JPH0374239B2 - - Google Patents

Description

BACKGROUND OF THE INVENTION Technical Field The present invention generally relates to novel oligonucleotide derivatives. More specifically, the present invention relates to oligonucleotide derivatives in which a primary amino group is introduced onto the 5'-terminal phosphate group of a nucleotide via a spacer of appropriate length. PRIOR ART In recent years, the scientific synthesis of nucleic acids has progressed dramatically through the introduction of new protecting groups and the development of new condensation methods such as the triester method and the phosphite method. In addition, with the rapid progress of genetic engineering, the scientific synthesis of nucleic acids has become important in this field. For example, useful substances are produced by synthesizing artificial genes and using genetic recombination (interferon: Nature, 281 ,
544 (1979), leukocyte-derived interferon:
Nature, 287 , 411 (1980)). In addition, probes for hybrid methods (Nucl, Acids Res. 9 , 897
(1981)), template DNA necessary for synthesizing double-stranded DNA from mRNA or single-stranded DNA using reverse transcriptase or DNA polymerase.
There are also examples of its application, such as its use as a complementary DNA fragment (primer) (Nucl. Acids Res. 8 , 4057 (1980)). In this way, organic chemical synthesis means for nucleic acids enable the synthesis of oligonucleotides with special sequences that cannot be isolated from living organisms, making a significant contribution to research in molecular biology, genetic engineering, and the like. The present inventors have so far synthesized various oligonucleotides using the solid phase method as a powerful synthesis method in the field of organic chemical synthesis of oligonucleotides, and have investigated its application. As a result of intensive efforts to develop resins for E and non-radioactive affinity probes, we discovered oligonucleotide derivatives that are useful intermediates for these structures. Affinity chromatography resins that have been developed or commercially available to date (Arch.
Biochem.Biophys., 168 , 561 (1974), J.
Biochem., 83 , 783 (1978), JP-A-52-25795,
No. 53-101396, No. 53-133283 and No. 55-
No. 36277) generally have poor specificity and reproducibility, and the synthesis is troublesome. Non-radioactive affinity probe (Proc. Natl.
Sci. USA. 78 , 6633-6637 (1981)),
There are problems such as difficulty in synthesizing cytosine derivatives and difficulty in synthesizing DNA with arbitrary and defined base sequences. In addition, the documents listed below in the synthesis of affinity resins have drawbacks such as the time-consuming process of synthesizing the ligand. J. Chromatog., 97 , 33 (1974Biochem.
Biophys. Acta, 304 , 231 (1973) Anal.
Biochem., 71 , 471 (1976) Summary of the Invention The present invention aims to solve the above-mentioned problems by adding a functional group (primary amino group) to a nucleotide that can bind other carriers only to the target product. The aim is to achieve this objective by means of an oligonucleotide derivative which is introduced via a spacer of appropriate length onto the 5'-terminal extension. Therefore, the oligonucleotide derivative according to the present invention is characterized by being represented by the following formula []. In addition, the method for producing the oligonucleotide derivative represented by the following formula [] according to the present invention includes protecting group R ² of the amino group on the 5'-end extension of the compound represented by the following formula [], COR of the 3'-end It is characterized by removing all of the protecting groups for ^{the 4} groups, the base moiety, and the phosphoric acid moiety. [However, m and n are each 0 or any natural number, R ¹ is a divalent straight-chain or branched hydrocarbon residue, and B is a base constituting the nucleotide (B and R ⁰ are (If there are more than one, they may be the same or different). [Effects] The oligodeoxyribonucleotides synthesized by the present inventors can avoid the disadvantages of the aforementioned affinity chromatography resins or non-radioactive affinity probes for nucleic acids, and have the following advantages. be. (b) Affinity resins and probes with any base sequence can be produced. (b) It is very easy to synthesize and can be synthesized in large quantities. (c) Since the oligonucleotide has a general amino group that is more reactive than other functional groups (hydroxyl group, phosphate group, amino group in the base moiety, etc.), the deprotected oligonucleotide can be used without purification. It can be used for condensation with a carrier. That is, it is possible to selectively bond to the amino group moiety by setting reaction conditions and the like. (d) Labeling substances such as carriers, biotin, haptens, enzymes, fluorescent substances, chemiluminescent substances, etc. can be selectively bound via the primary amino group, and it can be applied to non-radioactive affinity probes and primers. . DETAILED DESCRIPTION OF THE INVENTION Oligonucleotide derivative [] The oligonucleotide derivative according to the present invention is represented by the above formula []. In the formula, the symbol [Formula] is commonly used to indicate a deoxyribonucleoside residue excluding the 3'- and 5'-hydrogen groups of 2'-deoxyribonucleoside, and specifically, the following It is of structure. [Formula] Therefore, the oligonucleotide derivative according to the present invention is specifically represented by the following formula. Substituent B represents a base constituting a nucleotide, and is usually adenine, thymine, cytosine or guanine. When a plurality of Bs exist in the compound [], they may be the same or different. m and n each represent 0 or a natural number. The degree of polymerization of the oligonucleotide derivative of the present invention is expressed as m+n because fractions with a degree of polymerization of m and n are condensed in the preferred production method of the present invention (details will be described later). In that case, m is practically 0 to 6, especially 1 to 4, and n is practically 0 to 40, especially 0 to 20,
It is. The group R ¹ is the 5′- of the nucleotide part of the compound []
It is a divalent straight or branched hydrocarbon residue that connects the terminal phosphate group and the amino group. In particular, a linear or branched alkylene group having about 2 to 20 carbon atoms is suitable. Preferred R ¹ is an alkylene group having 2 to 6 carbon atoms. General Synthesis of Compound [] Compound [], the oligonucleotide derivative according to the invention, can be synthesized by any convenient method. One preferred method is to use a protecting group R ² for the amino group on the 5′-end extension of the compound represented by the following formula [],
It is characterized by removing all of the COR ⁴ groups at the 3'-terminus, the base moieties, and the protecting groups of the phosphoric acid moieties. [However, m and n are each 0 or any natural number, and m+n≧1, R ⁰ is a phenyl group usually substituted with a substituent that protects the phosphate group, and R ¹ is a divalent direct is a chain or branched hydrocarbon residue, R ² is a protecting group for the amino group,
The COR ⁴ group is a protecting group for the 3'-terminal hydroxyl group of the nucleotide, B' is a protected base constituting the nucleotide and is protected as necessary, and B is the base constituting the nucleotide. (If there are multiple B′ or B and R ⁰ ,
They may be the same or different). ] That is, this method introduces a primary amino group protected via the group R ¹ into the 5'-terminal phosphate group of the oligonucleotide derivative of the above formula [], that is, the oligodeoxynucleotide, and the base portion of the nucleotide and The phosphate group is protected and the hydrogen atom of the hydroxyl group bonded to the 3'-terminus is replaced with a ^COR4 group, and all of the protecting groups are removed. On the other hand, the compound of formula [] can be synthesized by a method consisting of introducing a protected primary amino group onto the 5'-hydroxyl extension of an oligonucleotide in which other functional groups are protected. FIG. 1 is a flowchart showing an example of this preferred synthetic method. The symbols in the flowchart have the following meanings (for details and meanings, please refer to
(As mentioned below). R ⁰ A substituent that protects the phosphate group, and usually an orthochlorophenyl group is used. R ¹ is a divalent straight or branched hydrocarbon residue. R ² A protecting group for an amino group, and a trifluoroacetyl group is usually used. R ³ is a substituent from which all other protecting groups can be easily removed under stable conditions to give a phosphoric acid diester, and a cyanoethyl group is usually used. COR ⁴ A 3'-hydroxyl protecting group used in conventional oligonucleotide synthesis methods. in particular,
A carrier in which R ⁴ is a lower alkyl group, an aryl group (especially a phenyl group or a methyl-substituted phenyl group), or a suitable spacer used in solid phase synthesis (polystyrene resin, polyamide resin)
There is something that is. R ⁵ A protecting group for the 5′-terminal hydroxyl group, and a dimethoxytrityl group is usually used. m 0 or any natural number. n 0 or any natural number. B indicates a base. B′ indicates a protected base, usually N ⁶ -benzoyladenine, N-isobutyrylguanine,
selected from ^N6 -benzoylcytosine and thymine (ie no protection required). Synthesis of compound [] The compound represented by formula [] can be synthesized by any purposeful method consisting of introducing a protected primary amino group onto the 5'-hydroxyl group extension of an oligonucleotide with other functional groups protected. It can be synthesized by a method. The method for synthesizing compound [] is described in one embodiment (first embodiment).
Figure) is shown below. In Figure 1, the 5'-hydroxyl group compound [0] is phosphorylated by the action of a phosphorylating agent (for example, phosphodithriazolide, phosphodichloride, or phosphobenzotriazolide), and then the amino group is protected. This compound is an aminoalkylene alcohol (NH ₂ −R ¹ −
The compound [ ] is obtained by condensing the amino group of OH) (which can be obtained by protecting the amino group of OH) with R ² . Note that compound [0] is an oligonucleotide and can be produced by a normal oligonucleotide synthesis method. On the other hand, a conventional oligonucleotide synthesis method, preferably the solid phase synthesis method of the present inventors (Tetrahedron
Letters 1979 , 3635 (1979), Nucleic Acids
Research 8 , 5473 (1980), Nucleic Acids Rese.
-arch 8 , 5491 (1980), Nucleic Acids
Research 8 , 5507 (1980), Nucleic Acids
Compound [] is synthesized according to Research Symposium Series 7 , 281 (1980). Compound [] can be obtained by condensing the compound ['] from which the protecting group at the 5' end has been removed with the previously synthesized compound [] using a condensing agent. Examples of the condensing agent include tosyl chloride, mesitylene sulfonyl chloride, mesitylene sulfonyl tetrazolide, and mesitylene sulfonyl nitro triazolide, with mesitylene sulfonyl nitro triazolide being preferred. For details of the reaction conditions, etc., please refer to the experimental examples described later. Synthesis of Compound [] Compound [] can be obtained by removing all the protecting groups from the above compound []. The protecting group COR ⁴ group, the orthochlorophenyl group in the phosphoric triester, and the acyl group in the base part were prepared using dioxane-water (9:1 (V/V) of 0.5M tetramethylguanidine-pyridine-2-carbaladoxime )) After treatment with a solution, it is removed by alkaline treatment (concentrated ammonia water). When R ² is a trifluoroacetyl group, it is sufficiently eliminated by treatment with ammonia, but when it is an orthonitrophenylsulfenyl group, a mercaptoethanol treatment is required. When another protecting group is used as R ² , it is also possible to add another treatment under conditions where the oligonucleotide moiety is stable. In addition, various methods for synthesizing oligodeoxyribonucleods are already known.
For details other than those mentioned above regarding the types of protecting groups, their introduction or removal, condensation, etc., please refer to books and reviews on chemical synthesis of nucleic acids, such as "Synthesis of Nucleosides and Nucleotides" (Maruzen 1977), "Nucleic Acid Organic Chemistry" (Kagaku Doujin 1979) ), “Nucleic Acids” (Asakura Shoten)
1979), Tetrahedron, 34 , 31 (1978), conjugation,
34, 723 (1978) and Area of Chemistry, 33 , 566
(1979) etc. Example Flow Chart A compound of the present invention (the compound shown in the figure) was prepared according to the flow chart in Figure 2. In Figure 2, the symbols have the following meanings: B' Benzoylated Adenine B Adenine DMTr Dimethoxytrityl p indicates the degree of polymerization of polystyrene. R ⁰ Orthochlorophenyl Et Ethyl CE - Cyanoethyl m 2 n 12 Synthesis experiment of compound [] (in Figure 2) 1-1 Dimethoxytrityladenosine/resin []
(Although the resin is only a carrier, the target compound supported on the resin does not differ in appearance from the resin itself, so the compound supported on the resin will be referred to simply as the resin below.) 300 mg (0.033 m
mol) isopropanol-methylene chloride (15:
85, V/V) After washing three times with 10 ml of solution, remove zinc bromide.
1.0M isopropanol-methylene chloride solution 8
ml for 5 minutes each (detritylation) four times to obtain a resin [ ]. The resin [] was washed three times with 10 ml of an isopropanol-methylene chloride solution, and a pyridine solution of 150 mg (0.1 mmol) of the dinucleotide [] was added thereto, and the system was made anhydrous by azeotroping, and mesitylenesulfonyl nitrilotriazolide (hereinafter referred to as
MSNT) 150 mg (0.5 mmol) and 2 ml of anhydrous pyridine were added and reacted (condensed) for 90 minutes.
After the reaction, wash with 10 ml of pyridine three times and remove the catalyst amount (approx.
10mg) dimethylaminopyridine (hereinafter referred to as DMAP)
acetic anhydride-pyridine (1:9, (V/V))
10 ml of the solution is added and reacted for 10 minutes to acetylate and protect the unreacted 5'-hydroxyl group, which is washed with pyridine to obtain compound ['] (n=2). Repeat the above operation 6 times to form a compound []
(n=12) is obtained. On the other hand, 5′-hydroxy-dinucleotide []
800 mg (0.71 mmol) and orthochlorophenylphosphoditriazolide were reacted for 2 hours in a solution of the latter in dioxane (1.0 mmol, 6 ml), followed by trifluoroacetyl-6-aminohexanol.
300 mg (1.4 mmol) and 115 mg (1.4 mmol) of 1-methyl-imidazole are added and the reaction is continued for an additional 2 hours. After the reaction, the solvent was distilled off and the residue was dissolved in chloroform, washed with water, 0.5M aqueous sodium dihydrogen phosphate, saturated aqueous sodium bicarbonate and 5% aqueous sodium chloride, and dissolved with anhydrous sodium sulfate. dry. After concentrating the chloroform layer, it is purified using a silica gel column (using chloroform containing 0 to 4% methanol as an eluent), and the eluate is concentrated and then added dropwise into pentane to obtain a powdery compound []. Compound synthesized above [] (n=12) 115 mg,
(3.45μmol) was detritylated in the same manner as above [], and 60mg (0.04μmol) of the compound []
mol) to triethylamine-pyridine-water (1:
After adding the treated (decyanoethylated) compound [] with 3 ml of 3:1, V/V) solution and making it anhydrous, 50 mg (0.2 mmol) of MSNT and 1 pyridine were added.
ml, reacted (condensed) for 90 minutes, washed with pyridine and methanol after the reaction, dried, and obtained a completely protected oligonucleotide derivative []
get. Oligonucleotide derivative [] 15mg to 0.5M
Tetramethylguanidine-pyridine-2-carbalaldoxime in dioxane-water (9:1,
Add 200 μl of (V/V) solution and place in a centrifuge tube at room temperature for 24 hours.
Allow time to react. After the reaction, add concentrated ammonia water (2.5
ml), seal, and react at 50°C overnight. After the reaction is completed, the solution is filtered, concentrated, dissolved in water, and extracted with ether. After concentrating the aqueous layer,
Sephadex G-50 (φ1.5×120cm, eluent
0.05M triethylammonium bicarbonate buffer PH
7.5) to obtain a pentadecaadenylic acid derivative [ ]. In addition, experiments 1-2, 1-3, 1-
Oligonucleotide derivatives 4, 1-5 and 1-6 were obtained. The base sequences and other details of the compounds of Experimental Examples 1-1 to 1-6 are shown in Table 1. [Table] [Table] However, in this table, A is adenine, T is thymine,
G represents guanine and C represents cytosine. The results of SEPHADEX and high performance liquid chromatography for Experimental Examples 1-1, 1-2 and 1-4 are shown in FIGS. 3-4, 5-6 and 7-8. These results show that the compound [] was produced.

[Brief explanation of drawings]

FIG. 1 is a flowchart showing an example of a method for synthesizing the compound of the present invention. FIG. 2 is a flowchart of the present compound shown in experimental examples. Figures 3, 5 and 7 show the elution patterns of Compound [] (Experimental Examples 1-1, 1-2 and 1-4, respectively) on Sephadex G-50. Figures 4, 6 and 8 show the compound []
(Experimental Examples 1-1, 1-2 and 1-4, respectively)
This figure shows the high performance liquid chromatography elution pattern of .

Claims (1)

[Scope of Claims] 1. An oligonucleotide derivative represented by the following formula []. [However, m and n are each 0 or any natural number, and m+n≧1, R ¹ is a divalent straight-chain or branched hydrocarbon residue, and B is a base constituting a nucleotide. (When a plurality of Bs exist, they may be the same or different.) 2. Claim 1, wherein the base B is selected from the group consisting of adenine, thymine, cytosine, and guanine. Oligonucleotide derivatives as described. 3. The oligonucleotide derivative according to claim 1 or 2, wherein ^R1 is a linear or branched alkylene group having 2 to 20 carbon atoms. 4 m is 0 or a natural number up to 6, n is 0 or
Claims 1 to 3 are natural numbers up to 40.
The oligonucleotide derivative according to any one of the above items.