JPS6362698B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to human chorionic gonadotropin (hereinafter referred to as
It is sometimes abbreviated as "hCG". ) related to enzyme immunoassay (hereinafter sometimes abbreviated as "EIA"). hCG is a type of protein hormone produced by trophoblastic cells that are formed at the same time as pregnancy, and promotes the secretion of progesterone. Detection of hCG is commonly used for early diagnosis of pregnancy. In addition, in recent years, the existence of hCG-producing malignant tumors, especially chorionic tumors, has been discovered, and hCG has been detected in the urine, blood, and cerebrospinal fluid of tumor patients, and qualitative and quantitative detection of this hormone can be used to diagnose these diseases. It was also found to be important for follow-up observation. Approximately 100Iu/
It is necessary to measure the following trace amounts of hCG, and the problem here is a protein hormone with a similar structure to hCG, namely luteinizing hormone (hLH).
It is abbreviated as. ) follicle stimulating hormone (hereinafter referred to as
It is abbreviated as "hFSH". ) and thyroid stimulating hormone (hereinafter abbreviated as "hTSH"). In particular, hLH has a high degree of similarity to hCG, and the physiological amount of hLH in urine is 100~
Since the amount can reach 150 Iu/h, it is necessary to immunologically distinguish between hCG and hLH in order to measure hCG in trace amounts of body fluids. Meanwhile, progress has been made in the chemical analysis of these protein hormones, and it has been found that their cross-reactivity is due to their α-subunit moieties, which have many common structures.
Therefore, attempts have been made to separate and purify the β-subunit of hCG (hereinafter referred to as "hCG-β"), which has relatively little similarity, and to produce anti-hCG-β antibodies to specifically detect hCG. There is. However, the separation and purification of hCG-β is complicated, and it is extremely difficult to avoid the mixture of hCG and the α-subunit of hCG (hereinafter abbreviated as “hCG-α”). Anti-hCG-
Cross-reactivity with hLH cannot be completely eliminated using β antibodies. Furthermore, the anti-hCG obtained in this way
The affinity of β antibody for hCG is that of anti-hCG antibody
This results in a decrease in sensitivity. However, a peptide consisting of about 30 amino acids at the C-end of hCG-β shows an amino acid sequence that does not exist in hLH, and it is believed that it is possible to completely distinguish hCG from hLH in this part. Ivy. Based on the results of such structural analysis, Matsuura
synthesized the C-terminal peptide of hCG-β, immunized rabbits to obtain hCG-specific antiserum, and performed radioimaging assay (hereinafter abbreviated as "RIA") using it. Endocrinology, Vol. 104 (1979), p. 396), satisfactory results have been obtained regarding specificity, but currently sensitivity is insufficient. Based on this technical background, the present inventors developed hCG
We have conducted extensive research on anti-hCG antibodies that can specifically and sensitively detect hCG, and have absorbed the anti-hCG antibodies obtained by immunizing animals with hCG using a carrier made by insolubilizing the synthetic C-terminal peptide of hCG-β. A new finding was obtained that the anti-hCG antibody obtained by this treatment can exhibit hCG specificity that does not cross-react with hLH, etc. Further research based on this new knowledge revealed that
We obtained the knowledge that enzyme immunoassay using an enzyme-labeled C-terminal peptide of hCG-β is extremely useful for the specific and highly sensitive detection of hCG, and as a result of further research, we completed the present invention. The present invention is based on (1) the general formula H-R-Pro-Ser-Asp-Thr-Pro-lle-
Leu-Pro-Gln-OH [] [In the formula, R is Ala1-Pro2-Pro3-Pro4-Ser5-
Leu6-Pro7-Ser8-Pro9-Ser10-Arg11-Leu12-P
ro13―
It represents 1 to 14 partial peptide chains containing Gly at position 14 of the peptide represented by Gly14. ] In an enzyme immunoassay for human chorionic gonadotropin using a peptide-enzyme condensate obtained by condensing a peptide represented by the following formula with a labeled enzyme, and (2) a peptide-enzyme condensate as a kind of reagent, the peptide- As an enzyme condensate, the general formula is H-R-Pro-Ser-Asp-Thr-Pro-lle-
Leu-Pro-Gln-OH [] [In the formula, R is Ala1-Pro2-Pro3-Pro4-Ser5-
Leu6-Pro7-Ser8-Pro9-Ser10-Arg11-Leu12-P
ro13
- Represents 1 to 14 partial peptide chains containing Gly at position 14 of the peptide represented by Gly14. An enzyme immunoassay method for human chorionic gonadotropin, which is characterized by using a peptide-enzyme condensate obtained by condensing a peptide represented by ] with a labeled enzyme. In this specification, Ala1-Pro2- represented by R in the peptide represented by the general formula []
Pro3-Pro4-Ser5-Leu6-Pro7-Ser8-Pro9-S
er10―
The 1 to 14 partial peptide chains containing Gly at position 14 of the peptide Arg11-Leu12-Pro13-Gly14 include, for example, Gly, Pro-Gly, Leu-Pro-Gly, Arg-
Leu―Pro―Gly, Ser―Arg―Leu―Pro―Gly,
Pro―Ser―Arg―Leu―Pro―Gly, Ser―Pro―
Ser―Arg―Leu―Pro―Gly, Pro―Ser―Pro―
Ser―Arg―Leu―Pro―Gly, Leu―Pro―Ser―
Pro―Ser―Arg―Leu―Pro―Gly, Ser―Leu―
Pro―Ser―Pro―Ser―Arg―Leu―Pro―Gly,
Pro―Ser―Leu―Pro―Ser―Pro―Ser―Arg―
Leu―Pro―Gly, Pro―Pro―Ser―Leu―Pro―
Ser―Pro―Ser―Arg―Leu―Pro―Gly, Pro―
Pro―Pro―Ser―Leu―Pro―Ser―Pro―Ser―
Arg―Leu―Pro―Gly, Ala―Pro―Pro―Pro―
Ser―Leu―Pro―Ser―Pro―Ser―Arg―Leu―
Examples include Pro-Gly. 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 Nomenclature 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. Ala: Alanine Pro: Proline Ser: Serine Leu: Leucine Arg: Arginine Cly: Glycine Asp: Aspartic acid Thr: Threonine Ila: Isoleucine Gln: Glutamine Glu: Glutamic acid Z: Benzyloxycarbonyl OBu ^t : t-Butyl ester HONB: N- Hydroxy-5-norbornene-2,3-dicarboximide ONB: N-hydroxy-5-norbornene-
2,3-Dicarboximide ester DMF: N,N'-dimethylformamide DCC: N,N'-dicyclohexylcarbodiimide DMSO: Dimethyl sulfoxide THF: Tetrahydrofuran HOBt: 1-hydroxy-benzotriazole OSu: N-hydroxysuccinimide ester ECDI : 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide CMCT: 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide meth-para-toluenesulfonate GLA: Glutaraldehyde m-MBHS: Metamaleimidobenzoyl-
N-Hydroxysuccinimide ester p-MCHS: paramaleimidomethylcyclohexane-1-carboxyl-N-hydroxysuccinimide ester The various peptides used in the present invention can be produced by known 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. As a means of such peptide synthesis, for example, “The
PePtides”, Volume 1 (1966), Schro¨der and
by Lubke, Academic Press, New York, US
A. Or "peptide synthesis", methods described in Izumiya et al., Maruzen Co., Ltd. (1975), such as azide method, chloride method, acid anhydride method, mixed anhydride method, DCC method, active ester method, Woodward method. Method using reagent K, carbodiimidazole method, redox method, DCC/additive (e.g., HONB,
Examples include the HOBt, HOSu) method. The labeling enzyme is preferably one that is stable and has a large specific activity, such as (1) carbohydrase [e.g., glycosidase (e.g., β-galactosidase, β-glucosidase, β-glucuronidase, β-fructosidase, α -galactosidase, α-glucosidase, α-mannosidase), amylase (e.g., α-amylase, β-
amylase, isoamylase, glucoamylase, Takaamylase A), cellulase, lysozyme], (2) amidase (e.g., urease, asparaginase), (3) esterase [e.g., cholinesterase (e.g., acetylcholinesterase), phosphatase (e.g., alkaline phosphatase),
self-atase, lipase], (4) nuclease (e.g. deoxyribonuclease, ribonuclease), (5) iron/porphyrin enzyme (e.g. catalase, peroxidase, cytochrome oxidase), (6) copper enzyme (e.g. tyrosinase, ascorbate oxidase) , (7) dehydrogenases (eg, alcohol dehydrogenase, malate dehydrogenase, lactate dehydrogenase, isocitrate dehydrogenase), and the like. When preparing the peptide-labeled enzyme condensate of the present invention, it is possible to utilize substituents present in each component of the peptide and labeled enzyme, such as amino groups, carboxyl groups, hydroxy groups, or sulfhydryl groups. can. These substituents contained in the peptide or labeled enzyme are activated by using various condensing reagents and bond with other reactive substituents to form a crosslink. In the condensation reaction between the peptide and the labeled enzyme,
Any condensation reagent that can be used for the condensation reaction between a peptide and an enzyme may be used. For example, (1) a reactive amino group on one of the peptide and the enzyme and a reactive carboxyl group on the other are combined in an aqueous solvent. Water-soluble carbodiimide reagents such as ECDI and CMCT, which are dehydrated and condensed with (2) N-
m-MBHS, which is reacted with an active ester of hydroxysuccinimide to form maleimide, and then reacted with an enzyme to bond to the sulfhydryl group of the enzyme.
maleimidation reagents for N-hydroxysuccinimide esters such as and p-MCHS, (3) succindialdehyde, which binds the reactive amino group of one of the peptide enzymes with the reactive amino group of the other;
Dialdehyde reagents such as GLA are used. Further, this condensation reaction can be carried out in an aqueous solvent, and the aqueous solvent can be appropriately selected from those known to be usable in the condensation reaction between a peptide and an enzyme. Examples include a phosphate buffer with a pH of 6 to 8, a borate buffer with a pH of 7 to 9, and the like. The reaction time for the condensation reaction between peptide and enzyme is usually
Although the time is 30 minutes to 20 hours, a short time of 30 minutes to 3 hours is preferable in consideration of the stability of the enzyme. However, for condensation reactions at low temperatures, reaction times ranging from 2 to 4 days can also be employed. Reaction temperature is usually about 4℃
The temperature is appropriately selected from the range of ~50°C, preferably approximately 15°C ~ 30°C. After the completion of this condensation reaction, the peptide-enzyme condensate is converted into Cephadex G100 or G200 (manufactured by Pharmacia Fine Chemicals) or Cephalose 6B or 4B (manufactured by Pharmacia Fine Chemicals).
It can be purified and fractionated by column chromatography using gels such as those manufactured by Chemicals Co., Ltd.). As an enzyme immunoassay method for hCG, (1)
A fixed amount of the peptide-enzyme condensate and a fixed amount of anti-hCG antibody in insoluble form are added to the test sample containing hCG, and either the peptide-enzyme condensate in the liquid phase or the reacted peptide-enzyme in the solid phase is prepared. hCG in the test solution by measuring the enzymatic activity of the condensate.
How to quantify [FEBS Letters (FEBS)
(2) Add a certain amount of peptide-enzyme condensate and a certain amount of soluble anti-hCG antibody to the hCG-containing test solution, and after competitively binding, Add a certain amount of an insoluble second antibody to the first antibody [for example, anti-rabbit immunoglobulin G (IgG) antibody when anti-hCG antibody was prepared using a rabbit], and use the liquid phase or A method for quantifying hCG in a test solution after measuring the enzyme activity in the solid phase (see the above Febus Letters), and (3) adding a certain amount of the peptide-enzyme condensate to the hCG-containing test solution in the same manner as in (2) above. After adding a certain amount of soluble anti-hCG antibody and competitively binding the second antibody, it is normal if the second antibody is an anti-animal (such as the same as the animal mentioned below) IgG antibody. Add a certain amount of animal serum (including those similar to the animals described below) to produce a precipitate, measure the enzyme activity in the liquid phase or solid phase in the same way as in (1), and Method for quantifying hCG [Clinica Chimica Acta, Vol. 67, p. 263 (1976)]
Applicable to In either method, when measuring enzyme activity in the liquid phase, it is advantageous to measure the activity in the solid phase, as contaminants present in the test solution may interfere with the measurement of the enzyme activity. be.
In addition, from the point of view of reproducibility and sensitivity, even trace amounts of anti-hCG
"Double antibody method" that allows measurement using antibodies, i.e.
Methods (2) and (3) are more preferably used. The test sample used in the EIA of the present invention may be any body fluid such as urine, serum, plasma, or cerebrospinal fluid, and urine or serum is particularly frequently used. The antibody used in this EIA may be any antibody that is reactive with the C-terminal peptide of hCG-β.
The antibodies include specific antibodies obtained by the method of the present invention.
hCG antibody, anti-hCG serum obtained by immunization with hCG,
Anti-hCG-β serum obtained by immunization with hCG-β,
Anti-C obtained by immunization with C-terminal peptide of hCG-β
Examples include end peptide serum and γ-globulin fractions prepared from these antisera. To produce the hCG-specific antibody of the present invention,
First, by inoculating an animal with hCG, anti-hCG antibodies are formed in the body fluid of the animal. The animal may be any animal other than humans, such as mammalian warm-blooded animals (e.g., rabbits, sheep, rats, mice, guinea pigs, cows, horses, pigs),
Birds (e.g., chickens, pigeons, ducks,
quail), cold-blooded animals (e.g. frogs), etc. Examples of carriers used in the production of the hCG-specific antibody of the present invention include gel particles (e.g., agarose gel [e.g., Sepharose 4B, Sepharose 6B (manufactured by Pharmacia Huain Chemical Co., Sweden)], dextran gel [e.g. Example, Cephadex G75, Cephadex
G100, Cephadex G200 (manufactured by Pharmacia Fine Chemicals)], polyacrylamide gel [e.g., Biogel P30, Biogel P60, Biogel P100 (Biorad Laboratories, Inc. (USA))], cellulose particles (e.g., Avicel (manufactured by Asahi Kasei)) Ion-exchanged cellulose (e.g. diethylaminoethyl cellulose, carboxymethyl cellulose)], physical adsorbent [e.g. glass (e.g. glass bulbs, glass rods, aminoalkyl glass bulbs, aminoalkyl glass rods), silicone pieces, styrenic resins (e.g. , polystyrene spheres, polystyrene particles)], ion exchange resins (e.g., weakly acidic cation exchange resins [e.g., Amberlite IRC-50 (ROHM,
Haas (USA)), Zeocarb 226 (Palmchitz (West Germany)), weakly basic anion exchange resins (e.g. Amberlite IR-4B, Dowex 3 (Dow Chemical (USA)), etc. can be mentioned. Known conventional methods can be applied to insolubilize peptides on carriers, and such means for synthesizing insolubilized peptides are described, for example, in "Metabolism", Vol. 8 (1971).
(2010), p. 696. Examples include the Promsyan method, the GLA method, and the DCC method, and preferably a method in which the peptide is bonded to a carrier activated with Bromsyan is used. Next, we immunized the animal with hCG and obtained the anti-inflammatory agent.
A body fluid, particularly serum, containing an hCG antibody is brought into contact with a peptide represented by the general formula [ ] insolubilized on a carrier. In this method, the IgG fraction is precipitated by salting out with sodium sulfate or ammonium sulfate, etc., and the IgG fraction is fractionated directly or further by column chromatography such as DEAE-cellulose, and then brought into contact with the above-mentioned insolubilized peptide to immobilize only the specific anti-hCG antibody. absorb into the phase. This results in hLH,
Other anti-hCG antibodies that cross-react with hFSH and hTSH can be removed. Then, the specific anti-hCG antibody absorbed on the solid phase is eluted. To perform this elution, use a low pH or high pH buffer (e.g. pH 2.3).
0.17M glycine-hydrochloric acid buffer, aqueous ammonia at pH 11) or buffers containing high salt concentrations (e.g. 6M
guanidine hydrochloride solution, 7M urea solution) were used,
Elute and separate the specific antibody fraction. The above operation may be carried out in a batch manner, but is preferably carried out in a column. The peptide to be insolubilized used in the production of the anti-hCG antibody of the present invention has the formula H-R-Pro-Ser-Asp-Thr-Pro-lle.
-Leu-Pro-Gln-OH [wherein, R is Ala1-Pro2-
Pro3-Pro4-Ser5-Leu6-Pro7-Ser8-Pro9-S
er10―
It represents 1 to 14 partial peptide chains containing Gly at position 14 of the peptide Arg11-Leu12-Pro13-Gly14. ] may be used, especially H-Ala-Pro-Pro-Pro-Ser-
Leu―Pro―Ser―Pro―Ser―Arg―Leu―Pro―
Gly―Pro―Ser―Asp―Thr―Pro―Ile―Leu―
A highly specific anti-hCG antibody can be recovered in good yield by using a carrier that combines the peptide () represented by Pro-Glu-OH with Sepharose 4B (manufactured by Pharmacia Huain Chemicals). The physical properties of this antibody are (1) Final dilution concentration 10-200IU/
ml, the ability to bind 10 to 100% of hCG-enzyme label, peptide()-enzyme label, peptide()-enzyme label, and peptide()-enzyme label each having an enzyme activity of approximately 2 μU. (2) The optimal pH for antigen binding activity is 6 to 9, (3) It is stable for more than a year when stored in a refrigerator, and (4) The molecular weight is approximately 14 to 17.
(5) It is easily soluble in aqueous solvents with a pH of 2 to 12, (6) Its electrophoretic mobility belongs to the γ-globulin fraction, (7) Figure 1 It has the ultraviolet absorption spectrum shown in (8) and the amino acid composition is 100% glycine.
Expressed in moles of each amino acid per mole, lysine
85-97, histidine 35-43, arginine 38-45,
Aspartic acid 110-132, Threonine 98-107,
Serine 118-135, glutamic acid 138-145, proline 92-134, glycine 100, alanine 73-79, valine 129-138, methionine 2-10, isoleucine 28
~37, leucine 100~112, tyrosine 38~48, phenylalanine 55~68, (9) each with two H
The chain and the L chain form a molecule through SS bonding. In addition, the second antibody used in EIA of the present invention is anti-hCG
Animal species used when preparing the antibody (e.g. rabbit)
IgG can be prepared by multiple injections into other animal species (eg, goats). The immunization method may use any of the conventional antibody preparation methods, such as Freund's complete adjuvant and about 20%
Approximately 4 mg of IgG subcutaneously administered every 3 weeks yields satisfactory antibodies. Also commercially available anti-rabbit
IgG serum (goat) (Miles Laboratory, USA) or the like may be used. The insoluble second antibody is the anti-antibody obtained as described above.
IgG serum was salted out and precipitated with sodium sulfate or ammonium sulfate, and the anti-IgG serum was fractionated using DEAE-cellulose column chromatography.
It is obtained by binding the IgG fraction to a solid phase activated with bromsyanide, such as cellulose or Cephadex (FEBS Letters, Vol. 15 (1971), p. 232).
It can also be obtained by physically adsorbing the second antibody onto the solid phase by contacting the IgG fraction of the antiserum with glass beads, silicon pieces, or polystyrene particles ("Chemistry and Biology"). , Vol. 14 (1976), p. 741). Compared to conventional methods, the method for producing a specific anti-hCG antibody of the present invention does not require complicated operations for preparing an immunogen, and provides a specific antibody with high sensitivity.
Furthermore, the enzyme immunoassay method of the present invention has been performed conventionally.
Compared to hCG detection methods, this method provides highly specific results and is therefore a highly useful hCG measurement method that is not interfered with by hLH, hFSH, or hTSH that coexist in the sample.
Its sensitivity makes it highly applicable to early diagnosis and prognosis management of diseases such as trophoblastic tumors. Therefore, the EIA of the present invention uses radioisotopes (hereinafter referred to as RI).
It is called. ), it can be carried out even in clinical laboratories where it is difficult to use the method, and hCG concentrations in urine and blood can be easily and specifically quantified. Furthermore, the peptide-enzyme condensate of the present invention is stable and easy to produce, unlike RI-labeled peptides that have a half-life.
It is possible to measure multiple samples and provides results with good reproducibility. In measuring hCG according to the present invention, it is preferable to use a human chorionic gonadotropin enzyme immunoassay kit comprising the following reagents. A test set for a quantitative method using an insolubilized second antibody mainly contains (1) an anti-human chorionic gonadotropin antibody (preferably an anti-human chorionic gonadotropin antibody obtained by the method of the present invention) in addition to a reaction system; (2) a moiety that binds an amount (preferably about 5-100%) of the peptide-enzyme condensate of the invention; (2) a portion (preferably about 0.1%) of the peptide-enzyme condensate of the invention; (3) 0 to 500 IU of standard human chorionic gonadotropin (4) A buffer (preferably pH 6) used for diluting the reagents and test samples in (1) to (3) above. ~9 phosphate buffer), (5) a fixed amount (an amount that can bind all of the first antibody in (1) above) of anti-animal IgG in an insoluble form (the carrier is the same as above); (6) a certain amount (preferably 1 to 100 μg) of the enzyme substrate, (7) a buffer used for washing the second antibody-bound carrier in (5) and dissolving the enzyme substrate in (6). (8) A buffer used to stop the enzyme reaction (preferably a carbonate buffer or a dilute caustic soda aqueous solution). Further, the above kit is preferably used, for example, by the following method. Reagent (1) 10-300μ, reagent (4) 100-500μ and standard human chorionic gonadotropin or test sample 5-5
After adding 100μ of reagent (2) and then adding 10 to 300μ of reagent (2), react at 0 to 40°C. After reacting for 1 to 72 hours, a certain amount of reagent (5) is added, thoroughly stirred, and further reacted at 0 to 40°C for 0.5 to 24 hours. After the reaction is complete,
Wash the second antibody-bound carrier with reagent (7), then add the reagent
(6) Add 10-1000μ to start the enzyme reaction. 20
After reacting at ~40℃ for 0.5 to 24 hours, reagent (8) 1
The reaction is stopped by adding ~5 ml. The absorbance or fluorescence intensity in the reaction solution is measured to determine the human chorionic gonadotropin concentration in the test solution. The present invention will be described in more detail below with reference to Examples and Reference Examples, but it goes without saying that these do not limit the scope of the present invention. In the following reference examples, thin layer chromatography uses a silica gel plate 60F ₂₅₄ manufactured by Merck Co., Ltd. and the following developing solvent, unless otherwise specified. Rf ¹ : Chloroform: Methanol = 95:5 Rf ² : Chloroform: Methanol: Acetic acid = 9:
1:0.5 Rf ³ : Ethyl acetate: Pyridine: Acetic acid: Water = 60:
20:6:10 Rf ⁴ :n-butanol:pyridine:acetic acid:water=
30:20:6:24 Rf ⁵ : Ethyl acetate: n-butanol: acetic acid: water = 1:1:1:1 Rf ⁶ : n-butanol: acetic acid: water = 12:3:5 Reference example 1 H-Ala ―Pro―Pro―Pro―Ser―Leu―Pro―
Ser―Pro―Ser―Arg―Leu―Pro―Gly―Pro
―Ser―ASp―Thr―Pro―Ile―Leu―Pro―
Gln-OH [hereinafter referred to as peptide (). ]
Production of [hCGβ(123-145) C-terminal peptide moiety] (a) Production of Z-Pro-Gln-OBu ^t : Z-Gln-
12.5 g of ^OBut is dissolved in 500 ml of methanol and catalytically reduced using palladium black as a catalyst. After filtering off the catalyst and distilling off the solvent, the residue is dissolved in 300 ml of ethyl acetate. In this Z-Pro-OH9.7
Z- synthesized from g, HONB8.4g, and DCC8.8g
Add 200ml of Pro-NOB ethyl acetate solution and stir for 5 hours. Add the reaction solution to 0.2N hydrochloric acid, 4%
Wash with sodium bicarbonate and water, and dry with anhydrous sodium sulfate. Ethyl acetate is distilled off, and petroleum benzine and a small amount of ether are added to the residue to form crystals, which are then recrystallized from the same solvent system. Yield 13.1g (81.5%), melting point 86-88℃, [α] ²⁶ _D
-64.8゜(c=0.5, methanol), Rf ¹ 0.42,
^Rf2 0.73. Elemental analysis C ₂₂ H ₃₁ O ₆ N ₃ : Calculated value C60.95;
H7.21; N9.69. Analysis value: C61.04; H7.20;
N9.49. (b) Production of Z-Leu-Pro-Gln-OBu ^t : Z-
Dissolve 13 g of Pro-Gln-OBu ^t in 500 ml of methanol and reduce in a hydrogen stream using palladium black as a catalyst. The catalyst was filtered off, the solvent was distilled off, the residue was dissolved in 300 ml of ethyl acetate, and Z-Leu-
Add an ethyl acetate solution of Z-Leu-ONB synthesized from 7.9 g of OH, 6.5 g of HONB, and 6.8 g of DCC,
Stir for 5 hours. Add the reaction solution to 0.2N-hydrochloric acid, 4
%-Sodium hydrogen carbonate solution, wash with water, dry over anhydrous sodium sulfate, and evaporate the solvent. Add petroleum benzine to the residue and filter it as a powder. Yield 10.6g (66.2%), melting point 74-77℃,
[α] ²³ _D −81.4° (c=0.6, methanol), Rf ¹ 0.38
，
^Rf2 0.66. Elemental analysis C ₂₈ H ₄₂ O ₇ N ₄ : Calculated value: C61.52;
H7.74; N10.25. Analysis value: C61.61; H7.94;
N9.92. (c) Production of Z-Ile-Leu-Pro-Gln-OBu ^t : Dissolve 10.6 g of Z-Leu-Pro-Gln-OBu ^t in 500 ml of methanol, perform catalytic reduction using palladium black as a catalyst, and then distill off the methanol. and dissolve the residue in 300 ml of ethyl acetate. Z-Ile to this
Add 200 ml of an ethyl acetate-dioxane mixed solution (1:1) of Z-Ile-ONB synthesized from 5.1 g of -OH, 4.2 g of HONB, and 4.4 g of DCC, and stir for 16 hours. The solvent was distilled off from the reaction solution, the residue was dissolved in 400 ml of ethyl acetate, and 0.2N hydrochloric acid, 4%
Wash with sodium bicarbonate and water, and dry with anhydrous sodium sulfate. The solvent was distilled off, petroleum benzine was added to the residue, and the powder was collected by filtration. Yield 12.1g (94.5%), melting point 78-80℃ (decomposition), [α] ²³ _D -87.0゜ (c = 0.42, methanol),
Rf ¹ 0.23, Rf ² 0.67. Elemental analysis C ₃₄ H ₅₃ O ₈ N ₅ Calculated value: C61.89;
H7.10; N10.62. Analysis value: C62.35; H8.31;
N10.16. (d) Production of Z-Pro-Ile-Leu-Pro-Gln-OBu ^t : Dissolve 12 g of Z-Ile-Leu-Pro-Gln-OBu ^t in 500 ml of methanol and use palladium black as a catalyst in a hydrogen stream. After reduction, the catalyst is filtered off and the solvent is distilled off. Dissolve the residue in 100ml of DMF, and add 4.7g of Z-Pro-OH and 3.0g of HOBt.
was added, cooled to 0℃, added 4.3g of DCC, and cooled to 0℃.
Stir for 4 hours at room temperature and 10 hours at room temperature. After the precipitate was filtered off and the solvent was distilled off, the residue was dissolved in 400 ml of ethyl acetate, washed with 0.2N hydrochloric acid, 4% aqueous sodium bicarbonate and water, and dried over anhydrous sodium sulfate. The solvent is distilled off, ether is added to the residue, heated, and the supernatant is removed. Ether is further added and the mixture is filtered as a powder. Yield 12.6g (91.5%), melting point 83-87℃ (decomposition), [α] ²³ _D -121.0゜ (c = 0.5, methanol),
Rf ¹ 0.31, Rf ² 0.84. Elemental analysis C ₃₉ H ₆₀ O ₉ N ₆ Calculated value: C61.88;
H7.99; N11.10. Analysis value: C62.04; H8.21;
N10.70. (e) Z―Thr―Pro―Ile―Leu―Pro―Gln―
Manufacture of OBu ^t : Z-Pro-Ilu-Leu-Pro-Gln
-Dissolve 12.5g of OBu ^t in 500ml of methanol and reduce in a hydrogen stream using palladium black as a catalyst. After filtering off the catalyst and distilling off the solvent, the residue is dissolved in 100 ml of DMF. In this solution, Z-
Add and dissolve Thr-OH4.2g, HOBt2.7g,
Cool to 0°C. Next, 3.7 g of DCC was added and the mixture was stirred at 0°C for 4 hours and at room temperature for 8 hours, and then the precipitate was filtered off. The solvent was distilled off and the residue was dissolved in ethyl acetate.
Extract to 400ml, wash with 0.2N hydrochloric acid, 4% aqueous sodium bicarbonate, and water, and dry over anhydrous sodium sulfate. The solvent was distilled off, ether was added to the residue, and the powder was collected by filtration. Yield 11.8g (86.8%), melting point 101-105℃,
[α] ²³ _D −124.3° (c=0.58, methanol),
Rf ¹ 0.20, Rf ² 0.68. Elemental analysis C ₄₃ H ₆₇ O ₁₁ N ₇ Calculated value: C60.19;
H7.87; N11.43. Analysis value: C59.54; H7.91;
N11.19. (f) Z―Asp（OBu ^t ―Thr―Pro―Ile―Leu―Pro
-Production of Gln-OBu ^t : Z-Thr-Pro-Ile-
Leu-Pro-Gln-OBu ^t 11.8g methanol 500ml
ml, and reduced in a hydrogen stream using palladium black as a catalyst. After filtering off the catalyst, the solvent was distilled off, and the residue was dissolved in 100 ml of DMF. Z to this
-ASp(OBu ^t )- Add 4.5 g of OH and 2.3 g of HOBt, dissolve, and cool to 0°C. Add 3.2 g of DCC and stir at C° for 4 hours and at room temperature for 10 hours, filter off the precipitate, and distill off the solvent. Add 150 ml of ethyl acetate to the residue, filter the precipitated gel, crystallize from ethyl acetate and ether, and filter with ether. Yield 12.15g (85.9%), melting point 94-96℃ (decomposition), [α] ²¹ _D -109.1゜ (c = 0.59, methanol),
Rf ¹ 0.13, Rf ² 0.47. Elemental analysis C ₅₁ H ₈₀ O ₁₄ N ₈・H ₂ O Calculated value:
C58.49; H7.89; N10.70. Analysis value: C58.60;
H8.07; N10.71. (g) Z―Ser―Asp(OBu ^t )―Thr―Pro―Ile―
Manufacturing of Leu-Pro-Gln-OBu ^t : Z-Asp
(OBu ^t )―Thr―Pro―Ile―Leu―Pro―Gln―
12 g of ^OBut is dissolved in 500 ml of methanol and reduced in a hydrogen stream using palladium black as a catalyst. After filtering off the catalyst and distilling off the solvent, the residue was collected together with 2.93 g of Z-Ser-OH and 2.0 g of HOBt.
Dissolve in 100 ml of DMF and cool to 0°C. to this
Add 2.8 g of DCC and stir at 0°C for 4 hours and at room temperature for 12 hours, filter off the precipitate, and distill off the solvent.
Dissolve the residue in 500 ml of ethyl acetate, wash with 0.2N hydrochloric acid, 4% aqueous sodium bicarbonate, and water.
After drying over anhydrous sodium sulfate, the solvent is distilled off. Add ethyl acetate and ether to the residue and filter it as a powder. Yield 11.7g (90.0%), melting point 111-115℃
(decomposition), [α] ²¹ _D −112.3° (c=0.63, methanol), Rf ¹ 0.06, Rf ² 0.31. Elemental analysis C ₅₄ H ₈₅ O ₁₆ N ₉ H ₂ O Calculated value:
C57.17; H7.73; N11.11. Analysis value: C57.34;
H7.77; N11.14. (h) Z―Pro―Ser―Asp (OBu ^t )―Thr―Pro―
Manufacture of Ile-Leu-Pro-Gln-OBu ^t : Z-Ser
-Asp(OBu ^t )-Thr-Pro-Ile-Leu-Pro-
11.6 g of Gln-OBu ^t is dissolved in 500 ml of methanol and reduced in a hydrogen stream using palladium black as a catalyst. After filtering off the catalyst and distilling off the solvent,
The residue along with 4.3g of Z-Pro-ONB
Dissolve in 100 ml of DMF and stir at room temperature for 16 hours. The solvent was distilled off, and the residue was extracted with 500 ml of ethyl acetate, washed with 0.2N hydrochloric acid, 4% aqueous sodium bicarbonate, and water, dried over anhydrous sodium sulfate, and then the solvent was distilled off. Add ether to the residue and filter it as a powder. Yield 11.1g (88.1%), melting point 117-120℃,
[α] ²¹ _D −119.2° (c=0.61, methanol),
^Rf2 0.45. Elemental analysis C ₅₉ H ₉₂ O ₁₇ N ₁₀ H ₂ O Calculated value:
C57.54; H7.69; N11.38. Analysis value: C57.44;
H7.69; N11.38. (i) Z―Gly―Pro―Ser―Asp (OBu ^t )―Thr―
Production of Pro-Ile-Leu-Pro-Gln-OBu ^t : Z
-Pro-Ser-Asp(OBu ^t )-Thr-Pro-Ile-
Leu-Pro-Gln-OBu ^t 10g methanol 500ml
ml and reduced in a hydrogen stream using palladium black as a catalyst. After the catalyst was filtered off, the solvent was distilled off. Residue with Z-Gly-ONB4g
Dissolve in 80ml of DMF and stir at room temperature for 12 hours.
The solvent was distilled off, the residue was dissolved in 500 ml of ethyl acetate, and 0.2N-hydrochloric acid, 4%-sodium hydrogen carbonate,
Wash with water, dry over anhydrous sodium sulfate, and evaporate the solvent. Dissolve the residue in 180ml of methanol,
Add 4.5 ml of hydrazine hydrate, stir at room temperature for 16 hours, and then distill off the solvent. The residue was dissolved in 500 ml of ethyl acetate, washed with water, dried over anhydrous sodium sulfate, and the solvent was distilled off. Add ether to the residue and filter it as a powder. Yield 7.35g (70.2%), melting point 131-135℃
(decomposition), [α] ²² _D −119.4° (c=0.35, methanol), Rf ² 0.27. Elemental analysis C ₆₁ H ₉₅ O ₁₈ N ₁₁ H ₂ O Calculated value:
C56.86; H7.59; N11.96. Analysis value: C56.65;
H7.68; N12.02. (j) Manufacturing of Z-Leu-Pro-OBu ^t : Z-Pro-
20.2 g of ^OBut is dissolved in 800 ml of methanol and reduced in a hydrogen stream using palladium black as a catalyst.
The catalyst was filtered off, the solvent was distilled off, the residue was dissolved in 300 ml of ethyl acetate, 26.3 g of Z-Leu-OSu was added, and the mixture was stirred for 16 hours. Add the reaction solution to 0.2N-hydrochloric acid,
Wash with 4% sodium bicarbonate, water, dry over anhydrous sodium sulfate, and evaporate the solvent to obtain an oil. Yield 27.6g (100%), Rf ¹ 0.71, Rf ² 0.78. (k) Production of Z―Arg―(NO ₂ )―Leu―Pro―OBu ^t : Add 13.8 g of Z―Leu―Pro―OBu ^t to methanol.
Dissolve in 500ml and use palladium black as a catalyst.
Reduction is carried out in a stream of hydrogen, the catalyst is filtered off and the solvent is distilled off. Residue and Z―Arg―(NO ₂ )―
Dissolve 11.7g of OH and 6.7g of HOBt in 200ml of DMF,
Cool to 0°C. Add 7.5g of DCC to this and bring it to 0°C.
The mixture was stirred for 4 hours at room temperature and for 12 hours at room temperature, the precipitate was filtered off, and the solvent was distilled off. 600% of the residue in ethyl acetate
ml, washed with 0.2N hydrochloric acid, 4% aqueous sodium bicarbonate, and water, dried over anhydrous sodium sulfate, and the solvent was distilled off. The residue is left to stand, and the precipitated crystals are filtered with ether and then recrystallized from methanol. Yield 12.4g (60.6%), melting point 170-172℃,
[α] ²⁶ _D −67.8° (c=0.48, methanol),
^Rf3 0.77. Elemental analysis C ₂₉ H ₄₅ O ₈ N ₇ Calculated value: C56.20;
H7.32; N15.82. Analysis value: C55.79; H7.16;
N15.85. (l) Production of Z―Ser―Arg―Leu―Pro―OBu ^t :
12.3 g of Z-Arg(NO ₂ )-Leu-Pro-OBu ^t was dissolved in 500 ml of methanol, 6.6 ml of 6N-hydrochloric acid was added, and then reduced in a hydrogen stream using palladium black as a catalyst, and the catalyst was filtered off. Afterwards, the solvent is distilled off. Residue with 2.8ml of triethylamine
Dissolve in 100ml of DMF and filter off the precipitated triethylamine hydrochloride. Add Z―Ser― to the filtrate.
Add 4.8g of OH and 5.4g of HONB and cool to 0℃.
Add 4.95 g of DCC and stir at 0°C for 4 hours and at room temperature for 16 hours. After the precipitate was filtered off and the solvent was distilled off, the residue was extracted with 500 ml of ethyl acetate, washed thoroughly with water saturated with sodium chloride, dried over anhydrous sodium sulfate, and the solvent was distilled off to leave a candy-like residue. get. ^Rf3 0.53. (m) Z―Pro―Ser―Arg―Leu―Pro―OBu ^t
Manufacture of: Z-Ser-Arg-Leu-Pro-OBu ^t 10.5
g was dissolved in 500 ml of methanol with 3 ml of 6N-hydrochloric acid, and reduced in a hydrogen stream using palladium black as a catalyst. The catalyst was filtered off, the solvent was distilled off, the residue was dissolved in 100 ml of DMF, and triethylamine was added.
Add 2.52ml to neutralize. After filtering off the precipitated triethylamine hydrochloride, Z-Pro-
Add 8.4 g of ONB, stir at room temperature for 16 hours, distill off the solvent, add 300 ml of ethyl acetate and 300 ml of salt-saturated water to the residue, and mix well. After standing still, the oily substance that precipitates in the water layer is collected, and ether is added to it to form a powder. Next, this powder is dissolved in methanol, insoluble matter is filtered off, the solvent is distilled off, ether is added again, and the powder is collected by filtration. Yield 8.45g (70.8%), melting point 115-120℃
(decomposition), [α] ²² _D −84.7° (c=0.53, methanol), Rf ³ 0.41. Elemental analysis C ₃₇ H ₅₈ O ₉ N ₈・HCl・H ₂ O Calculated value: C54.63; H7.56; N13.78; Cl4.36. Analytical values: C54.50; H7.70; N14.11; Cl4.21. (n) Z-Ser-Pro-Ser-Arg-Leu-Pro
-Manufacturing of OBu ^t : Z-Pro-Ser-Arg-Leu-
Dissolve 3.8g of Pro-OBu ^t in 200ml of methanol,
Reduction is carried out in a hydrogen stream using palladium black as a catalyst, the catalyst is filtered off, the solvent is distilled off, and the residue is dissolved in 30 ml of DMF. Add Z-Ser to this solution.
-DMF solution (10ml) of Z-Ser-ONB synthesized from 1.37g of OH, 1.24g of HONB and 1.30g of DCC
Add and stir at room temperature for 16 hours. After filtering off the precipitate, the solvent was distilled off, and ethyl acetate was added to the residue.
ml and 1 ml of acetonitrile to dissolve it, then add ether to form a precipitate, which is then powdered and collected by filtration. This powder was dissolved in 2 ml of a 1:1 mixture of Rf ³ solvent and ethyl acetate, and a silica gel column (5.6 x 9.0 cm) packed with the same solvent.
and develop with the same solvent. A portion of 365 ml to 746 ml is collected, the solvent is distilled off, and ether is added to the residue, which is filtered as a powder. Yield 2.12g (50.2%), melting point 130-135℃
(decomposition), [α] ²² _D −83.5° (c=0.38, methanol), Rf ³ 0.34. Elemental analysis C ₄₀ H ₆₃ O ₁₁ N ₉・HCl・H ₂ O Calculated value: C53.35; H7.39; N14.00; Cl3.94. Analytical values: C53.67; H7.45; N13.72; Cl3.52. (o) Production of Z-Ser-Leu-Pro-OBu ^t : Z
-Leu-Pro-OBu ^t 13.8g in methanol 700ml
and reduced in a hydrogen stream using palladium black as a catalyst. The catalyst was filtered off and the solvent was distilled off, and the residue was dissolved in 200 ml of acetonitrile along with 7.9 g of Z-Ser-OH and 8.9 g of HONB, and cooled to 0°C. Next, add 7.5 g of DCC and stir at 0°C for 4 hours and at room temperature for 16 hours. The precipitate was filtered off, the solvent was distilled off, and the residue was extracted into 500 ml of ethyl acetate.
0.2N-hydrochloric acid, 4%-sodium bicarbonate water,
Wash with water, dry over anhydrous sodium sulfate, and evaporate the solvent to obtain 17 g of an oil. Add this to 15ml of a mixed solvent of chloroform and methanol (200:3).
It is applied to a silica gel column (5.4 x 20 cm) packed with the same solvent, and developed with the same solvent.
A portion of 1300-2100 ml was collected and the solvent was distilled off to give an oil. Yield 12.2g (73.1%), Rf ¹ 0.38, Rf ² 0.69. (p) Z―Ser―Leu―Pro―Ser―Pro―Ser―
Manufacture of Arg-Leu-Pro-OBu ^t : Z-Ser-Pro
1.0 g of -Ser-Arg-Leu-Pro-OBu ^t was dissolved in 60 ml of methanol with 1.2 ml of N-hydrochloric acid, and reduced in a hydrogen stream using palladium black as a catalyst.
After filtering off the catalyst, distill off the solvent and remove the residue.
Dissolve in 20ml DMF. Z-Ser-Leu-Pro-
Dissolve 700 mg of OBu ^t in 7 ml of trifluoroacetic acid,
After 50 minutes, the solvent was distilled off, the residue was washed with a mixed solvent of ether and petroleum ether (1:2), the washing liquid was removed, and the remaining oil was evaporated under reduced pressure under a vacuum pump until it became a powder. After drying, it was immediately added to the above DMF solution together with 407 mg of HONB to dissolve, and the mixture was cooled to 0°C. 466 mg of DCC was added thereto, stirred at 0°C for 6 hours and at room temperature for 16 hours, the precipitate was filtered off, and the solvent was distilled off. residue
Rf ³ was dissolved in 5 ml of mixed solvent of ethyl acetate (4:1), applied to a silica gel column (3.6 x 9.0 cm) packed with the same solvent, and eluted with the same solvent.
A portion of ~572 ml was collected, the solvent was distilled off, ether was added to the residue, and the powder was collected by filtration. Yield 450 mg (33.8%), melting point 110-120℃ (decomposition), [α] ²⁴ _D -106.6゜ (c = 0.31, methanol),
^Rf5 0.71. (q) Manufacturing of Z-Pro-Pro-OBu ^t : Z-Pro
-Dissolve 10.1g of OBu ^t in 500ml of methanol, reduce it in a hydrogen stream using palladium black as a catalyst,
Filter off the catalyst, distill off the solvent, and remove the residue from Z-Pro.
-Ethyl acetate with 8.47g of OH and 5.4g of HOBt
Dissolve in 300ml and cool to 0°C. Next DCC7.5
The mixture was stirred at 0°C for 4 hours and at room temperature for 12 hours, and the precipitate was filtered off. Add filtrate to 0.2N hydrochloric acid, 4%
Wash with aqueous sodium bicarbonate and water, dry over anhydrous sodium sulfate, and evaporate the solvent. Filter the residue crystals with ether. Yield 9.85g (74.1%), melting point 94-96℃,
[α] ²² _D −116.9° (c=0.54, methanol),
Rf ¹ 0.58, Rf ² 0.72. Elemental analysis C ₂₂ H ₃₀ O ₅ N ₂ Calculated value: C65.65;
H7.51; N6.96. Analysis value: C65.42; H7.38;
N7.20. (r) Manufacturing of Z-Pro-Pro-Pro-OBu ^t : Z
-Dissolve 9g of Pro-Pro-OBu ^t in 300ml of methanol, reduce in a hydrogen stream using palladium black as a catalyst, filter off the catalyst, distill off the solvent, and dissolve the residue as Z-Pro-OH5.6g. , HOBt (3.63 g) in 250 ml of ethyl acetate and cooled to 0°C. Next, 5.1 g of DCC was added and stirred at 0°C for 6 hours and at room temperature for 16 hours, and the precipitate was filtered off. Add filtrate to 0.2N hydrochloric acid, 4% sodium bicarbonate water,
Wash with water, dry over anhydrous sodium sulfate, and evaporate the solvent. The precipitated crystals are filtered with ether. Yield 9.6g (85.9%), melting point 155-157℃,
[α] ²² _D ---176.0° (c=0.55, methanol),
Rf ¹ 0.40, Rf ² 0.69. Elemental analysis C ₂₇ H ₃₇ O ₆ N ₃・1/2H ₂ O Calculated value:
C63.76; H7.53; N8.26. Analysis value: C63.77;
H7.53; N8.62. (s) Production of Z-Ala-Pro-Pro-Pro-OBu ^t : Dissolve 5 g of Z-Pro-Pro-Pro-OBu ^t in 200 ml of methanol, reduce in a hydrogen stream using palladium black as a catalyst, After filtering off the catalyst, the solvent was distilled off and the residue was 2.23 g of Z-Ala-OH,
Dissolved in 20ml of DMF with 1.62g of HOBt at 0°C.
Cool to Add 2.27g of DCC to this solution and
Stir at ℃ for 4 hours and at room temperature for 12 hours, and filter off the precipitate. The solvent was distilled off and the residue was diluted with 3 ml of 2%
Silica gel column (3.7 x 10.5 cm) dissolved in methanol/chloroform and packed with the same solvent.
and develop with the same solvent. Collect 170-380ml portions, distill off the solvent, and remove 4.05g (71.0ml) of oily substance.
%). Rf ¹ 0.33, Rf ² 0.67. (t) Z―Ala―Pro―Pro―Pro―Ser―Leu
―Pro―Ser―Pro―Ser―Arg―Leu―Pro―
Manufacture of OBu ^t : Z-Ser-Leu-Pro-Ser-Pro
400 mg of -Ser-Arg-Leu-Pro-OBu ^t is dissolved in 80 ml of methanol with 0.8 ml of N-hydrochloric acid, and reduced in a hydrogen stream using palladium black as a catalyst. Filter off the catalyst, distill off the solvent, and remove the residue.
Triethylamine 0.11ml dissolved in DMF 10ml
Neutralize with water and filter off the precipitated triethylamine hydrochloride. Z―Ala―Pro―Pro―Pro―
Dissolve 233 mg of ^OBut in 2 ml of trifluoroacetic acid. After 50 minutes, the solvent is distilled off, ether is added to the residue, the powder is collected by filtration, and dried. this
Add 122 mg of HONB to the above DMF solution and dissolve, and cool to 0°C. DCC140 in this solution
After stirring at 0°C for 6 hours and at room temperature for 12 hours, the precipitate was filtered off and the solvent was distilled off. Ether is added to the residue, the powder is collected by filtration, and purified by reprecipitation from acetonitrile and ethyl acetate. Yield 430mg (82.1%), melting point 152-155℃ (decomposition), [α] ²⁴ _D -153.6゜ (c = 0.45, methanol),
Rf ³ 0.10, Rf ⁵ 0.54. Elemental analysis C ₇₂ H ₁₁₂ O ₁₉ N ₁₆・HCl・H ₂ O Calculated value: C55.42; H7.43; N14.37; Cl2.27. Analysis value: C56.21; H7.54; N14.04; Cl2.30. (u) Z-Ala-Pro-Pro-Pro-Ser-Leu
―Pro―Ser―Pro―Ser―Arg―Leu―Pro―
Gly-Pro-Ser-Asp(OBu ^t )-Thr-Pro-
Production of Ile-Leu-Pro-Gln-OBu ^t : Z-
Gly-Pro-Ser-Asp(OBu ^t )-Thr-Pro-
363 mg of Ile-Leu-Pro-Gln-OBu ^t is dissolved in 30 ml of methanol and reduced in a hydrogen stream using palladium black as a catalyst. The catalyst is filtered off, the solvent is distilled off and the residue is dissolved in 10 ml of DMF. Z-
Ala―Pro―Pro―Pro―Ser―Leu―Pro―Ser
-Dissolve 430 mg of Pro-Ser-Arg-Leu-Pro-OBu ^t in 3 ml of trifluoroacetic acid and leave at room temperature for 45 minutes. The solvent was distilled off, ether was added to the residue, the powder was collected by filtration, and after drying, it was added to the above DMF solution together with 103 mg of HONB to dissolve, and the powder was cooled to 0°C. Next, add 118 mg of DCC and stir at 0°C for 6 hours and at room temperature for 40 hours. The precipitate is filtered off, the solvent is distilled off, ether is added to the residue, the powder is collected by filtration, and further purified by reprecipitation from DMF and ether. Yield 700mg (95.5%), melting point 120-125℃ (decomposition), [α] ²³ _D -127.7゜ (c = 0.12, methanol),
Rf ⁵ 0.62, Rf ⁶ 0.44. Elemental analysis C ₁₂₁ H ₁₉₁ O ₃₄ N ₂₇・HCl・2H ₂ O Calculated value: C53.12; H7.80; N14.94; Cl1.40. Analytical values: C53.39; H7.62; N15.10; Cl1.54. (v) H-Ala-Pro-Pro-Pro-Ser-Leu
―Pro―Ser―Pro―Ser―Arg―Leu―Pro―
Gly―Pro―Ser―Asp―Thr―Pro―Ile―Leu
-Pro-Gln-OH [peptide ()]...hCGβ
(123-145) Production of C-terminal peptide moiety: Z-
Ala―Pro―Pro―Pro―Ser―Leu―Pro―Ser
―Pro―Ser―Arg―Leu―Pro―Gly―Pro―
Ser―Asp(OBu ^t )―Thr―Pro―Ile―Leu―
580 mg of Pro-Gln-OBu ^t is dissolved in 50 ml of methanol together with 0.45 ml of N-hydrochloric acid, and reduced in a hydrogen stream using palladium black as a catalyst. The catalyst was filtered off, the solvent was distilled off, 1 ml of water was added to the residue, the mixture was distilled off again, and the residue was dissolved in 10 ml of 90% trifluoroacetic acid water and left at room temperature for 60 minutes. The solvent was distilled off, 10 ml of water was added to the residue, it was dissolved, and the resin column of Amberlite IRA-140 (acetic acid type) (1
x 5 cm) and collect the eluate. resin to water 50
ml, and the washing solution is combined with the eluate and lyophilized to obtain 390 mg of white powder. Next, change this to N
- Apply to a Sephadex CH-20 column (2 x 83 cm) packed with acetic acid and develop with the same solvent.
A 70-100 ml section is collected and lyophilized, and further subjected to rechromatography on the same column, and a section containing the target product is collected and lyophilized to obtain a white powder. Yield 185 mg (35.2%), [α] ²³ _D −194.5° (c =
0.13, 0.1N-acetic acid), Rf ⁵ 0.06, Rf ⁴ (cellulose) 0.78. Amino acid analysis value (theoretical value) Arg1.00(1),
Asp1.00(1), Thr1.00(1), Ser3.74(4), Glu1.03
(1), Pro9.59(9), Gly0.98(1), Ala0.94(1),
Ile0.95(1), Leu2.97(3). Average recovery rate 79.3%. Reference example 2 H―Ser―Pro―Ser―Arg―Leu―Pro―Gly―
Pro―Ser―Asp―Thr―Pro―Ile―Leu―Pro
-Gln-OH [hereinafter referred to as peptide (). ]
Production of [hCGβ(130-145) C-terminal peptide moiety] Z-Gly-Pro-Ser-Asp (OBu ^t )-Thr-
Dissolve 2.16 g of Pro-Ile-Leu-Pro-Gln-OBu ^t in 100 ml of methanol, use palladium black as a catalyst,
Reduce in a hydrogen stream. The catalyst is filtered off, the solvent is distilled off and the residue is dissolved in 25 ml of DMF. Add Z―Ser―Pro―Ser―Arg―Leu―Pro― to this solution.
Z- obtained by treating 1.5g of OBu ^t with trifluoroacetic acid
Ser―Pro―Ser―Arg―Leu―Pro―OH
Add 1.22 g of HONB and cool to 0°C. next
Add 701 mg of DCC, stir at 0°C for 6 hours and at room temperature for 40 hours, filter off the precipitate, and distill off the solvent. Ethyl acetate and ether are added to the residue to form a precipitate, which is collected by filtration. This was dissolved in 5 ml of Rf ³ solvent, applied to a silica gel column (5.7 x 9 cm) packed with the same solvent, and developed with the same solvent. Collect portions of 534-914 ml, distill off the solvent, add ether to the residue, and filter. Yield 1.1g (33.9%), Rf ³ 0.20. Next, a portion of 70 mg of this protected peptide is dissolved in 10 ml of methanol and reduced in a hydrogen stream using palladium black as a catalyst, the catalyst is filtered off, and the solvent is distilled off. The residue was dissolved in 1 ml of 90% trifluoroacetic acid, the solvent was distilled off after 60 minutes, and 3 ml of water was added to the residue and dissolved. This aqueous solution is passed through a column (1 x 1 cm) of Amberlite IRA-410 (acetic acid type) and freeze-dried. Next, change this to N
-Dissolve in 0.5 ml of acetic acid, apply to a Sephadex LH-20 column packed with N-acetic acid, and develop with the same solvent. The sections containing the target product are collected and freeze-dried to obtain a white powder. Yield 22 mg (36.1%), [α] ²³ _D −159.3° (c=
0.15, 0.1N-acetic acid), Rf ⁵ 0.15. Amino acid analysis (theoretical value) Arg1.01(1), Asp1.01
(1), Thr0.98(1), Ser2.57(3), Glu0.96(1), Pro4.85
(5), Gly1.00(1), Ile0.96(1), Leu2.04(2). Average recovery rate was 81.1%. Reference example 3 H-Gly-Pro-Ser-Asp-Thr-Pro-Ile-
Leu-Pro-Gln-OH [hereinafter referred to as peptide ()
It is called. ] Production of [hCGβ (136-145) C-terminal peptide moiety] Z-Gly-Pro-Ser-Asp (OBu ^t )-Thr-
Dissolve 150 mg of Pro-Ile-Leu-Pro-Gln-OBu ^t in 2.5 ml of 90% trifluoroacetic acid and leave at room temperature for 60 minutes.
Next, the solvent is distilled off, and the residue is dissolved in 30 ml of 50% acetic acid and reduced in a hydrogen stream using palladium black as a catalyst. The catalyst was filtered off, the solvent was distilled off, and the residue was dissolved in water.
Dissolve in 10 ml and apply to Amberlite IRA-410 (acetic acid type) column (1 x 3 cm), then wash the resin thoroughly with water. Combine the eluate and wash solution and lyophilize. Cephadex filled with N-acetic acid
Apply to LH-20 column (2 x 83 cm) and elute with the same solvent. Aliquots of 90-105 ml are collected and lyophilized to obtain a white powder. Yield 70 mg (57.9%), [α] ¹⁹ _D −150.5° (c=0.
2,
0.1N-acetic acid), Rf ⁵ 0.29, Rf ⁴ (cellulose) 0.55. Amino acid analysis value: Asp1.02(1), Thr0.98(1),
Ser0.92(1), Glu1.04(1), Pro3.17(3), Gly0.99(1),
Ile0.99(1), Leu1.00(1). Average recovery rate 82.6%. Reference Example 4 Production of antibody Approximately 10,000 U/mg purified from human urine by a known method
Dissolve 1 mg of hCG in 1 ml of physiological saline and add Freund's complete adjuvant to this.
adjuvant, “Biochemistry of Immunity”, by Tachibana et al., Kyoritsu Shuppan Co., Ltd. (1967)), mix well to make an emulsion, and inject this into the muscles of both thighs and subcutaneously at several points on the back of rabbits. do. The above procedure is performed 5 times every 3 weeks, and one week after the final immunization, blood is collected and a pilot assay is performed. As a result, the C of hCG-β
An anti-peptide with strong affinity for end peptide-enzyme condensates.
Obtain hCG antibody. Example 1 Production of specific anti-hCG antibody 5 mg of the peptide () obtained in Reference Example 1 was
The solution was dissolved in 8 ml of 0.1 M NaHCO ₃ containing 0.5 M NaCl, added to 1 g of bromcyan-activated Sepharose 4B (manufactured by Pharmacia Huain Chemicals), which had been previously washed with N/1000 HCl, and stirred overnight at 5°C. After the reaction was completed, it was thoroughly washed with 0.1M NaHCO ₃ containing the same 0.5M NaCl, and then the pH was adjusted to 8 with HCl.
1 at room temperature by adding 10 ml of 0.5M ethanolamine.
After reacting for an hour, (1) 0.1M acetate buffer (PH4.0) containing 1M NaCl, (2) 0.1M borate buffer (PH8.0) containing 1M NaCl, and (3) 0.02M containing 0.15M NaCl.
It was washed sequentially with borate buffer (PH8.0) and packed into a column. 8 ml of the anti-hCG serum obtained in Reference Example 4 was subjected to salting-out precipitation using 1.5 g of anhydrous sodium sulfate, and the resulting γ-globulin fraction was added to the above peptide ().
It was applied to a coupled Sepharose 4B column (0.9 x 4 cm). 0.02M borate buffer (PH8.0) containing 0.15M NaCl
The column was washed with water to remove anti-hCG antibodies that cross-reacted with hLH, hFSH, and hTSH. then
By elution with 0.17M glycine-hydrochloric acid buffer (PH2.3), a specific anti-hCG antibody with strong affinity for hCG-β C-terminal peptide was obtained (protein amount 1.8
mg). The physical properties of this antibody will be described below. At a final dilution concentration of 80 ng/ml, this antibody has the ability to bind approximately 95% and 35% of the hCG-enzyme label and peptide()-enzyme label, respectively, with an enzymatic activity of approximately 2 μU. The optimal pH for antigen binding activity of this antibody is 6-9. This antibody is stable for over 1 year when stored refrigerated. It has a molecular weight of approximately 150,000 and contains approximately 3% sugar. Easily soluble in aqueous solvents with a pH of 2-12. In electrophoresis, γ- moves toward the cathode.
Belongs to the globulin fraction. See Figure 1 for the ultraviolet absorption spectrum. See Table 1 for amino acid analysis values. Other physical properties are those of immunoglobulin G (“Medical Immunology”, p. 61, Kikuchi et al., Nankodo (1976).
year)).

【table】

[Table] Number of moles of acid Example 2 Production of condensate of peptide () and β-D-galactosidase 2.3 mg of the peptide () obtained in Reference Example 1
0.5ml of 0.05M phosphate buffer (PH7.0) and DMSO
780 μg of m-MBHS was dissolved in 0.5 ml of this solution.
Add 50μ of THF containing 30% and react at 30°C for 30 minutes. Pour the reaction mixture into 0.02M phosphate buffer (PH6.3)
Sephadex G-15 column (0.9
x 55 cm) to separate excess reagent and maleimidized peptide. 0.5ml of the obtained maleimidized peptide solution was added to 0.02M phosphate saline buffer (PH7.5).
β-D-galactosidase solution diluted to (1
(mg/ml) gradually added to 0.5 ml and allowed to react overnight at 5°C with occasional shaking. After the reaction is complete, add 0.02M phosphate saline buffer (PH7.0)
The enzyme was purified by Sepharose 6B column chromatography, and the enzyme-containing fraction was collected to obtain a peptide-enzyme condensate. The physical properties of this condensate will be described below. In this condensate, the synthetic substrate O-nitrophenyl β-D-galactopyranoside used in EIA decomposes 4-methylumbelliferyl-β-D-galactopyranoside, producing O-nitrophenol and 4-methylumbeliferyl, respectively. Release Lifueron. Synthetic substrate 4-methylumbelliferyl-β-
When D-galactopyranoside is used, the Michaelis constant of this condensate shows the same value as the original β-D-galactosidase. The optimum pH for enzyme activity is 6.5 to 7.3. Approximately 90% or more of this condensate with enzyme activity has anti-inflammatory properties.
It has reactivity with hCG antibodies, and its immunoactivity and enzyme activity are stable for more than 4 months when stored under refrigeration. The molecular weight is approximately 550,000, and the peptide ()/enzyme molar ratio is approximately 7. It is easily soluble in aqueous solvents with a pH of 5 to 9. See Figure 2 for the ultraviolet absorption spectrum. See Table 2 for amino acid analysis values. Other physical properties are those of β-D-galactosidase (“The Enzymes”, Vol. 7 (1972), No. 617).
Page, Boyer, Academic Press, New York
-London).

[Table] Example 3 Production of condensate of peptide () and β-D-galactosidase Using 1.7 mg of peptide () obtained in Reference Example 2 in place of 2.3 mg of peptide () in Example 2, Peptide () was treated in the same manner as in Example 2.
A condensate of β-D-galactosidase and β-D-galactosidase was obtained. The physical properties of this condensate are the physical properties of the condensate obtained in Example 2, except for the following items ():
, , and ). The molecular weight is approximately 550,000, and the peptide()/enzyme molar ratio is approximately 9. Example 4 Production of condensate of peptide () and β-D-galactosidase Using 1.0 mg of peptide () obtained in Reference Example 3 in place of 2.3 mg of peptide () in Example 2, Example 2 A condensate of peptide () and β-D-galactosidase was obtained by treatment in the same manner as above. The physical properties of this condensate are the physical properties of the condensate obtained in Example 2, except for the following items ():
, , and ). The molecular weight is approximately 550,000, and the peptide()/enzyme molar ratio is approximately 11. Example 5 Production of a condensate of peptide (2) and alkaline phosphatase Add 1 ml of alkaline phosphatase solution (0.5 mg/ml) diluted in 0.1 M phosphate buffer (PH6.8) to the condensate obtained in Reference Example 1. Dissolve 1.2 mg of the peptide ().
Next, 0.1ml of 2% GLA solution was added and reacted for 60 minutes at room temperature, followed by 0.02M phosphate saline buffer (PH6.8).
Dialyze overnight at approximately 5°C. Furthermore, Sephadex G using the same phosphate saline buffer (PH6.8)
-100 column chromatography and fractionated the enzyme-containing fraction to obtain a peptide-enzyme condensate. The physical properties of this condensate will be described below. This condensate decomposes the synthetic substrates phenyl phosphate and p-nitrophenyl phosphate used in EIA, liberating phenol and p-nitrophenol, respectively. The optimum pH for enzyme activity is 9.3-10.2. Approximately 90% or more of this condensate with enzyme activity has anti-inflammatory properties.
It has reactivity with hCG antibodies, and its immunoactivity and enzyme activity are stable for more than 3 months when stored under refrigeration. The molecular weight is approximately 130,000, and the peptide()/enzyme molar ratio is approximately 13. Easily soluble in aqueous solvents with a pH of 6-11. See Figure 3 for the ultraviolet absorption spectrum. See Table 3 for amino acid analysis values. Other physical properties are those of alkaline phosphatase (“The Enzymes”, Vol. 4 (1971), No. 417).
Page, Boyer, Academic Press, New York
-London).

[Table] Number of moles Example 6 Production of condensate of peptide () and alkaline phosphatase Instead of 1.2 mg of peptide () in Example 5, 0.83 mg of peptide () obtained in Reference Example 2 was used. , and treated in the same manner as in Example 5 to obtain a condensate of peptide () and alkaline phosphatase. The physical properties of this condensate are the same as those of the condensate of Example 5 (,,,,, and) except for the following items (). The molecular weight is approximately 120,000, and the peptide()/enzyme molar ratio is approximately 11. Example 7 Production of condensate of peptide () and alkaline phosphatase In place of 1.2 mg of peptide () in Example 5, 0.49 mg of peptide () obtained in Reference Example 3 was used to produce the same as in Example 5. A condensate of peptide () and alkaline phosphatase was obtained by the same treatment. The physical properties of this condensate are the same as those of the condensate of Example 5 (,,,,,, and) except for the following items (). The molecular weight is approximately 120,000, and the peptide()/enzyme molar ratio is approximately 17. Reference Example 5 Production of condensate of hCG and β-D-galactosidase 1 mg of hCG (approximately 10,000 IU/mg) was dissolved in 1 ml of 0.05 M phosphate buffer (PH7.0), and 78 μg of m-
Add 50 μl of THF containing MBHS while stirring.30
Incubate for 30 minutes at °C. The reaction mixture is immediately passed through a Sephadex G-25 column (0.9 x 55 cm) to remove excess reagent. Elution is performed with 0.05M citrate buffer (PH5.5), and maleimidated hCG is fractionated. 2 ml of the maleimidated hCG solution is added dropwise to 0.5 ml of a β-D-galactosidase solution (1 mg/ml) diluted in 0.05 M phosphate saline buffer (PH 7.5), and the mixture is allowed to react at room temperature for 2 hours. After the reaction, the hCG-β-D-galactosidase condensate was adsorbed through a Concanavalin A-Sepharose 4B (Pharmacia Huain Chemicals Co., Ltd.) column (1 x 15 cm), and then 0.2Ma-methylmannoside was added.
Elute with 0.05M phosphate saline buffer (PH7.5) and pool the fractions showing hCG immunoreactivity as well as enzymatic activity. This active fraction was further purified by Sepharose 6B column chromatography using 0.02M phosphate saline buffer (PH7.0), and the enzyme-containing fraction was separated to obtain an hCG-enzyme condensate. Example 8 In a preliminary test, for each antiserum, 100μ of antiserum at various dilutions, 100μ of peptide-β-D-galactosidase condensate, assay buffer (0.5% human serum albumin, 0.5% ethylenediamine tetraacetate disodium)・
PH7.3 with _2H2O , 0.1% _NaN3 and 0.1M NaCl
Each peptide-β-D was
- Galactosidase condensate [having an appropriate predetermined enzymatic activity (approximately 20 μu/ml). ) is bound to the antiserum. next
2.5% anti-rabbit IgG antibody-conjugated cellulose suspension
Add 100μ and further react at 20°C for 4 hours.
This mixture was centrifuged and the precipitate was washed.
Enzyme substrate solution (0.1% bovine serum albumin, 0.1%
PH7.0 containing _NaN3 , 0.1M NaCl and _1mMgCl2
20 μg/ml of 4- in 0.02 M phosphate buffer
Suspend in 500μ of methylumbelliferyl-β-D-galactopyranoside solution and react overnight at room temperature. After the reaction is completed, the excitation wavelength is 365 nm, and the fluorescence wavelength is
The fluorescence intensity of 4-methylumbelliferon liberated as a result of enzymatic decomposition was measured at 450 nm. Next, in the case of quantifying hCG in the test solution, use the antiserum at a certain dilution determined as above and peptide-β.
-D-galactosidase condensate and each
100μ, test solution 50μ and Alcei buffer
250μ and then quantified as above. The results obtained are shown in Figures 4-5. Figure 4 shows hCG (indicated by 0) and
This is a standard curve for hLH (indicated by ×). Both systems were assayed using an hCG-enzyme label. From this result, in the system using a specific anti-hCG antibody, hCG
The detection sensitivity is almost the same as that of anti-hCG antibody system,
It can be seen that the cross-reactivity of hLH is less than 1/1000 of that of hCG. FIG. 5 is a standard curve of hLH in EIA using anti-hCG antibody. -Γ- indicates the results for the peptide ()-enzyme condensate, and -×- indicates the results for the hCG-enzyme condensate. This result shows that the cross-reaction of hLH in the system using the peptide()-enzyme condensate is extremely small compared to the system using the hCG-enzyme condensate. Example 9 As in Example 8, for each antiserum, antiserum concentrations at various dilutions (assay buffer 300μ,
Peptide-alkaline phosphatase condensate 100μ
A sample of each peptide-alkaline phosphatase condensate [having a predetermined appropriate enzymatic activity (approximately 4 mU/ml)] was prepared by keeping samples of 100 μl of antiserum and 100 μl of antiserum at 5° C. for two days and nights. ) is bound to the antiserum. Then anti-rabbit
IgG serum (commercially available antiserum diluted 10 times in advance)
50μ and 50μ of phosphate saline buffer (PH75) containing 2% normal rabbit serum are added and the resulting mixture is left overnight at 5°C. The precipitate generated by centrifugation was washed, and the enzyme substrate solution [1mMgCl ₂
2 mg/ml para-nitrophenyl phosphate dissolved in 0.05 M sodium carbonate buffer (PH 9.8)] and reacted overnight at 37°C.
After the reaction was completed, the absorption intensity of paranitrophenol liberated as a result of enzymatic decomposition was measured at 405 nm. Next, in the case of quantifying hCG in the test solution, 100μ each of the antiserum and the peptide-alkaline phosphatase condensate at a certain dilution determined as above.
was added to a mixture of 50μ of the test solution and 250μ of the assay buffer, and then quantified as described above. The results obtained are shown in FIG. Figure 7 is the same as Figure 5, using anti-hCG antibody.
This is a standard curve of hLH in EIA. -Γ- is the result of peptide ()-enzyme condensate, -×- is hCG-
The results for the enzyme condensates are shown separately. This result shows that the cross-reaction of hLH can be ignored in the system using the peptide()-enzyme condensate. Figure 6 shows standard curves of hCG (indicated by Γ) and hLH (indicated by be. In this assay system, hLH
The cross-reactivity of is almost negligible. Furthermore, it can be seen that the detection sensitivity of hCG is equivalent to the sensitivity obtained with the usual assay system shown in Fig. 4 (...Γ...). Reference Example 6 Using the hCG enzyme immunoassay kit described below, the hCG concentration in the urine or serum of normal subjects and pregnant women was measured by EIA according to the procedure described below. The results are shown in Table 4. Kit for enzyme immunoassay of hCG (1) A moiety that binds about 40% of the enzymatic activity of the peptide-β-D-galactosidase condensate added to the reaction system of the anti-human chorionic gonadotropin antibody obtained in Example 1, ( 2) A portion of the peptide ()-β-D-galactosidase condensate having an enzymatic activity of approximately 0.4 μU, (3) 0-100 IU of standard human chorionic gonadotropin, (4) Reagents of (1) to (3) above and 0.15M NaCl, 0.5% human serum albumin, used for diluting the test sample,
0.02M of PH7.3 containing 0.5% EDTA, 0.1% _NaN3
Phosphate buffer, (5) 2.5 (W/V) % suspension anti-rabbit IgG-conjugated cellulose, (6) 10 μg 4-methylumbelliferyl-β-D
- Galactoside, (7) Used for washing cellulose in (5) and dissolving enzyme substrates in (6). 0.1MNaCl, _1mMgCl2 , 0.1
% bovine serum albumin, PH7.0 containing 0.1% _NaN3
0.02M phosphate buffer, (8) 0.1M carbonate buffer at PH10.5. Procedure: To 100μ of reagent (1), add 250μ of reagent (4) and 50μ of standard human chorionic gonadotropin or test sample, then add 100μ of reagent (2), and react at 4°C. After reacting for 40 hours, add a 100μ suspension of reagent (5), stir thoroughly, and react at 20°C for an additional 4 hours. After the reaction is complete, wash the cellulose with reagent (7),
Next, add 500μ of reagent (6) to start the enzyme reaction. After reacting at 20°C for 16 hours, 3 ml of reagent (8) was added to stop the reaction. The fluorescence intensity in the reaction solution is measured to determine the human chorionic gonadotropin concentration in the test solution.

【table】

【table】 [Brief explanation of drawings]

Figure 1 shows the UV absorption spectrum of the specific anti-hCG antibody, Figure 2 shows the UV absorption spectrum of the peptide ()-β-D-galactosidase condensate, and Figure 3 shows the UV absorption spectrum of the peptide ()-alkaline phosphatase condensate. Figure 4 shows the standard curve of hCG and hLH, Figure 5 shows the standard curve of hLH, Figure 6 shows the standard curve of hCG and hLH, Figure 7 shows the standard curve of hLH, Represent each.

Claims (1)

[Claims] 1 General formula H-R-Pro-Ser-Asp-Thr-Pro-lle-
Leu-Pro-Gln-OH [wherein, R is Ala1-Pro2-Pro3-Pro4-Ser5-
Leu6-Pro7-Ser8-Pro9-Ser10-Arg11-Leu12-P
ro13―
It represents 1 to 14 partial peptide chains containing Gly at position 14 of the peptide represented by Gly14. A peptide-enzyme condensate obtained by condensing a peptide represented by ] with a labeled enzyme. 2 In an enzyme immunoassay for human chorionic gonadotropin using a peptide-enzyme condensate as a type of reagent, the peptide-enzyme condensate has the general formula H-R-Pro-Ser-Asp-Thr-Pro-lle-
Leu-Pro-Gln-OH [wherein, R is Ala1-Pro2-Pro3-Pro4-Ser5-
Leu6-Pro7-Ser8-Pro9-Ser10-Arg11-Leu12-P
ro13―
It represents 1 to 14 partial peptide chains containing Gly at position 14 of the peptide represented by Gly14. An enzyme immunoassay method for human chorionic gonadotropin, which is characterized by using a peptide-enzyme condensate obtained by condensing a peptide represented by the following formula with a labeled enzyme.