JP5526443B2 - Novel nucleoside derivative, polynucleotide containing the same, and method for identifying base using the same - Google Patents

Description

  The present invention relates to a novel nucleoside derivative, a polynucleotide containing the derivative, and a base identification method using the same.

Various methods have been developed so far to identify the type of nucleobase. For example, there are a method of identifying a base type based on the presence or absence of hybridization using a base-specific probe, and a method of identifying a base type based on the presence or absence of gene amplification using a base-specific primer.
Probe nucleic acids whose fluorescence intensity varies depending on the base to be paired have also been reported (Non-patent Documents 1 and 2). These probe nucleic acids are also expected to be used for single nucleotide polymorphism (SNP) detection and identification of gene silencing sites (sleeping sites). In particular, the latter is very useful and attracts attention for epigenetic research. However, in order to know whether the gene is activated or asleep, it is necessary to be able to distinguish the presence or absence of methylation, that is, the discrimination between a base called methylcytosine and a base called cytosine. In order to determine the presence or absence of cytosine methylation, a method of detecting by cytosine hybridization with a probe after treatment with a reagent such as bisulfite has been used. No probe nucleic acid has been known that can be used in a method that can detect the presence or absence of methylation only by making soybeans.

Okamoto A. et al, J Am Chem Soc. 2004; 126 (15): 4820-7. Rajendar B. et al. Bioorg Med Chem Lett. 2007; 17 (13): 3682-5.

  An object of the present invention is to provide a novel nucleoside derivative that can be used for the synthesis of a polynucleotide that can be used as a probe for discriminating the type of base at a polymorphic site or the presence or absence of methylation at a methylation site. .

  The present inventor has intensively studied to solve the above problems. As a result, they succeeded in synthesizing novel nucleoside derivatives, and by using these derivatives, they can be used as probes for discriminating the types of bases at polymorphic sites or the presence or absence of methylation at methylated sites. It has been found that a polynucleotide can be obtained, and the present invention has been completed.

式中、Ｒ¹は水素、炭素数１〜５のアルキル基またはアシル基であり、Ｒ²およびＲ³は独立して、水素、オリゴマー化のためのリン原子を含む置換基、リン酸基または水酸基の保護基である。
（２）（１）に記載のヌクレオシド誘導体のヌクレオシド残基を含むポリヌクレオチド。
（３）（１）に記載のヌクレオシド誘導体を含む、ポリヌクレオチド合成用試薬。
（４）（１）に記載のヌクレオシド誘導体を基質に用いて、ポリヌクレオチドを製造する方法。
（５）（２）に記載のポリヌクレオチドが固定化された、ポリヌクレオチド固定化担体。
（６）多型部位又はメチル化部位を含む標的核酸に対し、該標的核酸に相補的な配列を有し、該多型部位又はメチル化部位に相当する位置に（１）に記載のヌクレオシド誘導体のヌクレオシド残基を含むポリヌクレオチドをハイブリダイズさせ、ハイブリダイズ時の蛍光を測定することによって、多型部位の塩基の種類又はメチル化の有無を識別することを特徴とする、多型塩基またはメチル化の識別方法。 That is, the present invention is as follows.
(1) A nucleoside derivative represented by any one of the following general formulas (I) to (V).

In the formula, R ¹ is hydrogen, an alkyl group having 1 to 5 carbon atoms or an acyl group, and R ² and R ³ are independently hydrogen, a substituent containing a phosphorus atom for oligomerization, a phosphate group or It is a protecting group for a hydroxyl group.
(2) A polynucleotide comprising the nucleoside residue of the nucleoside derivative according to (1).
(3) A polynucleotide synthesis reagent comprising the nucleoside derivative according to (1).
(4) A method for producing a polynucleotide using the nucleoside derivative according to (1) as a substrate.
(5) A polynucleotide-immobilized carrier on which the polynucleotide according to (2) is immobilized.
(6) The nucleoside derivative according to (1), which has a sequence complementary to the target nucleic acid with respect to the target nucleic acid containing a polymorphic site or a methylation site, and which corresponds to the polymorphic site or the methylation site A polymorphic base or methyl, characterized by distinguishing the type of base at the polymorphic site or the presence or absence of methylation by hybridizing a polynucleotide containing a nucleoside residue and measuring fluorescence upon hybridization Identification method.

Since the nucleoside derivative of the present invention emits higher fluorescence when paired with methylcytosine than when paired with cytosine, by using the nucleoside derivative of the present invention, a methyl group is present at the 5-position of the paired base. A slight difference in structure, whether or not there is, can be distinguished by the intensity of fluorescence. Thus, the presence or absence of methylation can be detected without using a reagent such as bisulfite.
In addition, since the fluorescence intensity varies depending on the type of paired base, application to SNP (single nucleotide polymorphism) detection is also expected. Thereby, it is not necessary to prepare a plurality of probes corresponding to the type of paired base, so that the base can be easily identified.

The figure which shows the result of having measured the fluorescence spectrum by hybridizing each oligonucleotide which has an oligonucleotide containing the nucleoside derivative of this invention, and a complementary sequence to it, and each oligonucleotide containing various bases in the position which couple | bonds with this derivative. .

The present invention is described in detail below.
The nucleoside derivative of the present invention is represented by the general formula (I).

In the formula, R ¹ is hydrogen, an alkyl group having 1 to 5 carbon atoms or an acyl group, and R ² is hydrogen, a substituent containing a phosphorus atom for oligomerization, a phosphate group, or a protecting group for a 5 ′ hydroxyl group. R ³ is hydrogen, a substituent containing a phosphorus atom for oligomerization, a phosphate group, or a protecting group for a 3 ′ hydroxyl group.
Here, examples of the protecting group for the 5 ′ and 3 ′ hydroxyl groups include, but are not limited to, a dimethoxytrityl (DMTr) group, a benzoyl group, and an acetyl group.
The phosphoric acid group may be monophosphoric acid, diphosphoric acid, or triphosphoric acid.
The substituent containing a phosphorus atom for oligomerization is not particularly limited as long as it is a substituent that can be condensed with the 5 ′ or 3 ′ hydroxyl group of another nucleotide to form an oligomer. For example, (i-Pr) ₂ And NP (O (CH ₂ ) ₂ CN)-(amidide group).

  The nucleoside derivative of the present invention can be synthesized by the method shown in Examples described later. In addition, although the Example showed the synthesis example of the derivative of the said formula (I), it can obtain by the same method also about the other nucleoside derivative shown by general formula (II)-(V). For example, in the synthesis scheme described in the examples, instead of 2-Bromobenzoic acid, 2-Bromo-1-naphthoic acid, 2-Bromo-1-anthracenecarboxylic acid, 3-Bromo-2-naphthoic acid, or 3 By using -Bromo-2-anthracenecarboxylic acid, nucleoside derivatives represented by the general formulas (II) to (V) can be synthesized.

  A polynucleotide (also referred to as an oligonucleotide) can be synthesized using the nucleoside derivative of the present invention. The polynucleotide synthesized using the nucleoside derivative of the present invention may be single-stranded or double-stranded, but is preferably single-stranded for use as a nucleic acid probe. The synthesis method is not particularly limited and may be a chemical synthesis method, but a conventional oligonucleotide synthesis method using a DNA synthesizer or the like is preferable.

For example, in the general formulas (I) to (V), a compound having an amidite group at the 3′-position (R ³ ) is used, and the amidite compound corresponding to each of A, T, G, and C bases has a desired sequence. A polynucleotide containing the nucleoside derivative of the present invention (residue portion excluding R ² and R ³ ) can be obtained by sequential binding reaction as obtained.
The length of the polynucleotide is not particularly limited, but is preferably 15 to 50 bases, more preferably 20 to 40 bases. The position of the nucleoside derivative in the polynucleotide is not particularly limited, but it is preferably not located at both ends for use as a nucleic acid probe.

By using the polynucleotide of the present invention, the type of base at the polymorphic site can be identified, and the presence or absence of methylation at the methylated site can be identified.
Here, the polymorphism may be a single nucleotide polymorphism (SNP) or a multibase polymorphism. Further, it may be a base deletion. Among these, single nucleotide polymorphism is more preferable.
The methylation site means cytosine in the CpG sequence.

The procedure for identifying the type of base at the polymorphic site or the presence or absence of methylation is described below.
First, the target nucleic acid containing a polymorphic site or a methylation site has a sequence complementary to the target nucleic acid, and the nucleoside derivative (residue portion) of the present invention at a position corresponding to the polymorphism or methylation site A polynucleotide containing is prepared.
Then, this polynucleotide is hybridized with the target nucleic acid, and fluorescence at the time of hybridization is measured.
As shown in the examples, since the fluorescence intensity varies depending on the type of base at the polymorphic site, the type of base at the polymorphic site can be identified by examining the fluorescence intensity.
Further, since the fluorescence intensity varies depending on the presence or absence of methylation, the presence or absence of methylation can be identified by examining the fluorescence intensity.
The fluorescence measurement is preferably performed by exciting at 382 nm and measuring fluorescence at 400 to 480 nm.

In identifying polymorphism or methylation, a sample such as blood or cell containing the target nucleic acid may be used.
A polynucleotide containing the nucleoside derivative of the present invention is immobilized on a carrier such as a substrate or a bead to constitute a microchip, etc., and this is reacted with a sample such as blood or a cell or a nucleic acid-containing fraction obtained therefrom, By performing hybridization, multiple specimens can be processed.

  The following examples illustrate the present invention more specifically. However, the present invention is not limited to the following examples.

具体的手順を以下に示す。 <Synthesis of nucleoside derivatives>
Compounds 4 to 6 were synthesized according to the following synthesis scheme.

The specific procedure is shown below.

Vacuum-dried 2-Bromobenzoic acid (1.534 g, 7.63 mmol, FW201.02) was dissolved in dry DMF (30 ml) and K ₂ CO ₃ (556 mg, 4.02 mmol, FW138.21), Cu (52 mg, 0.818 mmol, FW63 .55) and 4-Iodobenzenamine (3.31 g, 15.1 mmol, FW219.02) were added and refluxed at 160 ° C. for 2 hours. The reaction mixture was evaporated under reduced pressure, the residue was dissolved in ethyl acetate, washed once with 1N hydrochloric acid, the organic phase was dried over magnesium sulfate, evaporated under reduced pressure, and silica gel column chromatography (Silica gel 60, 70-230 μm, 1% methanol / chloroform). ) To obtain a powdery compound 1. R _f value 0.69 [10% methanol / chloroform]

The yield and various spectra of Compound 1 were as follows.
Yield 2.49g Yield 96%
¹ H NMR (300 MHz, CDCl ₃ ) δ8.03 (1H, d), 7.64 (2H, m), 7.40 (2H, m), 7.02 (1H, d),
7.31 (1H, t), 6.79 (1H, s)
ESI-MS (negative mode) m / z [Attribution]
Found: 338.0, Calc .: 337.98 [(M−H) ⁻ ]

Pyrophosphate (15.3 ml) was added to the vacuum-dried compound 1 (830 mg, 2.45 mmol, FW338.98), and the mixture was refluxed at 90 ° C. for 1 hour. After cooling, ice-cold water was added and suction filtration was performed. The filtrate was extracted with dichloromethane, distilled off under reduced pressure, and purified by silica gel column chromatography (Silica gel 60, 70-230 μm, 0.5% methanol / chloroform) together with the filtered solid to obtain a powdered compound 2. R _f value 0.89 [5% methanol / chloroform]

The yield and various spectra of Compound 2 were as follows.
Yield 624mg Yield 79%
¹ H NMR (300 MHz, DMSO) δ 8.49 (1H, s), 8.21 (1H, d), 7.98 (1H, d), 7.73 (1H, s), 7.54 (1H, d), 7.39 (1H, d), 7.27 (1H, d)
ESI-MS (positive mode) m / z [Attribution]
Found: 322.0, Calc .: 321.97 [(M + H) ⁺ ]

Vacuum-dried compound 2 (1.68 g, 5.23 mmol, FW321.11) to 1,4-anhydro-3,5-bis-O- (tert-butyldimethylsilyl) -2-deoxy-D-erythro-pent-1-enitol (2.76 g, 8.00 mmol, FW344.64) was added and dissolved with degassed dry DMF (45 ml), and AS (ph) ₃ (322 mg, 1.05 mmol, FW306.25) was dissolved in Pd (oAc) ₂
(135 mg, 0.60 mmol, FW224.5) and degassed dry-DMF (15 ml) were added, and the mixture stirred for 30 minutes was added. Bu ₃ N (1.9 ml, 7.91 mmol, FW185.35) was added, and the mixture was refluxed at 90 ° C. for 1 hour, and then stirred in an ice bath for 30 minutes. TBAF (11 ml) and AcOH (1 ml) were added and stirred at room temperature overnight. Saturated multistory water was added, followed by extraction with ethyl acetate, distillation under reduced pressure, and purification by silica gel column chromatography (Silica gel 60, 70-230 μm, 0.5% methanol / chloroform) to obtain a powdered compound 3. R _f value 0.09 [5% methanol / chloroform]

The yield and various spectra of compound 3 were as follows.
Yield 625mg Yield 39%
¹ H NMR (300 MHz, CDCl ₃ ) δ7.59 (7H, m), 5.29 (1H, m), 4.06 (3H, d), 2.87 (1H, d), 2.61 (1H, d)
ESI-MS (positive mode) m / z [Attribution]
Found: 310.0, Calc .: 310.10 [(M + H) ⁺ ]
Found: 332.0, Calc .: 332.10 [(M + Na) ⁺ ]

Compound 3 (613 mg, 1.98 mmol, FW309.32) dried under vacuum was dissolved by adding AcOH (20 ml) and CH ₃ CN (20 ml), and NaBH (OAc) ₃ was added under ice cooling, followed by stirring at room temperature for 1 hour. Saturated multistory water was added, followed by extraction with ethyl acetate, evaporation under reduced pressure, and purification by silica gel column chromatography (Silica gel 60, 70-230 μm, 1% methanol / chloroform) to obtain powdered compound 4. R _f value 0.81 [10% methanol / chloroform]

The yield and various spectra of compound 4 were as follows.
¹ H NMR (300 MHz, CDCl ₃ ) δ8.35 (2H, m), 7.82 (1H, m), 7.68 (1H, m), 7.30 (2H, m), 7.27 (1H, t), 5.26 (1H , m), 4.38 (1H, d), 4.00 (1H, m), 3.73 (2H, d), 2.26 (1H, m), 2.05 (1H, m)
ESI-MS (positive mode) m / z [Attribution]
Found: 334.0, Calc .: 334.12 [(M + Na) ⁺ ]

Compound 4 (327 mg, 1.05 mmol, FW311.33) that had been vacuum-dried was dissolved in dry pyridine (4 ml), DMTr-Cl (500 mg, 1.48 mmol, FW338.83) was added, and the mixture was stirred at room temperature for 2 hours. Further DMTr-Cl (198 mg, 0.58 mmol, FW338.83) was added and stirred overnight. The reaction solution was distilled off under reduced pressure, and the residue was dissolved in dichloromethane and washed with saturated multistory water. The organic phase was then distilled off under reduced pressure, and silica gel column chromatography (Silica gel 60, 70-230 μm, 1: 1: 98 = TEA: methanol : Chloroform) to obtain powdered compound 5. R _f value 0.66 [10% methanol / chloroform]

The yield and various spectra of compound 5 were as follows.
Yield 656mg Yield 100%
¹ H NMR (300 MHz, CDCl ₃ ) δ 8.62 (1H, m), 8.45 (1H, d), 8.38 (1H, s), 7.74 (1H, d), 7.61 (1H, d), 7.45 (1H, d), 7.35-7.31 (6H, m), 7.28-7.16 (4H, m), 6.81 (4H, d), 5.28 (1H, m), 4.43 (1H, s), 4.09 (1H, s), 3.75 (6H, m), 3.33 (2H, d), 2.26 (1H, m), 2.06 (1H, m)
ESI-MS (positive mode) m / z [Attribution]
Found: 637.0, Calc .: 636.25 [(M + Na) ⁺ ]
Found: 653.0, Calc .: 652.25 [(M + K) ⁺ ]

Compound 5 (296 mg, 0.48 mmol, FW613.70) dried under vacuum was dissolved in dry CH ₂ Cl ₂ (2 ml), and DIPEA (210 μl, 1.21 mmol, FW129.29) and (i-Pr) ₂ NP ( Cl) O (CH ₂ ) ₂ CN (236 μl, 1.06 mmol, FW236.68) was sequentially added, and the mixture was stirred at room temperature for 30 minutes. Thereafter, dry MeOH (1 ml) was added and the mixture was further stirred for 30 minutes. After adding ethyl acetate treated with saturated multistory water, washing with saturated multistory water and distilling off under reduced pressure, the residue was purified by silica gel column chromatography (Silica gel 60, 70-230 μm, 1: 1: 98 = TEA: methanol: chloroform), Compound 6 in powder form was obtained by reprecipitation with hexane.

The yield and various spectra of compound 6 were as follows.
Yield 354 mg Yield 90%
¹ H NMR (300 MHz, CDCl ₃ ) δ 9.60 (1H, d), 8.43 (2H, d), 7.74 (1H, t), 7.51 (3H, m,),
7.33 (6H, d), 7.16 (3H, m), 6.79 (4H, d), 5.28 (1H, m), 4.54 (1H, s), 4.25 (1H, s),
3.85 to 3.49 (12H, m), 3.31 (2H, m), 2.61 (1H, m), 2.45 (1H, m), 1.30 to 1.01 (12H, m)
ESI-MS (positive mode) m / z [Attribution]
Found: 836.0, Calc .: 836.35 [(M + Na) ⁺ ]

<Synthesis of oligonucleotides containing derivatives>
Using compound 6, the polynucleotide of SEQ ID NO: 1 was synthesized using a DNA synthesizer.
ODN # X10
5'-ACTGTTGGC X TGTACAATGG-3 '(SEQ ID NO: 1)
X = Acridone derivative (base part of compound 4)

The following oligonucleotides were synthesized as hybridization partners.
cODN # Y11 (Y = A, C, G, T, mC, U, S)
5'-CCATTGTACA Y GCCAACAGT-3 '(SEQ ID NO: 2)
Y = A (adenine), C (cytosine), G (guanine), T (thymine), mC (5-methylcytosine), U (uracil), or S (base deletion; abasic)

<Hybridization experiment>
ODN # X10 was prepared with a phosphate buffer (final concentrations 0.01 M sodium phosphate (pH 7), 0.1 M NaCl, 0.1 mM EDTA · 2Na) to a concentration of 4 μM based on UV-visible absorption (Abs 260 nm). In addition, cODN # Y11 (Y = A, C, G, T, mC, U, S) is phosphate buffer solution (final concentration 0.01M sodium chloride) to a concentration of 4μM respectively based on UV-visible absorption (Abs260nm). phosphate (pH 7), 0.1M NaCl, 0.1 mM EDTA · 2Na). 35 μl of each was mixed and soaked in hot water at 90 ° C. and allowed to stand until it reached room temperature. Thereafter, fluorescence measurement was performed at 25 ° C. (Perkin ELMER LS 55 luminescence (fluorescence / phosphorescence) spectrophotometer, excitation wavelength 382nm)
The results are shown in FIG. According to this, the fluorescence intensity varied depending on the type of base pair, and was in the order of X / A> X / T> X / U> X / mC> X / C> X / G> X / S.

<Measurement of thermodynamic parameters of DNA duplex>
ODN # F10 (F = X, A, C, G, T) and cODN # Y11 (Y = A, C, G, T, mC, U, S) at a concentration of 50 μM based on UV-visible absorption (Abs260 nm) Respectively. 71 μl of ODN # F10 and cODN # Y11 were mixed and soaked in hot water at 90 ° C., and further allowed to stand until it reached room temperature.
Dilute the solution containing the DNA duplex so that the total concentration of ODN is 40, 12, 4, 3.5, 3, 2.5, 2, 1.5, 1 μM, and measure the melting temperature (T _m ) for each. It was. (Measurement temperature 80 → 20 → 80 ℃, temperature decrease / temperature increase rate 0.5 ℃ / min, measurement wavelength 260nm)
Thermodynamic parameters, ΔH (enthalpy change), ΔS (entropy change), and ΔG ₃₇ (free energy change at 37 ° C.) were determined from T _{m at} each ODN concentration (Table 1). As a result, it was found that no matter what base X (acridone derivative) is paired with, the stability of the duplex was not significantly affected. That is, it is considered that ODN forms a stable double chain under the conditions of fluorescence spectrum measurement.

ΔH, ΔS, ΔG _37, and ΔΔG ^* are enthalpy change, entropy change, free energy change at 37 ° C, and thermal stability of each DNA duplex and DNA duplex (F / Y =
C / G) is the difference from the thermal stability.

Claims (6)

A nucleoside derivative represented by any of the following general formulas (I) to (V).

In the formula, R ¹ is hydrogen, an alkyl group having 1 to 5 carbon atoms or an acyl group, and R ² and R ³ are independently hydrogen, amidide group , phosphate group , Dimethoxytrityl group, benzoyl group or acetyl group . is there. A polynucleotide comprising a nucleoside residue of the nucleoside derivative according to claim 1. A reagent for polynucleotide synthesis comprising the nucleoside derivative according to claim 1. A method for producing a polynucleotide using the nucleoside derivative according to claim 1 as a substrate. A polynucleotide-immobilized carrier on which the polynucleotide according to claim 2 is immobilized. 2. A nucleoside residue of the nucleoside derivative according to claim 1, wherein the nucleoside residue of the nucleoside derivative according to claim 1 has a sequence complementary to the target nucleic acid with respect to the target nucleic acid containing the polymorphic site or the methylation site, Identification of polymorphic bases or methylation characterized by hybridizing a polynucleotide containing a group and measuring fluorescence at the time of hybridization to identify the type of base at the polymorphic site or the presence or absence of methylation Method.

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