JPH0631309B2 - Novel 2'-5 'oligoadenylic acid compound and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel 2′-5 ′ oligoadenylic acid compound, a method for producing the same, and 2′-5 ′ oligoadenylic acid (hereinafter referred to as 2-5A) of the compound. Abbreviated) for use as a labeling compound with a radioactive substance ( ¹²⁵ I) in a radioimmunochemical assay method.

[Prior art]

The radioimmunoassay method (hereinafter abbreviated as RIA) utilizing the fact that an antigen labeled with a radioactive substance and an unlabeled antigen in a biological sample compete with and react with a specific antibody against the antigen is sensitive and specific. Is widely used as an excellent method for measuring trace substances. RIA requires the use of radioactive materials, and as a radioactive material for this purpose,
¹²⁵ I is used. ^For antigens that are difficult to label with ¹²⁵ I, radioactive substances such as ³ H, ¹⁴ C and ³² P are used for labeling. However, RIA using labeled ³ H, ¹⁴ C, ³² P, etc. must use a liquid scintillation counter, and the radioactive waste is accompanied by an organic solvent, and ³ H, ¹⁴ C The long half-life makes disposal of radioactive materials difficult. On the other hand, ³² P has a short radioactivity half-life, so there is a problem in its storage and handling.

Compared with this, when labeling with ¹²⁵ I, it is possible to measure with a well-type scintillation counter, so it is not necessary to use an organic solvent as a scintillator, and since the half-life of radioactivity is relatively short, treatment of radioactive waste is performed. Is easy. Labeling an antigen with ¹²⁵ I is relatively easy when it has a reactive group capable of introducing an iodine atom such as peptide, but when the antigen does not have a reactive group capable of introducing an iodine atom, a labeling antigen is used. As a result, it is necessary to prepare a derivative in which a reactive group capable of being iodinated is introduced into an antigenic site that does not inhibit binding to an antibody.

2-5A is a double-strand R by a 2-5A synthase that is induced in vivo by the antiviral action of interferon.
It is a substance produced using ATP as a substrate in the presence of NA. Therefore, measuring the concentration of 2-5A in blood is expected to be useful in knowing the function of the interferon-2-5A synthase system and also in the diagnosis of infectious diseases, and is of interest.

Meanwhile, as the RIA or radio Vine Day ring mediation Say of 2-5A, a method using a ³² P labeling of the antigen, Nature, 283, 13,189~192,1982; The Journal of
Biological Chemistry, 259 , 3, 1727 to 1730, 1984.

[Problems to be solved by the invention]

However, these methods have a low specific emission and thus have a low measurement sensitivity, cannot be used as a method for measuring 2-5A in blood, and have not been proposed at all. Moreover, as described above, since ³² _P is used, it is necessary to measure with a liquid scintillation counter, which is not suitable for application to clinical tests such as disease search. Also, 2
Methods for labeling of ¹²⁵ I into -5A itself is not known at present, yet because it is difficult to synthesize ¹²⁵ 2-5A derivatives for I-labeled, the RIA using ¹²⁵ I for labeling developed Not not.

[Means for solving problems]

In view of the above situation, the present inventors have made possible the labeling of ¹²⁵ I with 2-
As a result of intensive research to find a 5A derivative, the present invention has been completed by succeeding in synthesizing a novel 2-5A compound that can be used for labeling a radioactive substance ( ¹²⁵ I) in a radioimmunochemical assay method. .

The 2-5A compound of the present invention is 5'-triphospho (adenylyl 2'-5 ') ₂ adenosine-β-alanyl tyrosine methyl represented by the chemical structural formula [I] described in claim 1. It is an ester.

The novel 2-5A compound [I] according to the present invention has the constitution described in claim 2, namely, (a) a known compound 5'- represented by the chemical structural formula [II].
Monophospho (adenylyl 2'-5 ') ₂ adenosine is oxidized, for example by subjecting it to a periodate oxidation reaction, and then reacted with β-alanyl tyrosine methyl ester in dimethylformamide solution in the presence of sodium hydroxide,
5'-monophospho (adenylyl 2'-5 ') ₂ adenosine-β-alanyl tyrosine methyl ester step, (b) 5'-represented by the chemical structural formula [III] obtained in step (a) Monophospho (adenylyl 2′-5 ′) ₂ adenosine-β-alanyl tyrosine methyl ester was dissolved in a solvent such as dimethylformamide, and N, N′-carbonyldiimidazole was added in the presence of triethylamine and tri-n-octylamine. It is produced by reacting and then reacting with pyrophosphoric acid.

Hereinafter, the specific method for producing the novel 2-5A compound of the present invention, the analysis result of the obtained compound, and ¹²⁵ I when the obtained compound is assayed for 2-5A in blood by a radioimmunoassay method The usage method and the like used as the 2-5A compound for labeling will be described.

Example 1. Known compound represented by the chemical structural formula [II] 5'-monophospho (adenylyl 2'-5 ') ₂ adenosine 45 mg, 30
0.3 ml of an aqueous solution of μmole was mixed with 0.45 ml of a 0.1 M sodium periodate solution, and the mixture was stirred in an ice bath at 0 ° C. for 25 minutes.

The reaction mixture was added to a solution of 0.4 ml of a dimethylformamide: water (1: 1) mixture containing 33 μ of 1M sodium hydroxide, and β-alanyltyrosine methyl ester hydrobromide 3 was added.
0 μmole was added to the dissolved solution, and the mixture was stirred in a water bath at 0 ° C. for 15 minutes while maintaining the pH at 8.5 with 1M sodium hydroxide.

Next, 0.6 ml of 0.5 M sodium cyanohydroxyborate was added, and the mixture was stirred in a water bath at 0 ° C. for 1 hour while maintaining the pH at 6.5 with 1 M hydrochloric acid.

After confirming the production of the reaction product by high performance liquid chromatography (HPLC) using an RPC-5 column, this was analyzed by QA.
E-Sephadex A-25 (HC O ^- ₃ type) column (30 cm x 15 cm inner diameter) was attached, and a linear concentration gradient of 0.25 to 0.5 M bicarbonate / triethylammonium (800 ml /
(800 ml) was eluted with a buffer solution and the chemical structural formula [II
I '] corresponding to 5'-monophospho (adenylyl 2'-
5 ') 39 mg of triethylammonium salt of ₂ adenosine-β-alanyl tyrosine methyl ester (yield 75%)
Got

Analytical value UV: λmax 259 nm NMR: 8.25, 8.11, 8.02, 7.98, 7.96, 7.93 (s, 1H, H-2 and H-8) 7.03, 6.75 (d, J = 8.3Hz, 2H, tyrosine aromatetic proton) 6.13 (d, J = 3.7Hz, 1H, C-1'H) 5.95 (d, J = 3.4Hz, 1H, C-1'H) 5.47 (dd, J = 7.9Hz, 15Hz, 1H, C-1 ′ H) Triethylammonium salt of 5′-monophospho (adenylyl 2′-5 ′) ₂ adenosine-β-alanyl tyrosine methyl ester corresponding to the above-mentioned chemical formula [III] 38
mg and 24 μM are dissolved in 0.5 ml of pyridine, and water is distilled off azeotropically under reduced pressure. This operation was repeated 3 times, then dissolved in 0.5 ml of dimethylformamide, evaporated under reduced pressure, and then dissolved in 2 ml of dimethylformamide. 50μ in this solution
Of triethylamine and 30 μ of tri-n-octylamine were added, 70 mg of N, N′-carbonyldiimidazole and 0.4 mmole were added, and the mixture was stirred at room temperature for 2 hours. Then, 50 μm of dry methanol was added, and the mixture was stirred again at room temperature for 30 minutes. To this, tributylammonium pyrophosphate salt 0.
A solution prepared by dissolving 4 mmole in 0.8 ml of dry dimethylformamide was added, and the mixture was stirred at room temperature for one day. The reaction solution was gradually added dropwise to a mixed solution of 5 ml of ethanol, 5 ml of acetone and 0.5 ml of saturated sodium perchlorate acetone to form a white precipitate.

The white precipitate was washed with acetone and ether and then vacuum dried. The dried product was dissolved in water 25 ml, QAE-Sephadex ^{A-25 (HC ▲ O -} 3 ▼ type) column (30
cm × 15 mm inner diameter) and eluted with a linear concentration gradient of 0.33 to 0.67 M bicarbonate / triethylammonium (500 ml / 500 ml) to give a white product 5'-triphosphonate corresponding to the chemical structural formula [I]. 13.8 mg (yield 30%) of (adenylyl 2'-5 ') ₂ adenosine-β-alanyl tyrosine methyl ester was obtained.

Analytical value Thin layer chromatography (Rf value) (i) 0.49; stationary phase cellulose F, developing solvent / n-propanol: concentrated aqueous ammonia: water (55:10:35) (ii) 0.35; stationary phase cellulose F, Developing solvent / saturated ammonium sulfate solution: 0.1M sodium acetate: isopropanol (79:
19: 2) (iii) 0.11; stationary phase / PEI-cellulose F, developing solvent / 0.25M ammonium bicarbonate solution Example 2. (1) Preparation of 2-5A antiserum 5′-triphospho (adenylyl 2′-5 ′) ₃ , adenosine 31 mg, 15 μmole were dissolved in 0.4 ml of water, and 0.1 M periodate was added while stirring in an ice bath at 0 ° C. 0.4 ml of sodium acid salt was gradually added and stirred for 30 minutes. Next, bovine serum albumin (hereinafter referred to as B) was added to 0.4 ml of 0.2 M carbonate buffer (pH 9.2).
A solution in which 20 mg of SA was abbreviated was added, and the mixture was again stirred in an ice bath at 0 ° C. for 1 hour. After that, 0.55 sodium cyanoborohydride (0.65 ml) was added, and 1M phosphate buffer (pH 5.5) was added to maintain the pH at 6.5.
After stirring for 1 minute, the mixture was stirred overnight at 4 ° C. The reaction product was applied to a Sephadex G50 column (30 mm x 22 cm inner diameter) and eluted with 0.01 M phosphate buffer (pH 7.5), and the protein elution portion was collected to obtain a 1 mg / ml solution, which was used as an immunogen. did.

This was immunized into a rabbit according to a conventional method to obtain a 2-5A anti-rabbit serum stock solution. This antiserum stock solution was diluted with 0.05M phosphate buffer (pH 8.0) containing 0.25% BSA and 1% normal rabbit serum to 10,000
It was diluted twice to obtain a 2-5A antiserum solution.

(2) ¹²⁵ I-labeled 5'-triphospho (adenylyl 2'-
5 ') Preparation of ₂ adenosine-β-alanyl tyrosine methyl ester Example 1. 50 μg of the 5'-triphospho (adenylyl 2'-5 ') ₂ adenosine-β-alanyl tyrosine methyl ester (new 2-5A compound (I)) obtained in step 5 in 0.05M phosphate buffer (pH 7.5) Dissolve in 10 ml
Was labeled with ¹²⁵ I by the chloramine T method by a conventional method, and then purified with a Sephadex G10 column to obtain 1,200 Ci / m 2.
A ¹²⁵ I-labeled compound with a specific activity of mol was obtained.

With 0.05M phosphate buffer (pH8.0) containing 0.25% BSA 30,
It was diluted to 000 cpm / 0.2 ml and used as a ¹²⁵ I-labeled 2-5A solution ¹²⁵ I-labeled antigen.

Example. 3 2-5A measurement method in blood 2 ml of human blood is disodium ethylenediamine tetraacetate 2
100 μm of a plasma sample obtained by adding mg and mixing and immediately cooling and centrifuging at 4 ° C. was added to a test tube, and Example 2. 100 μ of the ¹²⁵ I-labeled 2-5A solution obtained in (2) of Example 2 and Example 2. of
200 µ of the 2-5A antiserum obtained in (1) was added, and the mixture was reacted at room temperature for a day.

Anti-rabbit α-globulin goat serum 0.1m was added to the obtained reaction solution.
l was added and left overnight, then centrifuged at 3,000 rpm to remove the supernatant by suction, and the amount of radioactivity in the precipitated portion was measured.

At the same time, human normal plasma was subjected to charcoal treatment, and disodium ethylenediaminetetraacetate was added to the obtained 2-5A free plasma at a rate of 4 mg / ml.
100 μl of 2-5A standard plasma (standard solution), which was added to each of the concentrations of 5,0.25, 1,4 and 16 ng / ml, was added to the test tube, and the measurement was conducted in the same manner as above. The standard curve shown in Figure 1 was obtained. Thus, the concentration of 2-5A in the plasma sample was determined.

Example 4. Measurement of 2-5A in blood of test patients having various diseases, 13 cases of patient blood having various diseases shown in the table below, and 15 cases of normal human blood as a control were carried out by the method shown in Example 3, respectively. 2-5A concentration was measured. The results are shown in Fig. 2.

[Effects of the Invention] An example of using the novel compound of the present invention as a ¹²⁵ I-labeling compound for 2-5A measurement by RIA has been described above.
This proves that the present invention makes it possible to measure the concentration of 2-5A in blood, and is useful for application to clinical tests such as disease search.

[Brief description of drawings]

FIG. 1 shows the third embodiment. Is a graph showing a standard curve of 2-5A measurement in blood, wherein the vertical axis represents ¹²⁵ I-labeled antigen binding rate (%),
The horizontal axis shows the 2-5A concentration (ng / ml). FIG. 2 shows the results of measuring the 2-5A concentrations of blood of patients with various diseases and normal human blood as a control.

Claims (2)

[Claims] 1. A chemical structural formula 5'-triphospho (adenylyl 2'-
5 ') ₂ adenosine-β-alanyl tyrosine methyl ester 2. (a) Chemical structural formula 5'-monophospho (adenylyl 2'-
5 ') ₂ adenosine is oxidized with an oxidant and then β-in dimethylformamide solution in the presence of sodium hydroxide.
Reacted with alanyl tyrosine methyl ester, 5'-monophospho (adenylyl 2'-5 ') ₂ adenosine-β-
Alanyl tyrosine methyl ester process, (b) Chemical structural formula 5'-monophospho (adenylyl 2'-
5 ') dissolving ₂ adenosine-β-alanyl tyrosine methyl ester in a solvent, reacting with N, N'-carbonyldiimidazole in the presence of triethylamine and tri-n-octylamine, and further reacting with pyrophosphoric acid Chemical formula characterized by 5'-triphospho (adenylyl 2'-
5 ') Method for producing ₂ adenosine-β-alanyl tyrosine methyl ester (I)