represented by (indicates a Cys group). Human PTH is a peptide hormone consisting of 84 amino acids, and in recent years it has been reported that measuring the blood concentration of the C-terminal side of PTH is important for diagnosing PTH-related diseases. [FP
Dibella etal; J.Cln.Endocrinol.Metab., 46 (4),
604 (1978)]. Therefore, the present inventors investigated the C-terminal fragment of human-PTH, 32 residues [h-PTH (53-
84)], 34 residues [h-PTH(51-84)], 39 residues [h
-PTH(46-84)], and further [Cys ⁴⁵ ]-h-PTH
(45−84) and [Tyr ⁴⁵ ]−h−PTH (45−84)
(Japanese Patent Application No. 187686, 1983;
126456), its h-PTH (46-84),
It has been found that [Cys ⁴⁵ ]-h-PTH (45-84) allows good quantification based on EIA using an antibody against the C-terminal fragment of human-PTH. Particularly preferably [Cys ⁴⁵ ]-h-PTH (45-
84) or a protein conjugate thereof, for example, a conjugate with bovine serum albumin (BSA), is used as an enzyme-labeled substance for EIA using a specific antibody obtained using the antigen as an enzyme-labeled substance using the following general formula [] R-Lys ⁵³ − Lys−Glu ⁵⁵ −Asp−Asn−Val−Leu
−Val ⁶⁰ −Glu−Ser−His−Glu−Lys ⁶⁵ −Ser
−Leu−Gly−Glu−Ala ⁷⁰ −Asp−Lys−Ala
−Asp−Val ⁷⁵ −Asp−Val−Leu−Thr−
Lys ⁸⁰ -Ala-Lys-Ser-Gln ⁸⁴ -OH [] (wherein, R is H or R ₁ -Rro ⁵¹ -Arg ⁵² - group,
R ₁ is H or R ₂ −Ala ⁴⁶ −Gly ⁴⁷ −Ser ⁴⁸ −Gln ⁴⁹ −
Arg ⁵⁰ - group, R ₂ is H or H-R ⁴⁵ ₃ - group, R ₃ is
It has been found that human-PTH or human-PTHC terminal fragments can be quantified favorably by using a labeled compound in which a peptide represented by Cys (indicating a Cys group) is labeled with an enzyme. The present invention was made based on the above findings, and involves adding an enzyme-labeled C-terminal fragment of human PTH and an antibody as a labeled antigen to a test solution containing human PTH or its C-terminal fragment in an immunoreaction medium. The following general formula [] R ₂ −Ala−Gly−Ser−Gln−Arg−Pro−Arg−
Lys−Lys−Glu−Asp−Asn−Val−Leu−
Val−Glu−Ser−His−Glu−Lys−Ser−
Leu−Gly−Glu−AlaAsp−Lys−Ala−Asp
−Val−Asp−Val−Leu−Thr−Lys−Ala
-Lys-Ser-Gln-OH [] (In the formula, R ₂ is H or H-R ₃ - group, R ₃
(represents a Cys group) is used to sensitize a non-human mammal to react with a specific antibody obtained, and then the bound part and the unbound free part are separated by the reaction. This is a method for measuring human-PTH or its C-terminal fragment in a test liquid, which is characterized in that the enzyme activity of the bound portion or the unbound free portion is then measured. First, the peptides represented by the general formula [] and the peptides represented by the general formula [] used in the present invention are produced by condensing individual amino acids or lower peptides in the amino acid order shown by the formula [], [], and then reacting with the condensation reaction. It is obtained by removing the protective group of the functional group on the side chain in the final step. As for the condensation order, it can be synthesized in any order as long as it is an amino acid sequence shown by the formulas [] and [], but C-
It is advantageous to synthesize from the terminal side. In addition, when synthesizing, it is preferable to use a condensation method such as the carbodiimide method, azide method, active ester method, or anhydride method, and in each step of the condensation, a method that does not cause racemization or a method that minimizes racemization is preferred. It is desirable to use a method, preferably an azide method,
Active ester method, Wu¨nseh method or Geiger method, especially N-ethyl-N'-3-dimethylaminopropyl-carbodiimide (WSCI) as condensing agent
A modified method using . In the synthesis, condensation is carried out sequentially using appropriately available protecting groups based on the synthetic techniques of the peptide field, and the synthetic techniques of the peptide field are widely used. Regarding the details of the synthesis, please refer to Japanese Patent Application No. 187680/1980 (Japanese Patent Application Laid-open No. 126456/1982), although it is not limited in any way. In this way, h-PTH (46-84), [Cys ⁴⁵ ]-h-PTH (45-
h-PTH (53-84) as a control to obtain a peptide represented by the general formula [] of 84), and a peptide or antibody of the general formula [] used as an enzyme label;
It is sufficient to obtain h-PTH (51-84). In this way, the peptide represented by the general formula [] (hereinafter abbreviated as peptide []) and the peptide represented by the general formula [] (hereinafter abbreviated as peptide []) were used to determine human PTH based on EIA.
Or, it measures its C-terminus/fragment, but first, it is used for PTH measurement.
They prepare all the reagents necessary to conduct EIA, such as antiserum, antibodies, and enzyme labels. First, when obtaining a specific antibody using the peptide [], the peptide [] may be used as is or
It is used as a conjugate with a protein such as BSA or its treated product with an alkali treatment or treatment with sodium lauryl sulfate and mercaptoethanol to cleave an intramolecular disulfide group, and is used in various mammals such as rabbits, rats, guinea pigs, and mice. All you have to do is to sensitize it by administering it to
For example, sensitization can be achieved by emulsifying the above-mentioned peptide [] or its protein conjugate in Freund's Complete Adjuvant, injecting this subcutaneously into guinea pigs, and administering this 4 to 7 times at 2-week intervals; Blood may be collected from an animal that has produced the desired antibody, and the blood may be centrifuged in a conventional manner to obtain the antiserum. Furthermore, this antiserum contains a sufficiently high concentration of specific antibodies, and may be stored as it is, or may be diluted as needed before use. Further, this antiserum may be used to obtain its specific antibody by conventional methods such as salting out, isoelectric precipitation, dialysis, chromatography, and gel filtration. In addition, in order to obtain a conjugate with the above protein, a polyfunctional reagent such as succinic aldehyde,
Aldehyde compounds such as glutaraldehyde and adipaldehyde, diisocyanate compounds such as hexamethylene diisocyanate and 2,4-toluene diisocyanate, and 3-(2'-benzothiazolyl-dithio)propionic acid succinimide ester (patent application) 53-85900 (Japanese Unexamined Patent Publication No. 55-17302)) 6-N[3-(2'-benzothiazolyldithio)propionyl]caproic acid succinimide ester, N-[2-(2'-pyridyldithio) )Ethyl]3-(2'-Benzothiazolyludithio)propionamide Patent Application No. 3507, 1983
Publication No. 94367), Japanese Patent Application No. 41737 (1983)
133382), Japanese Patent Application No. 133382 (Japanese Patent Application No. 1983-44589)
136261), maleimidobenzoic acid succinimide ester, N,N'-ethylene bismaleimide, bisdiazobenzidine, diethylmalonimidate, etc., and these polyfunctional reagents are effective in binding the peptide [] and protein used. They may be selected and used in consideration of the functional groups involved, such as amino groups, carboxyl groups, and thiol groups. Particularly preferably, the peptide [] is [ ^Cys45 ]-
By binding proteins using h-PTH (45-84) and a reagent that reacts well with thiol groups such as 3-(2'-benzothiazolyl-dithio)propionic acid succinimide ester as a polyfunctional reagent. , a good protein conjugate of peptide [] can be obtained based on the thiol group of the Cys ⁴⁵ group. In addition, for binding, the amount of the peptide [] of the present invention and protein, such as BSA, to be used is approximately 1 to 20 moles of peptide [] per mole of BSA. Furthermore, for the reaction, for example, pH7~
Add the required amount of peptide [ ] to the aqueous medium in step 8, then add the polyfunctional reagent to this and cool/cool normally.
After reacting at room temperature for 1 to 5 hours, purifying this as appropriate using a purification method such as gel filtration, adding BSA to this and reacting at room temperature for 1 to 5 hours,
After the reaction, it may be purified by means such as gel filtration or dialysis to obtain a conjugate of the peptide [ ] and a protein such as BSA. Furthermore, the above-mentioned specific antibody can be prepared by treating an insoluble carrier such as an insoluble protein carrier such as albumin or gelatin, agarose, cellulose or dextrin with epichlorohydrin or cyanogen bromide, and further treating with an amino group-introducing reagent thereof, as necessary. Insoluble semi-synthetic polymeric carrier, acrylonitrile, acrylic acid, acrylic ester, methacrylic acid, methacrylic ester,
An insoluble polymeric carrier such as a polymer or copolymer of vinyl alcohol, vinyl acetate, aminostyrene, acrylamide, ethylene, etc. may be used as an insolubilized antibody bound with a polyfunctional compound as described above. Furthermore, when obtaining an enzyme-labeled peptide [] used for EIA measurement, the first enzyme used is an oxidoreductase, a hydrolase, a transposase, a lyase, an isomerase, or a ligase. Examples include lactate dehydrogenase, maleate dehydrogenase, malate dehydrogenase, maltose dehydrogenase, lactate oxidase, maleate oxidase, glucose oxidase, choline oxidase, xanthine oxidase, amino acid oxidase, sarcosine oxidase, catalase, α-amylase, β- Examples include galactosidase, lysozyme, lipase, alkaline phosphatase, aminopeptidase, trypsin, papain, α-chymotrypsin, amidase, hexokinase, and glycerokinase. Furthermore, any spacer may be introduced in advance into these enzymes, such as dialdehydes such as glutaraldehyde, reactive derivatives such as ω-amino acid acid chloride, dialdehydes or dicarboxylic acid chlorides, hexamethylene diamine, decamethylene, etc. New aldehyde groups, amino groups, and thiol groups may be introduced using diamines such as amines, S-acetylmercaptosuccinic anhydride, dialdehydes, and spacer-introducing reagents such as 2-aminoethanethiol. When obtaining an enzyme label using such an enzyme, the peptide of the present invention [], the amino group possessed by the enzyme,
Based on hydroxyl groups, carboxyl groups, thiol groups, etc., and further introduced aldehyde groups, amino groups, thiol groups, carboxyl groups, etc., a peptide [mono-enzyme conjugate] is created using a polyfunctional reagent that can bind both groups. This is what you get. In addition, when producing an enzyme label that is such a peptide []-enzyme conjugate, for example, methanol, ethanol, acetone, dioxane,
The peptide of the present invention [ ] is reacted with a polyfunctional reagent at 0°C to 40°C in the presence of or in an organic solvent such as dimethylsulfoxide, dimethylacetamide, or tetrahydrofuran. In this case, the ratio to be used is equal to or more than the equimolar ratio of the polyfunctional reagent to the peptide [], and usually the equimolar ratio is used. After this reaction is completed, the enzyme may be purified if necessary, and an enzyme may be added thereto, and the reaction may be carried out preferably in a buffer having a stable pH for the enzyme.The amount of enzyme used is equal to that of the peptide. It is sufficient to use a molar amount. The product peptide []-enzyme conjugate obtained in the above manner may be purified and collected by purification means such as adsorption chromatography or gel filtration. The competition law of EIA is then used in implementing the invention. To give a detailed example of the competition method, first, a sample for which the content of human PTH is to be measured, an enzyme-labeled peptide [], and an antiserum or antibody obtained using the peptide [] are mixed with an immunoreaction medium, such as a phosphoric acid Incubate in a buffer solution or veronal buffer at about 4 to 5°C for about 1 to 3 days, and then separate the bound part B, which is the immune-bound part, from the free part F, which is not bound to B.
-F separation, preferably by adding normal serum from the same mammal as the one used for antiserum preparation and antiserum against that animal, incubating overnight, and then centrifuging at 3000 rpm for about 20 to 30 minutes. to separate B-F, and then measure the enzyme activity of the enzyme-labeled substance contained in the precipitate (B) or the F-F separation.
The enzyme activity in the liquid layer of the barrel may be measured. In addition, in the solid phase method, a competitive reaction is performed using an insolubilized antibody instead of the antiserum or antibody obtained using the peptide [] in the competitive method, and after the reaction, the B-F separation is performed, and similarly the enzyme All you have to do is measure the activity. In this way, by using an antibody obtained using peptide [] and an enzyme-labeled form of peptide [], human PTH or its C-terminal fragment in a sample can be quantified extremely accurately and easily. This is an excellent method, and in particular, [Cys ⁴⁵ ]-h-PTH (45-84) labeled with β-galactosidase was particularly preferred. Next, the present invention will be described in detail with reference to Examples and Reference Examples, but the present invention is not limited thereto. Example (1) Regarding antigens. h-PTH (53-
84), h-PTH (51-84), h-PTH (46-84),
and BSA − [Cys ⁴⁵ ] − obtained as follows.
h-PTH (45-84) was used as the antigen. Note that BSA-[Cys ⁴⁵ ]-h-PTH (45-84) was prepared as follows. 50 mg of BSA was dissolved in 10 ml of 0.1M phosphate buffer (PH8.0), and 6 mg of EDTA tetrasodium salt was added thereto. This was then added with 1.5 ml of a dimethylformamide solution containing 5 mg of 3-(2'-benzothiazolyl-dithio)propionic acid succinimide ester.
was stirred and reacted for 60 minutes under ice cooling, and after the reaction, 50 mg of [Cys ⁴⁵ ]-h-PTH (45-84) was added thereto. After reacting for 30 minutes under ice-cooling, adjust the pH to 7.0 and add Cephadex G-75 [containing 0.15M NaCl].
Filled with 0.01M phosphate buffer (PH7.2)] column (diameter 5cm x 50cm) from 350ml.
Collect the effluent fraction of 450 ml and add BSA-[Cys ⁴⁵ ]-h
-PTH (45-84) containing category was obtained [per 1 molecule of BSA, [Cys ⁴⁵ ]-h-PTH (45-84) has an average of 11 molecules bound]. (2) About antibodies Using each of the above antigens, antibodies were prepared at a concentration of 500 μg/ml.
A 0.01M phosphate buffer (PH7.0) containing 0.15M NaCl was prepared. 2.5 ml of each solution was taken, emulsified by adding 25 ml of Freund's Complete Adjuvant, and immunized by subcutaneous injection into the backs of 5 male guinea pigs (5 subcutaneous injections every 2 weeks). Then, two weeks after the final immunization, blood was collected from the heart, and antiserum against each antigen was obtained according to a conventional method. In addition, hereinafter, the antiserum against h-PTH (53-84) is abbreviated as A, the antiserum against h-PTH (51-84) is abbreviated as B, and the antiserum against h-PTH (46-84) is abbreviated as C. , BSA−[Cys ⁴⁵ ]−h−PTH(45−
Antiserum against 84) is abbreviated as D. (3) About the enzyme label: 4 mg of [Cys ⁴⁵ ]-h-PTH (45-84) in 1 ml.
Dissolved in 0.1M phosphate buffer (PH7.0) and added N[2-(2'-pyridyldithio)ethyl]3
- 0.9 ml of dimethylformamide containing 0.6 mg of (2′-benzothiazolyl-dithio)propionamide
was added, and the reaction was stirred at 0°C for 60 minutes. After the reaction, adjust the pH to 6.0 with hydrochloric acid, and add Sephadex G-25 using 0.1M phosphate buffer (PH6.0) as a medium.
Gel filtration with a column (1 cm x 50 cm) of 15 to 19
ml fractions were obtained {[Cys ⁴⁵ ] per ml -h-
Contains 660γ as PTH(45−84)}. Then β
- Add 20μ of the above fraction to 1.5ml of 0.1M phosphate buffer (PH8.0) containing 1.2mg of galactosidase,
After stirring for 60 minutes at 0°C, the reaction was carried out using a Sephadex G-100 column (1 cm x 50 cm).
0.01M phosphate buffer containing 0.15M NaCl (PH7.2)
The eluate was eluted and gel-filtered to obtain a 13-17 ml eluate fraction. The β-galactosidase labeled product in this fraction is [Cys ⁴⁵ ]-h-PTH (45-84):
The ratio of β-galactosidase was approximately 1:1 (hereinafter abbreviated as "LotA"). Dissolve 2 mg of h-PTH (53-84) in 1 ml of 0.1M phosphate buffer (PH8.0), and add 3-(2'-
0.2 mg of dimethylformamide containing 315 μg of benzothiazolyl-dithio)propionic acid succinimide ester was added, and the mixture was reacted with stirring at 0° C. for 60 minutes. After the reaction, this 8.5μ was added to 0.1M phosphate buffer (PH) containing 1mg of β-galactosidase.
7.0) and reacted at 0°C for 60 minutes. Next, the reaction solution was gel-filtered through a Sephadex G-100 column (1 cm x 50 cm).
Elution fractions of 14-17 ml were obtained. The β-galactosidase labeled substance of this product is h-PTH(53-84):β
- The ratio of galactosidase was approximately 1:1 (hereinafter abbreviated as "LotB"). (4) Regarding the measurement method, incubate 100μ of the sample solution, 100μ of the enzyme-labeled solution, 100μ of appropriately diluted antiserum, and 100μ of normal guinea pig serum (100-fold dilution) at 5°C for 24 hours, and then incubate with anti-guinea pig γ-globulin. Add 10μ of rabbit serum (10-fold dilution) and
℃, 24 hour ink bait training, 0.5M to this
After adding 3 ml of NaCl, the mixture was centrifuged at 3000 rpm for 15 minutes to collect the precipitate. Then, this precipitate was
0-nitrophenyl-β-galactoside 5mg/ml
Contains 0.1M phosphate buffer (0.1% sodium azide, 0.1% BSA, 20mM mercaptoethanol,
Contains 10% ethanol, PH6.7) 200μ plus 37
C. for 90 minutes, then 2.5 ml of 0.2M glycine buffer (PH 10.4) was added to stop the reaction, and the absorbance was measured at a wavelength of 420 nm. In addition, as a dilution medium for immune reaction,
0.1% sodium azide, 0.25% BSA,
A 0.01M phosphate buffer (PH7.2) containing 0.15M Nacl.5mMEDTA was used. The antibody titer (Bo/T) of each antiserum was measured using LotA as an enzyme label. The results were as shown in Table 1.

[Table] Each antiserum was used at a 1000-fold dilution. Using LotB as an enzyme label, the above C
Antibody titer (Bo/T) of antiserum and D was measured. The results were as shown in Table 2.

[Table] Each antiserum was used at a 2000-fold dilution. A calibration curve was created using the antiserum. The antisera used were A-2 (600-fold dilution);
B-2 (500-fold dilution) C-2 (1500-fold dilution), D-
A calibration curve for h-PTH (53-84) was created using 2 (2000-fold dilution) and LotB as an enzyme label. As a result, the results are as shown in Figure 1 for A-2, as shown in Figure 2 for B-2, as shown in Figure 3 for C-2, and as shown in Figure 3 for D-2. was as shown in Figure 4. As a result of the above, the antiserum obtained using the peptide of the present invention [] gives a good calibration curve, and therefore human-PTH or its C-terminal fragment can be measured well by EIA. It was hot. Reference example 1 h-PTH (53-84); H-Lys-Lys-Glu-
Asp−Asn−Val−Leu−Val−Glu−Ser−His−
Glu−Lys−Ser−Leu−Gly−Glu−Ala−Asp−
Lys−Ala−Asp−Val−Asp−Val−Leu−Thr−
Production example of Lys-Ala-Lys-Ser-Gln-OH. (1) BOC−Lys(Z−Cl)−Lys(Z−Cl)−Glu
(OBzl)−Asp(OBzl)−Asn−Val−Leu−Val
−Glu(OBzl)−Ser(Bzl)−His−Glu(OBzl)−
Lys(Z−Cl)−Ser(Bzl)−Leu−Gly−Glu−
(OBzl)-Ala-Asp(OBzl)-Lys(Z-Cl)-Ala-Asp
(OBzl)−Val−Asp(OBzl)−Val−Leu−Thr
(Bzl)−Lys(Z−Cl)−Ala−Lys(Z−Cl)−
Ser(Bzl)-Gln-OBzl [1] [Amino acid analysis;
Thr0.92(1)Ser1.60(3), Glu4.06(5), Gly0.85(1),
Ala3.00(3), Val3.33(4), Leu2.48(3), Lys5.41
(6), His0.70(1)] 3.49g (0.6mM) and 3ml of anisole in 25ml of anhydrous hydrogen fluoride (HF) at 0℃
was added under cooling and stirred for 75 minutes. After reaction, HF
was distilled off under reduced pressure, and ether was added to the residue. The resulting precipitate was collected and dissolved in 50ml of 0.1N acetic acid.
It was passed through a Dowex x 1 column (2.7 x 35 cm). The effluent was freeze-dried to obtain 2.18 g of crude product. This was dissolved in 50 ml of 8M urea aqueous solution, adjusted to pH 9.5 with aqueous ammonia, and then left for 50 minutes. Next, this solution was charged to a CM-cellulose column (4.4 x 12 cm) packed with 8M urea aqueous solution, and about 100ml of 0.01M ammonium acetate aqueous solution (PH4.5) was discharged, and then 0.01M ammonium acetate aqueous solution (PH4.5) was charged. ) Elution was performed using a linear concentration gradient from 700 ml to 700 ml of a 0.1 M aqueous ammonium acetate solution (PH4.5), followed by elution with 300 ml of a 0.2 M aqueous ammonium acetate solution (PH4.5).
The eluate was fractionated into 13.5 ml portions, and each fraction was
−Category C ₁ of 30th to 50th line, _C2 and 120 of 56th to 119th line measured by Lowry method (500 nm)
~150th segment _C3 eluate was obtained. Each fraction was desalted by passing it through a Sephadex LH-20 column. The effluent was fractionated into 85 ml portions, and each fraction was measured in the same manner as above. Category C ₁ is
Pass through a 3.4 x 113 cm column and divide the 31st to 40th sections.
L ₁ , 41st to 44th segment L ₂ and 45th to 54th segment L ₃ were obtained. Segment _C2 was passed through a 3.4 x 120 cm column to obtain the 35th to 45th segment _L1 , the 46th to 52nd segment _L2 , and the 53rd to 60th segment _L3 . classification
_C3 was passed through a 3.4 x 120 cm column to obtain the 31st to 44th sections _L1 and the 45th to 52nd sections _L2 . Each section was freeze-dried to obtain the following components.

[Table] (2) Purification of C ₂ L ₂ Dissolve 565 mg of the above C ₂ L ₂ in 5 ml of 0.1N acetic acid,
Charge a CM-cellulose column (4.4 x 70 cm) and add 0.01M ammonium acetate aqueous solution (PH
4.5) Elution was performed using a linear concentration gradient from 500 ml to 500 ml of a 0.1 M ammonium acetate aqueous solution (PH4.5). The eluate was fractionated into 6.0ml portions, and each fraction was
Measured by Folin-Lowry method: 113th to 136th segment C ₂ L ₂ − C ₁ , 137th to 151st segment C ₂ L ₂ −
_C2 and the 152nd to 190th sections _{C2L2 - C3} were obtained. Each fraction was desalted by passing it through a Sephadex LH-20 column. The effluent was fractionated into 5.2 ml portions.
Each fraction was measured in the same manner as above. classification
C ₂ L ₂ −C ₁ is passed through a 3.4 x 120 cm column and 55 to 72
The main division _{C2L2 - C1L1} and _the 73rd to 80th divisions _{C2L2 - C1L1 were} obtained. Division C ₂ L ₂ −C ₂ is 3.4×
Pass through a 110cm column and divide the 50th to 59th sections C ₂ L ₂
−C ₂ L ₁ and the 60th to 67th sections C ₂ L ₂ −C ₂ L ₂ were obtained. Segments C ₂ L ₂ - C ₃ are passed through a 3.4 x 110 cm column, and C ₂ L ₂ - C ₃ L ₁ and 61 of the 45th to 60th sections are passed through a 3.4 x 110 cm column.
~72nd segment C ₂ L ₂ −C ₃ L ₂ was obtained. Each section was freeze-dried to obtain the following components. C ₂ L ₂ −C ₁ L ₁ 108.7mg C ₂ L ₂ −C ₁ L ₂ 95.9mg C ₂ L ₂ −C ₂ L ₁ 53.6mg C ₂ L ₂ −C ₂ L ₂ 44.1mg C ₂ L ₂ −C ₃ L ₁ 97.0mg C ₂ L ₂ −C ₃ L ₂ 95.1 mg (3) Purification of C ₂ L ₂ −C ₁ L ₁ Dissolve the above C ₂ L ₂ −C ₁ L ₁ in 1 ml of 0.1N acetic acid,
Charge a CM-cellulose column (2.0 x 15 cm) and add 0.01M ammonium acetate aqueous solution (PH
4.5) Elution was performed using a linear concentration gradient from 300 ml to 300 ml of a 0.1 M ammonium acetate aqueous solution (PH4.5). The eluate was fractionated into 7.4ml portions, and each fraction was
46 measured by Folin-Lowry method (500nm)
~56th segment _{C2L2 - C1L1 -} C was obtained. This was lyophilized, charged to a Sephadex LH-20 column (3.0 x 90 cm), and eluted with 0.1N acetic acid. After elution, fractionate into 6.0 ml portions and measure using the same method as above to determine the 25th to 35th fraction C ₂ L ₂ −
_{C1L1 -} CL was obtained. This is freeze-dried and h-
90.0 mg of PTH (53-84) was obtained. TLC; R ₉ = 0.76 1 spot Amino acid analysis; Asp4.45(5), Thr0.92(1),
Ser2.16(3), Glu4.94(5), Gly0.96(1), Ala3,
Val3.96(4), Leu2.92(3), Lys6.22(6), His1.01(1) Reference example 2 h-PTH(51-84); H-Pro-Arg-Lys-
Lys−Glu−Asp−Asn−Val−Leu−Val−Glu−
Ser−His−Glu−Lys−Ser−Leu−Gly−Glu−
Ala−Asp−Lys−Ala−Asp−Val−Asp−Val−
Production example of Leu-Thr-Lys-Ala-Lys-Ser-Gln-OH. (1) P(52−54); AOC−Arg(Tos)−Lys(Z−
Regarding BOC-Lys(Z-Cl)-Lys(Z-Cl)-PAC [Cl)-Lys(Z-Cl)-PAC[2]
(Melting point 72-75℃, elemental analysis value [C ₄₁ H ₅₀ O ₁₀ N ₄ Cl ₂
As, C=59.22%, H=5.98%, N=6.81
%) to 14.65g (18mM) was added TFA50ml and stirred at room temperature for 30 minutes. After the reaction, TFA was distilled off under reduced pressure, and ether was added to the residue. The resulting precipitate was collected, dissolved in 10 ml of DMF, and AOC-
Arg(Tos)-OH9.19g (1.2xM)-HOBT2.92
g (1.2 times M) and 3.95 ml of WSCI (1.2 times M) were added and stirred at room temperature overnight. After the reaction, DMF was distilled off under reduced pressure, and the residue was dissolved in 300 ml of ethyl acetate and washed three times each in the order of 5% sodium bicarbonate solution, 1N hydrochloric acid, and water. The ethyl acetate layer was dried over anhydrous sodium sulfate, dried under reduced pressure, and recrystallized from ethyl acetate-ether to obtain 14.46 g (yield: 69.6%) of [2]. Melting point: 79-82℃ TLC; Rf ₇ = 0.76 Elemental analysis [as C ₅₅ H ₇₀ O ₁₃ N ₈ SCl] C% H% N% Measured value 57.06 6.26 9.82 Calculated value 57.23 6.11 9.71 (2) P (51-54 )；BOC−Pro−Arg(Tos)−Lys
Regarding (Z-Cl)-Lys(Z-Cl)-PAC [3]. [2] 40 ml of TFA was added to 14.43 g (12.5 mM) and stirred at room temperature for 20 minutes. After the reaction, TFA was distilled off under reduced pressure, ether was added to the residue, and the resulting precipitate was collected and dissolved in 80 ml of DMF. to this
HOBT2.03g (1.2x M), BOC-Pro-
OH3.23g (1.2xM) and WSCI2.75ml (1.2
M) was added, and the mixture was stirred at room temperature overnight. After the reaction,
DMF was distilled off under reduced pressure and the residue was dissolved in 300ml of ethyl acetate.
After dissolving in water, the solution was washed with 5% sodium bicarbonate solution and 1N hydrochloric acid solution in this order. After drying the ethyl acetate layer with anhydrous sodium sulfate,
It was dried under reduced pressure and the residue was recrystallized twice from ethyl acetate-ether [37] 14.71g (yield 95.1%)
I got it. Melting point: 90-93℃ TLC; Rf ₇ = 0.64 Elemental analysis [as C ₅₉ H ₇₅ O ₁₄ N ₉ SCl ₂ ] C% H% N% Measured value 57.33 6.20 9.79 Calculated value 57.27 6.11 10.19 (3) P (51− 54);BOC−Pro−Arg(Tos)−Lys
Regarding (Z-Cl)-Lys(Z-Cl)-OH [4]. 20g of zinc powder/30ml of acetic acid [37] 6.19g
A solution of (5mM) dissolved in 40ml of acetic acid was added, and the mixture was stirred at room temperature for 2 hours. After the reaction, the zinc powder was separated, the liquid was concentrated under reduced pressure, and the residue was extracted by adding 5% aqueous sodium bicarbonate and ether. The separated aqueous layer was adjusted to pH 2 with 1N hydrochloric acid, and then extracted with ethyl acetate. The ethyl acetate layer was washed three times with water, dried over anhydrous sodium sulfate, dried under reduced pressure, and the residue was recrystallized from ethyl acetate-ether to give 5.40 g (yield 96.5) of [4].
%) was obtained. Melting point: 110-113℃ TLC; Rf ₄ = 0.43 Elemental analysis [as C ₅₁ H ₆₉ O ₁₃ N ₉ SCl ₂ ] C% H% N% Measured value 54.56 6.46 11.22 Calculated value 54.73 6.21 11.27 (4) P (51- 84);BOC−Pro−Arg(Tos)−Lys
(Z-Cl)-Lys(Z-Cl)-Glu(OBzl)-Asp
(OBzl) −Asn−Val−Leu−Val−Glu(OBzl)
−Ser(Bzl)−His−Glu(OBzl)−Lys(Z−Cl)
−Ser(Bzl)−Leu−Gly−Glu(OBzl)−Ala−
Asp(OBzl)−Lys(Z−Cl)−Ala−Asp(OBzl)
Val−Asp(OBzl)−Val−Leu−Thr(Bzl)−
Lys(Z-Cl)-Ala-Lys(Z-Cl)-Ser(Bzl)
Regarding -Gln-OBzl [5]. BOC−Glu(OBzl)−Asp(OBzl)−Asn−Val
−Leu−Val−Glu(OBzl)−Ser(Bzl)−His−
Glu(OBzl)−Lys(Z−Cl)−Ser(Bzl)−Leu−
Gly−Glu(OBzl)−Ala−Asp(OBzl)−Lys(Z
−Cl) −Ala−Asp(OBzl) −Val−Asp(OBzl)
−Val−Leu−Thr(Bzl)−Lys(Z−Cl)−Ala
-Lys(Z-Cl)-Ser(Bzl)-Gln-OBzl[6]
[Amino acid analysis; Asp4.11(5), Thr0.92(1),
Ser1.59(3), Glu3.99(5), Gly0.83(1), Ala3,
Val3.28(4), Leu2.49(3), Lys4.08(4), His0.71
(1)] 50 ml of TFA was added to 7.83 g (1.5 mM) and stirred at room temperature for 60 minutes. After the reaction, TFA was distilled off under reduced pressure, and ether was added to the residue. The resulting precipitate was collected and dissolved by adding 120 ml of DMF and 120 ml of NMP. Add to this HOBT0.30g (1.5x M), [4] 2.52
g (1.5 times M) and WSCI 0.41 ml (1.5 times M) were added, stirred at room temperature for 2 days, the reaction solution was added to ice water, and the resulting precipitate was washed with water, then methanol was added and heat treated. After cooling, the insoluble matter was collected, and the above heat treatment was repeated twice, followed by washing with ether to obtain 8.20 g (yield: 87.9%) of [5]. Amino acid analysis; Asp4.09(5), Thr1.05(1),
Ser2.30(3), Glu4.08(5), Pro0.52(1), Gly0.88
(1), Ala3, Val3.42(4), Leu2.53(3), Lys5.11
(6), His0.69(1), Arg0.51(1) (41) h-PTH (51-84) 3.73 g in 40 ml of anhydrous HF while cooling to 0°C [5]
(0.6mM) and 4ml of anisole were added and stirred for 60 minutes. After the reaction, HF was distilled off under reduced pressure, ether was added to the residue, and the resulting precipitate was collected.
Dissolve in 50 ml of 0.1N acetic acid and pass through a Dowex x 1 column (acetate type - 2.7 x 33 cm).
The effluent was freeze-dried to obtain 2.41 g of crude product. This was dissolved in 50 ml of 8M urea aqueous solution, adjusted to pH 10.0 with aqueous ammonia, and then left for 80 minutes. Next, this solution was charged to a CM-cellulose column (4.2 x 11.5 cm) packed with 8M urea aqueous solution, and after urea was flushed out with 0.01M ammonium acetate aqueous solution (PH4.5), 0.01M ammonium acetate aqueous solution (PH4.5) was charged. 5) Elution was performed using a linear concentration gradient from 700 ml to 700 ml of a 0.1 M aqueous ammonium acetate solution (PH4.5), followed by elution with 250 ml of a 0.2 M aqueous ammonium acetate solution (PH4.5). After elution, fractionate into 8.5 ml portions, and each fraction is
Measured by Lowry method (500nm): 30th to 63rd division C ₁ , 105th to 150th division C ₂ and 151~
A 195th eluate of category _C3 was obtained. Section C ₂ and Section C ₃ were desalted by passing through a Sephadex LH-20 column. The effluent was fractionated into 5.2 ml portions.
Segment C ₂ was passed through a 3.4×110 cm column to obtain 51st to 63rd sections C ₂ L ₁ and 64th to 80th sections C ₂ L ₂ . Section C ₃ was passed through a 3.4 x 120 cm column and 50
~69th division C ₃ L ₁ and 70th to 78th division
C ₃ L ₂ was obtained. Each section was freeze-dried to obtain the following components.

[Table] Dissolve 375 mg of the above C ₂ L in ₄ ml of 0.1 N acetic acid, charge it to a CM-cellulose column (2.0 x 31 cm), and add 0.01 M ammonium acetate aqueous solution (PH
4.5) 500ml ~ 0.2M ammonium acetate aqueous solution (PH
4.5) Elution was performed using a 500 ml linear concentration gradient. The eluate was fractionated into 7.5 ml portions to obtain the 85th to 103rd fractions C ₂ L ₂ -C. Secure this classification
Desalting was carried out through an LH-20 column (3.0 x 123 cm). The effluent is fractionated into 6 ml portions and divided into 34th to 42nd sections.
_{C2L2 - CL1} and 43rd to 50th sections _{C2L2 - CL2} were obtained. Each section was freeze-dried to obtain the following components. C ₂ L ₂ −CL ₁ 80.0mg Amino acid analysis; Asp4.95(5), Thr0.98(1),
Ser2.47(3), Glu5.14(5), Gly1.01(1), Ala3(3),
Val4.05(4), Leu3.02(3), Lys6.16(6), His0.96(1),
Arg0.97(1), Pro1.06(1) C ₂ L ₂ −CL ₂ 197.6g Amino acid analysis; Asp5.00(5), Thr0.93(1),
Ser2.30(3), Glu5.17(5), Gly1.01(1), Ala3(3),
Val4.03(4), Leu3.00(3), Lys6.15(6), His0.95(1),
Arg1.01(1), Pro1.04(1) Dissolve 195 mg of the above C ₂ L ₂ -CL ₂ sections in 2 ml of 0.1N acetic acid, and add it to a CM-cellulose column (2.0 x 15 cm).
and elution was performed using a linear concentration gradient of 300 ml of 0.01 M ammonium acetate aqueous solution (PH 4.5) to 300 ml of 0.2 M ammonium acetate aqueous solution (PH 4.5). The eluate was fractionated into 6.4 ml portions, and the 55th to 64th sections C ₂ L ₂ −CL ₂ −C ₁ and the 56th to 72nd sections
_{C2L2 - CL2} - _C2 was obtained. Securely index each category
Desalting was carried out through an LH-20 column. Category C ₂ L ₂ −
CL ₂ −C ₁ was passed through a 3.0 x 123 cm column, and the effluent was
Fractionate into 7.4ml portions and divide into 31st to 38th sections C ₂ L ₂ −CL ₂
−C ₁ L ₁ and the 39th to 43rd divisions C ₂ L ₂ −CL ₂ −C ₁ L ₂ were obtained. Freeze-dry the division C ₂ L ₂ −CL ₂ −C ₁ L ₁ and h−
77.2 mg of PTH (51-84) was obtained. TLC; R ₁₀ = 0.89 1 spot reference example 3 h-PTH (46-84); H-Ala-Gly-Ser-Gln
−Arg−Pro−Arg−Lys−Lys−Lys−Glu−Asp
−Asn−Val−Leu−Val−Glu−Ser−His−Glu
−Lys−Ser−Leu−Gly−Glu−Ala−Asp−Lys
−Ala−Asp−Val−Asp−Val−Leu−Thr−Lys
Production example of -Ala-Lys-Ser-Gln-OH. (1) P(49−50); BOC−Gln−Arg(Tos)−OMe
Regarding [7]. H-Arg(Tos)-OMe・HCl11.37g
(30mM) and 13.21g (1.2x M) of BOC-Gln-ONP were dissolved in 200ml of DMF and cooled to 0℃.
After adjusting the pH to 7 with NMM, the mixture was stirred overnight. After the reaction, DMF was distilled off under reduced pressure, and the residue was dissolved in chloroform, and then washed three times with 5% aqueous sodium bicarbonate, twice with 1N hydrochloric acid, and three times with water. The chloroform layer was dried with anhydrous sodium sulfate, and chromatography was performed on a silica gel column packed with chloroform. The chloroform layer was flushed with chloroform-ethanol-ethyl acetate, and when the target product began to elute, the chloroform-
Elution was performed with ethanol-ethyl acetate (1:1:1). Corresponding fractions were collected and concentrated under reduced pressure. The residue was dissolved in ethyl acetate, and while cooling at 0°C, hexane was added to crystallize it to obtain [7]. yield
11.86g Melting point; 103-107℃ (2) P(48-50); BOC-Ser(Bzl)-Gln-Arg
Regarding (Tos)-OMe [8]. [7] 50 ml of TFA was added to 9.39 g (16.5 mM) and stirred at room temperature for 20 minutes, and then TFA was distilled off under reduced pressure. Ether was added to the residue, and the resulting precipitate was collected and dissolved in 50 ml of DMF. in this solution
HOBT3.24g (1.45x M), BOC−Ser(Bzl)−
OH7.07g (1.45xM) and WSCI4.39ml
(1.45 times M) was added and stirred at room temperature overnight. After the reaction, DMF was distilled off under reduced pressure, and the residue was dissolved in 300 ml of ethyl acetate, and then washed with 5% sodium bicarbonate, 1N hydrochloric acid, and water in this order. The ethyl acetate layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was crystallized twice from ethyl acetate-ether.
81.0%). Melting point: 97-102℃ Elemental _analysis [as _C34H49O10N7S・1 _{/ 2H2O} ] _C % H% N% Measured value 54.07 6.90 13.29 Calculated value 53.95 6.66 13.00 (3) P (46−50 )；BOC−Ala−Gly−Ser(Bzl)−
Gln−Arg(Tos)−OMe

Regarding [9]. [8] Add 50ml of TFA to 9.72g (13mM),
Stirred at room temperature for 30 minutes. After the reaction, TFA was distilled off under reduced pressure, and ether was added to the residue. The resulting precipitate was taken and dissolved in 100 ml of DMF, and 3.84 g of BOC-Ala-Gly-OH (1.2 times M) was added to this solution.
HOBT2.11g (1.2xM) and WSCI2.85ml
(1.2xM) was added and stirred at room temperature for 2 days. After the reaction, DMF was distilled off under reduced pressure, the residue was dissolved in 200 ml of ethyl acetate, and then washed with water. After drying with anhydrous sodium sulfate and concentrating under reduced pressure, the residue was diluted with ethanol.
Crystallized twice in ether

[9] 10.46 g (yield 91.9%) was obtained. Melting point: 154-157℃ Elemental analysis [as C ₃₉ H ₅₇ O ₁₂ N ₉ S] C% H% N% Measured value 53.21 6.90 14.38 Calculated value 53.74 6.56 14.39 (4) P (46-50); BOC-Ala- Gly−Ser(Bzl)−
About Gln−Arg(Tos)−NHNH ₂ [10].

[9] 9.64 g (11 mM) was dissolved in 50 ml of ethanol, 6.4 ml of 50% NH ₂ NH ₂ was added thereto, and the mixture was stirred at room temperature overnight. 100 ml of ethanol was added to the reaction solution to remove insoluble matter. Add this to ethanol
The suspension was suspended in 100 ml, heated, cooled, and filtered to obtain 9.02 g (yield: 93.6%) of [10]. Melting point: 178-180℃ Elemental analysis [as C ₃₃ H ₅₇ O ₁₁ N ₁₁ S] C% H% N% Measured value 52.18 6.86 16.65 Calculated value 52.10 6.56 17.59 (5) P (46−54); BOC−Ala− Gly-Ser (Bzl)
−Gln−Arg(Tos)−Pro−Arg(Tos)−Lys(Z
-Cl)-Lys(Z-Cl)-PAC [11]. [3] 8.04g (6.6mM) described in Reference Example 2
After adding 40 ml of TFA and stirring at room temperature for 20 minutes,
TFA was distilled off under reduced pressure. Ether was added to the residue, the resulting precipitate was collected and the crude H-Pro-
Arg(Tos)−Lys(Z−Cl)−Lys(Z−Cl)−
Obtained PAC/TFA. On the other hand, [10] 6.83g (7.3mM) was dissolved in 30ml of DMF, and 5.42ml (23.4mM) of a 4.32N hydrogen chloride dioxane solution and 1.10ml (8.09mM) of isoamyl nitrile were added to this while cooling at -50°C. ,−
Stirred at 20°C for 20 minutes. Then the above H-Pro-
After adding Arg(Tos)-Lys(Z-Cl)-PAC・TFA and adding Et ₃ N 5.46ml (39mM) at -35℃,
Stirred at 0-5°C for 2 days. After the reaction, DMF was distilled off under reduced pressure, and the residue was dissolved in 300 ml of chloroform, followed by washing in the order of 5% aqueous sodium bicarbonate, 1N hydrochloric acid, and water. Dry the chloroform layer with anhydrous sodium sulfate,
Concentrate under reduced pressure and purify with ethanol-ether and chloroform-ether [11]
13.42g was obtained. TLC; Rf=0.64 [chloroform-methanol-acetic acid (83:18:3.5)] Elemental analysis [as C ₉₂ H ₁₂₀ O ₂₂ N ₁₈ Cl ₂ S・3H ₂ O] C% H% N% Measured value 54.68 6.21 12.72 Calculated value 54.72 6.29 12.49 Amino acid analysis; Ser0.65(1), Glu1.10(1), Pro1
(1), Gly1.02(1), Ala1.00(1), Lys1.89(2), Arg2.02
(2) (6) P-(46-54); BOC-Ala-Gly-Ser(Bzl)
−Gln−Arg(Tos)−Pro−Arg(Tos)−Lys(Z
-Cl)-Lys(Z-Cl)-OH [12]. 15g of zinc powder/30ml of acetic acid [11] 5.31g of acetic acid
40 ml of solution was added and stirred at room temperature for 2 hours. After the reaction, the zinc powder was separated and the liquid was concentrated under reduced pressure. Ether was added to the residue, and the resulting precipitate was purified once with ethanol-ether and twice with ethanol-ethyl acetate to obtain 4.41 g (87.7% yield) of [12]. TLC; Rf ₄ = 0.22 Elemental analysis [as C ₈₄ H ₁₁₄ O ₂₂ N ₁₈ S ₂ Cl ₂・2H ₂ O] C% H% N% Measured value 52.89 6.16 13.22 Calculated value 53.12 6.26 13.28 Amino acid analysis; Ser0.88( 1), Glu1.11(1),
Pro1.02(1), Gly1.02(1), Ala1(1), Lys2.00(2),
Arg2.09(2) (6) P(46−84); BOC−Ala−Gly−Ser(Bzl)−
Gln−Arg(Tos)−Pro−Arg(Tos)−Lys(Z−
Cl)−Lys(Z−Cl)−Glu(OBzl)−Asp(OBzl)
−Asn−Val−Leu−Val−Glu(OBzl)−Ser
(Bzl)-His-Glu(OBzl)-Lys(Z-Cl)-Ser
(Bzl)−Leu−Gly−Glu(OBzl)−Ala−Asp
(OBzl)−Lys(Z−Cl)−Ala−Asp(OBzl)−
Val−Asp(OBzl)−Val−Leu−Thr(Bzl)−
Lys(Z-Cl)-Ala-Lys(Z-Cl)-Ser(Bzl)
Regarding −Gln−OBzl [13]. [6] 10.44g (2mM) described in Reference Example 2
After adding 80 ml of TFA and stirring at room temperature for 60 minutes,
TFA was distilled off under reduced pressure. Ether was added to the residue, the resulting precipitate was dissolved in a mixture of 160 ml of DMF and 160 ml of NMP, and the mixture was cooled to 0°C [12] 4.28
After adding 0.32 g of HOBT (1.2 times M) and 0.44 ml of WSCI (1.2 times M), the mixture was stirred at room temperature for 2 days. After the reaction, DMF was distilled off under reduced pressure.
After adding ice water to the residue, the resulting precipitate was collected. Add 200ml of methanol to this and heat.
The operation of removing insoluble matter after cooling was repeated twice to obtain 12.17 g (yield: 87.4%) of [13]. (7) h-PTH (46-84) Anhydrous HF 60ml Cooled to 120℃ [13] 4.18g
(0.6mM) and 10ml of anisole were added and stirred for 60 minutes. After the reaction, HF was distilled off under reduced pressure, and ether was added to the residue. Collect the resulting precipitate and add 10%
It was dissolved in 50 ml of acetic acid, passed through a Dowex x 1 column (acetate type, 2.5 x 24 cm), and the effluent was freeze-dried to obtain 2.82 g of a crude product. This was dissolved in 50 ml of 8M urea aqueous solution (PH9.0) and left at room temperature for 60 minutes. Then this solution
Charge a CM-cellulose column (2.0 x 33 cm) packed with 8M urea aqueous solution, and add 500ml to 0.3M of 0.01M ammonium acetate aqueous solution (PH4.5).
Elution was performed using a linear concentration gradient of 500 ml of ammonium acetate aqueous solution (PH4.5). Eluate is 7.5
Fractionate by ml, each fraction is Folin-Lowry
(500nm) 1st to 22nd division _C1 ,
23rd to 45th division C ₂ , 46th to 80th division C ₃ ,
And the 81st to 120th eluate of category _C4 was obtained.
Each fraction was desalted by passing it through a Sephadex LH-20 column. Division C ₁ was suitable for a 3.0×120 cm column, and the effluent was fractionated into 7.5 ml portions to obtain the 28th to 42nd divisions C ₁ L. Division C ₂ reached 3.4×120 ml, and the effluent was fractionated into 7.6 ml portions to obtain the 33rd to 40th division C ₂ L ₁ and the 41st to 62nd division C ₂ L ₂ .
Section C ₃ passes the effluent through a 3.0 x 120 cm column.
It was fractionated into 6.0 ml portions to obtain the 31st to 40th sections C ₃ L ₁ and the 41st to 51st sections C ₃ L ₂ . Category C ₄ is
The effluent was passed through a 3.4 x 120 cm column and fractionated into 7.5 ml portions to obtain ₄ L of the 35th to 48th fractions. Freeze-dry each section to obtain 312 mg for C ₁ L section and ₁ C ₂ L section.
142.3mg, _{C2L 2} divisions 1380mg, _{C3L 1} division 104mg,
510 mg of C ₃ L ₂ fraction and 130 mg of C ₄ L fraction were obtained. Dissolve 1380 mg of the above C ₂ L ₂ sections in 13 ml of 0.1N acetic acid and apply it to a CM-cellulose column (4.3 x 6.0 cm).
Elution was performed using a linear concentration gradient from 500 ml of 0.01 M ammonium acetate aqueous solution (PH 4.5) to 500 ml of 0.3 M ammonium acetate aqueous solution (PH 4.5). The eluate was fractionated into 7.6 ml portions, and the 40th to 50th sections C ₂ L ₂ - and the 53rd to 77th sections C ₂ L _2
−C2 was obtained. Sephadex LH-20 for each category
It was desalted by passing it through a column. The division C ₂ L ₂ −C ₁ is
Pass through a _{2.9 x} 120 cm column and fractionate _the effluent _{into 8.0 ml portions .} Obtained. classification
C ₂ L ₂ - C ₂ is passed through a 3.4 x 120 cm column, the effluent is fractionated into 8.0 ml portions, and the 34th to 44th sections C ₂ L ₂ -
_{C2L1 and 45th} to 53rd sections _{C2L2 - C2L2} were obtained. Freeze-dry each section to obtain C ₂ L ₂ −C ₁ L ₁ section 79.0
mg, C ₂ L ₂ −C ₁ L ₂ divisions 455 mg, C ₂ L ₂ −C ₂ L ₁ division
_157.3 mg and 551.7 mg _{of C2L2} - _C2L2 fraction were obtained. Amino acid analysis of C ₂ L ₂ −C ₂ L ₂ division; Asp4.65
(5), Thr0.97(1), Ser3.47(4), Glu5.99(6),
Pro0.93(1), Gly1.96(2), Ala4(4), Val3.97(4),
Leu3.01(3), Lys6.13(6), His0.93(1), Arg1.96(2) Dissolve the above C ₂ L ₂ −C ₂ L ₂ division in 5 ml of 0.1N acetic acid and prepare CM-cellulose. Column (4.2 x 7.0cm)
The elution was performed using a linear concentration gradient from 300 ml of 0.01M ammonium acetate aqueous solution (PH4.5) to 0.3M ammonium acetate aqueous solution (PH4.5). The eluate was fractionated into 8.0 ml portions to obtain the 39th to 45th fractions C ₂ L ₂ -C ₂ L ₂ -C. This fraction was desalted by passing it through a Sephadex LH-20 column (2.9 x 120 cm). The effluent was fractionated into 8.0 ml portions, and the 24th to 30th divisions C ₂ L ₂ −C ₂ L ₂ −CL ₁ ,
31st to 35th division C ₂ L ₂ −C ₂ L ₂ −CL ₂ and 36~
The 39th segment C ₂ L ₂ −C ₂ L ₂ −CL ₃ was obtained. Freeze-dry each portion to obtain 200 mg of C ₂ L ₂ −C ₂ L ₂ −CL ₂ portions and ₁ portion of C ₂ L ₂ −C ₂ L ₂ −CL [h-PTH (46−
84)] 94.9 mg was obtained. TLC; Rf ₉ = 0.76 Amino acid analysis; Asp4.86(5), Thr0.99(1),
Ser3.5(4), Glu6.17(6), Pro0.96(1), Gly1.97(2),
Ala4, Val4.02(4), Leu2.93(3), Lys6.05(6),
His0.90(1), Arg1.87(2) Reference example 4 [Tyr ⁴⁵ ] h-PTH (45-84); H-Tys-Ala
−Gly−Ser−Gln−Arg−Pro−Arg−Lys−Lys
−Glu−Asp−Asn−Val−Leu−Val−Glu−Ser
−His−Glu−Lys−Ser−Leu−Gly−Glu−Ala
−Asp−Lys−Ala−Asp−Val−Asp−Val−Leu
Production example of -Thr-Lys-Ala-Lys-Ser-Gln-OH. (1) P(45−84); BOC−Tyr(Bzl−Cl ₂ )−Ala−
Gly−Ser(Bzl)−Gln−Arg(Tos)−Dro−Arg
(Tos)−Lys(Z−Cl)−Lys(Z−Cl)−Glu
(OBzl)−Asp(OBzl)−Asn−Val−Leu−Val
−Glu(OBzl)−Ser(Bzl)−His−Glu(OBzl)−
Lys(Z−Cl)−Ser(Bzl)−Leu−Gly−Glu
(OBzl)-Ala-Asp(OBzl)-Lys(Z-Cl)-
Ala−Asp(OBzl)−Val−Asp(OBzl)−Val−
Leu−Thr(Bzl)−Lys(Z−Cl)−Ala−Lys(Z
-Cl)-Ser(Bzl)-Gln-OBzl [14]. [13] 6.27g (0.9mM) described in Reference Example 3
60 ml of TFA was added and stirred at room temperature for 60 minutes. After the reaction, TFA is distilled off under reduced pressure, and ether is added to the residue. The resulting precipitate was collected to obtain 6.30 g of a BOC-free compound. 2.10g (0.3mM) of the above BOC removed product was added to DMF35
ml and NMP (35 ml), and add 0.16 _g (1.2
times M), HOBT0.05g (1.2 times M) and
After adding 0.07 ml of WSCI (1.2 times M), the mixture was stirred at room temperature overnight. After the reaction, DMF was distilled off under reduced pressure.
Ice water was added to the residue, and the resulting precipitate was collected to obtain 2.00 g of [14]. (2) [Tyr ⁴⁵ ]-h-PTH (45-84) 2.00 g of [14] in 20 ml of anhydrous HF while cooling to 0℃
(0.27mM) and 1.0ml of anisole, 60
Stir for a minute. After the reaction, HF was distilled off under reduced pressure,
Ether was added to the residue. Collect the resulting precipitate, dissolve it in 20 ml of 0.1N acetic acid, and add Dowex
1 column (acetate type, 2.5×15 cm), and the effluent was freeze-dried to obtain 1.37 g of crude product. This was dissolved in 50 ml of 8M urea aqueous solution (PH9.5) and left at room temperature for 60 minutes. Then this solution
Charge a CM-cellulose column (4.3 x 8.0 cm) packed with 8M urea aqueous solution, and add 400ml to 0.3M 0.01M ammonium acetate aqueous solution (PH4.5).
Elution was performed using a linear concentration gradient of 400 ml of ammonium acetate aqueous solution (PH4.5). 6.5 after elution
Fractionate by ml, each fraction is measured by Folin-Lowry method (500nm), and the 30th to 43rd division _C1 ,
The 51st to 68th sections _C2 , the 69th to 83rd sections _C3 , and the 84th to 100th sections _C4 were obtained. Each fraction was desalted by passing it through a Sephadex LH-20 column. Section C ₁ passes through a 3.4 x 120 cm column and the effluent is fractionated into 7.5 ml portions to obtain 25th to 41st sections C ₁ L. Section C ₂ passes through a 3.0 x 120 cm column and the effluent is fractionated into 7.5 ml. The 25th to 36th fractions C ₂ L were obtained. Section C ₃ is passed through a 2.9 x 120 cm column, and the effluent is fractionated into 8.0 ml portions.
C ₃ L ₁ and 28th to 38th sections C ₃ L ₂ were obtained. Section C ₄ passes through a 2.9 x 95 cm column and the effluent is 7.6
Fractionate by ml and divide into 20th to 25th sections C ₄ L ₁ and
The 26th to 30th segment _C4L2 was obtained _. Freeze-dry each section to obtain 521.6 mg for C ₁ L section, 230.2 mg for C ₂ L section,
C ₃ L ₁ category 41.8 mg, C ₃ L ₂ category 222.4 mg, C ₄ L ₁ category
74.3 mg and 48.9 mg of C ₄ L ₂ fractions were obtained. Dissolve the above C ₂ L fraction in 3 ml of 0.1N acetic acid,
Charge a CM-cellulose column (2.1 x 25 cm) and add 0.01M ammonium acetate aqueous solution (PH
4.5) Elution was performed using a linear concentration gradient from 300 ml to 300 ml of a 0.3 M ammonium acetate aqueous solution (PH4.5). The eluate was fractionated into 8.0 ml portions to obtain the 30th to 36th fractions C ₂ L-C. This fraction was desalted by passing it through a Sephadex LH-20 column (2.9 x 90 cm). Fractionate the effluent into 8.0ml portions,
Freeze-dry the 20th to 29th sections [Tyr ⁴⁵ ]−
163.3 mg of h-PTH (45-84) was obtained. TLC; Rf ₉ = 0.75 Amino acid analysis; Asp4.86(5), Thr1.02(1),
Ser3.51(4), Glu6.05(6), Pro0.93(1), Gly1.90
(2), Ala4(4), Val4.00(4), Leu2.93(3), Tyr0.88
(1), Lys6.02(6), His0.86(1), Arg1.81(2) Reference example 5 [Cys(Acm) ⁴⁵ ]-h-PTH(45-84);H-
Cys(Acm)−Ala−Gly−Ser−Gln−Arg−Pro−
Arg−Lys−Lys−Glu−Asp−Asn−Val−Leu−
Val−Glu−Ser−His−Glu−Lys−Ser−Leu−
Gly−Glu−Ala−Asp−Lys−Ala−Asp−Val−
Asp−Val−Leu−Thr−Lys−Ala−Lys−Ser−
Example of manufacturing Gln-OH. (1) P(45−84); BOC−Cys(Acm)−Aa−Gly
−Ser(Bzl)−Gln−Arg(Tos)−Pro−Arg
(Tos)−Lys(Z−Cl)−Lys(Z−Cl)−Glu
(OBzl)−Asp(OBzl)−Asn−Val−Leu−Val
−Glu(OBzl)−Ser(Bzl)−His−Glu(OBzl)−
Lys(Z−Cl)−Ser(Bzl)−Leu−Gly−Glu
(OBzl)-Ala-Asp(OBzl)-Lys(Z-Cl)-
Ala−Asp(OBzl)−Val−Asp(OBzl)−Val−
Leu−Thr(Bzl)−Lys(Z−Cl)−Ala−Lys(Z
-Cl)-Ser(Bzl)-Gln-OBzl [15]. 4.20 mg of the remaining BOC removed product obtained in Reference Example 4
(0.6mM) was dissolved in a mixture of 70ml of DMF and 70ml of NMP, and 0.10g of HOBT (1.2
After adding 0.20 g (1.2 times M) of BOC-Cys(Acm)-OH (1.2 times M) and 0.13 ml (1.2 times M) of WSCI, the mixture was stirred at room temperature overnight. After the reaction, DMF was distilled off under reduced pressure, ice water was added to the residue, and the resulting precipitate was collected. This was suspended in ethanol, heated, cooled, and then the insoluble matter was removed. Repeat this operation twice [15] 4.07g (yield 95.0%)
I got it. (2) [Cys (Acm) ⁴⁵ ]-h-PTH (45-84) anhydrous
4.00g in HF60ml cooled to 0℃ [15]
(0.57mM) and 10ml of anisole, 60
Stir for a minute. After the reaction, HF was distilled off under reduced pressure,
Ether was added to the residue. Collect the resulting precipitate, dissolve it in 40ml of 20% acetic acid, and add Dowex x 1.
column (acetate type, 2.8 x 35 cm),
The effluent was lyophilized. Add this to 8M urea aqueous solution.
After dissolving in 50 ml and adjusting the pH to 9.0 with aqueous ammonia, it was left for 30 minutes. This solution was then diluted to 8M
CM-cellulose filled with urea aqueous solution (3.4×
35 cm), and elution was performed using a concentration gradient of 700 ml of 0.01 M ammonium acetate aqueous solution (PH4.5) to 700 ml of 0.3 M ammonium acetate aqueous solution (PH4.5). The eluate was fractionated into 8.0ml portions, and each fraction was measured using the Folin-Lowry method (500nm).
A 25th to 35th segment _C1 , a 36th to 45th segment _C2 , and a 46th to 84th segment _C3 were obtained. Each fraction was desalted by passing it through a Sephadex LH-20 column. Section C ₂ is passed through a 3.0 x 120 cm column, the effluent is fractionated into 8.0 ml portions, and the 27th to 33rd sections C ₂ L ₁
and the 34th to 40th _{divisions C2L2} were obtained. Category C ₃
is passed through a 3.4 x 120 cm column and the effluent is 8.0 ml.
The 35th to 47th fractions C ₃ L ₁ and 48
~53rd segment _C3L2 was obtained _. Freeze-dry each section to obtain 148 mg of C ₂ L ₁ section, 620 mg of C ₂ L ₂ sections, and 620 mg of C ₃ L _1.
212 mg of fraction and 605 mg of C ₃ L ₂ fraction were obtained. Dissolve the ₂ portions of C ₂ L in 6 ml of 0.1N acetic acid,
This is a CM-cellulose column (5.0 x 12 cm)
and elute with a linear concentration gradient of 400 ml of 0.0 M aqueous ammonium acetate solution (PH4.5) to 400 ml of 0.3 M aqueous ammonium acetate solution (PH4.5), and fractionate the eluate into 6.0 ml portions of 110 to 126 The main classification C ₂ L ₂ -C was obtained. This was desalted by passing it through a Sephadex LH-20 column (4.0 x 120 cm). The effluent was fractionated into 8.0 ml portions, 38 to 54
The main segment C ₂ L ₂ −CL was obtained. Freeze-dry this section to obtain [Cys (Acm) ⁴⁵ ]-h-PTH (45-84).
246.8mg was obtained. TLC; Rf ₉ = 0.74 Amino acid analysis; Asp4.91(5), Thr0.98(1),
Ser3.50(1), Glu6.11(6), Pro0.98(1), Gly1.98
(2), Ala4(4), Val4.04(4), Cys0.42(0.5),
Leu2.90(3), Lys5.99(6), His0.87(1), Ars1.87(2) Reference example 6 [Cys ⁴⁵ ]-h-PTH (45-84); H-Cys-Ala
−Gly−Ser−Gln−Arg−Pro−Arg−Lys−Lys
−Glu−Asp−Asn−Val−Leu−Val−Glu−Ser
−His−Glu−Lys−Ser−Leu−Gly−Glu−Ala
−Asp−Lys−Ala−Asp−Val−Asp−Val−Leu
Production example of -Thr-Lys-Ala-Lys-Ser-Gln-OH. [Cys(Acm) ⁴⁵ ]-h-PTH obtained in Reference Example 5
88 mg (0.02 mM) of (45-84) was dissolved in 2 ml of 50% acetic acid, and 57.24 mg (0.18 mM) of mercuric acetate was added thereto, followed by stirring at room temperature for 70 minutes. Then β-
3.4 ml of mercaptoethanol was added, and the mixture was stirred at room temperature for 24 hours. The reaction solution was centrifuged, and the supernatant was charged to a Sephadex LH-20 column (3.5 x 42 cm) and eluted with 0.1M acetic acid. The eluate was fractionated into 5 ml portions, the 9th to 14th fractions that were positive for ninhydrin reaction were collected, and this was lyophilized to give [Cys ⁴⁵ ]-h-
76.1 mg of PTH (45-84) was obtained. TLC; Rf ₉ =0.73 In addition, the abbreviations described in this specification have the following meanings. Gln; L-glutamine Ser; L-serine Lys; L-lysine Ala; L-alanine Thr; L-threonine Leu; L-leucine Val; L-valine Asp; L-aspartic acid Glu; L-glutamic acid Gly; glycine His ; L-histidine Asn; L-asparagine Arg; L-arginine Pro; L-proline Tyr; L-tyrosine Cys; L-cysteine BOC; t-butyloxycarbonyl AOC; t-amyloxycarbonyl Z-Cl; o-chlorobenzyl Oxycarbonyl Bzl; benzyl Tos; tosyl OMe; methyl ester OEt; ethyl ester OBzl; benzyl ester OSU; N-hydroxysuccinimide ester ONP; p-nitrophenyl ester PAC; phenacyl ester Acm; acetamidomethyl TosOH; p- Toluenesulfonic acid TFA; trifluoroacetic acid _Et3N ; triethylamine TBA; tribenzylamine NMM; N-methylmorpholine HOBT; 1-hydroxybenzotriazole DMF; dimethylformamide THF; tetrahydrofuran NMP; N-methyl-2-pyrrolidone MeOH; methanol EtOH; Ethanol BuOH; Butanol ether; Diethyl ether WSCI; N-ethyl, N-3-dimethylaminopropyl-carbodiimide Also used thin layer chromatography (TLC)
The carrier and developing solvent are as follows. Support: Silica Gel G manufactured by Merck & Co., Ltd. Developing solvent: 1; CHCl ₃ -MeOH-acetic acid (95:5:3) 2; 〃 (85:15:5) 3; 〃 (85:10:5) 4; 〃 (80) :25:2) 5; Benzene-ethyl acetate (1:1) 6; Benzene-ethyl acetate (2:1) 7; CHCl3 _- EtOH-ethyl acetate (5:2:5) 8; (10:1) :5) Support: Cellulose manufactured by Merck & Co., Ltd. Developing solvent: 9; BuOH-pyridine-acetic acid-water
(2:2:2:3) 10;BuOH-pyridine-acetic acid-water
(1:1:1:2)

[Brief explanation of drawings]

Figures 1, 2, 3 and 4 show calibration curves.

Claims (1)

[Scope of Claims] 1. In a test solution containing human PTH or its C-terminal fragment, the following general formula [] R ₂ −Ala−Gly−Ser−Gln−Arg−Pro−Arg−
Lys−Lys−Glu−Asp−Asn−Val−Leu−
Val−Glu−Ser−His−Glu−Lys−Ser−
Leu−Gly−Glu−Ala−Asp−Lys−Ala−
Asp−Val−Asp−Val−Leu−Thr−Lys−
Ala-Lys-Ser-Gln-OH [] (wherein, R ₂ is H or H-R ₃ - group, R ₃
(represents a Cys group) is used to sensitize a non-human mammal to react with a specific antibody obtained, and then the bound part and the unbound free part are separated by the reaction. 1. A method for measuring human-PTH or its C-terminal fragment in a test liquid, which comprises the steps of: and then measuring the enzymatic activity of the bound portion or the unbound free portion. 2 The antibody is such that R ₂ is an H-R ₃ - group and R ₃ is a Cys
The measuring method according to claim 1, which is an antibody obtained using a peptide represented by the general formula [ ], which is a group. 3 In the antibody, R ₂ is an H-R ₃ - group, and R ₃ is a Cys
The assay method according to claim 1, which is an antibody obtained using a peptide-protein conjugate represented by the general formula [ ], which is a group. 4 The enzyme-labeled C-terminal fragment of human-PTH has the following general formula [] R-Lys-Lys-Glu-Asp-Asn-Val-Leu-
Val−Glu−Ser−His−Glu−Lys−Ser−
Leu−Gly−Glu−Ala−Asp−Lys−Ala−
Asp−Val−Asp−Val−Leu−Thr−Lys−
Ala-Lys-Ser-Gln-OH [] (wherein, R is H or R ₁ -Pro-Arg- group, R ₁
is H or R ₂ -Ala-Gly-Gln-Arg- group, R ₂
2. The measuring method according to claim 1, which is an enzyme-labeled peptide of the peptide represented by H or H- _R3- group, _R3 is Cys group. 5 The peptide represented by the general formula [] is R ₂
5. The measuring method according to claim 4, wherein the peptide is a peptide represented by the general formula [ ], in which is an H-R ₃ - group and R ₃ is a Cys group. 6. The measuring method according to claim 1, wherein the enzyme used for enzyme labeling is β-galactosidase.