JPS61109797A - Labeled nucleotide and labeled polynucleotide - Google Patents

Abstract

NEW MATERIAL:A compound bearing a labeled purine base analog of formula I (X<1>, X<2>, X<3> are C, N; Y, Z are H, hydroxyl, amino group; F is fluorescent labeling compound) where 7-deazapurine is excluded. USE:A clinical diagnostic reagent used in identification of labeled genes, a reagent in biotechnology for extracting genes. PREPARATION:For example, 2'-deoxyaribonucleoside transferase and thymidine are allowed to act on 4-amino-3(2-aminoethyl)-pyrazolo[3,4-d]pyrimidine and further phosphorus oxychloride is allowed to react with the product in anhydrous trimethyl phosphate to give 5'-monophosphate of formula III. The product is converted into 5'-triphosphate, which is used to effect nick translation whereby a polynucleotide bearing the group of formula I is obtained.

Description

Detailed Description of the Invention (Industrial Application Field) The nuclear phantom base of the present invention has the general formula CI) (wherein, hydrogen atom,
A hydroxyl group or an amino group, F represents a fluorescent labeling compound. ) labeled purine base analogs (but
Labeled nucleotides or labeled deoxynucleotides (hereinafter referred to as
(collectively referred to as labeled nucleotides), and labeled/IJ! containing one or more of each of the above-mentioned labeled end nucleotides or labeled deoxyrime nucleotides. J
/nucleotides or labeled polydeoxynucleotides (hereinafter collectively referred to as labeled polynucleotides), labeled polynucleotides having a specific base sequence obtained by the present invention can be used to identify and extract target genes. It can be widely used in the fields of genetic engineering, clinical diagnosis, food, etc. (Snowscape of invention) As genetic engineering research has become more active in recent years, the detection of genes necessary for the production of M substances has become increasingly important. Polynucleotides with a specific base sequence tV have come to be used.

For example, in the field of clinical diagnosis, immunological and biological methods are currently used; however, although the test time for the former is generally short (a few minutes), cross-reactivity and interference effects often give unclear results. Moreover, the latter has drawbacks such as requiring a long time for culturing.

On the other hand, polynucleotide arrows, which diagnose diseases by detecting genes, generally require several hours to test, but the detection sensitivity is low and the specificity is very high.
It also has the advantage of having extremely small errors and can detect not only infectious diseases but also latent viruses that cannot be detected by immunological methods, so there is a demand for the development of polynucleotides suitable for this purpose.

(Prior Art) / When detecting a target gene by a hybridization method using IJ nucleotides, it is necessary to label the polynucleotide. Labeling is broadly divided into methods using radioisotopes and optical labeling methods.

Currently, the method using the radioactive isotope 5P is the most used method), and Japanese Patent Application Laid-open No. 170496/1983 describes the method using the radioactive isotope 5P (
A phosphate group labeled with 5P is introduced into the terminal group of the polynucleotide using r-"p) and kinase, and the film is exposed to β-rays emitted from "P, and the presence of the target gene is detected by the black spots. A method is disclosed. Although labeling with radioactive isotopes is superior in terms of sensitivity, it requires skill in handling and handling.

■O! is subject to strict legal regulations, requires special equipment and measuring equipment, and is only available in limited locations. ! I have a health problem.

■There is a problem with the disposal of waste after use. ■Since pre-purchases are made according to the half-life, the expiry date of the capsule is limited by this. There are other difficulties. In response to this, a photolabeling method was proposed as a labeling method that does not use radioactive isotopes,
The publication discloses a method using chemiluminescence, bioluminescence, and fluorescence. However, as a photolabeling method, di-0-triphosphate t--which has pyotin attached to its 0-5 position via an amide bond as a thymidine or chrysine analogue--is incorporated into a polynucleotide using an enzymatic two-translation method. Methods for labeling and detecting target genes (Proceedings of Nati
onal person cad@my of Sal@nc@
ofth@Unit@d 8tat@of Am@ri
EA, Vol. 80, p. 4045 (1983)] has only been put into practical use. (Problem to be solved by the invention) When labeling 11 nucleotides by optical labeling, polynucleotides are It is necessary to introduce a labeling compound so as not to impair the ability to hybridize with the gene. However, when performing full optical labeling of the nucleic acid base, it is possible to introduce a labeling compound into the 0-5 position with a pyrimidine base, but it is stereochemically possible to introduce a label compound into the 0-5 position with a purine base. Am 0 only in 8th and N-7th place, but C-
When a labeling compound is introduced at the 8-position, the base and sugar assume a Byll conformation, so if the doublet is right-handed, the hybridization ability is lost; when the labeling compound is introduced at the N-7 position, the nitrogen becomes tetragonal. It is extremely difficult to photolabel purine bases because the so-called depurination reaction, in which the glycosidic bond between the sugar and the base moiety is broken, tends to occur. Therefore, photolabeling of the nucleic acid base portion is effective only in the case of male rimidine bases, and when the base sequence of the polynucleotide consists only of purine bases, it is difficult to photolabel.

Furthermore, compared to polynucleotides labeled with radioactive isotopes, photolabeled polynucleotides generally have lower detection sensitivity and therefore require a larger amount of the target gene, making it difficult to research and speed up genetic engineering.

Practical problems remain in clinical tests that require continuous testing, and in order to further increase detection sensitivity, it is necessary to consider more sensitive labeling compounds and increase the number of labeled sites.

(Means for Solving the Problems) The present inventors have improved the detection sensitivity of photolabeled 9-rinucleotides, and have repeatedly investigated the possibility of photolabeling the nucleobase portion, particularly purine bases. As a result, the general formula [! ] (In the formula, x", x", x", y, z > y and r represent the same meanings as above.) , 7-deazapurine)) as a nucleobase, the intended purpose can be achieved by using nucleotides and polynucleotides,
This completes the present invention.

The labeled nucleotides and labeled polynucleotides of the present invention have the general formula (
1) (In the formula, a functional group bonded directly or through a hydrocarbon chain is represented by By reacting a covalent fluorescent labeling compound with a functional group of a nucleotide or polynucleotide having a purine base analog having a functional group at the -7 position,
The O-7 position of a purine base analog is fluorescently labeled. Thiol groups, amino groups and carzenyl groups are commonly used as functional groups to be reacted with covalently bonded fluorescent labeling compounds, and are attached via a hydrocarbon chain or directly to the O-7 position of the purine base analog. . These functional groups may cause side reactions during polyscleotide synthesis, so it is desirable to protect them with a protecting group.
Any protecting group may be used as long as it can be removed in the final deprotection step. In addition, any covalently bonded fluorescent labeling compound can be used as long as it has a specific covalent bonding property with the above-mentioned functional group, such as dancing aziridine, 5-
Iodoacetamide fluorescein, 1-prosopaiman, N-Cp-(2-benzimidazolyl) phenylcomaleimide, 4-pro7methyl, 7-medoxycoumarin, dansylhydrodidine, fluorescein-5-thiosemical/sito, etc. These are examples, but are not limited to four.

Purine base analogs in the present invention, such as 4-amino-3(2-aminoethyl)-virazolo(3,4-d)pyrimidine represented by the formula [厘]
of the Am*rlcan Oh@m1aal
8ocisty * Volume 81, page 2456 (19
59), it can be synthesized using malononitrile and human didine as starting materials, and the compound represented by formula (1) is treated with 7-zooxylyse nucleoside transferase and thymidine. By doing so, 1-/-])-]deoxylysilyl represented by the formula (ff) can be obtained in good yield. In addition, this 1-β-D-deoxylysyl unit is '1'h* Jotlrnal of Or
ganl* Chamistry, Volume 39, No. 36
Formula (1) can also be obtained by the synthesis method described on page 54 (1974).
) is derived from 3. After protecting the amino group and 5'-hydroxyl group of the silizecyl in the 1-β-D-deo obtained in this way with a protecting group, the triester method or phosphite triester method was used in place of deoxyadenosine. Let it be a polynucleotide.

Alternatively, as a method for producing polynucleotides using an enzymatic method, 1-β-D-deoxyly2yl is reacted with phosphorus oxychloride in trimethyl phosphate anhydride to form a dermonophosphate represented by formula (V). After that, Oh@m
1meh@B@rieht* r Volume 94, No. 1612
Page (1981) ith@Journal of
th@Amer1eanOh@m1eal 8oai@
ty, p. 649 (1961) to obtain dartriphos7ate represented by the formula [■], and perform nick translation using this instead of deoxyadenosine triphosphate to obtain a polynucleotide. You can also do that.

In the aqueous solution of polynucleotide thus obtained,
Polynucleotides are easily fluorescently labeled by dropping an acetone solution of a fluorescently labeled compound such as danzyl cloide. Separation and purification of this fluorescently labeled polynucleotide is carried out using a high performance liquid chroma dog 2 fee)
Complete quickly.

(Effects of the Invention) By using purine base analogs in place of purine bases in nucleotides and polynucleotides, light and Labeling becomes possible for all nucleobases, improving detection sensitivity and solving constraints on the base composition of polynucleotides.

(Example) Example 1 ■ Synthesis of 4-amino-3-(2-aminoethyl)-12zolo(3,4-d)pyrimidine 331 malononitrile and 11.511 hydrazine were added to 90 mj of ethanol and kept for 8 hours. After refluxing, the reaction solution was concentrated and allowed to stand overnight. The resulting precipitate was dissolved in hot water and treated with activated carbon to give white needle-like crystals of 3-cyanomethyl-4-cyano-5-
Aminopyrazole 6, obtained ON'. Next, formamide 21 was added to this, and after heating at 200 to 210 C for 4 hours, the reaction solution was dissolved in a 17 um aqueous solution, treated with activated carbon, and adjusted to pH with hydrochloric acid. 1. C, -
The precipitate formed by standing overnight was filtered and dried to form a powdery 4-amino-3-cyanomethylpyrazolo (a, +-d).
4.71'ft of pyrimidine was obtained. Furthermore, this t″2
7) Suspend in ammonia-saturated ethanol together with catalyst 3I, and react in an autoclave at 110C for 4 hours while maintaining the hydrogen pressure at 130i pressure or higher. After the reaction, the catalyst was filtered off and the solvent was distilled off.
(N hydroxide) Dissolved in 17um aqueous solution, pHf with hydrochloric acid:
The precipitate formed after the H reduction was filtered and dried to obtain 3.411t of 4-amino-3-(2-7onoethyl)-pyrazolo(3,4-a)pyrimidine in powder form. .

■ 4-amino-3-(2-aminoethyl)-1-β-
Synthesis of D-deoxylyl 1g Silpyrazolo C3,4-d]pyrimidine Well-dried 4-amino-3-(2-aminoethyl)-
Pyrazolo (3,4-d)pyrimidine 10.9 with anhydrous vinegar @
50- and anhydrous pyridine 501j were added, refluxed for 40 minutes, cooled to room temperature, the solvent was distilled off under reduced pressure, dissolved in methanol, and evaporated again under reduced pressure. To the obtained residue, 30.9 g of ice water was added to form a precipitate. The mixture was separated from t-a, dissolved in 4001j hot water, treated with activated carbon, and cooled to obtain crude crystals. The crude crystals were dissolved in an aqueous IJ cum solution, treated with activated carbon, and pII adjusted with acetic acid, then left overnight to form a precipitate t-
g and dried to obtain crystals 8.2 IIf of 4-acetamino-3-(2-acetaminoethyl)-pyrazolo(3,4-d)pyrimidine.

Next, this was heated with 2.81 g of anhydrous potassium cyanide and 20011 Ej of anhydrous nitromethane, and 1311 t of 0-acetyl-β-D-deoxylysylclotide was further added thereto, and the mixture was reacted under reflux for 2 hours. After the reaction was completed, the solvent was distilled off and the resulting residue was dissolved in ethyl acetate 30011j.
This is washed, dehydrated, concentrated, and separated using a silica gel column. Collect the 7-lactone containing the target compound, evaporate the solvent, dissolve the residue in methanol saturated with ammonia, leave it at room temperature for 3 hours, evaporate the solvent, and dissolve the resulting residue in a small amount of hot water. This was added dropwise to ethyl acetate, and the resulting powder was filtered and dried to obtain powdered 4
-amino-3-(2-aminoethyl)-1-β-D-deoxylysilpi2zolo(a,+-a)pyrimidine OH
N O Theoretical value (to): 49.0 6.1 2 & 6 16
．． 3 Actual measurement value (4): 4 & 9! L9 2&9
1 & 4■ Synthesis of polydeoxyrinucleotides 4-amino-3-(2-aminoethyl)- by the method described in Protein Nuclear Enzyme, Vol. 25, p. 227 (1980)
1-β-D-deoxylyzecylze2zolo[3,4-d)
The amino group of pyrimidine was benzoylated, then the 5'-position of deoxyrimase was dimethoxytritylated, and the 3'-position was phosphorylated to synthesize a heptamine represented by the formula [■]. Next, trimer block-0-T was synthesized by a separately known method (in the formula, 0 and T represent the unit nucleotides forming the trimer block by their base residues, and the base residue 0 is cytosine). , T is the condensation of thymine t-wawasu) and the seven-termer represented by the formula ``t'', Nuc
Lola Cuds Rei Tsuru Reh, Volume 8, No. 54
73 (1981) by the method described in 11 times,
The base sequence is A'-o-a-r-A'-o-o-r-A'
-o-o-T-r (wherein,
(2-aminoether)-pyrazolo(a,+-a)pyrimidine, 0 and T have the same meanings as above. ) Is it? Lysooxyly dinucleotide (s of2!:
body) t-synthesized.

■ Fluorescent labeling of polydeoxyri I nucleotide (decamer) 100D (
260mm)'io, 5M sodium borate buffer (p
Hs, o) a, s- and acetonitrile 1. (l
Dissolve in 100ml of danzyl chloride a7tone solution (201nIl/100JIj) and shake well.
Drop Ll. After stirring for 10 minutes, the reaction solution was concentrated under reduced pressure, and the fluorescently labeled polydeoxylyse nucleotides were purified by reverse phase column chromatography. KoO
The silicate nucleotide in the gt photolabeled polydeoxynucleotide is 250
! l! ! It is excited by light around 1, and strong fluorescence at 530 to 540 nm is measured.

Procedural Amendment Work, March S, 1985, Patent Application No. 231847, 1985, 2, Name of the invention Labeled nucleotides and labeled polynucleotides 3, Relationship with the person making the amendment Case Patent applicant: Chuo-ku, Tokyo Kyobashi 2-17-4 Organic Synthetic Pharmaceutical Industry Co., Ltd. Representative Masao Tamashige 4, Agent 6 Column 7 of “Detailed Description of the Invention” of the specification to be amended, Contents of the amendment (L) Description r on page 6, lines 7-8 (Proceed
ings...(omitted)・=of AmericaJ
ionalAcademy of 5eience o
f the United 5tate of A+*e
rica)”.

(2) rTheJou on page 10 of the same book, lines 5-4 from the bottom
rnal-(omitted)-5ociety J's description in "The Journal of the American Chemical Society"
e-rican Chemical 5ociety
)” is corrected.

(3) rThe Jo on page 11 of the same book, lines 10-9 from the bottom
urnal...(omitted)...Chemistry
J's description in "The Journal of Organic Chemistry"
nic Chemistry)”.

(4) r Chemisch on page 12, lines 6-7 of the same book
e - (omitted)... 5ociety The description of J is "Chemische Beric
ht, e), Volume 94.

Page 1612 (1961) or The Journal of the American Chemical Society (The
Journal of the American
Chemical Society) 41.

(5) rNucleic A on page 18, lines 8-9 of the same book
The description of c1dsResearchJ has been changed to
Acid Research (NucLeie Ac1ds Re
5earch)”.

that's all

Claims (1)

[Claims] 1. As a nucleic acid base, the general formula [I] ▲ Numerical formula, chemical formula, table, etc.▼ [I] (In the formula, X^1, X^2 and X^3 are each a carbon atom or nitrogen Y and Z each represent a hydrogen atom, a hydroxyl group, or an amino group, and F represents a fluorescent labeling compound.
7-deazapurine). 2. As a nucleic acid base, there are general formulas [I] ▲mathematical formulas, chemical formulas, tables, etc.▼[I] (In the formula, X^1, X^2, and X^3 each represent a carbon atom or a nitrogen atom, and Y and Z (respectively represents a hydrogen atom, hydroxyl group, or amino group, and F represents a fluorescent labeling compound.)
A labeled polyribonucleotide or a labeled polydeoxyribonucleotide, characterized in that it contains one or more labeled ribonucleotides or labeled deoxyribonucleotides each having a compound (excluding 7-deazapurine).