JPS6163692A - Novel 2'-5' oligoadenylic acid compound, preparation thereof and antigen for 125i-labeling composed of same used for 2'5' oligoadenylic acid measurement by radioimmunoassay - Google Patents

Abstract

NEW MATERIAL:Methyl ester of 5'-triphospho (adenylyl 2'-5')2 adenosine-beta- alanyltyrosine, expressed by formula I. USE:An antigen for labeling with <125>I, used to measure 2'-5' adenylic acid with RIA (radioimmunoassay). PREPARATION:5'-Monophospho (adenylyl 2'-5')2 adenosine expressed by formula II is oxidized with an oxidizing agent (example; periodic acid, etc.) and further reacted with methyl ester of beta-alanyltyrosine in DMF (dimethyl formamide) solvent in the presence of sodium hydroxide to obtain methyl ester of 5'- monophospho (adenylyl 2'-5')2 adenosine-beta-alanyltyrosine. The resultant compound is then reacted with N,N'-carbonyldiimidazole in a solvent such as DMF in the presence of triethylamine and tri-n-octylamine, and further reacted with pyrophosphoric acid. Labeling is carried out in 0.05M phosphoric acid buffer solution (7.5pH) using the <125>I by chloramine T method.

Description

Detailed Description of the Invention [Industrial Field of Application] The present invention relates to a novel 2'-5' oligoadenylate compound, a method for producing the same, and a method for producing a 2'-5' oligoadenylate compound using the compound using a radioimmunological assay method. This invention relates to (1) labeling antigen for measuring adenylic acid (hereinafter abbreviated as 2-5 people).

[Prior art]

The radioimmunoassay method (hereinafter referred to as RI), which utilizes the competitive reaction between an antigen labeled with a radioactive substance and an unlabeled antigen with an antibody specific to that antigen, has extremely high sensitivity and specificity. It is widely used as a method for measuring trace substances.

It is essential for people with RI to use radioactive substances, and general Kl2sI is used as this radioactive substance. 1
For antigens that are difficult to label with 25I, H, C,
Radioactive substances such as P are used as labels. However, RIA using substances labeled with 3g, 14c, 33P, etc. requires the use of a liquid scintillation counter, and the radioactive waste is accompanied by an organic solvent.
Since R1!4c has a long half-life of radioactivity, it is difficult to dispose of the radioactive material. On the other hand, the half-life of 4P's radioactivity is too short, so there are problems in its storage and handling.

In comparison, when labeling with It's easy.

Labeling an antigen with 1251 is relatively easy when the antigen has a reactive group capable of introducing an iodine atom, such as a peptide, but if the antigen does not have a reactive group capable of introducing an iodine atom, the labeling antigen Therefore, it is necessary to create a derivative in which a reactive group that can be iodinated is introduced into an antigen site that does not inhibit binding with an antibody.

2-5A is converted into double-stranded R by 2-5A synthase induced by the antiviral action of interferon in vivo.
It is a substance produced using human TP as a substrate in the presence of NA. Therefore, measuring the blood 2-5A concentration is expected to be useful in understanding the functions of the interferon/2-5 synthase system and in diagnosing infectious diseases, and is of interest.

By the way, as a 2-5 person RIA or radiobinding assay, Nature + 288.13 is a method that uses 32P to label the antigen.

189-192.1982: Th@Journal
of Biological Chemistry,
259, 3, 1727-1730.1984, etc.

[Problem that the invention seeks to solve]

However, these methods have low measurement sensitivity due to low specific radiation, and cannot be used as a method for measuring blood in 2 to 5 people, and no proposals have been made to date. Moreover, as mentioned above, since 32P is used, it is necessary to measure with a liquid scintillation capacitor, and the reality is that it is not suitable for application to clinical tests such as searching for diseases. Also, 2
- Since there is currently no known method for labeling K1 itself, and the synthesis of a 2-5 derivative for I labeling is difficult, it is difficult to use RI with ① as the labeled antigen.
A has not been developed.

Means for determining problem C] In view of the above circumstances, the present inventors have determined that
As a result of intensive research to find a -5Au conductor, it was found that the I
We have succeeded in synthesizing a new yt2-5 A compound that can be used as a labeling antigen, and have completed the present invention.

The 2-5 compound of this invention is 51-triphospho(
adenylyl 2'5') * athenosine-β-7 ranyltyrosine methylesterteal.

The novel 2-5 compound CI) according to the present invention has the structure described in claim 2, that is, (a) chemical ζ; the known compound 5'-monophospho(
Adenylyl 2'5') S adenosine is oxidized, for example, by subjecting periodine to an oxidation reaction, and then β-
React with alanyltyrosine methyl ester to form 5'-monophospho(adenylyl 2'5')*adenosine-β
- Step of producing alanyltyrosine methyl ester, (b) 5'-monophospho(adenylyl 2'-5') represented by the chemical structural formula (1) obtained in step (a)! Produced by dissolving adenosine-β-alanyltyrosine methyl ester in a solvent such as dimethylformamide, reacting with N,N'-carbonyldiimidazole in the presence of triethylamine-n-octylamine, and further reacting with hyrophosphoric acid. It is something that will be done.

Hereinafter, the specific method for producing the novel 2-5 compound of the present invention, the analysis results of the obtained compound, and the method for measuring the obtained compound in the blood of 2-5 persons using the radioimmunological assay method will be described. We will explain how to use it as an antigen for summer labeling.

Example 1゜Known compound represented by chemical structural formula (II) 5'-monophospho(adenylyl 2'5')t adenosine 45m
f.

0.3 ml of 304 mole water bath solution and 0.3 ml of water bath solution. IM 29 sodium uio, 4sy was mixed and stirred in a 0°C water bath for 25 minutes.

The reaction mixture was mixed with dimethylformamide:water (1:1) in a bath solution of 33 pt of 1M aqueous liquefied sodium in 4 ml of dimethylformamide:water (1:1), and 30 μmol of β-alanyltyrosine methyl ester hydrobromide was added to the solution. In addition, I.M.
The mixture was stirred for 15 minutes in a water bath at 0° C. while maintaining the water at 9% sodium hydroxide and H8.5.

Then 0.6 m of 0.5 M sodium cyanoborate
The mixture was stirred for 1 hour in a water bath at 0° C. while keeping the pH at 6.5 with 1M hydrochloric acid.

After confirming the formation of the reactant by high performance liquid chromatography (HPLC) using an RP C-5 column, this was
AE-Sephadex A-25 (HCos flll)
Column (30anX I 5g inner diameter), 0.25
Elution with a linear concentration gradient of ~0.5 M bicarbonate/triethylammonium (SOO/80 Q d ) buffer yielded a 5'-monophospho(adenylyl 2'-5') structure corresponding to the chemical structure (13). 39 pieces of triethylammonium salt of adenosine-β-alanyltyrosine methyl ester (yield: 75 pieces) were obtained.

Analysis value U: λmax 259 nm N 8.25.8.11.8. O2,7,98,7,
96,7,93 (9, IH, H-2 and H-8)? , 03.6.75 (d, J -8, 3Hz, 2H.

Tyrosine aromatic ink proto;/) 6.13 (d, J -3,7Hz, L H, C-1
'H) 515 (d, J -3,4Hz, I HlC
-1'H)5.47 (dd, J -7,9■Z,1
5Hz, 15, C-1'H) The chemical structural formula Cff1. Triethylammonicum salt 38 K, 24 μM of 5'-monophospho(adenylyl 2'5')*adenosine-β-arr-retiq-syn methyl ester corresponding to 1 was dissolved with 0.5 g J IC of pyridine, and water was removed under reduced pressure. Distill off azeotropically. This operation was repeated three times, and then 0.5 dl of dimethylformamide was dissolved in C4, distilled off under reduced pressure, and then dissolved in 211Ij of dimethylformamide. Add 5σμto triethylamine and 3
After adding 0 μt of tri-n-octylamine, N,N
'-Carbonyldiimidazole 70 kg, 0.4 mmol
and stirred at room temperature for 2 hours.

Next, add 50 pt of dry methanol and boil again at room temperature for 30 pt.
Stir for a minute. A solution of 0.4 mmole of triptylammonicum pyrophosphate dissolved in 0.8 mm dry dimethylformamide was added to the mixture, and the mixture was stirred for one day and night at room temperature. The reaction solution was gradually added dropwise to a mixed solution of 5 d of ethanol, 5 d of acetone, and 0.5 d of acetone saturated with sodium perchlorate to form a white precipitate.

This white precipitate was washed with acetone and ether and then dried under vacuum. This dried product was dissolved in 25 dK of water and QAE-
Sephadex A 25 (HCOs type) column (30
crnX 15m inner diameter), 0.33 to 0.67
A linear concentration gradient of bicarbonate/triethylammonium (
500d1500d), chemical structural formula (1)
A white product corresponding to K 5'-)IJ phospho(adenylyl 2'5')*adenosine-β-alanyltyrosine methyl ester 13.8 µ (115130 qb>) was obtained.

Analysis value Thin layer coomadography (Rf value) (1) 0.49; Stationary phase Cellulose F, developing solvent/n-globanol: a Aqueous ammonia: Water (55:10:35) (1) 0.35 ; Stationary phase: Cellulose F, developing solvent: saturated ammonium sulfate solution: 0.1M sodium acetate:isopropanol (79:19:2) (Ill) 0.11
: Stationary phase/PEI-Cellulose F, developing solvent/0.25
M ammonium bicarbonate solution Example 2゜(1) Preparation of 2-5A antiserum 5-triphospho(adenylyl 2/5/) 1 athenosine 31 μmole, 15 μmole was dissolved in 0.4 d of water.
0.1 M periodic acid with stirring in a water bath at 0 °C.
4 d was gradually added and stirred for 30 minutes. Then 0.2
M+7) Carbonate buffer (PH9,2) Bovine serum albumin (hereinafter abbreviated as BSA) 20”ltN in OO,4d
The dissolved solution was added and stirred again in a 0°C water bath for 1 hour. Thereafter, 0.5M shea/hydrogen borohydride 065 d was added, and LM glue 7 tons buffer (pqs, s
) was added and stirred for 3θ minutes in a water bath while maintaining the temperature at H6.5, and then stirred overnight at 4°C.The reaction product was applied to a Sephadex G50 column (30α x 22 sea bream inner diameter), and the reaction product was applied to a Sephadex G50 column (30α x 22 sea bream inner diameter). 01M c'r!I phosphate buffer (pH 7,5)
The eluted portion of the protein was collected to obtain a solution of 1 protein/lIt.

This was used to immunize rabbits in a conventional manner to obtain a 2-5 anti-rabbit serum stock solution. This antiserum stock solution was mixed with 0.25% B3A and 1%
0.05M containing normal rabbit serum! J acid buffer (p) 1
8.0) to prepare a 2-5A antiserum solution.

(2) 125I mark Rs'-)liphospho(adenylyl 2'
5') Preparation of ta+'/n-β-alanyltyrosine methyl ester Example 1. 5'-)! 7 Phospho(adenylyl 2'5')*Adenosine-β-alanylthiamine methyl ester solution at 0.05 M! J acid buffer (P
H7,5) to 507'f/d, use this 10#t to label 1251 using the chloramine T method in a conventional manner, and then apply it to a Sephatics GIQ column to 1.200 f/d.
A lL4-specific compound containing a specific activity of C1/mmot was obtained.

0.05M phosphorus-grade buffer containing 0.25-BSA (pl
30.000 cpm/0.2nlVC at t8.0)
It was diluted and given the lul label m 2-5 A 74 branch.

Example 3 2-5 All in dish assay method Human blood a2mK ethylenediamine 46ffi disodium 2F? 9 was added to 1, and the plasma sample 100/2 obtained by immediately refrigerating and centrifuging at Vc4℃ was tested M'! 7 K) M, 100 μt of the I-labeled 2-5A solution obtained in Example 2.0(2) and Example 2. 200 µt of the 2-5A antiserum obtained in (1) was added to the mixture, and the mixture was incubated at Koon.

The resulting reaction solution was added with 0.0% anti-rabbit α-guprin goat serum.
After adding 1 d and leaving it for one night, the mixture was centrifuged at 3,000 rpm, the supernatant was removed by suction, and the amount of radioactivity in the sediment was measured.

At the same time, normal human plasma was treated with charcoal, and disodium ethylenediaminetetraacetate was added to the resulting 2-5A free plasma at a rate of 4 Mf/d.
0,0625,0,25,1,4 and 16n? /
100 μt each of 2-5 people's standard blood springs (standard solution) added to the test tube so that the temperature of Obtained. Thereby, the degree of 2-5 Ad in the plasma sample was determined.

Example 4 Measurement of 2-5A in the blood of test patients with various diseases The results shown in Example 3 were carried out for 13 cases of patient blood with various diseases shown in the table below and 15 cases of normal human blood as a control. The 2-5AQ degree of each was determined by the method. The results are shown in Figure 2.

〔Effect of the invention〕

Above, an example in which the novel compound of the present invention is used as a 1251-labeled antigen for measurement in 2 to 5 people by RIA has been described, and thereby the present invention enables measurement of blood concentration in 2 to 5 people, It has been proven to be useful and suitable for application to clinical tests such as searching for diseases.

[Brief explanation of drawings]

FIG. 1 is a graph showing the standard curve of blood 2-5A measurement in Example 3, where the vertical axis is the Vc125I-labeled antigen binding rate (
%), and the 2-5 concentration (nf/ml) is shown on the horizontal axis. FIG. 2 shows the results of measuring the 2-5A concentration using the blood of patients with various diseases and the blood of a normal person as a control. Hiroaki Sawai, normal person, chickenpox Figure 2, s.・ ・ Subject disease name

Claims (3)

[Claims] (1) Chemical structural formula ▲ Numerical formulas, chemical formulas, tables, etc. ▼... 5'-triphospho(adenylyl 2'-5'
)_2 Adenosine-β-alanyltyrosine methyl ester (2) (a) Chemical structural formula ▲ Numerical formulas, chemical formulas, tables, etc. are available ▼... [II] 5'-monophospho (adenylyl 2'-5'
)_2 adenosine is oxidized with an oxidizing agent and then reacted with β-alanyltyrosine methyl ester in dimethylformamide solution in the presence of sodium hydroxide to form 5'-mono(adenylyl 2'-5')_2 adenosine-β -Process of producing alanyltyrosine methyl ester, (b)Chemical structural formula ▲ Numerical formulas, chemical formulas, tables, etc. ▼...[III] 5'-monophospho(adenylyl 2'-5')
)_2 Adenosine-β-alanyltyrosine methyl ester was dissolved in a solvent, and triethanolamine and tri-n-
5'-Triphosphor expressed by the chemical structural formula ▲ Numerical formulas, chemical formulas, tables, etc. are available ▼ which is characterized by reacting with N,N'-carbonyldiimidazole in the presence of octylamine and further reacting with pyrophosphoric acid. (Adenylyl 2'-5'
)_2 Method for producing adenosine-β-alanyltyrosine methyl ester (3) Chemical structural formula ▲ Numerical formulas, chemical formulas, tables, etc. are available ▼ 5'-triphospho(adenylyl 2'-5'
)_2 ^1^2^5 for measuring 2'-5' oligoadenylic acid by a radioimmunochemical measurement method characterized by consisting of adenosine-β-alanyltyrosine methyl ester
Antigen for I labeling