JPH0435480B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates generally to 2,4-dinitrophenyl nucleotide derivatives. More specifically, the present invention provides 2,4-dinitrobenzene bonded to a portion other than the base of a nucleotide.
4-dinitrophenyl nucleotide derivatives. PRIOR ART Poly or oligonucleotide derivatives, which can be detected by special antibodies or enzymes without the use of radioactive isotopes, are of interest as affinity probes for nucleic acids. In recent years, Vincent et al. have developed a DNA probe in which a 2,4-dinitrophenyl (hereinafter abbreviated as DNP) group is attached to a nucleic acid (Nucl.
Acids Res., 10 6787-6796 (1982)). They use DNP derivatives of adenosine triphosphate (ATP)
incorporated into the DNA strand and has a complementary base sequence
After hybridization with DNA, rabbit antiserum against DNP and sheep antiserum against peroxidase-labeled rabbit immunoglobulin type G (IgG) are sequentially added to detect the target DNA. The DNA strand used here is a fragment extracted from nature. However, according to the knowledge of the present inventors, the DNP-nucleotide derivatives produced in this manner have the following problems. (b) Contains DNP in the base portion of the nucleotide, which causes a change in the melting temperature (Tm value) specific to the oligonucleotide used. (b) DNA with an arbitrary and defined base sequence
is difficult to synthesize. For these reasons, the current DNP-nucleotide derivatives have a narrow range of application and limited usefulness. SUMMARY OF THE INVENTION The present invention aims to provide a solution to the above-mentioned problems, and uses a DNP-nucleotide derivative in which 2,4-dinitrobenzene is bonded to a specific site in addition to the nucleotide base of a specific oligodeoxyribonucleotide. The goal is to achieve this goal. Therefore, the DNP-nucleotide derivative according to the present invention is characterized by being a DNP-oligodeoxyribonucleotide represented by the following formula []. [However, m and n are each 0 or any natural number, R ¹ is a divalent linear or branched hydrocarbon residue, and B is a base constituting a nucleotide (if there is more than one B) they may be the same or different). [Effects] The DNP-oligodeoxyribonucleotide synthesized by the present inventors can avoid the disadvantages of the non-radioactive affinity probe for nucleic acids, and
It has the following advantages. (a) Since the base portion of the nucleotide does not contain DNP, it is stable without any change in melting temperature (Tm value). (b) DNP-oligonucleotides with any base sequence can be synthesized. (c) A short oligomer is sufficient as a probe. (d) It is very easy to synthesize, can be synthesized in large quantities, and can be stored for a long time. (e) It can also be used as a primer (DNA fragment during template synthesis). Recently, Itakura et al. have been diagnosing genetic diseases of β-globin using a synthetic oligonucleotide with a chain length of 19 (Proc. Natl. Acad. Sci. USA; 80 , 278
-282 (1983), has shown that synthetic oligonucleotides, which can detect even a single difference in base sequence in a gene, are useful for analyzing various genetic diseases. Although they used a detected radioisotope ( ^32P ) of a synthetic oligonucleotide, it would be obvious that it would be very useful to use the DNP-nucleotide derivatives developed by the present inventors instead. That is, for example, as mentioned above, DNP-oligonucleotide can be useful as a affinity probe for non-radioactive nucleic acids or as a primer, and its detection methods include antibody precipitation, enzyme immunoactivity assay, and fluorescence. The DNP-nucleotide derivatives of the present invention can be visualized in various ways, such as by optical staining, and the DNP-nucleotide derivatives of the present invention are also useful compared to radioactive probes ( ^32P ) in terms of risk of exposure, cost, waste disposal, and storage stability. The DNP group can be used with commercially available rabbit antiserum (e.g. Miles Laboratories, Code No. 61-006-
1) Alternatively, it can be easily detected with a monoclonal antibody against DNP. Because of these advantages, the present invention
It is also conceivable to expand the usage of DNP-nucleotide derivatives. DETAILED DESCRIPTION OF THE INVENTION DNP nucleotide derivative [ ] The DNP nucleotide 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'-hydroxyl groups of 2'-deoxyribonucleoside, and specifically the following structure belongs to. 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 DNP oligonucleotide derivative of the present invention is expressed as m+n because fractions having 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~
6, especially 1 to 4, n is practically 0 to 40, especially 0
~20. The group R ¹ is a divalent straight-chain or branched hydrocarbon residue that connects the nucleic acid moiety and the DNP moiety of the compound [ ]. 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 [], ie the DNP-nucleotide derivative according to the present invention, can be synthesized by any convenient method. One preferred method is to add 2,
It is characterized in that a DNP-oligodeoxyribonucleotide represented by the following formula [] is obtained by binding 4-dinitrobenzene. [However, m and n are each 0 or any natural number, R ¹ is a divalent linear or branched hydrocarbon residue, and B is a base constituting a nucleotide (if there is more than one B) they may be the same or different). ] That is, in this method, DNP is added to the amino group of the oligonucleotide derivative of the above formula [], in which a primary amino group is introduced into the 5′-terminal phosphate group of the oligonucleotide via the group ^R1 . It consists of combining. On the other hand, the compound of formula [] can be synthesized by a method consisting of synthesizing an oligonucleotide and introducing a primary amino group onto the 5'-hydroxyl group extension of the resulting oligonucleotide. 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 hydrocarbon residue. R ² is a protecting group for the 5′-terminal hydroxyl group, and a dimethoxytrityl 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. R ⁴ A protecting group for the amino group, and a trifluoroacetyl group is usually used. q Any natural number smaller than n. m 0 or any natural number. n 0 and 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). A carrier via a spacer, which is usually one of the following. (p is the degree of polymerization of styrene) Synthesis of Compound [ ] Generally, oligonucleotide synthesis methods include triester method, phosphite method, and their respective solid phase and liquid phase methods. The present inventors have already established a technique for producing oligonucleotides using a solid phase method, and the following method of the present inventors is preferable for the synthesis of compound []. Tetrahedron Letters 1979 , 3635 (1979) Nucleic Acids Research 8 , 5473 (1980) Nucleic Acids Research 8 , 5491 (1980) Nucleic Acids Research 8 , 5507 (1980) Nucleic Acids Research Symposium Series
7, 281 (1980) In addition, the oligonucleotide synthesized above
As a method for introducing a primary amino group into a 5'-hydroxyl group via a phosphoric acid group, that is, a method for synthesizing compound [], there is, for example, the method described in Japanese Patent Application No. 138136/1983 by the present inventors. The method for synthesizing the compound [] in one embodiment is as follows. That is, as shown in Figure 1, by condensing the compound [] with the protecting group R ³ removed and the compound [] with the protecting group R ² removed, and repeating these operations, Then, compound [] is synthesized. The method for synthesizing the oligonucleotide compound [] is known as described above. On the other hand, a compound of formula [] is synthesized according to the method of the present inventors (see Japanese Patent Application No. 57-138136). That is, by removing R ² of the compound []
An amino alcohol compound R in which a 5'-hydroxyl group compound is phosphorylated by the action of a phosphorylating agent (for example, phosphoditriazolide, phosphodichloride, or phosphodibenzotriazolide, etc.), and then the amino group is protected. ² −NH−R ¹ −OH
[This compound is an omega-amino alcohol ( _NH2
-R ¹ OH) by protecting the amino group with R ² ], the compound [ ] can be obtained (see the specification for details). The protecting group R ³ of this compound [] is removed and the compound [] is condensed with the compound [] from which the protecting group R ² has been removed to synthesize the compound []. The condensation can be carried out essentially in the same manner as the condensation used in the synthesis of compound []. By removing all the protecting groups of the compound [] synthesized in this way, the compound [] can be obtained. The protecting group CO, the ortho-chlorophenyl group in the phosphotriester, and the acyl group in the base moiety were replaced with 0.5M tetramethylguanidine-pyridine-2-carbaladoxime dioxane-water (9:1, (V/V)). ) solution and then removed by alkaline treatment (concentrated aqueous ammonia). When R ⁴ is a trifluoroacetyl group,
Treatment with ammonia can sufficiently remove the group, but in the case of an orthonitrophenylsulfenyl group, treatment with meltcaptoethanol is required. When another protecting group is used as R ⁴ , it is also possible to add another treatment under conditions where the oligonucleotide moiety is stable. Various methods for synthesizing oligodeoxyribonucleotides are already known, and details other than those mentioned above regarding the types of protecting groups, their introduction or removal, condensation, etc., can be found in books and reviews on chemical synthesis of nucleic acids, such as "Nucleoside. "Synthesis of Nucleotides""Maruzen1977)"
"Nucleic acid organic chemistry" (Kagaku Doujin 1979), "Nucleic acids" (Asakura Shoten 1979), Tetrahedron, 34 , 3143 (1978),
Unionization, 34 , 723 (1978) and Chemistry Area, 33 ,
566 (1979) etc. Synthesis of compound [] DNP-oligodeoxyribonucleotide (compound []) can be obtained by bonding 2,4-dinitrobenzene to the primary amino group on the 5'-terminal extension of the above compound []. can. The bond between the two is 1 of 2,4-dinitrobenzene.
It can be carried out by any method capable of realizing the formation of a C--N bond between the - position and the amino group of the compound []. The bonding between the two is generally through deH-X condensation between the former derivative, ie, DNP-X (X is a 1-substituent), and an amino group. As X, halogen is preferable. Preferred derivatives in which X is halogen, i.e. 1-halogeno-2,4-dinitrobenzene, generally do not react with the amino group of the base portion of the oligonucleotide, but only with the primary amino group on the 5'-terminal extension. This is because it reacts selectively and the reaction operation is simple. In particular, 1-fluoro-2,4-dinitrobenzene is commercially available and easily available, and the reaction with the amino group of the compound [] proceeds under mild reaction conditions. The reaction between 1-halogeno-2,4-dinitrobenzene and the compound [] can be carried out in a homogeneous solution (the solvent is, for example, a hydrous alcohol) or a heterogeneous solution (the solvent is, for example, water), using a hydrogen halide scavenger. (e.g., sodium bicarbonate, triethylamine, potassium hydroxide, etc.)
It can be carried out at a temperature of about 50°C. The desired product may be recovered, for example, by extraction.
Regarding DNP conversion, please refer to appropriate reviews such as "Experimental Chemistry Course, Chemistry of Proteins, p. 118"
(published by Maruzen Co., Ltd. in 1976), etc. Experimental Example 1 Flow Chart A compound of the present invention (compound shown in the figure) was produced according to the flow chart shown in FIG. In Figure 2, the symbols have the following meanings: B Benzoylated Adenine B Adenine DMTr Dimethoxytrityl R ⁰ orthochlorophenyl Et ethyl CE-cyanoethyl m 2 n 12 2 Synthesis experiment 1-1 of compound [] (shown in Figure 2) Dimethoxytrityladenosine/resin []
(Although the resin is just a carrier, the target compound supported on the resin looks the same as the resin itself, so the compound supported on the resin will be simply referred to as resin below.) 300 mg (0.033 mmol) )
isopropanol-methylene chloride (15:85,
After washing 3 times with 10 ml of V/V) 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 15 mg (0.1 mmol) of the dinucleotide [] was added thereto, and the system was made anhydrous by azeotroping, and mesitylenesulfonyl nitrotriazolide ( below
MSNT) 150 mg (0.5 mmol) and 2 ml of anhydrous pyridine are 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,
(3.45 μmol) was detritylated in the same manner as above [], and 60 mg of the compound []
(0.04 mmol) was treated (decyanoethylated) with 3 ml of triethylamine-pyridine-water (1:3:1, V/V) solution [ ] to make it anhydrous, and then 50 mg (0.2 mmol) of MSNT and 1 ml of pyridine were added. was added and reacted (condensed) for 90 minutes. After the reaction was completed, the mixture was washed with pyridine and methanol and dried to obtain a completely protected oligonucleotide derivative [ ]. Oligonucleotide derivative [] 15mg to 0.5M
Tetramethylguanidine-pyridine-2-carbaladoxime in dioxane-water (9:1, (V/
V) Add 200 μl of the solution and incubate for 24 hours at room temperature in a centrifuge tube. After reaction, add concentrated ammonia water (2.5ml)
Add, seal, and react at 50℃ 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 was 0.05M
triethylammonium bicarbonate buffer PH7.5)
Desalt and purify pentadecaadenylic acid derivative []
I got it. Experiments 1-2, 1-3 and 1 were also carried out in the same manner.
-4-like oligonucleotide derivatives were obtained.
Table 1 shows the compounds synthesized above. [Table] However, in this table, A is adenine, T is thymine,
G represents guanine and C represents cytosine. The results of high performance liquid chromatography of these four compounds are shown in FIG. A to D are diagrams for compounds of Experiments 1-1 to 1-4, respectively. 3 Production experiment 2-1 of 2,4-dinitrophenyl-pentadecaadenylic acid [] Approximately 1.0OD of the pentadecaadenylic acid derivative [] synthesized in Experiment 1-1 above was added to 10 μl of 0.1M aqueous sodium bicarbonate solution (PH8.3) 1-fluoro-2,4-dinitrobenzene in ethanol solution (50 mg/ml) (5 μl (large excess)) and reacted at 37°C for 2 hours. Added 30 μl of water and diluted with 150 μl of ether.
Removal of the reagents yields 2,4-dinitrophenyl-pentadecaadenylic acid. The reaction was confirmed by high performance liquid chromatography. At that time, in order to compare the reactivity, the oligonucleotide synthesized above was deprotected and obtained.
A compound having a 5'-hydroxyl group [] is similarly reacted with 1-fluoro-2,4-dinitrobenzene (control experiment 3-1). Compounds [] synthesized in Experiments 1-2, 1-3, and 1-4 are also subjected to the same operations as in Experiment 2-1 to produce each compound []. 2-2, 2-3, 2-4 of this experiment, respectively.
shall be. In addition, for reaction comparison, a compound [] with a 5'-hydroxyl group was also prepared, and the compound [] and 1-
and fluoro-2,4-dinitro-benzene. The experiments at this time are Experiment 3-2,
3-3 and 3-4. The compounds prepared in Experiment 2 and Control Experiment 3 are shown in Table 2. [Table] However, in this table, A is adenine, T is thymine,
G represents guanine and C represents cytosine. The above results are shown in FIG. 4 (results of high performance liquid chromatography). FIG. 4 shows the elution pattern of high performance liquid chromatography. In the figure, 1 is a chromatogram of the compound itself before reaction, and 2 is a chromatogram of the compound reacted with 1-fluoro-2,4-dinitrobenzene. A is experiment 3-
1 is a compound of formula [], B is a compound of formula [] in Experiment 2-1, and C is a compound of formula [] in Experiment 3-2.
D is the compound with formula [] in Experiment 2-2, E is the compound with formula [] in Experiment 3-2, F is the compound with formula [] in Experiment 2-3, and G is the compound with formula [] in Experiment 3. -4 shows a chromatogram of a compound of the formula [], and H shows a chromatogram obtained when the above operation was performed on a compound of the formula [] in Experiment 2-3.
Note that the numerical value above the peak indicates the retention time. From these results, 5′− shown in Eq.
Compounds with hydroxyl groups (I-1, H-1 in Figure 4,
Ho-1 and To-1) are 1-fluoro-2,4
- It can be seen that there is no reaction with dinitrobenzene (Fig. 4 I-2, H-2, H-2, and T-2).
2). On the other hand, oligonucleotide derivatives []
When it is reacted with 1-fluoro-2,4-dinitrobenzene, the elution pattern of high performance liquid chromatography changes, and the peaks of the raw material (Figure 4 Row-1, Ni-1, He-1 and Chi-1) change. -1) has disappeared, and new compounds (Figure 4 Ro-2, Ni-2, He-2 and Chi-2) have been formed by reacting with 1-fluoro-2,4-dinitrobenzene. I understand. That is, a compound having a primary amino group []
is a compound that selectively reacts with 1-fluoro-2,4-dinitrobenzene and has a 5'-hydroxyl group []
It turns out that there is no reaction at all. The peaks shown in FIG. 4 that are eluted at a retention time of about 5 minutes are considered to be 2,4-dinitrophenol. In the above, high performance liquid chromatography uses JASCO HPLC System Tri-Roter.
Measurements were carried out under the following conditions. Column: μ-Bondapak C18 (Waters) Flow rate: 2ml/min Eluent: 20mM- containing acetonitrile
TEAA buffer (PH7.2) Concentration: Acetonitrile concentration 6-14%/16
minutes (continue 14% after 16 minutes)

[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 method for producing the compound of the present invention shown in experimental examples. FIGS. 3A to 3D are diagrams showing the results of high performance liquid chromatography of the compound [] shown in the experimental example. FIG. 4 is a diagram showing the elution pattern of high performance liquid chromatography.

Claims (1)

[Scope of Claims] 1. A 2,4-dinitrophenyl nucleotide derivative, which is a 2,4-dinitrophenyl oligodeoxyribonucleotide represented by the following formula []. [However, m and n are each 0 or any natural number, R ¹ is a divalent linear or branched hydrocarbon residue, and B is a base constituting a nucleotide (if there is more than one B) they may be the same or different). 2. The 2,4-dinitrophenyl nucleotide derivative according to claim 1, wherein base B is selected from the group consisting of adenine, thymine, cytosine and guanine. 3. The 2,4-dinitrophenyl nucleotide derivative according to claim 1 or 2, wherein R ¹ 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.
2,4-dinitrophenyl nucleotide derivative according to any one of paragraphs.