JPS63130599A - Modified nucleotide - Google Patents

Abstract

NEW MATERIAL:The compound of formula I (P is phosphoric acid segment; S is sugar segment; B is nucleic acid base segment; Sig is chemical segment bonded to the nucleic acid base segment B through covalent bond; R is a spacer segment linking the nucleic acid base segment B and the chemical segment Sig; P is bonded to 5'-site of S; 2'- and 3'-sites of S are dideoxy type of hydrogen; B is pyrimidine derivative or 7-deazapurine derivative). USE:A probe for detecting the presence of particular DNA or RNA molecule. PREPARATION:The compound of formula I can be produced e.g. by converting a compound of formula II (X is mono-, di- or triphosphoric acid; Y is OH, etc.; Z is OH, etc.) to dideoxy compound, reacting with a mercury salt to obtain a mercury compound of formula III, reacting the compound with a chemical group of R in the presence of a Pd catalyst and reacting the product with Sig.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to modified nucleotides. More specifically, one aspect of the present invention provides for easily detectable nucleic acid substances when bound to and/or incorporated into the substance.

It relates to chemically modifying or labeling polynucleotides and/or oligonucleotides including nucleotides and DNA.

(Prior art) Conventionally, for detection of ifi gene, sequence analysis, etc., one example is:
lH2+4(, 32p, 3SS or +251
Genes have often been labeled with radioactive isotopes such as However, although labeling with these radioactive isotopes is excellent in sensitivity, it has the following drawbacks when used.

l) Skill is required for handling.

2) It is subject to strict legal regulations, requires special equipment and measuring equipment, and can only be handled in limited locations.

3) You have a health problem.

4) There is a problem with disposal after use.

5) Since pre-purchasing is done in conjunction with the half-life period, the date of the experiment is constrained by this.

Therefore, instead of gene detection and sequence analysis methods using radioisotopes, which have the drawbacks mentioned above, we have developed a stable and biologically safe high-precision detection and sequence analysis method that does not require special equipment or equipment. Its appearance is awaited. Development of gene labeling reagents to meet the demands of such techniques is progressing.

For example, JP-A-59-62600 discloses modified nucleotides labeled with 5 (particularly biotin).

However, this modified nucleotide is not a dideoxy form, that is, its 3' end is not a deoxy form, and therefore it is thought to be difficult to selectively label only one on the 3' side of the gene in question. . Therefore, polynucleotides labeled with this modified nucleotide and/or
Alternatively, the oligonucleotide cannot be subjected to gene sequence analysis using tools such as the Maxam-Gilbert method. As described above, conventional modified nucleotides are not sufficient to easily and accurately label the gene of interest and to achieve the broad objectives of various genetic studies.

(Problems to be Solved by the Invention) An object of the present invention is to provide a novel labeled dideoxyribonucleotide derivative used for gene labeling.

Another object of the present invention is the above-mentioned labeled dideoxy+J
; iF sreotide derivatives). 1 millinucleotide and/or oligonucleotide.

(Means for Solving the Problems) To achieve the above object, one labeled modified nucleotide of the present invention has the following general formula: P-5-B-R-S
ig where P is a phosphate moiety, S is a sugar moiety, B is a nucleobase moiety, Sig is a chemical moiety covalently bonded to the nucleobase moiety B, and R is a link between the nucleobase moiety B and the chemical moiety Sig The phosphoric acid moiety P is bound to the 5'-position of the sugar moiety S;
is a dideoxy type in which the 2°-position and the 3°-position are hydrogen; the nucleobase moiety B is a pyrimidine derivative or a 7-deazapurine derivative; In some cases, it binds to the 1″-position of the sugar moiety S by its N-1 position, or by its N-9 position when the B is a 7-deazapurine Li 8;
The spacer moiety R is attached to the 5-position when the nucleobase moiety B is a pyrimidine derivative, or to the 7-position when the B is a 7-deazapurine derivative; When bound to the nucleobase moiety B, it can signal itself, thereby self-detecting itself or making its presence known.

In addition, one polynucleotide and/or oligonucleotide of the present invention has a chemical moiety Si in the above general formula.
g is a chemical group containing at least one carbon atom. Labeled using at least one labeled modification reagent.

The present invention will be explained in detail below.

It has been found that the compound shown by the following structure is widely used as a probe in biochemical research and genetic recombination technology.

Here, B represents a pyrimidine derivative or a 7-deazapurine derivative, which is a nucleobase covalently bonded to the C-1'' position of the sugar moiety. When B is the former, the sugar is attached to the N-1 position of the nucleobase. while in the latter case the sugar is attached to the N-9 position of the nucleobase.Sig is the reporter group for detection and is a chemical group containing at least one carbon atom.R is Reporter Group Sig and Nucleic Acid Base B
This is a spacer group that connects the When B is a pyrimidine derivative, R is attached to the 5-position of the pyrimidine derivative, whereas when B is a 7-deazapurine derivative, R is attached to the 7-deazapurine derivative.
- It is attached to the 7-position of the deazapurine derivative. X represents mono-, di-, or triphosphoric acid. Y and Z are hydrogen.

The labeled dideoxynucleotide derivatives of the invention shown in the structure above must have the following properties:

1) Scales that serve as substrates for enzymes such as polymerase and terminal deoxynucleotidyl transferase.

2) The 5゛hydroxyl group must be a phosphoric acid ester.

3) The 3' end must be a deoxy form to stop the reaction.

4) The polymerase reaction can be stopped without error at adenine, guanine, cytosine, or thymine (or uracil).

5) The labeling substance (reporter group Sig) and spacer group R must not interfere with hybridization or reactions by enzymes such as polymerase and terminal deoxynucleotidyl transferase.

The reporter group Sig for detection is thus attached to a sterically permissive site of the nucleotide unit to be modified thereby. here.

A sterically permissive site of a nucleotide unit is a position on the nucleobase of the nucleotide unit at which the nucleotide unit is modified by attachment of a substituent to that position; , hybridization of the product oligonucleotide with complementary nucleic acid components is not significantly hindered, and the presence of the substituent makes the nucleotide useful as a substrate for enzymes such as polymerases and terminal deoxynucleotidyl transferases. It means a position and structure that does not interfere with function. Such sterically permissive sites are the C-7 position of 7-deazapurine derivatives and the C-5 position of pyrimidine derivatives.

On the other hand, sites that should not be modified include N-1 of the adenine base and NH at the 6th position, and N-1 of the guanine base.
, Ni+□ at the 2nd position and O at the 0-6th position, O at the 2nd position of the cytosine base, Nlh at the N-3 and 4th position, and N at the thymine base.
Examples include -3 and 0 at the 4th position.

In general, substitutions with heteroatoms (N or O) should be avoided. In addition, when a substituent is introduced at the C-8 position of a purine derivative, the base and sugar take a syn-type coordination position, so
If the double fold is cloth-wrapped (normal or NA), it loses the ability to form hybrids and is generally not preferred. Furthermore, when it is introduced into the N-7 position of a purine derivative, the nitrogen becomes quaternary, and the glycosidic bond between the sugar and the base moiety is easily broken, resulting in a so-called depurination reaction (Japanese Patent Laid-Open No. 61-57595). .

Substitution 5R as a spacer group for modifying a nucleotide unit with a reporter group Sig characterized in that it can function as one or more reporter groups or can be linked to one or more reporter groups have. In the present invention, the substituent R capable of bonding with the reporter group Sig can generally be said to exhibit nucleophilic properties. Examples of such substituents include primary amino groups, aromatic amino groups, thiol groups, carboxyl groups, and hydroxyl groups. In substituted pyrimidine or 7-deazapurine bases, the substituent R contains one or more carbon atoms. From the above, it is preferable that the substituent (spacer group) R takes the form of a carbon chain represented by the following chemical formula.

Here, R1 is hydrogen or a lower alkyl group of Cl-6.

R2 is C, R2, Y, and Z is a polyvalent heteroatom. Here, n is 0 to 20. Y is hydrogen or at least one amide group, substituted amide group, nitrophenyl group, substituted nitrophenyl group, substituted amino group, ester group,
A substituted carboxyphenyl group, a substituted aminoalkylphenyl group, or a substituted aminobenzenesulfonic acid group.

The pyrimidine bases modified in the present invention are:

The C-5 position is substituted, and one representative example is shown by the following structural formula. Uracil and cytosine base derivatives.

The 7-deazapurine base modified in the present invention is:

The C-7 position is substituted, and two representative examples are 7-diazaadenine and 7-diazaguanine base derivatives shown in the following structural formulas.

(Left space below) Regarding the reporter group Sig, at least one reporter group has functional coloring.

It is a fluorescent, diluminescent, radioactive, or Gantt-recognizing reporter group. Examples include fluorescein, rhodamine, acridinium salts, nitrophenyl, dinitrophenyl, benzenesulfonyl, vitamins, luminol, isoluminol, luciferin, dioxecune, dioxamide or biotin, and substitutes or adducts thereof.

Representative examples of fluorescent labeling substances include fluorescein isothiocyanate (FITC), eosin isothiocyanate (EITC), tetramethylrhodamine isothiocyanate (TMRITC), substituted rhodamine isothiocyanate (XRITC), and 7-fluoro-4-nitroxy-2-oxa. -1,3-diazole (NBD
F) etc.

The reporter group Sig is bound to the dideoxy compound via a spacer group R.

In addition, a chelating group such as ethylenediaminetetraacetic acid group was bonded to the dideoxy compound using the reactive amino group of spacer group H, and a substance with long-lived fluorescence such as europium was bonded to it. using things,
By measuring delayed or long-lived fluorescence,
Significantly reduces bank ground and improves signal-to-noise ratio (
It is also possible to perform good measurements of S(ratio). as needed.

The reporter portion may include a magnetic component bound or attached, such as a magnetic oxide or magnetic iron oxide, and thus
Such polynucleotides can be detected by magnetic methods. Furthermore, it is also possible to bind a substance such as biotin and then use a detection system with high detection sensitivity such as a biotin-avidin system.

The labeled dideoxynucleotide derivative of the present invention as described above is produced by the following method.

1) A compound having the following structure, where Y=On and Z=
Oh.

or Y=Otl and Z=H, or Y=H and Z=O
First, a compound in which H is converted into a dideoxy form with y=z·II.

2) The dideoxy compound is reacted with a mercury salt in a suitable solvent to produce a mercury compound having the following structure.

(Left below) 3) The mercury compound and the chemical group represented by R are reacted in the presence of a palladium catalyst to produce a compound having the following structure. Here, N is a reactive terminal functional group or Si
It is g.

4) When N is not Sig, the compound is reacted with Sig to synthesize a compound having the following structure.

The modified nucleotide of the present invention synthesized by the above method can be used to produce a specific DNA or RNA molecule by using a polynucleotide and/or oligonucleotide labeled thereby as a probe and utilizing a hybridization reaction.

In particular, it is possible to determine the presence of specific DNA or RNA molecules of biological origin, such as for example 5 viruses, bacteria, fungi, yeast or mammals. It can also be used for diagnosis of genetic diseases, etc. As an alternative to fluorescence methods for visualization of hybridized probes, peroxidase, alkaline or acid phosphatase, or β-
Directs enzymes such as galactosidase to the site of hybridization.

Therefore, it is also possible to enzymatically convert a soluble substrate into an insoluble colored precipitate for detection.

Furthermore, since the modified nucleotides of the present invention can be selectively labeled only on the 3"-side of polynucleotides and/or oligonucleotides, the labeled polynucleotides and/or oligonucleotides can be labeled using the Maxam-Gilbert method. It is also possible to subject it to gene sequence analysis using methods such as.

When using the modified nucleotide of the present invention as a label for gene sequence analysis, there are many different detection methods for detecting labeled molecules separated by molecular weight (length) by polyacrylamide gel electrophoresis. Basically, the device described in Japanese Patent Application Laid-Open No. 60-220860 can be used.

Furthermore, existing equipment or improved versions thereof can be used to detect labeled molecules separated using HPLC. Typical detection methods are shown below.

1) Detection of fluorescence excited by different wavelengths of light for different dyes.

2) Detection of fluorescence excited by light having the same wavelength for different dyes.

3) Elution of molecules from the gel and detection by chemiluminescence.

4) Detection by absorption of light by molecules.

5) Magnetic detection by magnetism.

6) Detection with affinity substance.

The method for detection is not limited to the one exemplified here.
It is obvious that various methods are included.

Examples of fluorescent materials have already been described, but they may also be combined with chemiluminescence-exciting agents, such as dioxyethanedione, and passed directly to a detector that can measure the emitted light. In this case.

It does not require an excitation light source, the background signal is much lower than in fluorescence, and the signal-to-noise ratio (
(S/N ratio) becomes high, and sensitivity improves. Furthermore, although the method using absorbance is simple in terms of equipment, it has the drawback of essentially low sensitivity. A method using an affinity substance is a relatively simple detection method.

Another use of the modified nucleotides of the invention is to develop or provide a range of chemical agents with specific biological effects, including therapeutic and cytotoxic effects. in general,
Dideoxy derivatives of nucleotides have cell growth inhibiting activity, and some are also used as anticancer agents. As part of the reporter of the present invention, it is also possible to actively bind toxins, rather than substances for detecting the presence of substances as already mentioned. Such nucleotide derivatives either inhibit the synthesis of DNA and/or RN tl in the body or cells, or they attack and actually kill the cancer.

Otherwise, they can be introduced or administered into a human body or animal so as to be incorporated into the DNA and/or RNA components of the body or cells to inhibit unwanted cell growth.

(Left below) (Examples) The examples described below are illustrative of various aspects of the invention, but are in no way intended to limit the scope specifically stated in the claims.

Fluorescently or biotinylated 2' according to Figure 1
, 3'-dideoxy-5-(3-amino)-allyluridine-5''-triphosphate will be described.

100Tn1 of 10mmo+ 2°-deoxyuridine
was dissolved in anhydrous pyridine, 15 mmol of dimethoxytrityl chloride (DMTr-CI) was added, and the mixture was heated at room temperature.

Stirred for 18 hours. After diluting the solution with 500-chloroform, 0. Extracted with IN sodium bicarbonate. The chloroform phase was dried and evaporated to dryness under reduced pressure.

2''-deoxy-5''-0-dimethoxytrityl uridine was obtained by silica gel column chromatography using a chloroform-methanol stenope gradient. The yield was 83%.

The synthesis of dideoxy compounds shown in the following sections (1-2) to (1-5) is carried out by the method of Llorwitz et al. (J.

P., lorwitz, et al., Nucl.
eosides, nucleotides + nails, 3
045 (1962)).

2′-deoxy-5″ obtained in this Example (1-1)
9.5 mmol of methanesulfonyl chloride (MsCl) was slowly added dropwise to a solution of 7 mmol of -0-dimethoxytrityluridine in pyridine under ice cooling.

After reacting for 24 hours under water cooling, 1 ml of water was added to stop the reaction. This was slowly dropped into 800 ml of ice water, and the resulting precipitate was filtered off, washed with water, and air-dried.

Dissolve the pale yellow product in a small amount of acetone and redissolve.

Slowly drop it into ice water and filter out the colorless precipitate 2.
Recovered. The yield was 85%.

2′-deoxy-3′-0-mesyl-5°-0-dimethoxytrityl uridine obtained in section (1-2) 1, 2
mmo 1, 0.24M potassium-tert-
After adding 101nl of a dimethyl sulfoxide solution of butoxide (KOtBu) and reacting at room temperature for 1 hour, the mixture was dropped into 250ml of ice water. After neutralization with dilute acetic acid, the precipitate was centrifuged, washed with water, and air-dried. To 15-m of this dissolved in ethanol, 80-m of benzene was added and concentrated under reduced pressure.

Water was completely removed azeotropically. Benzene was further added and the mixture was concentrated under reduced pressure again, and the resulting precipitate was recrystallized from ethanol. The yield was 80%.

(1-4) Anvil processing of 2°,3”-dideoxy-2°-uridinene - 5′-O-dimethoxytrityl-2′,3′-dideoxy- obtained in section (1-3) 2゛-uridinene 1 m
20 mol of chloroform solution under water cooling, 1.
Slowly add 1 part dry hydrogen chloride gas under stirring,
Detritylation was carried out for about 30 minutes under water cooling. The generated precipitate was filtered, washed with chloroform, and then recrystallized from benzene-ethanol (90:10v/v). The yield was 60%.

(1-5) People with 2', 3'-'iboxyuridine:
2゛,3''-dideoxy-2 obtained in section (1-4)
゛-Uridinene 0.5 mmol dioxane? Liquid 25
-, 115 μm of 10% palladium-carbon catalyst was added, and the mixture was reduced at 25° C. for 1 hour under 1 atmosphere of hydrogen pressure.

After the reaction, the catalyst was filtered off, and the filtrate was dried under reduced pressure.

The residue was recrystallized from benzene-petroleum ether.

The yield was 80%.

The triphosphorylation shown in sections (1-6) and (1-7) below was performed using the method of Ru5sell et al. (J, Org, Chem
, 8(12), 4889 (1969)).

To 20 d of anhydrous tetrahydrofuran (T) solution containing 1 mmol of 2',3'-dideoxyuridine obtained in section (1-5), 4 mmol of triimidazole phosphinisochloride and 100 μl of 1,1'-carbonyl were added. Diimidazole was added, and the mixture was stirred overnight at room temperature to react. Then, triethylamine 2 and water 5-
was added and stirred for an additional 30 minutes.

350-DEAE-5ephadex A-25(H
CO3-type) and linear gradient end (0-0
, 2M) At 41? I was able to get over it. The main peak eluate was evaporated to dryness under reduced pressure and then azeotroped with methanol to desalt.

The yield was 55%.

Phosphorylation can also be performed in a first order manner.

5-1.2 mmol of phosphorous phosphochloride in anhydrous dioxane, 2.4 mmol of (1-11
)-1,2,4-triacylide and 2.0 mmol
of triethylamine was mixed and stirred at 10-15°C for 30 minutes and at room temperature for 1 hour. To this, 2',3'-dideoxyuridine l mmo obtained in section (1-5)
1 was added and stirred at room temperature for 1 hour. To this was added 5 ml of water under water cooling, and the 9 reaction mixture was stirred at room temperature for 18 hours. The reaction solution was purified by silica gel column chromatography eluting with a step gradient of chloroform-methanol. It was converted into the sodium salt by stirring in Dowex 50 (Na'' type) and 10 d of water at room temperature for 4 hours. The yield was 55%.

Triphosphorylation of 2′,3″-dideoxyuridine-sea monophosphate was performed according to the method of D. E. Hoard et al. (J. Am. Chem. Soc., a7
(8), 1785 (1965)).

Anhydrous tributylammonium salt of 2',3'-dideoxyuridine-5'-monophosphate obtained in section (1-6) 0.1 mmol of cimitylformamide (
A DMF solution 1- containing 0.5 mmol of 1.1''-carbodiimidazole was added to the DMF) solution 1-, and the mixture was reacted at room temperature for 5 hours.
After stirring at room temperature for 0 min, an onp solution of 0.5 mmol of tributylammonium pyrophosphate 5Pn
1 was added, and the mixture was stirred at room temperature overnight. DM the generated precipitate
After washing with F, the supernatant was treated with an equal amount of methanol, and concentrated under tJji pressure. The residue was treated with 60-DEAE-
It was charged to Sephadex A-25 (HCO+- type) and eluted with linear gradient (0-0.4M) 11 of triethylamine carbonate buffer (pH 7.5). The main peak eluate was dried under reduced pressure and then azeotroped with methanol to desalt it. The yield was 75%.

5-Mercurylated-2',3'-dideoxyuridine-5'-triphosphate was prepared by the method of Dale et al. (Bio
chemistry.

Kai, 2447-2457 (1975)).

2",3"-dideoxyuridine-5"-triphosphate 11!1Illol obtained in section (1-7) was added to 0.1M sodium acetate buffer (pH 6,0).
100-, 5 mmol of mercury acetate was added thereto, and the mixture was reacted at 50°C for 4 hours. Thereafter, 9 mn+ol of lithium chloride was added to the solution under water cooling, and the mixture was extracted several times with ethyl acetate to remove mercuric chloride. When the amount of mercury in the ethyl acetate extract was measured using 4,4"-bis(dimethylamino)thiobenzophenone, the mercury conversion rate of nucleotides was 90 to 95%. Then, 3 times the amount of mercury was added to the aqueous phase. Water-cooled ethanol was added to form a precipitate, which was centrifuged to recover the precipitate. This precipitate was washed with water-cooled ethanol 9 times, then with diethyl ether, and then air-dried. The yield was 93%.

Allyl amination of 5-mercury-2'', 3'-dideoxyuridine-5''-triphosphate was carried out by Berg
The method of Strom et al. (J, Hachim, Chem, Soc
, 98.1587 (1976)).

5-Mercurylation-2°, 3′-dideoxyuridine-5′-triphosphate obtained in section (1-8) 0.5
mmol in 0.1 M sodium acetate buffer y-(
Dissolved in pl(5,0)25-. To this was added 3-2M aqueous solution of allylamine acetate, and then 4-potassium palladium chloride (KzPdC14) dissolved in water was added.
．． 5 mmol was added. The solution gradually turned black, and metallic mercury precipitated on the walls of the vessel. After reacting for 24 hours at room temperature, one reaction solution was filtered with a 0.45μ membrane filter.
Metal mercury was removed. The filtrate was treated with 100-DEAR-Se.
Charge phadexA-25 and add 0.1M sodium acetate buffer (pif 5.0) 10
After washing with triethylamine carbonate buffer (pH
7,5) linear gradient end (0,1M → 0.6M
) It was eluted at 1 f. The main beak eluate was dried under reduced pressure and then azeotroped with methanol to desalt it. Next, this eluate was fractionated and analyzed by 005 (C-18) reverse phase high performance liquid chromatography using 0.5M triethylamine carbonate buffer (pH 7.5). The yield was 65%.

2',3'-dideoxy-5 obtained in section (1-9)
0.1 mmol of -(3-amine)-ruuridine-5'-triphosphate was added to 0.1 M carbonate buffer (
pH 9,0) Dissolve in 20ml and add 20mg/-
Add 2n11 of a DMF solution of XRITC and incubate at room temperature.
Stir for an hour.

Made me react. Add 3Qml of DEAE-5e to the reaction solution.
Charge phadex^-25 (IIco3-type) and add 500 mN of linear gradient (0.1 M → IM) of triethylamine carbonate buffer (pH 7.5).
It was eluted with Main peak elution fraction (0.6-0.7
(eluted at a salt concentration of M) was dried to solidification under reduced pressure, and then azeotropically distilled with methanol to desalt. The yield was 55%.

2",3"-diodeoxy- obtained in section (1-9)
0.1 mmol of 5-(3-amino)-allyluridine-5'-triphosphate in 0.1 M sodium carbonate buffer (pH 8.5), 0.2 mmol of N-hydroxylsuccinimide biotin ester
1 of DMF solution was added and reacted for 4 hours at room temperature.Then, 1 reaction solution was added to 30- of DEAE-Sephad.
ex A-25 (HCO3-type) and eluted with linear gradient (0.1M-0.9M) 5001n1 of triethylamine carbonate buffer (pH 7.5). The main peak elution fraction with a salt concentration of around 0.6M was dried under reduced pressure, and then azeotroped with methanol to desalt it. The yield was 60%.

Fluorescently labeled or biotinylated 2' according to Figure 2.
, 3'-dideoxy-5-(3-amino)-allylcytidine-5'-triphosphate will be described.

10 mmol of 2°-deoxycytidine was dissolved in 100-anhydrous pyridine + 15 mmol of dimethoxytrityl chloride was added, and the mixture was stirred at room temperature for 18 hours. After diluting the solution with 500-chloroform.

Extracted with 0, IN sodium bicarbonate. The chloroform phase was dried and evaporated to dryness under reduced pressure. 2"-deoxy-5"-
〇-dimethoxytritylcytidine was obtained. Yield is 87%
Met.

The synthesis of dideoxy compounds shown in sections (2-2) to (2-5) below is carried out by the method of Horwitz et al. (J.

P. Hortmitz, et al., Nucl.
eosides, nvcleotides.

Rev. 3045 (1962)).

2′-deoxy-5′ obtained in Nijimoto Example (2-1)
9.5 mmol of methanesulfonyl chloride was slowly added dropwise to a solution of 7 mmol of -0-dimethoxytritylcytidine in 20- of pyridine under ice cooling. After reacting for 24 hours under water cooling, 1 ml of water was added to stop the reaction. Slowly drop this into sooml of ice water.

The obtained precipitate was filtered, washed with water, and air-dried. Dissolve the pale yellow product in a small amount of acetone and again.

Slowly drop it into ice water in step 11, and filter out the colorless precipitate.
Recovered. The yield was 80%.

2′-deoxy-3′-0-mesyl-5”-〇-dimethoxytritylcytidine obtained in Section (2-2) 1.21
1111101, 0.24M potassium-ter
Dimethyl sulfoxide solution of t-butoxide IQ
Add ml.

After reacting at room temperature for 1 hour, the mixture was dropped into 250ml of ice water. After neutralizing with dilute acetic acid, the precipitate was centrifuged.

After washing with water, it was air-dried. To 15-m of this dissolved in ethanol, 80-m of benzene was added and concentrated under reduced pressure.

Water was completely removed azeotropically. Benzene was further added and the mixture was concentrated under reduced pressure again, and the resulting precipitate was recrystallized from ethanol. The yield was 75%.

5′-0-dimethoxytrityl-2′,3″-dideoxy-2′-cytidinene obtained in section (2-3) 1 m
Add mol of chloroformin at night for 20 d, under water cooling, 1.
1 equivalent of dry hydrogen chloride gas is slowly added under stirring,
Detritylation was carried out for about 30 minutes under water cooling. After filtering the generated precipitate and washing with chloroform.

It was recrystallized from benzene-ethanol (90:10v/v). The yield was 63%.

(2-5) 2', 3'-dideoxycytidine person: Dioxane solution 2 of 0.5 mmol of 2'', 3'-dideoxy-2°-cytidinene obtained in section (2-4)
5-, add 10% palladium-carbon catalyst to 115■
Addition.

Reduction was carried out at 25° C. for 1 hour under 1 atmosphere of hydrogen pressure.

After the reaction, the catalyst was filtered off, and the filtrate was dried under reduced pressure.

The residue was recrystallized from benzene-petroleum ether.

The yield was 80%.

The triphosphorylation shown in sections (2-6) and (2-7) below was performed using the method of Ru5sell et al. (J, Org, C
hem,.

8 (12), 4889 (1969)).

(2-6) 2', 3'-'iboxycytidine-5'-
Input of monophosphate: 1 mmol of 2',3''-dideoxycytidine obtained in section (2-5) in anhydrous tetrahydrofuran (THF)
411o1 of triimidazole phosphinic oxide and 100x of 1,1'-carbonyldiimidazole were added to solution 20-, and the mixture was stirred overnight at room temperature to react. Next, triethylamine 2- and 5- were added, and the mixture was further stirred for 30 minutes.

350-DEAR-5ephadex A-25(l
IcO3- form) and linear gradient end (0-
0,2M) 4 I! It was eluted with The main peak eluate was evaporated to dryness under reduced pressure and then azeotroped with methanol to desalt. The yield was 61%.

Phosphorylation can also be performed in a first order manner.

1,2 mmol 1 of phosphorous phosphochloride, 2.4 mmol of (1
-H)-1,2,4-triacylide and 2.0 mm
Mix 3 ol of triethylamine and heat at 10-15℃.
Stirred for 0 minutes and then at room temperature for 1 hour. To this (2-5)
1 mm of 2”,3”-dideoxycytidine obtained in Section
ol was added and stirred at room temperature for 1 hour. Under water cooling,
Water was added and the two reaction mixtures were stirred at room temperature for 18 hours. The reaction solution was purified by silica gel column chromatography eluting with a Stetsop gradient of chloroform-methanol. Dowex50 (Na” type)
and 10- in water at room temperature for 4 hours to convert it into a sodium salt. The yield was 55%.

Triphosphorylation of 2", 3'-dideoxycytidine-5"-monophosphate is D, E, 1load
This was carried out using the method of et al. (mentioned above).

Add 0.1 mmol of the anhydrous tributylammonium salt of 2', 3'-dideoxycytidine-5'-monophosphate obtained in section (2-6) to 1 d of DMF solution.

1.1-carbodiimidasol 0,5mmo
o of l? 8 liquid l- of IF' was added and reacted at room temperature for 5 hours. Add 0.8111 moI of methanol,
After stirring for 0 min at room temperature, 5 ml of a DMF solution of 0.5 mmol of tributylammonium picophosphate
was added and stirred at room temperature overnight. DMF the generated precipitate
After washing, the supernatant was treated with an equal amount of methanol,
Concentrate under reduced pressure. Add 5Qml of the residue to DEAE-3ep
Charge hadex＾-25 (IICO3-type),
Elution was carried out with 1 l of a linear gradient (0-0.4 M) of triethylamine carbonate buffer (ptl 7.5). The main peak elution needle was dried under reduced pressure and then azeotroped with methanol to desalt it. The yield was 85%.

Rainbow 5-mercurylated-2',3'-dideoxycytidine-5'-triphosphate was prepared by the method of Dale et al. (Bio
-chemistry, 44.2447-2457
(1975)).

0.1 mmol of 2', 3'-dideoxycytidine-5'-triphosphate obtained in section (2-7) was added.
1M sodium acetate buffer (ptl 6.0) 10
0mj! 5 mmol of mercury acetate was added thereto, and the mixture was reacted at 50° C. for 4 hours. Thereafter, 9 mmol of lithium chloride was added to the solution under water cooling, and the mixture was extracted several times with ethyl acetate to remove mercuric chloride. The amount of mercury in the ethyl acetate extract was 4.4゛-bis(dimethylamino)
When measured using thiobenzophenone, the mercuryation rate of the nucleotide was 90 to 95%. Next, 3 times the amount of water-cooled ethanol was added to the aqueous phase to form a precipitate, which was centrifuged to collect the precipitate.

This precipitate was washed with water-cooled ethanol, then with diethyl ether, and then air-dried. The yield was 90%.

5-Mercurylation-2°, 3°-dideoxycytidine-5'
- Allyl amination of triphosphate was performed by Berg
The method of Strom et al. (J, Hachim, Chem, Soc
, 98.1587 (1976)).

5-Mercurylated-2゛,3゜-dideoxycytidine-5゛-triphosphate obtained in section (2-8) 0.5m
mol in 0.1M sodium acetate buffer (pH 5
,0) dissolved in 25-. To this was added 3 ml of a 2M allylamine acetate aqueous solution, and then 0.5 ml of potassium palladium chloride (K, PdC1n) dissolved in 4-water was added.
mmol was added. The solution gradually turned black, and metallic mercury precipitated on the walls of the vessel. After reacting at room temperature for 24 hours, one reaction solution was filtered through a 0.45μ membrane filter.

Metal mercury was removed. The filtrate was treated with 100-DEAE-3e.
Charge the phadexA-25 and set it to 0.1? I
Sodium acetate buffer y - (pH 5,0) 100m
After washing with triethylamine carbonate pamphlet (pH7
,5) linear gradient end (0,1M-0,6M)
It was eluted with 11.

The main peak eluted bone was dried under reduced pressure and then azeotroped with methanol to desalt it. This eluate was then subjected to ODS
(C-18) In reverse phase high performance liquid chromatography, 0
．． 5M triethylamine carbonate buffer (ρ117.5
) was used for fractionation and purification. The yield was 60%.

(2-xoB -hy2゛3'-゛ideoxy-5
Input of -(3-amino)-alcytidine-5゛-to1 phosphate: 2',3''-dideoxy-5 obtained in section (2-9)
0.1 mmol of -(3-amino)-lucitidine-5'-triphosphate was added to 0.1 M carbonate buffer (
pH 9,0) Dissolved in 20td and added 20mg/m
! (7) Add 2 ml of the BITCODMF solution to the solution at 2 temperatures and stir for 4 hours.

Made me react. The reaction solution was diluted with 30-DEAE-Seph.
It was charged to adexA-25 (HCO, - type) and eluted with a linear gradient (0.1M-IM) 500- of triethylamine carbonate buffer (pH 7.5). The main peak eluted bone (eluted at a salt concentration of 0.6 to 0.7 M) was dried under reduced pressure and then azeotroped with methanol to desalt it. The yield was 53%.

2゛,3''-dideoxy-5 obtained in section (2-9)
-(3-Amine)-To a solution of 0.1 mmol of lucitidine-5'-triphosphate in 10 mt' of O, 1M sodium carbonate buffer (pFf 8.5), 0.2 mmol of N-hydroxylsuccinimide biotin ester was added.
1 of DMF solution 2- was added and allowed to react at room temperature for 4 hours.
Charge dexA-25 (lIcO3-type) and linear gradient end (0.1M-0.9M) of triethylamine carbonate buffer (987,5) 500#! 1! in? I was able to get a 6. The main peak elution fraction near the salt concentration of 0.6j was dried under reduced pressure.

Then, it was azeotroped with methanol to desalt it. The yield was 62%.

Fluorescently labeled or biotinylated 2” according to Figure 3.
, 3'-dideoxy-7-(3-amino)-allyl-
A method for synthesizing 7-diazaguanosine-5''-triphosphate will be explained.

2' shown in the following sections (3-1) and (3-2).

Synthesis of dideoxy-7-zoazag-7nosine.

The method of Winkeler et al. (J, Org, Chem.
26.3119 (1983)) by phase transfer glycosylation.

3 mmol of 2-amino-4-methoxy-7H-pyrrolo(2,3-d)pyrimidine was dissolved in 20 ml of benzene-dimethoxyethane (4:1) containing 0.3 mmol of methyltrioctylammonium chloride and 50% sodium hydroxide. The mixture was emulsified by stirring in 20rn1 of the aqueous solution for 30 minutes. One drop of a benzene solution containing 4.3 mmol of 1-chloro-2,3-dideoxy-5-0-1-luo-erythrobentofuranose (30 mmol) was added to the mixture under stirring to cause a reaction. This suspension was diluted with 100% water and dichloromethane.

The organic phase was concentrated under reduced pressure. This was dissolved in 203ml of methanol and treated with concentrated aqueous ammonia at room temperature for 24 hours. It was concentrated under reduced pressure, and the aqueous phase was adjusted to pH 4 with hydrochloric acid. The precipitated p-toluoic acid was removed by filtration. The filtrate was concentrated to dryness under reduced pressure. This was suspended in 100 methanol and refluxed for 10 minutes. Inorganic salts were filtered off, and the filtrate was concentrated under reduced pressure. Residue in 30-methanol? 8, charged to a 100 column silica gel column, and eluted with dichloromethane-methanol (95:5). The yield was 45%.

2-Amino-7-(2) obtained in this Example (3-1)
',3''-dideoxy-β-D-erythropentofuranosyl)-4-methoxy-7H-pyrrolo(2,3-d
) 1.5 pyrimidine O1 was suspended in 15 ml of anhydrous toluene, and 2.6 m of p-sodium-thiocresolate was suspended in 15 ml of anhydrous toluene.
mol and 2.6 mmol of hexamityl phosphor triamide were added. This was refluxed under nitrogen for 3 hours. Add 5Qml of water to this.

Washed twice with 50° each of chloroform. The aqueous phase was adjusted to p) 12 with 2N hydrochloric acid, and further extracted twice with 30-chloroform each. After neutralizing the aqueous phase, under reduced pressure.

It was concentrated by evaporation. This was recrystallized from 10-m water.

The yield was 88%.

The triphosphorylation shown in sections (3-3) and (3-4) below was performed by a chemical method according to the method of Ru5sell et al. (supra).

2°,3”-dideoxy-7 obtained in section (3-2)
-To a solution of 1 mmol of diazaguanosine in anhydrous tetrahydrofuran (THF), 4 n+mol of triimidazole phosphinic oxide and 100 mmol of triimidazole phosphinic oxide are added.
Add 1'-carbonyldiimidazole and leave at room temperature.

The reaction mixture was stirred overnight. Next, 1 ml of triethylamine and 5 ml of water were added, and after further stirring for 30 minutes, 350. d DEAE-Sephad
ex A-25 (1ICO3-type) and eluted with a linear gradient (0→0.2M) 41 of triethylamine carbonate buffer (pH 7,5). Main peak? The volume was evaporated to dryness under reduced pressure and then azeotroped with methanol to desalt. The yield was 48%.

Phosphorylation can also be performed in a 5-order manner.

1.2 mmol of phosphorous phosphochloride, 2.4 mmol of (1-H
)-1,2,4-) rear silide and 2. Mix 1 mol of triethylamine and heat at 10-15℃ for 30 minutes.
The mixture was stirred at room temperature for 1 hour. To this was added 1 mmol of 2'',3'-dideoxy-7-deazaguanosine obtained in section (3-2), and stirred at room temperature for 1 hour.To this was added 5 ml of water under water cooling to complete one reaction. The solution was stirred at room temperature for 18 hours.The reaction solution was purified by silica gel column chromatography eluting with a Stetsop gradient of chloroform-methanol.

In Dowex 50 (Na” type) and Long water,
The mixture was stirred at room temperature for 4 hours and converted into a sodium salt. The yield was 58%.

(3-4) 2゛, 3′-゛Ibu Oxy Two Begins Inukyu 2
', 3'-dideoxy-7-diazagua, noson-5'-monophosphate triphosphorylation is D, E
.

This was carried out by the method of Troad et al. (mentioned above).

2”,3”-dideoxy-7 obtained in section (3-3)
Anhydrous tributylammonium salt Q of deazaguanosine-5'-monophosphate was added to 1 ml of a 1 mmol DMF solution at 1.1° -carbodiimidiso-
1 ml of a DMF solution containing 0.5 mmol I was added, and the mixture was allowed to react at room temperature for 5 hours. 0.8 mmo of methanol
After stirring at room temperature for 30 minutes, add triptylammonium pyrophosphate, 5 mmol of DMF? Yong? Drink 5 ml at night.

Stir overnight at room temperature. After washing the generated precipitate with DMF, it was treated with an equal amount of methanol together with the supernatant.

Concentrate under reduced pressure. The residue was added to 6Qml of DEAE-5ep.
Charge hadexA-25 (llcO3-type),
Triethylamine carbonate buffer (pH 7,5) linear gradient (O → 0.4M) 11? I was able to get an 8. The eluate from the main peel was dried under reduced pressure and then azeotroped with methanol to desalt it. The yield was 81%.

2°,3′-dideoxy-7-mercurylated-7-deazaguanosine-5′-triphosphate was prepared by the method of Dale et al. (Biochemistry, 14.2447).
-2457 (1975)).

2'',3''-dideoxy-7 obtained in section (3-4)
-Deazaguanosine-5”-triphosphate 1mm
ol in O, 1M sodium acetate buffer y-(pH 6,
The solution was dissolved in 0.0100 g of water, and 5 mmol of mercury acetate was added thereto.

The reaction was carried out at 50°C for 4 hours. Thereafter, 9 mmol of lithium chloride was added to the solution under water cooling, and the mixture was extracted several times with ethyl acetate to remove mercuric chloride. When the amount of mercury in the ethyl acetate extract was measured using 4.4''-bis(dimethylamino)thiobenzophenone, the mercury conversion rate of nucleotides was 90 to 95%.
A double amount of water-cooled ethanol was added to form a precipitate, which was centrifuged to collect the precipitate. This precipitate was washed with water-cooled ethanol, then with diethyl ether, and then air-dried. The yield was 96%.

Allyl amination of 2′,3″-dideoxy-7-mercurylated-7-deazaguanosine-5′-triphosphate was performed according to the method of Bergstrom et al. (J, Am, C
hem.

Soc, 98.1587 (1976)).

2',3'-dideoxy obtained in section (3-5) = 7
-0.5 mmol of mercurylated-7-deazaguanosine-5'-triphosphate was dissolved in 0.1 M sodium acetate buffer (pH 5,0). 2M for this
Add 31n1 of allylamine acetate aqueous solution of 9 then 4
Potassium palladium chloride (KzP) dissolved in ml of water
dCI4) 0.5 mmol was added. The solution gradually turned black, and metallic mercury precipitated on the walls of the vessel. After reacting at room temperature for 24 hours, the 9 reaction solutions were filtered through a 0.45μ membrane filter to remove metal mercury. 100ml of filtrate
DEAE-Sephadex A-25 with 0.19 sodium acetate buffer (pH
After washing with 100 ml of triethylamine carbonate buffer (pH 7,5), linear gradient end (0
, 1M→0.6M) was eluted at 1p. Main peak eluted bone was dried under reduced pressure, then azeotroped with methanol,
Desalted. Next, this eluate was fractionated using 0DS (C-18) reverse phase high performance liquid chromatography using 0.5M) ethylamine carbonate buffer (pH 7.5).
Purified. The yield was 60%.

2'',3''-dideoxy-7 obtained in section (3-6)
-(3-amino)-allyl-7-diazagua/Schiff -
0.1M of 5'-1-liphosphate 0.1mmol
The mixture was dissolved in 20 ml of carbonate buffer (pH 9,0), and 20 mg/- of TMRITC in DMF solution 2- was added thereto, and the mixture was stirred at room temperature for 4 hours to perform one reaction. The reaction solution was added to 3Qml of D1 AE-Sephadex A-25 (
IIco: l-type).

Linear gradient of triethylamine carbonate buffer (pH 7.5) (0.1M → IM) in 500ml?
I was able to get over it. Main beak eruption bone (0.6~0.7M
(eluted at a salt concentration of ) was dried to dryness under reduced pressure, and then azeotroped with methanol to desalt. The yield was 52%.

2', 3'-dideoxy- obtained in section (3-6)
7-(3-amino)-allyl-7-diazaguanosine-
Add 0.1 mmol of 5'-triphosphate to 10 m2 of 0.1 M sodium carbonate buffer (pH 8.5) solution.

N-hydroxyl succinimide biotin ester 0.
Add 2 ml of 2 mmol DMF solution and incubate at room temperature for 4
React for 2 hours, then add 1 reaction solution to 30 ml of DEAE.
-5 ephadex A-25 (HCO+- type) and triethylamine carbonate buffer (pH 7,5
) linear gradient end (0,1M-0,9M)5
It was eluted at 00ml. The main peak eluted bone with a salt concentration of around 0.6M was dried under reduced pressure, and then azeotroped with methanol to desalt it. The yield was 71%.

Moreover, 7-diazaguanosine BaR form may be synthesized using pre-Q1 nucleoside as a raw material. Pre-Q1 nucleoside has a primary amino group bonded to the 7-position carbon via medilene as a spacer, and is simply 2', 3
It is sufficient to simply use the dideoxy form at the ``-position'' and bond triphosphate to the 5''-position.

It is easy to react and is advantageous for synthesis.

Fluorescently labeled or biotinylated 2' according to Figure 4
, 3'-dideoxy-7-(3-amino)-allyl-7-diazaadenosine-5'-triphosphate will be described.

2゛, 3 shown below from (t-1) to (4-5)
°-dideoxy-7-diazaadenosine (2°, 3″
-dideoxypercidin) synthesis.

K, Anzai et al.'s method (Agr, l3io1.Ch
Em., 37.345-348 (1973)).

200 mj of 10 mmol of tuberculin! of anhydrous pyridine, to which first 20 mmo + dimethoxytrityl chloride was added under stirring at room temperature, and 1
After 8 hours, 10 mmol of dimethoxytrityl chloride was further added, and the mixture was stirred for an additional 3 days to carry out 9 reactions.

Add the reaction solution to ice-cold sodium carbonate solution.

The product was extracted with chloroform and the solution was then evaporated to dryness under reduced pressure. This was recrystallized from ethyl acetate. The yield was 33%.

Elimination of the vicinal diol was performed using the method of E.vedejs et al., Org, Chem, 39 (25),
3641 (1974)).

To a suspension of 1 mmol of 5'-0-dimethoxytrityl percidine obtained in Section (4-1) of this example in pyridine-toluene (1:2) was added 1.1 mmol of N,N'-thiocarbonylbisimidazole. Add 1 mmol and add 2
Stirred at room temperature for an hour. The solvent was removed under reduced pressure and the residue was washed with ether. The thiocarbonate of 5'-0-dimethoxytrityl percidine was recrystallized from methanol. The yield was 78%.

0.7 mmol of the thiocarbonate of 5"-0-dimethoxytritylubercidine obtained in section (4-2) was refluxed in 3o-isopropyl iodide under nitrogen for 5 hours.

The solvent was removed under reduced pressure, and the residue was dissolved in 90% ethanol. 1 g of zinc dust was added thereto, and the mixture was stirred at room temperature for 18 hours. The precipitate was removed and the filtrate was evaporated to dryness under reduced pressure and recrystallized from methanol. The yield was 33%.

Dissolve 0.5 mmol of 5'-0-dimethoxytrityl-2',3'-dideoxy-2'-7-zoazaadenosinene obtained in section (4-3) in dioxane; nightly in 25 ml.

115 μm of 10% palladium-carbon catalyst was added, and the mixture was reduced at 25° C. for 1 hour under 1 atmosphere of hydrogen pressure.

After the reaction, the catalyst was filtered off, and the filtrate was dried under reduced pressure.

The residue was recrystallized from benzene-petroleum ether.

The yield was 80%.

20 ml of chloroform solution of 1 mmol of 5''-0-dimethoxytrityl-2', 3'-dideoxy-2'-7-diazaadenosine obtained in section (4-4)
1.1 equivalents of dry hydrogen chloride gas was stirred under water cooling,
The mixture was added slowly and detritylation was carried out for about 30 minutes under water cooling. The generated precipitate was filtered, washed with chloroform, and then recrystallized from benzene-ethanol (90:10 v/v). The yield was 67%.

The triphosphorylation shown in sections (4-6) and (4-7) below was performed according to the method of Ru5sell et al. (supra).

It was done by chemical method.

2゛,3''-dideoxy-7 obtained in section (4-5)
- Diazaadenosine 1 mmol anhydrous tetrahydrofuran (TIIF)? 8 solution 20-, 4 mmol of triimidazole phosphinisochloride and 10
Add 0 parts of 1.1'-carponyldiimidal and leave at room temperature.

The reaction mixture was stirred overnight. Next, triethylamine 2d and water 5- were added, and after further stirring for 30 minutes, 350-DEAR-Sephadex A-25 (
llCO*-type) and linear gradient end (0
-0.2M)4β. The main peak eluate was evaporated to dryness under reduced pressure and then azeotroped with methanol to desalt. The yield was 58%.

Phosphorylation can also be performed in a 5-order manner.

1.2 mmol phosphorous phosphochloride, 2.4 mmol (
1-H)-L2,4-triacylide and 2.0 mm
Mix 3 ol of triethylamine and incubate at 10-15 °C.
Stirred for 0 minutes and then at room temperature for 1 hour. To this (4-5)
1 mmol of 2', 3'-dideoxy-7-diazaadenosine obtained in Section 1 was added, and the mixture was stirred at room temperature for 1 hour. Water 5- was added to this under ice cooling, and the two reaction solutions were stirred at room temperature for 18 hours. The reaction solution was subjected to silica gel column chromatography using a step gradient of chloroform-methanol? Moriyama Seshime refined it.

Dowex 50 (Na" type) and io- were stirred in water at room temperature for 4 hours to convert to the sodium salt. The yield was 53%.

2”,3′-dideoxy-7-deazaadenosine-5
゛-Triphosphorus M conversion of monophosphate is D, E.

This was carried out by the method of 11oard et al. (mentioned above).

2', 3'-dideoxy- obtained in section (4-6)
0.1 mmol of anhydrous tributylammonium salt of 7-diazaadenosine-5゛-monophosphate [)MF
) Yong? In the evening, 1 ml of a DMF solution containing 0.5 mmol of 1,1'-carbodiimidasol was added, and the mixture was allowed to react at room temperature for 5 hours. After adding 0.8 mmol of methanol and stirring at room temperature for 30 minutes, 0.5 mmol of tributylammonium pyrophosphate [1MF solution 5
- was added, and the mixture was stirred at room temperature overnight. DM the generated precipitate
After washing with F, the supernatant was treated with an equal amount of methanol and concentrated under reduced pressure. The residue was dissolved in 6Qml of DEAE-Se.
Phadex A-25 (IICO, - type) was charged and triethylamine carbonate buffer (pi(7,5)
It was eluted with Linear Gradient (0-0,4M) 31. The main beak eluate was dried under reduced pressure and then azeotroped with methanol to desalt it. The yield was 81%.

(4-8) 2', U゛-°iboxy-2 ≦ He says to the person who is Z2 So 2 Kojeng esfuate J: 2'', 3'-dideoxy-7-mercury-7- Deazaadenosine-5'-triphosphate was synthesized according to the method of Date et al. (supra).

2°, 3′-dideoxy-7 obtained in section (4-7)
-Diazaadenosine-5”-triphosphate l m
7-mol in 0.1M sodium acetate buffer (pH 6
,0)100m1! 5 mmol of mercury acetate was added to this.

The reaction was carried out at 50°C for 4 hours. Thereafter, 9 mmol of lithium chloride was added to the solution under water cooling, and the mixture was extracted several times with ethyl acetate to remove mercuric chloride. When the amount of mercury in the ethyl acetate extract was measured using 4,4'-bis(dimethylamino)thiobenzophenone, the mercury conversion rate of nucleotides was 90 to 95%. Then add 3 to the aqueous phase.
Add twice the amount of water-cooled ethanol.

A precipitate was formed, which was centrifuged to collect the precipitate. This precipitate was washed with water-cooled ethanol and then with diethyl ether, and then air-dried.

The yield was 89%.

(4-9) 2°, 3°-'iboxy-(3-amino)-allyl-7--'azaadenosine 2'', 3'-dideoxy-7-mercurylated-7-deazaadenosine-5 ′
=Allyl amination of triphosphate is performed by Berg
This was carried out according to the method of Strom et al. (mentioned above).

2°, 3゛-dideoxy-
0.5 mmol of 7-mercurylated-7-deazaadenosine-5'-triphosphene was dissolved in 25 ml of O.LM sodium acetate buffer (pH 5,0). 2 for this
Add 3- of allylamine acetate aqueous solution of M and then 4 m
Potassium palladium chloride (KzPdC) dissolved in l of water
la) 0.5...mol was added. The solution gradually turned black, and metallic mercury precipitated on the walls of the vessel. After reaction for 24 hours at room temperature.

The reaction solution was filtered using a 0.45μ membrane filter to remove metal mercury. Pour the filtrate into 100 ml of DEAE
-Charge Sephadex A-25 and O
, LM sodium acetate buffer (1)H5,O)
After washing with 1001111.

Triethylamine carbonate buffer (pH 7,5) linear gradient (0,1M-0,6M) at 11?
I was able to get over it. The main peak eluate was dried to dryness under reduced pressure.
Then, it was azeotroped with methanol to desalt it. Next, this eluate was fractionated and purified by 0DS (C-18) reverse phase high performance liquid chromatography using 0.5M 1-ethylamine carbonate buffer (pl+7.5). Yield is 6
It was 1%.

2', 3'-dideoxy- obtained in section (4-9)
7-(3-amino)-allyl-7-diazaadenosine-
5° - 0.1 mmol of triphosphate was dissolved in 20 - O, 1M carbonate buffer (pH 9,0), and 2
A DMF solution of 0 mg/- FITC (2rn1) was added, and the mixture was stirred at room temperature for 4 hours to perform one reaction. 30m of reaction solution
1 DEAE-3ephadexA-25 (tlcO3
- type) and a linear gradient end (0, IM → I
M) eluted at 500-.

The main beak eluate was dried at a salt concentration of 0.6 to 0.7 M and then azeotroped with methanol.

Desalted. The yield was 58%.

2', 3'-dideoxy- obtained in section (4-9)
7-(3-amino)-allyl-7-diazaadenosine-
5”-triphosphini) 0.1 mmol O, 1M
Sodium carbonate buffer (pH 8,5) solution 10-.

N-hydroxyl succinimide biotin ester 0
, 2mmol I of DMF solution 2- was added, and the mixture was incubated at room temperature for 4
The reaction solution was allowed to react for 1 hour, and then the reaction solution was mixed with 30 DEAE-
Charge Sephadex^-25 (HCO, - type) and linear gradient end (0.1M → 0.9M) of triethylamine carbonate buffer (pH 7.5) 50
It was eluted at 0-. The main peak eluted bone with a salt concentration of around 0.6M was dried under reduced pressure.

Then, it was azeotroped with methanol to desalt it. The yield was 63%.

Example 4 of DNA. Using FITC-fluorescently labeled 2',3'-dideoxy-7-(3-amino)-allyl-7-diazaadenosine-5'-triphosphate obtained in section (4-10), DNA was labeled under the following conditions.

100mM sodium cacodylate (pH 7,2), 8m
M Mgct2゜1mM 2-mercaptoethanol, 1
00μg/ml DNA and 0.5mM FITC
Labeled-2',3'-dideoxy-7-(3-amine)-allyl-7-diazaadenosine-5''-triphosphate was added with 1 unit of terminal deoxynucleotidyl transferase and incubated at 37°C. Allow to react for 1 hour.

A fluorescently labeled dideoxy compound was attached to the 3°-end of the DNA. After the reaction was completed, the fluorescently labeled DNA was separated by gel filtration using Sephadex G100.

From the fluorescence intensity of FITC, it was found that one fluorescent label per DNA molecule was labeled.

g6: Biotin labeling of DNA Example 4. Using biotin-labeled 2'',3''-dideoxy-7-(3-amino)-allyl-7-diazaadenosine-5''-triphosphate obtained in section (4-11), DNA labeling was performed under the following conditions.

100mM sodium cacodylate (pH 7, 2), 8
mM MgC1z.

lff1M2-mercaptoethanol, 100μg/
m1DNA and 0.5mM biotin labeling-2
', 3'-dideoxy-7-(3-amino)-allyl-7-diazaadenosine-5''-triphosphate.

1 unit of terminal deoxynucleotidyl transferase was added and reacted at 37°C for 1 hour.
A biotin-labeled dideoxy compound was attached to the 3'-end of the DNA. After the reaction, Sephadex G1
Biotin-labeled DNA was separated by gel filtration at 0.00.

Biotin-labeled DNA was transferred onto a nitrocellulose filter by lpg, lopg, 1100p, lng,
A biotin-avidin detection kit manufactured by Amajam was used to spot and fix 10 ng, 1000 g and 1 μg each.

The detection sensitivity was investigated using As a result, we were able to clearly distinguish those with spots above TOPG, but
Those with lpg spotted could only barely be distinguished, and therefore the detection sensitivity was estimated to be several pg/sub.

(Effects of the Invention) According to the labeled dideoxyribonucleotide derivative, which is a modified nucleotide of the present invention, a gene can be selectively labeled only on its 3''-side.

Moreover, simple and highly accurate marking is possible.

Therefore, polynucleotides and/or oligonucleotides labeled with the modified nucleotides of the present invention are
They can be useful probes in determining the presence of specific DNA or RNA molecules or in diagnosing genetic diseases, and can also be used in gene sequence analysis.

Additionally, one modified nucleotide of the present invention can be used to actively bind a toxin to a portion of its reporter.

This opens up prospects for the development of chemical agents 2, such as anticancer agents, with specific biological effects.

4. Sting's ζ゛-now
1 is a schematic diagram showing the synthesis of 5-(3-amine)-allyluridine-5°-triphosphate.

Figure 2 shows the labeling of the present invention -2'',3''-dideoxy-
1 is a schematic diagram showing the synthesis of 5-(3-amino)-allylcytidine-5′-triphosphate.

Figure 3 shows the labeling of the present invention -2',3'-dideoxy-
7-(3-amino)-allyl-7-diazaguanosine-
Figure 2 is a schematic diagram showing the synthesis of 5'-triphosphate.

FIG. 4 is a schematic diagram showing the synthesis of labeled-2',3'-dideoxy-7-(3-amino)-allyl-7-diazaadenosine-5''-triphosphate of the present invention.

that's all

Claims (1)

[Claims] 1. A labeled modified nucleotide having the following general formula: P-S-B-R-Sig, where P is a phosphate moiety, S is a sugar moiety, B is a nucleobase moiety, Sig is a chemical moiety covalently bonded to the nucleobase moiety B, and R is a chemical moiety covalently bonded to the nucleobase moiety B and the chemical moiety Sig
the phosphate moiety P is bound to the 5'-position of the sugar moiety S; the sugar moiety S is the spacer moiety that binds the 2'
- and 3'-positions are hydrogen in the dideoxy type; the nucleobase moiety B is a pyrimidine derivative or a 7-deazapurine derivative, and when the nucleobase moiety B is a pyrimidine derivative, the N-1 position is Therefore, the B is also 7-
In the case of deazapurine derivatives, depending on the N-9 position,
When the nucleobase moiety B is a pyrimidine derivative, the spacer moiety R is bonded to the 1'-position of the sugar moiety S;
and, if said B is a 7-deazapurine derivative, to its 7 position; and said chemical moiety Sig can signal itself when bound to said nucleobase moiety B, thereby self-detect or make its presence known. 2. In the general formula, the chemical moiety Sig is the nucleobase moiety B, and when the nucleobase moiety B is a pyrimidine derivative, at the C-5 position, and when the B is a 7-deazapurine derivative; The modified nucleotide according to claim 1, wherein is linked at the C-7 position. 3. In the general formula, the chemical moiety Sig is selected from the group consisting of an electron-dense component, a magnetic component, an enzyme component, a hormone component, a vitamin component, a radioisotope component, a metal-containing component, a fluorescent component, and an antigen-antibody component. The modified nucleotide according to claim 1, which is composed of a component. 4. Polynucleotide and/or oligonucleotide labeled using at least one labeled modification reagent having the following general formula: P-S-B-R-Sig where P is a phosphate moiety , S is a sugar moiety, B is a nucleobase moiety, Sig is a chemical moiety covalently bonded to the nucleobase moiety B, and R is the nucleobase moiety B and the chemical moiety Sig
the phosphate moiety P is bound to the 5'-position of the sugar moiety S; the sugar moiety S is the spacer moiety that binds the 2'
- and 3'-positions are hydrogen in the dideoxy type; the nucleobase moiety B is a pyrimidine derivative or a 7-deazapurine derivative, and when the nucleobase moiety B is a pyrimidine derivative, the N-1 position is Therefore, the B is also 7-
In the case of deazapurine derivatives, depending on the N-9 position,
When the nucleobase moiety B is a pyrimidine derivative, the spacer moiety R is bonded to the 1'-position of the sugar moiety S;
or, if said B is a 7-deazapurine derivative, at its 7 position; and said chemical moiety Sig is a chemical group containing at least one carbon atom; It can signal itself, thereby self-detecting itself or making its presence known. 5. In the general formula, the chemical moiety Sig is the nucleobase moiety B, and when the nucleobase moiety B is a pyrimidine derivative, at the C-5 position, and when the B is a 7-deazapurine derivative; The polynucleotide and/or oligonucleotide according to claim 4, wherein is linked at the C-7 position. 6. In the general formula, the chemical moiety Sig is selected from the group consisting of an electron-dense component, a magnetic component, an enzyme component, a hormone component, a vitamin component, a radioisotope component, a metal-containing component, a fluorescent component, and an antigen-antibody component. 5. The polynucleotide and/or oligonucleotide according to claim 4, which is composed of the following components.