JPH01294674A - Pseudouridine derivative - Google Patents

Abstract

NEW MATERIAL:The pseudouridine derivative of formula I [either one of R<1>, R<2> and R<3> is R-CO-A-CO- and the others are H; A is alkylene; R is hydroxyl or (4-hydroxyphenyl) lower alkylamino which may have (lower alkyl-protected) carboxyl group bonded to the lower alkyl group]. EXAMPLE:5'-Mono-succinyl-pseudouridine. USE:Useful as an immunogen, especially its hapten for the preparation of a specific antibody useful for the immunoassay of pseudouridine. The determination of cancer can be effectively performed by the use of the present compound. PREPARATION:The objective compound of formula I can be produced by using the pseudouridine of formula II as a starting raw material and reacting with a compound of formula III in an inert solvent (e.g., benzene) in the presence of a basic compound (e.g., sodium carbonate) preferably at 20-25 deg.C.

Description

[Detailed description of the invention] 5 Emperor's League The present invention relates to novel pseudouridine derivatives.

Early detection is essential for the treatment of cancer and cancer, and research and development of various gamma markers have been carried out for this purpose, and even now, research and development of various cancer markers, including the development of the above-mentioned markers, is still in progress. Various attempts have been made to develop techniques for early detection.

Generally, when a living body becomes cancerous, the metabolism of t-RNA becomes active, and its consumption in the living body becomes relatively faster than that of DNA, resulting in an increase in the amount of t-RNA produced in the living body. Moreover, this t-RNA is a dogma of life science.
As is clear from the NA-mRNA/tRNA-protein (enzyme) process, various general tumor markers that have been proposed in the past, such as AFP (alphafetoprotein, Proc, 5outh Afr, As5oc)
, Pathol, , E.

67 (1969): Bull, Soc, Chi
m, Biol,, 49.

1389-1398 (1967)) is a protein, it is produced faster in the body than these. Therefore, the t-RNA has a very high potential as a marker that enables early detection of cancer compared to currently known general tumor markers.

However, in practice, measuring t-RNA in tissues is extremely difficult and almost impossible. On the other hand, the above t-RN
Pseudouridine (Pse
udouridine) is not metabolized in the metabolic pathway of the living body and is excreted in the urine as it is.

If a simple method that can accurately measure pseudouridine in urine could be established, it would indirectly measure t-RNA.
The establishment of a new measurement system using pseudouridine as a tumor marker can be expected to contribute to the early detection of cancer as a substitute for t-RNA measurement.

On the other hand, it has been confirmed that pseudouridine exists in the blood and urine, and there are also reports that the levels in cancer patients are about an order of magnitude higher than the amounts in the blood and urine of healthy people ( Cancer.

2457-2464 (19B2): Cancer,
5 Yu.

1571-1575 (198B>).

However, conventional immunoassay methods that generally use antibodies (
immunoassay) is well known, and although attempts have been made to measure such pseudouridine using this immunoassay, the antibodies themselves that can be used in this measuring method are still unknown. Moreover, even if you try to create antibodies by using the amino group of the base part of pseudouridine according to the usual antibody production method or by using the aldehyde group generated by periodine decomposition of the sugar chain part, these methods will not be able to produce pseudouridine. The basic skeleton properties of the antibody are destroyed, and the resulting antibody itself loses its specificity, making it difficult to perform accurate measurements using such antibodies. Currently, a method using high-performance liquid chromatography is known as a method for measuring pseudouridine, but this method is not only simple but also accurate, making it an effective method for early diagnosis of basolateral cancer. I can't find any practicality as such.

In view of the above-mentioned current situation, the present inventors have carried out extensive research with the ultimate goal of establishing a diagnostic technique or measurement system for early detection of cancer using pseudouridine as a marker. In the process, the above-mentioned pseudouridine immunoassay (immunoassay) was performed.
ay) antibodies against said pseudouridine that can be used for
We succeeded in synthesizing an immunogen for producing the antibody and a new derivative of the immunogen as a hapten, thereby completing the present invention.

That is, the present invention relates to the general formula [wherein R1, R2 and R3 are any of the groups R-Co
-A-Go-, and the others represent hydrogen atoms. In addition, in the above group, A represents a lower alkylene group, and R represents a hydroxyl group, or (4-hydroxyphenyl) lower Indicates an alkylamino group. ] This relates to a pseudouridine derivative represented by the following.

Hereinafter, R1 in general formula (1) will not represent the derivative of the present invention.
represents the group R-Co-A-Co-, "2'-derivative", R2 is the above-mentioned substituent is called "3'-derivative", and R3 is the above-mentioned substituent is called "5-derivative". '-derivative'
Distinguish. In addition, among the substituents of each of the above derivatives, the lower alkylene group defined by A includes, for example, methylene, ethylene, trimethylene, 2-methyltrimethylene, 2,2-dimethyltrimethylene, 1-methyltrimethylene, methylmethylene. , ethylmethylene, tetramethylene, pentamethylene, hexamethylene groups, etc. with 1 or more carbon atoms
6 straight or branched alkylene groups are included. The lower alkyl group in the lower alkylamino group (4-hydroxyphenyl) with or without a carboxyl group that may be protected by a lower alkyl group in the lower alkyl moiety defined by R includes, for example, methyl, ethyl, propyl. , isopropyl, butyl, isobutyl, tert-
Butyl groups and the like are included. As a specific example of the (4-hydroxyphenyl) lower alkylamino group that does not have a substituent on the alkyl moiety defined by R above, tyramine residue (
Tyramyl group) [HO-Ce Ha -CH2CH2NH
], and as a specific example of a (4-hydroxyphenyl) lower alkylamino group having a carboxyl group in the lower alkyl moiety, tyrosine residue (tyrosyl group) OOH [HO-C6H4-CH2CH-NH-], further lower Specific examples of lower alkylamino groups (4-hydroxyphenyl) having a carboxyl group protected by a lower alkyl group in the alkyl moiety include the above tyrosine residues protected by a lower alkyl group [tyrosine (lower alkyl ester) residues] ], for example, methyltyrosyl, ethyltyrosyl, etc., respectively.

The derivatives of the present invention are useful as immunogens, particularly haptens, for producing specific antibodies useful in pseudouridine immunoassays. That is, the derivative of the present invention can be used to easily produce a specific antibody against pseudouridine according to a general method from an immunizing antigen obtained by combining the derivative as a hapten with a common carrier protein, and the antibody can be used for immunoassay of pseudouridine by using the derivative. , which in turn is useful for cancer measurements.

In particular, the derivatives of the present invention can be used at the 2'-position, 3'-position and 5'-position of the sugar chain.
' is characterized by having a substituent represented by a dicarboxylic acid or a specific functional group derived therefrom, particularly a succinyl group, and based on this, as seen in conventional antibody production methods, , the specificity as an antigen can be maintained without destroying the basic skeleton of pseudouridine, and the desired specific antibody can be produced. In addition, the carboxyl group or phenolic hydroxyl group in the substituent group of the above-mentioned derivatives of the present invention generally has high reactivity with carrier proteins, and their bonding is easy, and it is said that induction into a labeled form of 125I is simple. It also has characteristics. In particular, in the above formula (1), R is a lower alkylamino group (4-hydroxyphenyl) with or without a carboxyl group that may be protected by a lower alkyl group in the lower alkyl moiety, especially a tyramine residue, tyrosine The derivatives of the present invention, which are residues (tyrosyl group) and tyrosine (lower alkyl ester) residues, are suitable for labeling with labeling compounds such as the above-mentioned 1251, as described below, and derivatives labeled with such labeling agents. is useful as a labeled antigen in pseudouridine immunoassays.

The method for producing the derivative of the present invention will be described in detail below.

Among the wood derivatives, a compound having a substituent in which R is a hydroxyl group can be prepared by using, for example, pseudouridine represented by the formula as a starting material, in the presence of a basic compound in an inert solvent suitable for this, the compound having the general formula [In the formula, A is the same as above] It can be produced by reacting a dicarboxylic acid anhydride represented by the following.

In the above, the inert solvent is a usual one, such as benzene, chloroform, methylene chloride, carbon tetrachloride,
Dioxane, tetrahydrofuran, pyridine, etc. can be used. Common basic compounds such as sodium carbonate, potassium carbonate, pyridine, and triethylamine can be used.

As the dicarboxylic anhydride in the above reaction, for example, succinic anhydride, maleic anhydride, etc. can be used. The amount of the dicarboxylic anhydride to be used relative to pseudouridine is:
Usually at least equimolar amount, preferably equimolar to about 3
It is preferable to select from a range of about twice the molar amount. The reaction temperature is generally about 4°C to 37°C, preferably about 20 to 25°C.
℃, and the reaction is completed in about 10 to 24 hours.

Further, among the derivatives of the present invention, compounds in which R has a substituent in the lower alkyl moiety which is a (4-hydroxyphenyl) lower alkyl group with or without a capoxyl group that may be protected with a lower alkyl group include, for example, the above-mentioned compounds. It can be produced by reacting the derivative of the present invention, in which R is a hydroxyl group, with an appropriate amine providing the above-mentioned specific R group, typically tyramine, tyrosine, tyrosine lower alkyl ester, etc. .

This reaction can be carried out according to a conventional amide bond forming reaction. More specifically, the above reaction is carried out in a suitable solvent such as dioxane, dimethylformamide, tetrahydrofuran, methyl ethyl ketone, etc., more preferably dioxane, using a reagent used in a conventional peptide bond forming reaction, such as N,N-dicyclohexylcarbodiimide. (
DCC), N-ethyl-N'-dimethylaminocarbodiimide, 1-ethyl-3-diisopropylaminocarbodiimide, 3-(2-morpholinyl-4-ethyl)carbodiimide, etc., more preferably carried out using a dehydration condensation agent such as DCC. can. The reaction temperature is, for example, about 0°C to 30°C,
Preferably, the temperature is about 0 to 10°C, and the reaction is generally carried out at 1
It takes about 2 hours or more to complete.

After the above reaction is completed, the derivative of the present invention can be isolated and purified from within the reaction system by conventional separation means such as solvent extraction, recrystallization, column chromatography, etc.

The thus obtained derivative of the present invention can be used as a hapten, and a desired immunizing antigen can be obtained by binding an appropriate carrier to the hapten in the presence or absence of a hapten-carrier binding reagent.

In the production of the above-mentioned immunizing antigen, a wide range of high-molecular natural or synthetic proteins commonly used in the production of antigens can be used as carriers.

Examples of the carrier include animal serum albumins such as horse serum albumin, bovine serum albumin, rabbit serum albumin, human serum albumin, and sheep serum albumin; horse serum globulin, bovine serum globulin, rabbit serum globulin, human serum globulin, and sheep serum. Animal serum globulin such as globulin; animal thyroglobulin such as equine thyroglobulin, bovine thyroglobulin, rabbit thyroglobulin, human thyroglobulin, ovine thyroglobulin; equine hemoglobulin, bovine hemoglobulin, rabbit hemoglobulin,
Animal hemoglobulins such as human hemoglobulin and sheep hemoglobulin; keyhole limb hemocyanin (
KLH> and other animal hemocyanins: Proteins extracted from roundworms (Ascaris extract, JP-A-56-1641)
Publication No. 4, J, I++ueun, 111. 26
0-268 (1973), J, lm5un, 1
22, 302-30B (1979), J. Imm.
un, 98.893-900 (1967) and Am
, J. Physiol, 199.575-578 (
196G) or further purified products thereof); examples include polylysine, polyglutamic acid, lysine-glutamic acid copolymers, and copolymers containing lysine or ornithine.

As the hapten-carrier binding reagent, a wide variety of those commonly used for preparing antigens can be used.

Specifically, reagents used in the usual peptide bond-forming reaction that creates an amide bond between an amino group and a carboxyl group,
For example, N,N-dicyclohexylcarbodiimide (DC
C>, dehydration condensation agents such as carbodiimides such as N-ethyl-N'-dimethylaminocarbodiimide, 1-ethyl-3-diisopropylaminocarbodiimide drohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide, etc. can be mentioned. As the hapten-carrier binding reagent, a combination of a diazonium arylcarboxylic acid such as p-diazonium phenylacetic acid and a conventional peptide bond forming reaction reagent, such as the dehydration condensation agent described above, can also be used.

The reaction for producing the above-mentioned immunizing antigen can be carried out according to a conventional method, and is generally carried out at 0 to 40°C in an aqueous solution or a normal buffer solution with a pH of about 6 to 9, preferably a buffer solution with a pH of about 6 to 9.
, preferably near the chamber. The reaction usually takes about 2
It will be completed in about 5 hours.

In the above, the ratio of the hapten, the hapten-carrier binding reagent, and the carrier to be used can be determined as appropriate, but the ratio of the carrier to the hapten is usually 5 to 20 times I! degree, preferably 10-2
About 0 times the weight, and 1 to 10 times the hapten-carrier binding reagent
It is best to use about double the mole. Through the above reaction, a desired immunizing antigen consisting of a hapten-carrier complex in which the derivative (hapten) of the present invention and a carrier are bound is obtained.

The antigen obtained after the reaction is processed according to conventional methods, such as dialysis,
It can be easily isolated and purified by gel filtration, fractional precipitation, etc.

Using the immunizing antigen thus obtained, a desired antibody (anti-pseudouridine antibody) having specific reactivity against pseudouridine can be produced. The antibody can be produced according to a general method, for example, by administering the above-mentioned immunizing antigen to a mammal, producing the desired antibody (polyclonal antibody) in vivo, and collecting it; Hybridomas are created between plasma cells (immune cells) from an immunized mammal and mammalian plasmacytoma cells, and clones that produce the desired antibody (monoclonal antibody) are selected and cultured.
The latter method is particularly preferred.

In any of the above methods, there are no particular restrictions on the mammal used for antibody production, and various animals such as rabbits, guinea pigs, mice, and rats can be used, but especially in the case of producing the monoclonal antibodies mentioned above, It is generally preferable to select and use mice, rats, etc. in consideration of compatibility with the plasmacytoma cells used for cell fusion.

In the above method for producing antibodies, immunization can be carried out by a conventional method, for example, by administering the immunizing antigen to a mammal by intravenous, subcutaneous, subcutaneous, intraperitoneal injection, or the like. More specifically, the immunizing antigen is administered to the test animal several times every 2 to 14 days, if desired in combination with a conventional adjuvant, so that the total dose is about 10 to 100 μQ for mice, for example. This can be done by doing. The antibody is collected by collecting blood from the immunized animal one to two weeks after the final administration, centrifuging the blood, and separating the serum.

Furthermore, as the immune cells used in the production of the monoclonal antibody, it is preferable to use spleen cells excised about 38 days after the final administration.

As the mammalian plasmacytoma cell as the other parent cell to be fused with the above-mentioned immune cell, there are various known mammalian plasmacytoma cells,
For example, E)3 (p3/X63-AQ8) (Natur
e, 256, 495-497 (1975)), p3
-Ul (Current Topics inM
icrobiology and lmmunolo
gy, Danyu, 1-7 (197B>), N5-1
(Eur, J. Immunol.

6, 511-519 (197B)), MPC-1
1 (Cell, Dan, 405-415 (197B>
), 5P210 (Nature, 2Lf:L,
269-270 (197B)], FO (J, Iu
unol, Meth, J branch.

1-21 (198G>), x63.6.5.3. (J
.

Imn+uno1. ．． 123.1548-1550 (
1979) ) Sl 94 (J, Exp, Me
d,,148. 313-323 (1978)), and R210 in rats (Nature, 277.
131-133 (1979)) W myeloma cells can be used.

The above-mentioned fusion reaction between immune cells and plasmacytoma cells can be carried out using known methods, such as the method of Milstein et al.
, VOl, 73°E) I) 3 (1981)).

More specifically, the above fusion reaction is carried out using a common fusion promoter,
For example, it is carried out in a normal medium in the presence of polyethylene glycol (PEG), Sendai virus (HVJ), etc., and an adjuvant such as dimethyl sulfoxide is added to the medium as necessary to further increase the fusion efficiency. You can also do it. The ratio of immune cells to plasmacytoma cells used is the same as in conventional methods; for example, the ratio of immune cells to plasmacytoma cells is usually about 1 to 10 times that of plasmacytoma cells. As the medium for the fusion reaction, various types of medium commonly used for the proliferation of the plasmacytoma cells, such as RPMI-1640 medium,
Examples include MEM medium and other media commonly used for this type of cell culture, and the secondary medium is usually fetal bovine serum (Fe2
) and other serum replenishers are recommended. For fusion, a predetermined amount of the above immune cells and plasmacytoma cells are mixed well in the above medium, and a PEG solution pre-warmed to about 37°C, for example, one with an average molecular weight of about 100 to 6000, is added to a normal medium. This is done by adding and mixing at a concentration of 30 to 60 W/v%. Thereafter, the desired hybridoma is formed by repeating the steps of sequentially adding an appropriate medium, centrifugation, and removing the supernatant.

The resulting hybridomas are isolated using standard selection media,
For example, this can be carried out by culturing in HAT medium (a medium containing hypoxanthine, aminopterin, and thymidine). Culture in the HAT medium may be carried out for a sufficient period of time, usually from several days to several weeks, to kill cells other than the target hybridoma (unfused cells, etc.). The hybridoma thus obtained is subjected to the search for the desired antibody and single cloning by the usual limiting dilution method.

Searching for target antibody-producing strains can be performed, for example, by ELISA method (Eng.
vall, E,, Metb, Enzyn+o1. ,
70.419-439 (1980)), plaque method, spot method, agglutination reaction method, offtelony (Quch
terlony) method, radioimmunoassay (RIA)
Various methods commonly used for detecting antibodies such as
"Hybridoma method and monoclonal antibodies", published by R&D Planning Co., Ltd., pp. 30-53, March 1982.
5th of the month], and the above-mentioned immune antigen can be used for this search.

The thus obtained hybridoma producing the desired monoclonal antibody can be subcultured in a normal medium and can be stored for a long period of time in liquid nitrogen.

The desired antibody can be collected from the above-mentioned hybridoma by culturing the hybridoma according to a conventional method and obtaining the culture supernatant, or by administering the hybridoma to a mammal compatible with the hybridoma and allowing it to proliferate. The method used is to obtain the information as follows. The former method is suitable for obtaining highly purified antibodies, and the latter method is suitable for mass production of antibodies.

The antibody obtained as described above can be further purified by conventional means such as salting out, gel filtration, and affinity chromatography, thereby producing the desired anti-pseudouridine antibody.

The resulting antibody has specific reactivity to pseudouridine, and its use can therefore be used to accurately measure the amount of pseudouridine present in various specimens, such as blood and urine, by immunoassay. By comparing these measured values, cancer screening and diagnosis can be performed.

The present invention also provides a method for diagnosing cancer and a kit for diagnosing cancer.

The measurement of pseudouridine according to the present invention requires the use of the above-mentioned specific antibody, and its basic operation is performed using conventional immunoassay methods, such as radioimmunoassay (
RIA> method, enzyme immunoassay (EIA), etc. can be followed. The operations, procedures, etc. in each of these immunoassay methods are not particularly different from those generally employed, and can be based on, for example, known direct methods, indirect methods, competitive methods, etc.

In the above, body fluids such as blood and urine can preferably be used as the specimen.

In the above method, the standard antigen (standard) is
Purified antigens as described above can be used. When an insolubilization method (method using an insolubilized antigen or antibody) is employed, the standard antigen or antibody is produced by chemically or physically reacting with an insoluble carrier according to a conventional method. Examples of the insoluble carrier include cellulose powder, Sephadex, Sepharose, polystyrene, filter paper, carboxymethyl cellulose, ion exchange resin, dextran, plastic film, plastic tube, nylon, glass beads, silk, polyamine-methyl vinyl ether-maleic acid, etc. Polymers, amino acid copolymers, ethylene-maleic acid copolymers, etc. can be used. Insolubilization can be carried out using the diazo method as a covalent bond method, the peptide method (acid amide derivative method, carboxychloride resin method, carbodiimide resin method, maleic anhydride derivative method, isocyanate derivative method,
cyanogen bromide activated polysaccharide suspension, cellulose carbonate derivative method, method using condensation reagents, etc.), alkylation method,
Carrier bonding method using a crosslinking reagent (using geltaraldehyde, hexamethylene isocyanate, etc. as a crosslinking reagent), chemical reaction such as carrier bonding method using UO+ reaction; or ion bonding method using a carrier such as an ion exchange resin;
This is carried out by a physical adsorption method using porous glass such as glass beads as a carrier.

As the labeled antigen or labeled antibody, those obtained by labeling the standard antigen or antibody described above with various labeling agents such as ordinary radioactive substances, enzyme labeling substances, fluorescent substances, etc. can be used. Here, the radioactive substances include radioactive iodine, etc., and the fluorescent substances include fluorescein isothiocyanate (FI
TC), tetramethylrhodamine isothiocyanate (TRITC), substituted rhodamine isothiocyanate (XRITC), rhodamine B isothiocyanate, dichlorotriazine fluorescein (DTAF)
etc., and as an enzyme labeling substance, peroxidase (PO
X), microperoxidase, chymotrypsinogen, procarboxypeptidase, glyceraldehyde-3-phosphate dehydrogenase, amylase, phosphorylase, D-nase, P-nase and the like. The labeling method using these labeling agents also follows a conventional method.
z mutual A.

9349-9351 (1979):Natur
e, 194°495 (1962): Fluorescent antibody method, Medical Chemistry Experiment Course No. 4.263-270:
ACta, EndOCrinOI.

5upp1. ．． 168.206 (1972): P
roc, Natl.

Acad, Sci, USA, Mutual, 713 (1
967) etc.).

The measurement and quantification of pseudouridine in a specimen (body fluid) in the above method is carried out by immunoreacting the specimen with any of the standard antigen, insolubilized antigen, insolubilized antibody, labeled antigen, or labeled antibody. At this time, the solvent used in the measurement system is a normal solvent that does not have a negative effect on the reaction.
For example, preferred examples include buffers having a pH of about 4 to 8, such as citrate buffer, phosphate buffer, Tris-hydrochloric acid buffer, and acetate buffer. Furthermore, the immunoreaction conditions during the measurement are not particularly limited, and can be the same as those used in ordinary measurement methods of this type. That is, the immune reaction is generally carried out at a temperature of 45°C or lower, preferably about 4 to 40°C, for 1 to 40 hours.

Separation of the conjugate and free form (B-F) after the completion of the immune reaction also follows known methods; for example, when an insolubilization method is adopted,
This can be carried out by separating the solid phase and liquid phase by means such as centrifugation, furnace separation, washing, and decantation. In other cases, conventional methods such as the dextran-activated charcoal method and the second antibody method may be followed.

The procedure for measuring pseudouridine according to the present invention will be described in detail by taking the liquid phase method as an example. The method consists of (1) first combining a test substance in a sample to be measured and a certain amount of antibody with a constant amount of labeled antigen; (2) First, the amount of the test substance is quantified by performing a competitive reaction with the amount of the test substance, then performing B-F separation by the dextran-charcoal method (DeXtran-ChaCOal), and measuring the activity of one of the labeling agents. A substance and a certain amount of labeled antigen are competitively reacted with a fixed amount of antibody, and then B-F is separated by a second antibody method, and the activity of one of the labeled antigens is measured to quantify the amount of the test substance. This can be carried out by methods such as

A particularly convenient method for carrying out the above measurement method is a method using a kit for measuring the amount of pseudouridine in body fluids such as blood and urine. It is important that such a kit contains an antibody that specifically makes an antigen-antibody reaction with pseudouridine, that is, an anti-pseudouridine antibody. Stabilizers and/or preservatives, such as glycerol-Japanese serum protein, can be added to the antibody reagent. Preferably, the antibody reagent is lyophilized, and the kit can include a water-soluble or water-miscible solvent. Additionally, the antibody reagent can be supplemented with a buffer to maintain a constant pH of the reconstituted reagent system and/or a preservative and/or stabilizer to prevent the sample from deteriorating before use. . Although the buffer solution is not an essential component of the kit reagent, it is necessary to adjust the pH to 4 to 8 when performing the above measurement method.
It is preferable to use one that is of a certain degree. Although the reconstitution agent preferably contains water, part or all of the water may be replaced by a water-miscible solvent. Water-miscible solvents are well known to those skilled in the art and, for example, glycerin, alcohols, glycols, glycol ethers, etc. can be used.

The pseudouridine derivative of the present invention, which is used as a hapten, enables the production of an immunizing antigen, and an anti-pseuduuridine antibody can be obtained from the immunizing antigen, and by using the antibody,
The immunoassay method allows the amount of pseudouridine in body fluids to be measured very easily, with high accuracy, and with high sensitivity. By comparing this measured value with the value of a healthy person, it is possible to screen for cancer in a subject and /or diagnosis is possible.

In particular, immunoassay methods using the above-mentioned antibodies are extremely effective for early detection of cancer compared to those using existing cancer markers.

! --111 In order to explain the present invention in more detail, examples of production of the derivative of the present invention, preparation of an immunizing antigen, and immunization examples will be given below as examples.

Example 1 Pseudo-lysine-succinyl ■ Pseudouridine (manufactured by Sigma >240m).

(1mM) was dissolved in 2-3mG of dimethylformamide (Wako Pure Chemical Industries, Ltd.), 51 mIQ of triethylamine (manufactured by Wako Pure Chemical Industries, Ltd.) was added and mixed, and then succinic anhydride 200+eo (2mM) was dissolved in dioxane 5 A solution dissolved in - was added, and after being left at room temperature for about 24 hours, it was concentrated under reduced pressure.

Next, the reaction solution was subjected to high performance liquid chromatography to separate and purify the target compound.

DEAE-5PW (manufactured by TOSOH) was used as the column, and a concentration gradient of ammonium acetate from 20 mM to 0.5M was used for elution.

The resulting fraction group was analyzed by ultraviolet absorption spectrum (OD265) to obtain four fractions.

■ By subjecting the above fraction to NMR analysis,
One of them was confirmed to be a single fraction of 5'-mono-succinyl-pseudouridine (5'-derivative of the present invention).

The NMR analysis diagram of the fraction is shown in Figure 1.
The main NMR data are as follows.

7, 51 (d.J-0.8>, 4.65 (dd.

J-12.7.2.0>, 4.31 (dd.J-12
, 7.2.0>, 4.22 (dd, J-12, 7.2
．． 0>, 4.18 (t.J-4.7>4, 08 (
m. Hx2), 2.57 (t, J-6, 7.Hx2>
, 2.46 (t, J-6.7.

HX2) The fractions were collected to obtain mono-succinyl-pseudouridine. The yield was 21.2%.

The above NMR data is assigned as follows. That is, 2.57 and 2. 46CI)l (triple signal (t), coupling constant (J) - 6.7.2 protons)
The signal indicates ester-linked maleic acid. 4.31 and 4.22 CI) 1 (double, double (
d, d) J-12.7. Two protons) are chemically shifted to the 5'-position hydroxyl group, indicating that maleic acid is bonded to the 5'-position hydroxyl group. . 4
．． The signal of 65CD-(d,d, J-4.7) is 1
The proton at the ′-position is shown. The signal at 7.51 cp- indicates the proton of the base. From these facts, succinylation at the 5' position is confirmed.

In addition, as a result of infrared analysis, 1670 and 156
An increase in absorption near 0 crl was observed, and from this, an ester bond was confirmed.

Furthermore, the presence of a uracil group was confirmed by ultraviolet analysis based on maximum absorption at 260.

Furthermore, it was confirmed that the 5'-derivative of the present invention obtained above was a single substance, as it showed a single peak in DEAE and reversed phase high performance liquid chromatography (HPLC).

■ Another fraction among the fractions obtained in (■) above (20 μM) was added with 24 μM of nityrosine ethyl ester (Peptide Research Group), 00 μM of 1DCCI, and 10 μM of dioxane, left at 10°C for 24 hours, and concentrated under reduced pressure. .

The obtained reaction concentrate was subjected to silica gel reverse phase high performance liquid chromatography using ODS120T (manufactured by TOSOH) (eluent: 2% acetonitrile + 0.1
%TFA→100% acetonitrile).

Thus, pseudouridine-2'-ethyltyrosyl-succinylated product (2'-derivative of the present invention) was isolated as a fraction eluting at a retention time of 8.68 minutes. The yield of this product from the starting material, pseudouridine, was about 11.6%.

Furthermore, by the same chromatography, pseudouridine-3'-ethyltyrosyl-succinylated product (3'-derivative of the present invention) was isolated as a fraction eluting at a retention time of 9.58 minutes. The yield of this product from the starting material, pseudouridine, was about 18.3%.

As a result of subjecting each of these to NMR analysis (during D20), the main peaks were as follows.

Kiori 12-membered 7.52 (d, J-0,4Hz, base proton) 7.
02 (d, J-8,3Hz, proton at 2°6 position of tyrosine) 6.73 (d, J-8,3Hz, proton at 3°5 position of tyrosine) 5.18 (dd, J=6.7Hz, Proton at 2' position of sugar) 4.61 (dd, J-0,4Hz, proton at 1' position of sugar) 4.49 (m>2.50,2.60 (t, J-13,9Hz.

Proton of succinyl group) Mizu and Mame Unini Kasa Naoson 7.58 (d, J-0,4Hz, base proton) 7.
05 (d, J-8,3Hz, proton at 2°6 position of tyrosine) 6.75 (d, J-8,3Hz, proton at 3°5 position of tyrosine) 5.02 (dd, J-5,6Hz, Proton at 3' position of sugar) 4.54 (dd, J-0,4Hz, proton at 1' position of sugar) 4.49 (m>2.50,2.60 (t, J-13,9Hz.

Proton of succinyl group) Example 2 10 μM of 5'-mono-succinyl-pseudouridine obtained in ① of Example 1 and 12 μM of tyramine (manufactured by Wako Pure Chemical Industries, Ltd.)
is dissolved in dioxane 5-, to which DCC 5-
Add 0 μM, leave at 10°C for 24 hours, and after the reaction is complete,
It was concentrated under reduced pressure.

Then, the obtained reaction solution was subjected to reverse phase high performance liquid chromatography on a silica gel column using 120 Toso 0DS (eluent: 2% acetonitrile + 0.1% TFA →
100% acetonitrile) to obtain the desired 5'-tyramine-succinate-pseudouridine at a retention time of 18.56 minutes.

The NMR analysis diagram (in CD30D) of the obtained compound is shown in the second
As shown in the figure.

Example 3 125I-Na (manufactured by Amajam Co., Ltd. > 0.5 mC1 50
mM phosphate buffer (pH 7, 4>10.1 solution, derivative of the present invention [2'-ethyltyrosyl-succinate-pseuduridine or 3'-ethyltyrosyl-succinate-pseuduridine derivative obtained in Example 1, ① or Examples) A solution of 20μQ of 5'-tyramine-succinate-pseudouridine obtained in step 2 in the same buffer and 20μQ of chloramine T (manufactured by Wako Pure Pharmaceutical Co., Ltd.) in 10μQ of the same buffer was mixed for 1 minute. Add sodium metabisulfite to the mixture at a ratio of 20 μQ/20 μQ to stop the reaction.

Next, after adding 200 μQ of 2 tJV of KI, the desired iodized products of each of the present derivatives were obtained by column chromatography using Toso ODS120T.

Example 4 100 μQ of pseudolysine rabbit serum 100 μQ of the 1251-labeled pseudouridine-5'-tyramine-succinylated product obtained in Example 3 (ca, 20,000 CI) and 50 mM phosphate buffered saline (PBS% pH7,4,
Mix 300 μQ (containing 0.1% BSA) and incubate at 4°C for 24 hours.
Let stand for ~48 hours. Next, after adding 500 μQ of dextran charcoal, centrifugation is performed at 3000 rpm for 30 minutes, the precipitate is separated by decantation, and the labeling activity is counted using a γ-counter (manufactured by Aloka).

In this way, the labeling activity of the conjugate of labeled antigen and antibody can be measured.

Example 5 5'-monosuccinia L/- prepared in Example 1 (■)
2mQ (8.4mM) of gin and 11mo of bovine serum albumin (BSA) were dissolved in 50mM phosphate buffered saline (PBS, pH 7.4), and N,N-
Dicyclohexylcarbodiimide (DCC) was added, and a coupling reaction was carried out at 25°C. This-
After standing for day and night, the reaction solution was transferred to a dialysis membrane and incubated in distilled water for 4 hours.
Dialysis was carried out overnight at ℃ to obtain the immunizing antigen.

This coupling reaction was confirmed by ultraviolet (UV) absorbance, which showed an increase in absorption near O and D26 Onm.

Next, 200 μQ of the immunizing antigen prepared above was dissolved in phosphate buffered saline (PBS) (pH 7.4), and the resulting solution 111Q was mixed with an equal volume of Freund's comblete adjuvant (Freund C0Ipleteadjuvant).
vant, DIFCD LabOratOrieS, D
etrOit.

HiChil; Jan tJsA) at a ratio of 1:1 and suspended. An amount of the resulting suspension containing 100 μQ of antigen was taken and subcutaneously administered to a 12-week-old domestic rabbit. From then on,
The same amount of the same solution was administered subcutaneously in the mouth for 2 weeks, and 2 weeks later, the same solution containing 100 μg of antigen was administered subcutaneously.

After immunization by administering the antigen, blood was collected from the immunized animal and centrifuged to obtain the desired antibody (antiserum).

[Brief explanation of the drawing]

FIG. 1 is an NMR analysis diagram of the derivative of the present invention obtained in Example 1, and FIG. 2 is an NMMR analysis diagram of the derivative of the present invention obtained in Example 2.
It is an R analysis diagram. (that's all)

Claims (1)

[Claims] [1] General formula ▲ Numerical formula, chemical formula, table, etc. ▼ [In the formula, R^1, R^2 and R^3 are any one of the groups R
-CO-A-CO- is shown, and the others are hydrogen atoms. In addition, in the above group, A represents a lower alkylene group, and R represents a hydroxyl group, or (4-hydroxyphenyl) lower Indicates an alkylamino group. ] A pseudouridine derivative represented by