JP5946170B2 - Deazapurine nucleoside derivatives, deazapurine nucleotide derivatives and polynucleotide derivatives - Google Patents

Description

  The present invention relates to a deazapurine nucleoside derivative in which a fluorescent molecule is introduced into the C7 position of 7-deazapurine nucleoside via a double bond or a triple bond. The present invention also relates to a deazapurine nucleotide derivative including the deazapurine nucleoside derivative, and further to a polynucleotide derivative in which at least one nucleotide is substituted with the deazapurine nucleotide derivative.

Many compounds that change the fluorescence emission wavelength depending on the surrounding polar environment have been reported. There are also reports on similar fluorescent nucleosides.
The inventors of the present application previously also introduced a compound in which a fluorescent molecule is introduced into the C8 position of the purine base via a double bond or a triple bond (a compound represented by the following formula; hereinafter referred to as “C8 position substituted purine base derivative”). Reported that the fluorescence emission wavelength varies greatly depending on the surrounding polar environment (for example, JP 2011-37796 A (Patent Document 1), Tetrahedron Letters 51 (2010) 2606-2609 (non-patent document). Reference 1).)

[Wherein, ring A ¹ and ring A ² are each independently the same or different and are divalent or trivalent aromatic rings, and X ¹ and X ² are each independently independent and are the same or different. , A carbon-carbon double bond (—C═C—) or a carbon-carbon triple bond (—C≡C—), and Y ¹ and Y ² are each independently of each other, the same or different, an acyl group, alkoxycarbonyl group, a carboxylic acid amide group, a cyano group, a thiol group, a halogen atom, a hydroxyl group, a nitro group, a substituent selected from the group consisting of amino group and C ₁ -C ₂₀ hydrocarbon group, m及n is Are each independently the same or different and are 1 or 2, and R ¹ and R ² are each independently the same or different and are a hydrogen atom or a hydroxyl group. ]
Compounds that change the fluorescence emission wavelength due to the difference in the surrounding polar environment can detect changes in the polar environment of the target substance by the difference in color, so it is possible to investigate the probe, protein for gene detection, or the local polar environment in the cell It is expected to be used as a probe for industrial use. For example, by preparing a probe DNA in which a fluorescent nucleoside is introduced on the opposite side of the base to be identified in the DNA, and using the change in the surrounding polar environment due to the difference in match-mismatch with the opposite base, the type of the target base can be determined. Such a compound is useful as a probe for gene detection because it can be identified by the difference in color.
However, in general, when a substituent is introduced at the C8 position of a purine base, the substituent and the sugar-phosphate backbone of the DNA collide, and in addition, the sugar and base of the nucleoside have a syn from the normal anti conformation. It is known that the double helix structure of DNA is destabilized when a nucleoside having a substituent introduced at the C8 position is used, since it tends to invert to the conformation. When the DNA double helix structure is destabilized, the surrounding polar environment changes due to the influence of the destabilization. Therefore, the change in the surrounding polar environment due to the difference in match-mismatch between the fluorescent nucleoside and the facing base cannot be accurately detected. There is a case.
The previous C8-substituted base derivative was excellent in terms of optical characteristics, but in order to put it into practical use as a probe for gene detection with higher accuracy, improvement of the molecule is required.
By the way, Synthesis, June 1996, 726-730 (Non-patent Document 2) describes a compound in which a phenyl group is introduced via a triple bond at the C7 position of 7-deazaadenosine. The present invention relates to a method for synthesizing 7-alkynyl-2′-deoxytubercidin, and no attention is paid to the change in the fluorescence emission wavelength of the compound and the use as a fluorescent probe.

JP 2011-37796 A (Patent Document 1)

Tetrahedron Letters 51 (2010) 2606-2609 Synthesis, June 1996, 726-730 Tetrahedron Letters 52 (2011) 4726-4729

  Under such circumstances, development of a novel fluorescent nucleic acid that does not destabilize the double helix structure of DNA even when introduced into DNA while maintaining the good optical properties of conventional molecules and a probe using the same are required. It has been.

In order to solve the problems of the prior art, the present inventors have developed a novel fluorescent nucleic acid using 7-deazapurine nucleoside having a hydrogen bonding pattern similar to that of adenosine or guanosine, which is a natural nucleoside constituting DNA. Tried. Since the 7th position of adenosine or guanosine is a nitrogen atom, chemical modification at the 7th position is usually impossible. However, in 7-deazaadenosine or 7-deazaguanosine, the 7th position is CH. Chemical modification with is possible. In addition, since position 7 of the purine base is located in the main groove of DNA when introduced into DNA, and a large space is available, destabilization of the double helix structure of DNA as when C8 position is modified Is not expected. Furthermore, the previous compound is designed to introduce electron-donating and electron-withdrawing substituents into the molecule in order to increase the change in fluorescence emission wavelength due to the difference in the surrounding polar environment. The base played the role of an electron donating group. However, as a result of molecular calculations, it was predicted that 7-deazapurine base showed the same degree of electron donating as natural nucleobase. It can be said that this is a preferred molecular design.
Based on these predictions, the inventors of the present application synthesized a compound obtained by introducing an electron-donating fluorescent molecule through a triple bond so as to effectively form a conjugated structure at the C7 position of 7-deazaadenosine. (Tetrahedron Letters 52 (2011) 4726-4729 (Non-Patent Document 3), see the following formula).

However, although this compound can greatly change the fluorescence emission wavelength, it cannot sufficiently stabilize the double helix structure of DNA when introduced into DNA.
Therefore, as a result of further intensive studies, the inventors of the present application have found that when the compound represented by the formula (I) or (II) is introduced into DNA while maintaining the good optical properties of the conventional nucleoside derivative, It has been found that the heavy helical structure is not destabilized, and the present invention has been completed.

That is, the present invention relates to the following deazapurine nucleoside derivatives, deazapurine nucleotide derivatives, polynucleotide derivatives in which at least one nucleotide is a deazapurine nucleotide derivative, and probes containing the polynucleotide derivatives.
[1] The following formula (I) or (II):

[Wherein, ring A ¹ and ring A ² are each independently the same or different and are C _{6 to} C ₁₈ aromatic rings,
X ¹ and X ² are each independently the same or different and are a carbon-carbon double bond (—C═C—) or a carbon-carbon triple bond (—C≡C—),
Y ¹ and Y ² are independent of each other, and are the same or different and are each a C _{1 to} C ₁₀ acyl group, a C _{1 to} C ₁₀ alkoxycarbonyl group, a carboxylic acid amide group (—C (═O) —NR ^a R ^b R ^a and R ^b are each independently the same or different and are a hydrogen atom or a C _{1 to} C ₁₀ alkyl group.), A cyano group, a halogen atom, a nitro group, and a substituent. An amino group (represented by —NR ^a R ^b , where R ^a and R ^b are each independently the same or different and are a hydrogen atom or a C ₁ -C ₁₀ alkyl group), a hydroxyl group, and a thiol group. And a substituent selected from the group consisting of C _{1 to} C ₁₀ alkyl groups,
m and n are each independently the same or different and are 0, 1 or 2,
R ¹ and R ² are each independently the same or different and are a hydrogen atom or a hydroxyl group,
However, the case where ring A ¹ is a phenyl group, X ¹ is a carbon-carbon triple bond (—C≡C—), m is 0, and R ¹ is a hydrogen atom is excluded. ]
A deazapurine nucleoside derivative represented by:
[2] The deazapurine nucleoside derivative according to [1], wherein R ¹ and R ² are hydrogen atoms.
[3] The ring A ¹ and the ring A ² are independent of each other and are the same or different and are selected from the group consisting of a phenylene group, a naphthylene group, an anthrylene group, and a phenanthrylene group, [1] or [2 ] Deazapurine nucleoside derivative of description.
[4] The deazapurine nucleoside derivative according to any one of [1] to [3], wherein the ring A ¹ and the ring A ² are naphthylene groups.
[5] The substituents Y ¹ and Y ² are independent of each other and are the same or different from each other, and are a C _{1 to} C ₁₀ acyl group, a C _{1 to} C ₁₀ alkoxycarbonyl group, a carboxylic acid amide group (—C (═O ) is represented by ^-NR a ^{R b, R a} and ^{R b} are each independently of one another, identical or different, a hydrogen atom or a _C 1 _{-C 10} alkyl group.), a cyano group, a halogen atom, a nitro group and The deazapurine nucleoside derivative according to any one of [1] to [4], which is selected from the group consisting of thiol groups.
[6] The deazapurine nucleoside derivative according to any one of [1] to [5], wherein the substituents Y ¹ and Y ² are cyano groups.
[7] The deazapurine nucleoside derivative according to any one of claims 1 to 4, which is represented by any one of the following formulas I (a) and II (a).

[Wherein, Y ¹ and Y ² are as defined above. ]
[8] The deazapurine nucleoside derivative according to any one of [1] to [7], wherein the fluorescence emission wavelength changes in response to a change in polar environment.
[9] The following formula (III) or (IV):

[Wherein, ring A ¹ and ring A ² are each independently the same or different and are C _{6 to} C ₁₈ aromatic rings,
X ¹ and X ² are each independently the same or different and are a carbon-carbon double bond (—C═C—) or a carbon-carbon triple bond (—C≡C—),
Y ¹ and Y ² are independent of each other, and are the same or different and are each a C _{1 to} C ₁₀ acyl group, a C _{1 to} C ₁₀ alkoxycarbonyl group, a carboxylic acid amide group (—C (═O) —NR ^a R ^b R ^a and R ^b are each independently the same or different and are a hydrogen atom or a C _{1 to} C ₁₀ alkyl group.), A cyano group, a halogen atom, a nitro group, and a substituent. An amino group (represented by —NR ^a R ^b , where R ^a and R ^b are each independently the same or different and are a hydrogen atom or a C ₁ -C ₁₀ alkyl group), a hydroxyl group, and a thiol group. And a substituent selected from the group consisting of C _{1 to} C ₁₀ alkyl groups,
m and n are each independently the same or different and are 0, 1 or 2,
R ¹ and R ² are each independently the same or different and are a hydrogen atom or a hydroxyl group,
s and t are independent of each other, and are the same or different and are 1, 2 or 3,
However, the case where ring A ¹ is a phenyl group, X ¹ is a carbon-carbon triple bond (—C≡C—), m is 0, and R ¹ is a hydrogen atom is excluded. ]
A deazapurine nucleotide derivative represented by:
[10] The deazapurine nucleotide derivative according to [9], wherein R ¹ and R ² are hydrogen atoms.
[11] A polynucleotide derivative obtained by substituting at least one nucleotide in the polynucleotide with the deazapurine nucleotide derivative described in [9] or [10].
[12] A probe comprising the polynucleotide derivative according to [11].

According to the present invention, a novel deazapurine nucleoside derivative obtained by introducing a fluorescent molecule through a double bond or triple bond at the C7 position of 7-deazapurine base, and phosphoric acid is ester-bonded to the deazapurine nucleoside derivative. And a polynucleotide derivative in which at least one nucleotide is substituted with the deazapurine nucleotide derivative.
The preferred deazapurine nucleoside derivative of the present invention can change the fluorescence emission wavelength depending on the difference in the surrounding polar environment. Such a polynucleotide derivative into which the deazapurine nucleoside derivative of the present invention is introduced can detect a change in the surrounding polar environment by a change in color. It can be used as a probe for investigating a typical polar environment.

A graph showing the fluorescence emission wavelength of a solution obtained by dissolving the deazapurine nucleoside derivative (5b) which is an example compound of the present invention in various solvents, and a table showing the fluorescence emission wavelength and the amount of change thereof (Table 1A), Tm value when various complementary strands (ODN2 (N) (N = A, G, C, T)) were added to the oligonucleotide DNA (ODN1 (5b)) containing the deazapurine nucleoside derivative (5b) of the present invention And a table (Table 1B) showing the amount of change. A graph showing the fluorescence emission wavelength of a solution obtained by dissolving the deazapurine nucleoside derivative (5c), which is an example compound of the present invention, in various solvents, and a table showing the fluorescence emission wavelength and its variation (Table 2A), Tm value when various complementary strands (ODN2 (N) (N = A, G, C, T)) were added to oligonucleotide DNA (ODN1 (5c)) containing the deazapurine nucleoside derivative (5c) of the present invention And a table (Table 2B) showing the amount of change. A graph showing the fluorescence emission wavelength of a solution obtained by dissolving the compound (C1) which is a comparative example compound in various solvents, a table showing the fluorescence emission wavelength and the amount of change thereof (Table 3A), and a compound which is a comparative example compound Table showing the Tm value and the amount of change when various complementary strands (ODN2 (N) (N = A, G, C, T)) were added to the oligonucleotide DNA (ODN1 (C1)) containing (C1) ( Table 3B). A graph showing the fluorescence emission wavelength of a solution obtained by dissolving the compound (C2) as a comparative example compound in various solvents, a table (Table 4A) showing the fluorescence emission wavelength and its variation, and a compound as a comparative example compound Table showing the Tm value and the amount of change when various complementary strands (ODN2 (N) (N = A, G, C, T)) were added to oligonucleotide DNA (ODN1 (C2)) containing (C2) ( Table 4B). A graph showing the fluorescence emission wavelength of a solution obtained by dissolving the compound (C3) as a comparative example compound in various solvents, a table (Table 5A) showing the fluorescence emission wavelength and its variation, and a compound as a comparative example compound Table showing Tm value and amount of change when various complementary strands (ODN2 (N) (N = A, G, C, T)) were added to oligonucleotide DNA (ODN1 (C3)) containing (C3) ( Table 5B). A graph showing the fluorescence emission wavelength of a solution obtained by dissolving the compound (C4) as a comparative example compound in various solvents, a table (Table 6A) showing the fluorescence emission wavelength and its variation, and a compound as a comparative example compound Table showing the Tm value and the amount of change when various complementary strands (ODN2 (N) (N = A, G, C, T)) were added to oligonucleotide DNA (ODN1 (C4)) containing (C4) ( Table 6B). 2 is a photograph of fluorescence emission of a solution obtained by dissolving a deazapurine nucleoside derivative (5b), which is an example compound of the present invention, in various solvents.

  Hereinafter, the deazapurine nucleoside derivative, deazapurine nucleotide derivative, polynucleotide derivative, probe and the like of the present invention will be described in detail.

The deazapurine nucleoside derivative of the present invention is a compound represented by the following formula (I) or (II).

[Wherein, ring A ¹ and ring A ² are each independently the same or different and are C _{6 to} C ₁₈ aromatic rings,
X ¹ and X ² are each independently the same or different and are a carbon-carbon double bond (—C═C—) or a carbon-carbon triple bond (—C≡C—),
Y ¹ and Y ² are independent of each other, and are the same or different and are each a C _{1 to} C ₁₀ acyl group, a C _{1 to} C ₁₀ alkoxycarbonyl group, a carboxylic acid amide group (—C (═O) —NR ^a R ^b R ^a and R ^b are each independently the same or different and are a hydrogen atom or a C _{1 to} C ₁₀ alkyl group.), A cyano group, a halogen atom, a nitro group, and a substituent. An amino group (represented by —NR ^a R ^b , where R ^a and R ^b are each independently the same or different and are a hydrogen atom or a C ₁ -C ₁₀ alkyl group), a hydroxyl group, and a thiol group. And a substituent selected from the group consisting of C _{1 to} C ₁₀ alkyl groups,
m and n are each independently the same or different and are 0, 1 or 2,
R ¹ and R ² are each independently the same or different and are a hydrogen atom or a hydroxyl group,
However, the case where ring A ¹ is a phenyl group, X ¹ is a carbon-carbon triple bond (—C≡C—), m is 0, and R ¹ is a hydrogen atom is excluded. ]

  The deazapurine nucleoside derivative of the present invention is designed to introduce a fluorescent molecule through a carbon-carbon double bond or a carbon-carbon triple bond at the C7 position of the 7-deazapurine skeleton. The preferred deazapurine nucleoside derivative of the present invention changes in the fluorescence emission wavelength in response to changes in the surrounding environment, for example, the polar environment, so that it can be used as a probe for identifying changes in the surrounding environment (polar environment) by color change It is.

In the deazapurine nucleoside derivative of the present invention, the pentose linked to the 7-deazapurine base may be 2-deoxyribose or ribose, and R ¹ and R ² are independent of each other, and are the same or different, Or it is a hydroxyl group. When the deazapurine nucleoside derivative of the present invention is introduced into a nucleotide and used as a DNA type probe, R ¹ and R ² are preferably hydrogen atoms, and when used as an RNA type probe, R ¹ and R ² are hydroxyl groups. Is preferred.

X ¹ and X ² are each independently the same or different and are a carbon-carbon double bond (—C═C—) or a carbon-carbon triple bond (—C≡C—). X ¹ and X ² are preferably carbon-carbon triple bonds in order to further enhance the conjugated structure.

Ring A ¹ and Ring A ² are each independently the same or different and are C _{6 to} C ₁₈ aromatic rings. The aromatic ring is not particularly limited as long as it can form a conjugated structure with a deazapurine base via a carbon-carbon double bond or a carbon-carbon triple bond. Examples of such an aromatic ring include a phenylene group, a naphthylene group, an anthrylene group, a phenanthrylene group, and a pyrenylene group. Among these, a phenylene group, a naphthylene group, an anthrylene group, and a phenanthrylene group are preferable, and a naphthylene group is particularly preferable.

Ring A ¹ and ring A ² may have one or two substituents Y ¹ or Y ² .
The substituents Y ¹ and Y ² are independent of each other and are the same or different, and are a C _{1 to} C ₁₀ acyl group, a C _{1 to} C ₁₀ alkoxycarbonyl group, a carboxylic acid amide group (—C (═O) —NR ^a. represented by R ^{b, R a} and ^{R b} are each independently of one another, identical or different, is a hydrogen atom or a _C 1 _{-C 10} alkyl group.), a cyano group, a halogen atom, a nitro group, a substituent Yes An amino group (which is represented by —NR ^a R ^b , R ^a and R ^b are each independently the same or different and are a hydrogen atom or a C ₁ -C ₁₀ alkyl group), a hydroxyl group, selected from the group consisting of thiol groups, and _C 1 _{-C 10} alkyl group.
Among these, the substituents Y ¹ and Y ² are preferably electron-withdrawing groups. When the substituents Y ¹ and Y ² are electron-withdrawing groups, between the deazapurine base moiety and the ring A ¹ and the ring A ² having the substituent Y ¹ or Y ² through a double bond or a triple bond Thus, since the “push-pull” structure of conjugated electrons is formed, the change in the fluorescence emission wavelength can be further increased, and the identification by the color of the fluorescence emission can be made easier. Specifically, it is represented by a C ₁ -C ₁₀ acyl group, a C ₁ -C ₁₀ alkoxycarbonyl group, a carboxylic acid amide group (—C (═O) —NR ^a R ^b , and R ^a and R ^b are independently the same or different, is a hydrogen atom or a C ₁ -C ₁₀ alkyl group.), a cyano group, a halogen atom, a substituent selected from the group consisting of a nitro group and a thiol group. A cyano group is particularly preferable.
The substituents Y ¹ and Y ² may be bonded at any position of the aromatic ring. In order to further stabilize the conjugated structure, it is preferable that at least one of them is bonded to the carbon-carbon double bond or the carbon-carbon triple bond in the para position.

  m and n are each independently the same or different, and are 0, 1 or 2, with 1 being preferred.

In the present specification, the “C ₁ -C ₁₀ acyl group” is preferably C ₁ -C ₁₀ alkylcarbonyl, more preferably C ₁ -C ₆ alkylcarbonyl, and still more preferably C ₁ -C ₄ alkylcarbonyl. Examples of acyl groups include, but are not limited to, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, benzoyl, methylbenzoyl, dimethylbenzoyl, methylethylbenzoyl, diethylbenzoyl, benzylcarbonyl, and the like.

In the present specification, _"C 1 _{-C 10} alkoxycarbonyl group" is _preferably C 1 -C ₆ alkoxycarbonyl _group, C 1 -C ₄ alkoxycarbonyl group is more preferable. Examples of alkoxycarbonyl groups include, but are not limited to, methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, pentyloxycarbonyl, and the like.

In the present specification, the “carboxylic amide group” is a group represented by —C (═O) —NR ^a R ^b . Wherein, ^{R a} and ^{R b} are each independently of one another, identical or different, represent a hydrogen atom or a _C 1 _{-C 10} alkyl group, preferably a hydrogen atom or a _C 1 -C ₆ alkyl group, a hydrogen atom or a _{C 1} -C ₄ alkyl group is more preferable.

  In the present specification, examples of the “halogen atom” include fluorine, chlorine, bromine and iodine.

In the present specification, _"C 1 _{-C 10} alkyl group" may be linear, may be _branched, preferably C 1 -C ₆ alkyl _group, C 1 -C ₄ alkyl group is more preferable. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, dodecanyl and the like.

In the present specification, the “amino group optionally having substituent (s)” is represented by —NR ^a R ^b . Wherein, ^{R a} and ^{R b} are each independently of one another, identical or different, is a hydrogen atom or a _C 1 _{-C 10} alkyl group. Examples of the amino group which may have a substituent include, but are not limited to, an amino group, a dimethylamino group, a methylamino group, a diethylamino group, and an ethylamino group.

Preferred deazapurine nucleoside derivatives of the present invention include compounds represented by the following formula I (a) or II (a). The definitions of Y ¹ and Y ^{2 in} the formula are the same as above.

  As described above, in the deazapurine nucleoside derivative of the present invention, the fluorescence emission wavelength is changed by introducing a fluorescent molecule into the C7 position of the deazapurine skeleton via a double bond or a triple bond to form a conjugated structure. Designed. The degree of change in the fluorescence emission wavelength varies depending on the surrounding polar environment. By using the deazapurine nucleoside derivative of the present invention, it is possible to identify the difference in the surrounding polar environment by the change in the color of fluorescent light emission. For example, the deazapurine nucleoside derivative of the present invention can be used as a gene detection probe that identifies the type of facing base based on the difference in local polar environment in DNA or RNA. The deazapurine nucleoside derivative of the present invention can be used as a probe for investigating a protein or a local polar environment in a cell.

  The deazapurine nucleoside derivative of the present invention can be easily produced by introducing a fluorescent molecule at the C7 position such as 7-deazaadenosine or 7-deazaguanosine.

The deazapurine nucleoside derivative represented by the formula (I) of the present invention can be produced, for example, according to the following scheme A.

<Process (a)>
First, compound 1 is dissolved in a solvent, tetrakistriphenylphosphine palladium (0), copper iodide, trimethylsilylacetylene, and triethylamine are added, and the mixture is stirred at 40 to 60 ° C. for 1 to 4 hours. Thereafter, the reaction solvent is concentrated under reduced pressure, and the residue is purified by silica gel column chromatography to obtain compound 2.
Trimethylsilylacetylene is preferably used in an equimolar or slightly excess amount relative to Compound 1, and the amount used is preferably 1.2 to 1.5 (molar amount) relative to Compound 1.
Tetrakistriphenylphosphine palladium (0) and copper iodide may be used in catalytic amounts, and 0.01 to 0.1 (molar amount) with respect to Compound 1 is preferable.
Triethylamine is preferably used in a range of 5 to 10 (molar amount) relative to Compound 1.
Solvents include ether solvents such as tetrahydrofuran and diethyl ether, halogenated hydrocarbons such as methylene chloride and o-dichlorobenzene, amides such as N, N-dimethylformamide (DMF), sulfoxides such as dimethyl sulfoxide, benzene, toluene, Aromatic compounds such as xylene can be used.

<Step (b)>
Next, compound 2 is dissolved in a solvent, tetrabutylammonium fluoride is added, and the mixture is stirred at room temperature (20 to 25 ° C.) for 1 to 3 hours. Thereafter, a liquid separation operation is performed, and the organic layer from which the reaction product has been extracted is concentrated under reduced pressure. The residue is purified by silica gel column chromatography to obtain compound 3.
The amount of tetrabutylammonium fluoride used is preferably in the range of 1.1 to 1.2 (molar amount) relative to compound 1.
As the solvent, the same solvent as used in the step (a) can be used.

<Step (c)>
Next, the compound 3 and the compound 4 are dissolved in a solvent, tetrakistriphenylphosphine palladium (0), copper iodide, and triethylamine are added, and the mixture is stirred within a range of 50 to 60 ° C. for 1 to 4 hours. Thereafter, the reaction solvent is concentrated under reduced pressure, and the residue is purified by silica gel column chromatography to obtain the target compound (compound represented by the formula (I ′)).
Compound 3 is preferably used in an equimolar or slightly excess amount relative to Compound 4. Compound 4 is a known compound and can be easily produced by the method described in Synthesis, June 1996, 726-730 (Non-patent Document 2). Commercial products may also be used. For example, “7-Deaza-7-iodo-2′-deoxyadenosine” manufactured by 2Daybiochem Co., Ltd., etc. can be used.
Tetrakistriphenylphosphine palladium (0) and copper iodide may be used in catalytic amounts, and 0.01 to 0.1 (molar amount) with respect to compound 4 is preferable.
Triethylamine is preferably used in the range of 5 to 10 (unit: molar multiple) with respect to compound 4.
As the solvent, the same solvent as used in the step (a) can be used.

  Any of the above reactions is preferably performed in a nitrogen or argon atmosphere.

In the above, X ¹ in formula (I) is a carbon - has been described a process for the preparation of a compound is carbon triple bond, X ¹ is carbon in formula (I) - preparation of compounds a carbon double bond This is the same as Scheme A except that tetravinyltin (IV) is used in place of trimethylsilylacetylene in step (a).

The deazapurine nucleoside derivative represented by the formula (II) of the present invention can be produced according to the following scheme B.

  Steps (a) and (b) of Scheme B are the same as steps (a) and (b) of Scheme A, respectively. Step (c) in Scheme B is the same as Step (c) in Scheme A except that Compound 4 ′ is used in place of Compound 4. Compound 4 'is a known compound and can be easily produced by the method described in Synthesis, June 1996, 726-730 (Non-patent Document 2). Commercial products may also be used. For example, “7-Deaza-7-iodo-2′-deoxyguanosine” manufactured by 2Daybiochem Co., Ltd., etc. can be used.

  Since the deazapurine nucleoside derivative of the present invention can be produced by a simple method using 7-deazanucleoside as described above, it is highly practical.

Next, the nucleotide derivative of the present invention will be described.
The nucleotide derivative of the present invention is a derivative of the above-described deazapurine nucleoside derivative of the present invention in which phosphoric acid is ester-bonded, and has the following formula (III) or (IV):

[Wherein, ring A ¹ and ring A ² are each independently the same or different and are C _{6 to} C ₁₈ aromatic rings,
X ¹ and X ² are each independently the same or different and are a carbon-carbon double bond (—C═C—) or a carbon-carbon triple bond (—C≡C—),
Y ¹ and Y ² are independent of each other, and are the same or different and are each a C _{1 to} C ₁₀ acyl group, a C _{1 to} C ₁₀ alkoxycarbonyl group, a carboxylic acid amide group (—C (═O) —NR ^a R ^b R ^a and R ^b are each independently the same or different and are a hydrogen atom or a C _{1 to} C ₁₀ alkyl group.), A cyano group, a halogen atom, a nitro group, and a substituent. An amino group (represented by —NR ^a R ^b , where R ^a and R ^b are each independently the same or different and are a hydrogen atom or a C ₁ -C ₁₀ alkyl group), a hydroxyl group, and a thiol group. And a substituent selected from the group consisting of C _{1 to} C ₁₀ alkyl groups,
m and n are each independently the same or different and are 0, 1 or 2,
R ¹ and R ² are each independently the same or different and are a hydrogen atom or a hydroxyl group,
s and t are independent of each other, and are the same or different and are 1, 2 or 3,
However, the case where ring A ¹ is a phenyl group, X ¹ is a carbon-carbon triple bond (—C≡C—), m is 0, and R ¹ is a hydrogen atom is excluded. ]
Indicated by

^A 1 in formula (III) or ^{A 1,} A ² in ^{(IV), X 1, X} 2, Y 1, Y 2, R 1, R 2, m, n of formula (I) or (II), A ² , X ¹ , X ² , Y ¹ , Y ² , R ¹ , R ² , m, and n are synonymous, and preferred examples are also the same.
Each of s and t is 1, 2 or 3, but 1 or 3 is preferable, and 3 is particularly preferable because it can be easily introduced into a polynucleotide derivative.

  The monophosphate (s, t = 1) of the nucleotide derivative (III) or (IV) of the present invention is usually prepared by using the deazapurine nucleoside derivative (I) or (II) of the present invention as a solvent such as trimethyl phosphite. It can be easily synthesized by adding water after adding phosphorus oxychloride and reacting at 0 ° C. The triphosphate (s, t = 3) can be synthesized in the same manner as the monophosphate except that a step of adding tributylammonium pyrophosphate (diphosphate) is added before adding water. it can.

  In addition, the triphosphate of the nucleotide derivative (III) or (IV) of the present invention can be easily introduced into DNA using a PCR method, and at least one nucleotide in the polynucleotide is the nucleotide derivative of the present invention. Substituted polynucleotide derivatives of the invention can be obtained. Alternatively, as shown in Example 2, the deazapurine nucleoside derivative of the present invention is reacted with N, N-dimethylformamide diethyl acetal, and then a catalytic amount of 4,4′-dimethoxytrityl chloride is added and reacted. The resulting compound is further reacted with 2-cyanoethyltetraisopropylphosphoramidite in the presence of triethylamine, and the resulting amidite is directly subjected to an automatic DNA synthesizer to obtain the polynucleotide derivative of the present invention. be able to.

  In the present invention, the polynucleotide derivative may be an oligonucleotide derivative. The number of bases of the oligonucleotide derivative of the present invention is not particularly limited, and is preferably 2 to 1000, for example, more preferably 2 to 200, and particularly preferably 2 to 100.

  The polynucleotide derivative of the present invention can be used as a base-identifying fluorescent nucleobase (probe for gene detection) for identifying the type of facing base by utilizing the difference in local polar environment in DNA, or in a protein or intracellular It can be used as a probe for investigation of the local polar environment.

  When the probe of the present invention is hybridized with the target DNA and fully matched, the facing base of the target DNA can be identified by the difference in color of fluorescence emission of the probe due to the change in polar environment.

  EXAMPLES Hereinafter, although this invention is demonstrated based on an Example, this invention is not restrict | limited to the following Example.

According to the following scheme 1, the deazapurine nucleoside derivative of the present invention was synthesized.

Preparation of Compound (2a) Compound (1a) (500 mg, 2.41 mmol) was dissolved in anhydrous DMF (5 ml), tetrakistriphenylphosphine palladium (0) (27.9 mg, 0.024 mmol), copper iodide (4.6 mg, 0.024 mmol) ), Trimethylsilylacetylene (409 μl, 2.89 mmol) and triethylamine (0.5 ml) were added, and the mixture was stirred at 60 ° C. for 1 hour. Thereafter, the reaction solvent was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (hexane only) to obtain Compound (2a) (0.4207 g, 77.67%).

Preparation of Compound (2b) Compound (1b) (500 mg, 2.15 mmol) was dissolved in anhydrous DMF (5 ml), tetrakistriphenylphosphine palladium (0) (24.9 mg, 0.021 mmol), copper iodide (4.1 mg, 0.021 mmol) ), Trimethylsilylacetylene (365 μl, 2.58 mmol) and triethylamine (0.5 ml) were added, and the mixture was stirred at 60 ° C. for 1 hour. Thereafter, the reaction solvent was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (hexane: ethyl acetate = 3: 1) to obtain compound (2b) (0.5068 g, 94.35%).

Preparation of Compound (2c) Compound (1c) (100 mg, 0.34 mmol) was dissolved in anhydrous DMF (5 ml), tetrakistriphenylphosphine palladium (0) (38.7 mg, 0.034 mmol), copper iodide (6.4 mg, 0.034 mmol) ), Trimethylsilylacetylene (71.1 μl, 0.50 mmol) and triethylamine (0.5 ml) were added, and the mixture was stirred at 60 ° C. for 1 hour. Thereafter, the reaction solvent was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (hexane: ethyl acetate = 3: 1) to obtain compound (2c) (0.100 g, quantitative).

Preparation of Compound (3a) Compound (2a) (400 mg, 1.78 mmol) was dissolved in THF (10 ml), tetrabutylammonium fluoride (557 μl, 1.96 ml) was added, and the mixture was stirred at room temperature for 3 hours. After confirming the disappearance of the raw material by thin layer chromatography (TLC), liquid separation operation was performed (ethyl acetate / 10% HCl aqueous solution), and the organic layer was extracted under reduced pressure. The residue was purified by silica gel column chromatography (hexane only) to obtain compound (3a) (0.2699 g, 94.58%).

Preparation of Compound (3b) Compound (2b) (453 mg, 1.81 mmol) was dissolved in THF (17 ml), tetrabutylammonium fluoride (620 μl, 2.18 ml) was added, and the mixture was stirred at room temperature for 2 hours. After confirming the disappearance of the raw material by thin layer chromatography (TLC), liquid separation operation was performed (ethyl acetate / 10% HCl aqueous solution), and the organic layer was extracted under reduced pressure. The residue was purified by silica gel column chromatography (hexane: ethyl acetate = 5: 1) to obtain compound (3b) (0.3211 g, 99.18%).

Preparation of Compound (3c) Compound (2c) (100 mg, 0.374 mmol) was dissolved in THF (8 ml), tetrabutylammonium fluoride (161 μl, 1.12 ml) was added, and the mixture was stirred at room temperature for 3 hours. After confirming the disappearance of the raw material by thin layer chromatography (TLC), liquid separation operation was performed (ethyl acetate / 10% HCl aqueous solution), and the organic layer was extracted under reduced pressure. The residue was purified by silica gel column chromatography (hexane: ethyl acetate = 3: 1) to obtain compound (3c) (0.0928 g, quantitative).

Preparation of compound (5a) Compound (4) (100 mg, 0.36 mmol) and compound (3a) (42.1 mg, 0.43 mmol) were dissolved in DMF (5 ml) and tetrakistriphenylphosphine palladium (0) (42.1 mg, 0.036 mmol) ), Copper iodide (2.1 mg, 0.010 mmol) and triethylamine (5 ml) were added, and the mixture was stirred at 60 ° C. for 1 hour. The reaction solvent was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (methanol: chloroform = 1: 10) to obtain compound (5a) (0.1343 g, 91.99%).
¹ H-NMR (DMSO-d ₆ , 400 MHz) δ 2.24 (ddd, J = 2.6, 5.8, 13.1 Hz, 1H), 2.54 (m, 1H, overlapped with DMSO), 3.52-3.64 (complex, 2H), 3.86 (m, 1H), 4.38 (m, 1H), 5.09 (m, 1H), 5.31 (d, J = 4.0 Hz, 1H), 6.54 (m, 1H), 6.87 (br, 2H), 7.57-7.60 (complex, 2H), 7.66-7.69 (complex, 2H), 7.94-7.97 (complex, 4H), 8.23 (s, 1H)

Preparation of Compound (5b) Compound (4) (150 mg, 0.39 mmol) and Compound (3b) (84.8 mg, 0.478 mmol) were dissolved in DMF (5 ml), and tetrakistriphenylphosphine palladium (0) (46.0 mg, 0.04 mmol) ), Copper iodide (7.6 mg, 0.04 mmol) and triethylamine (0.5 ml) were added, and the mixture was stirred at 60 ° C. for 1 hour. The reaction solvent was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (methanol: chloroform = 1: 30) to obtain compound (5b) (0.1400 g, 84.00%).
¹ H-NMR (DMSO-d ₆ , 400 MHz) δ 2.24 (ddd, J = 2.8, 6.0, 13.2 Hz, 1H), 2.54 (m, 1H, overlapped with DMSO), 3.55-3.63 (complex, 2H), 3.86 (m, 1H), 4.38 (m, 1H), 5.12 (m, 1H), 5.32 (d, J = 4.1 Hz, 1H), 6.54 (dd, J = 6.0, 7.8 Hz, 1H), 6.88 (br , 2H), 7.82-7.86 (complex, 2H), 7.98 (s, 1H), 8.09-8.14 (complex, 2H) 8.18 (s, 1H), 8.34 (m, 1H), 8.61 (m, 1H)

Preparation of compound (5c) Compound (4) (50 mg, 0.13 mmol) and compound (3c) (28.5 mg, 0.15 mmol) were dissolved in DMF (5 ml), and tetrakistriphenylphosphine palladium (0) (76.8 mg, 0.07 mmol) ), Copper iodide (12.6 mg, 0.07 mmol) and triethylamine (0.3 ml) were added, and the mixture was stirred at 55 ° C. for 1 hour. The reaction solvent was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (methanol: chloroform = 1: 10) to obtain compound (5c) (0.0490 g, 84.98%).
¹ H-NMR (CD ₃ OD, 400 MHz) δ 2.35 (ddd, J = 2.7, 6.0, 13.4 Hz, 1H), 2.66 (ddd, J = 5.9, 8.0, 13.4 Hz, 1H), 3.05 (s, 6H ), 3.73 (dd, J = 3.6, 12.1 Hz, 1H), 3.81 (dd, J = 3.3, 12.1 Hz, 1H), 4.02 (m, 1H), 4.53 (m, 1H), 6.51 (dd, J = 6.0, 8.0 Hz, 1H), 6.92 (d, J = 2.6 Hz, 1H), 7.22 (dd, J = 2.6, 9.1 Hz, 1H), 7.39 (dd, J = 1.6, 8.6 Hz, 1H), 7.61 ( d, J = 8.6 Hz, 1H), 7.66-7.68 (complex, 2H), 7.85 (m, 1H), 8.11 (s, 1H)

According to the following scheme 2, the deazapurine nucleoside derivative of the present invention was introduced into the DNA strand.

Production of Compound (6a) Compound (5a) (180 mg, 0.449 mmol) was dissolved in DMF (5 ml), DMF diethyl acetal (95 μl, 0.585 mmol) was added, and the mixture was stirred at 60 ° C. for 2 hours. After confirming the disappearance of the starting material by thin layer chromatography, the reaction solvent was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (methanol: chloroform = 1: 30) to obtain compound (6a) (0.2033 g, 99.40%).
¹ H-NMR (CDCl ₃ , 400 MHz) δ 2.28 (m, 1H), 2.98 (ddd, J = 5.1, 9.2, 13.1 Hz, 1H), 3.07 (s, 3H), 3.19 (s, 3H), 3.76 (m, 1H), 3.96 (dd, J = 1.6, 12.4 Hz, 1H), 4.18 (m, 1H), 4.71 (m, 1H), 6.27 (dd, J = 5.6, 9.2 Hz, 1H), 7.40 ( s, 1H), 7.42-7.48 (complex, 2H), 7.54 (dd, J = 1.5, 8.5 Hz, 1H), 7.71-7.79 (complex, 3H), 8.00 (m, 1H), 8.41 (s, 1H) , 8.68 (s, 1H)

Preparation of Compound (6b) Compound (5b) (120 mg, 0.282 mmol) was dissolved in DMF (5 ml), DMF diethyl acetal (55 μl, 0.338 mmol) was added, and the mixture was stirred at 60 ° C. for 2 hours. After confirming the disappearance of the starting material by thin layer chromatography, the reaction solvent was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (methanol: chloroform = 1: 30) to obtain compound (6b) (0.1248 g, 95.00%).
¹ H-NMR (CDCl ₃ , 400 MHz) δ 2.29 (m, 1H), 3.04 (ddd, J = 5.1, 9.4, 13.4 Hz, 1H), 3.14 (s, 3H), 3.22 (s, 3H), 3.78 (m, 1H), 3.98 (dd, J = 1.7, 12.6 Hz, 1H), 4.19 (m, 1H), 4.74 (m, 1H), 6.26 (dd, J = 5.6, 9.4 Hz, 1H), 7.42 ( s, 1H), 7.58 (dd, J = 1.4, 8.4 Hz, 1H), 7.65 (dd, J = 1.5, 8.3 Hz, 1H), 7.81 (complex, 2H), 8.01 (m, 1H), 8.17 (m , 1H), 8.41 (s, 1H), 8.76 (s, 1H)

Production of Compound (6c) Compound (5c) (60 mg, 0.135 mmol) was dissolved in DMF (3 ml), DMF diethyl acetal (26 μl, 0.162 mmol) was added, and the mixture was stirred at 60 ° C. for 2 hours. After confirming the disappearance of the starting material by thin layer chromatography, the reaction solvent was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (methanol: chloroform = 1: 10) to obtain compound (6c) (0.0640 g, 95.08%).
¹ H-NMR (CDCl ₃ , 400 MHz) δ 2.27 (m, 1H), 3.07-3.15 (complex, 10H), 3.22 (s, 3H), 3.80 (m, 1H), 4.01 (dd, J = 1.7, 12.6 Hz, 1H), 4.21 (m, 1H), 4.79 (m, 1H), 6.24 (dd, J = 5.6, 9.5 Hz, 1H), 6.87 (d, J = 2.6 Hz, 1H), 7.16 (dd, J = 2.6, 9.1 Hz, 1H), 7.35 (s, 1H), 7.46 (dd, J = 1.6, 8.6 Hz, 1H), 7.58 (d, J = 8.6 Hz, 1H), 7.64 (d, J = 9.1 Hz, 1H), 7.88 (m, 1H), 8.43 (s, 1H), 8.74 (s, 1H)

Preparation of Compound (7a) Compound (6a) (200 mg, 0.439 mmol) was dissolved in anhydrous pyridine (8 ml), 4,4′-dimethoxytrityl chloride (193 mg, 0.571 mmol) was added, and the mixture was stirred at room temperature for 6 hours. . After confirming the disappearance of the raw material by thin layer chromatography, it was distilled off under reduced pressure to carry out a liquid separation operation (ethyl acetate / sodium bicarbonate aqueous solution), and the organic layer was recovered and repeatedly distilled off under reduced pressure. The residue was purified by silica gel chromatography (methanol: chloroform / triethylamine = 30: 1/10 ml) to obtain compound (7a) (0.3101 g, 93.17%).
¹ H-NMR (CDCl ₃ , 400 MHz) δ 2.50 (ddd, J = 4.4, 6.4, 13.6 Hz, 1H), 2.59 (m, 1H), 3.12 (s, 3H), 3.24 (s, 3H), 3.36 (dd, J = 4.8, 10.2 Hz, 1H), 3.44 (dd, J = 4.4, 10.2 Hz, 1H), 3.72 (s, 6H), 4.11 (m, 1H), 4.61 (m, 1H), 6.76 ( m, 1H), 6.82 (m, 2H), 6.85 (m, 2H), 7.19 (m, 1H), 7.30 (m, 2H), 7.34 (m, 2H), 7.36 (m, 2H), 7.44-7.52 (complex, 4H), 7.54 (dd, J = 1.6, 8.5 Hz, 1H), 7.57 (s, 1H), 7.76-7.83 (complex, 3H), 7.99 (m, 1H), 8.48 (s, 1H), 8.75 (s, 1H)

Preparation of Compound (7b) Compound (6b) (100 mg, 0.208 mmol) was dissolved in anhydrous pyridine (10 ml), 4,4′-dimethoxytrityl chloride (91.6 mg, 0.270 mmol) was added, and the mixture was stirred at room temperature for 6 hours. did. After confirming the disappearance of the raw material by thin layer chromatography, it was distilled off under reduced pressure to carry out a liquid separation operation (ethyl acetate / sodium bicarbonate aqueous solution), and the organic layer was recovered and repeatedly distilled off under reduced pressure. The residue was purified by silica gel chromatography (methanol: chloroform / triethylamine = 30: 1/10 ml) to obtain compound (7b) (0.1800 g, quantitative).
¹ H-NMR (CDCl ₃ , 400 MHz) δ 2.52 (ddd, J = 4.4, 6.3, 13.5 Hz, 1H), 2.60 (m, 1H), 3.16 (s, 3H), 3.25 (s, 3H), 3.37 -3.45 (complex, 2H), 3.72 (s, 6H), 4.12 (m, 1H), 4.64 (m, 1H), 6.75 (m, 1H), 6.82 (m, 2H), 6.85 (m, 2H), 7.19 (m, 1H), 7.30 (m, 2H), 7.35 (m, 2H), 7.37 (m, 2H), 7.45-7.47 (complex, 2H), 7.60-7.65 (complex, 3H), 7.82-7.84 ( complex, 2H), 7.98 (m, 1H), 8.20 (m, 1H), 8.48 (s, 1H), 8.79 (s, 1H)

Preparation of Compound (7c) Compound (6c) (80 mg, 0.160 mmol) was dissolved in anhydrous pyridine (6 ml), 4,4′-dimethoxytrityl chloride (70 mg, 0.209 mmol) was added, and the mixture was stirred at room temperature for 6 hours. . After confirming the disappearance of the raw material by thin layer chromatography, it was distilled off under reduced pressure to carry out a liquid separation operation (ethyl acetate / sodium bicarbonate aqueous solution), and the organic layer was recovered and repeatedly distilled off under reduced pressure. The residue was purified by silica gel chromatography (methanol: chloroform / triethylami = 25: 1/10 ml) to obtain compound (7c) (0.1032 g, 80.53%).
¹ H-NMR (CDCl ₃ , 400 MHz) δ 2.48 (ddd, J = 4.6, 6.4, 13.6 Hz, 1H), 2.58 (m, 1H), 3.07 (s, 6H), 3.10 (s, 3H), 3.22 (s, 3H), 3.35 (dd, J = 5.0, 10.1 Hz, 1H), 3.43 (dd, J = 4.6, 10.1 Hz, 1H), 3.72 (s, 6H), 4.10 (m, 1H), 4.59 ( m, 1H), 6.74 (m, 1H), 6.82 (m, 2H), 6.85 (m, 2H), 6.88 (d, J = 2.4 Hz, 1H), 7.16 (m, 1H), 7.20 (m, 1H ), 7.30 (m, 2H), 7.34 (m, 2H), 7.36 (m, 2H), 7.42-7.46 (complex, 3H), 7.51 (s, 1H), 7.57 (d, J = 8.6 Hz, 1H) , 7.63 (d, J = 9.1 Hz, 1H), 7.84 (m, 1H), 8.47 (s, 1H), 8.71 (s, 1H)

Preparation of Compound (8a) Compound (7a) (70 mg, 0.0923 mmol) was dissolved in anhydrous acetonitrile (1.5 ml), triethylamine (1 ml) and 2-cyanoethyldiisopropylchlorophosphoramidite (150 μl) were added, and room temperature was added. Stir for 1 hour. After confirming the disappearance of the raw materials by thin layer chromatography, liquid separation was performed (ethyl acetate / sodium hydrogen carbonate aqueous solution), and the organic layer was collected and repeatedly distilled off under reduced pressure. The residue was dissolved in 600 μl of anhydrous acetonitrile and filtered through Cosmonis filter S. The filtrate was distilled off under reduced pressure to obtain the target compound (8a) as a crude product. The crude product of compound (8a) was used as it was for DNA synthesis.

Preparation of Compound (8b) Compound (7b) (100 mg, 0.1277 mmol) was dissolved in anhydrous acetonitrile (2.0 ml), triethylamine (1 ml) and 2-cyanoethyldiisopropylchlorophosphoramidite (150 μl) were added, and room temperature was added. Stir for 1 hour. After confirming the disappearance of the raw materials by thin layer chromatography, liquid separation was performed (ethyl acetate / sodium hydrogen carbonate aqueous solution), and the organic layer was collected and repeatedly distilled off under reduced pressure. The residue was dissolved in 600 μl of anhydrous acetonitrile and filtered through Cosmonis filter S. The filtrate was distilled off under reduced pressure to obtain the target compound (8b) as a crude product. The crude product of compound (8b) was used as it was for DNA synthesis.

Preparation of Compound (8c) Compound (7c) (60 mg, 0.0749 mmol) was dissolved in anhydrous acetonitrile (2.0 ml), triethylamine (1 ml) and 2-cyanoethyldiisopropylchlorophosphoramidite (150 μl) were added, and at room temperature. Stir for 1 hour. After confirming the disappearance of the raw materials by thin layer chromatography, liquid separation was performed (ethyl acetate / sodium hydrogen carbonate aqueous solution), and the organic layer was collected and repeatedly distilled off under reduced pressure. The residue was dissolved in 600 μl of anhydrous acetonitrile and filtered through Cosmonis filter S. The filtrate was distilled off under reduced pressure to obtain the target compound (8c) as a crude product. The crude product of compound (8c) was used as it was for DNA synthesis.

DNA synthesis Each of the obtained compounds (8a), (8b) and (8c) was dissolved in 600 μl of acetonitrile and converted to a DNA strand using an automatic DNA synthesizer (manufacturer: Applied Biosystems, model number: 3400 DNA synthesizer). Introduced.

[1] Measurement of fluorescence spectrum For the compounds (5b) and (5c) obtained in Example 2, various solvents (methanol (MeOH), ethanol (EtOH), 2-propanol (2-prOH), N, Fluorescence spectrum (λmax) and its change amount when dissolved in N-dimethylformamide (DMF), acetonitrile (MeCN), tetrahydrofuran (THF), ethyl acetate (AcOEt), chloroform (CHCl ₃ ), water (water) ( Δλem max) was measured according to the following procedure.
Compounds (5b) and (5c) were dissolved in each solvent to prepare a concentration of 10 μM (total volume: 1 ml). 600 μl was transferred to a quartz cell, and the fluorescence spectrum (λmax) was measured using a spectrofluorometer (manufacturer: Shimadzu Corporation, model number: RF-5300PC). The amount of change (Δλem max) was calculated from λmax for each solvent.
The measurement results are shown in the graphs and tables (Tables 1A and 2A) of FIGS. 1 and 2, respectively. For reference, changes in the fluorescence spectrum when the C8-substituted base derivative and the compound described in Tetrahedron Letters 52 (2011) 4726-4729 (Non-patent Document 3) were dissolved in various solvents were observed in the same manner. The measurement results are shown in the graphs and tables (Tables 3A to 6A) of FIGS.

As shown in the graphs and tables of FIGS. 1 and 2, when the deazapurine nucleoside derivatives (5b) and (5c) of the present invention are dissolved in various solvents, the fluorescence emission wavelength varies depending on the type of solvent (polar environment). changed. Particularly in the compound (5b), the change in the fluorescence emission wavelength could be further increased. This is because the compound (5b) has an electron-withdrawing cyano group in the substituent Y ¹ and the conjugated electron “push” is connected between the deazapurine base and the biphenyl ring having a cyano group via a triple bond. This is considered to be because the change of the fluorescence emission wavelength can be made more effective in response to the change of the polar environment because the “-pull” structure is formed.
Even when the compounds (C1), (C2) and (C3), which are C8 substituted base derivatives, and the compound (C4) described in Tetrahedron Letters 52 (2011) 4726-4729 (Non-patent Document 3) are used, The fluorescence emission wavelength changed in response to changes in the polar environment.

[2] Evaluation of thermal stability of DNA conformation
The Tm value (melting temperature) is an indicator of the thermal stability of the intrinsic DNA conformation under specific conditions. Various complementary strands (N = A) to oligonucleotide DNAs (ODN1 (X), X = 5b, 5c) obtained by introducing the compounds (8b) and (8c) of the present invention obtained in Example 2 into DNA strands, respectively. , G, C, T), the Tm value and the amount of change ΔTm were measured according to the following procedure, and the thermal stability of the DNA double helix structure was evaluated.
The DNA solution prepared to a concentration of 2.5 μM is transferred to an 8-series microcell and measured using a UV-visible spectrophotometer (manufacturer: Shimadzu Corporation, model number: UV-2550) in the range of 4 to 90 ° C. Was used to calculate the Tm value. Further, the amount of change ΔTm was calculated based on the Tm value of the natural oligonucleotide DNA.
The measurement results are shown in Tables 1B and 2B of FIGS. For reference, the Tm value and the amount of change ΔTm thereof were also measured in the same manner for the C8-substituted base derivative and the compound described in Tetrahedron Letters 52 (2011) 4726-4729 (Non-patent Document 3). The measurement results are shown in Tables 3B to 6B in FIGS.

The base sequence of the oligonucleotide DNA used in this measurement is as shown in the table below.

  As shown in Tables 1B and 2B, when the compounds (5b) and (5c) of the present invention are introduced into DNA strands, the amount of change in the Tm value is small, and the DNA duplex is similar to natural nucleobases. Showed stability. In particular, when the compound (5b) of the present invention is introduced into the DNA strand, the amount of change in the Tm value is zero when it matches the opposite base of the complementary strand (that is, when ODN1 (5b) / ODN2 (T)). Showed the same DNA duplex stability as natural nucleobases. On the other hand, as shown in Tables 3B to 6B, when the comparative compounds (C1) to (C4) were used, the amount of change in the Tm value was large and the DNA double helix structure was destabilized.

  As shown in the above results, the deazapurine nucleoside derivative of the present invention can change the fluorescence emission wavelength in response to the difference in the surrounding polar environment and destabilize the double helix structure of DNA. Therefore, a probe in which the deazapurine nucleoside derivative of the present invention is introduced to the opposite side of the base to be identified in DNA is prepared, and the change in the surrounding polar environment due to the difference in match-mismatch with the opposite base is utilized. The target base type can be identified by the difference in fluorescence emission color. Since the probe of the present invention can be identified not by the light emission intensity but by the difference in the color of the fluorescent light emission, it can be easily used by a general person as a test kit. In addition, since the degree of surrounding polarity can be indicated by color, it can also be used as a probe for investigating the local polar environment in proteins and cells.

  The deazapurine nucleoside derivative of the present invention can change the fluorescence emission wavelength due to the difference in the surrounding polar environment, and does not destabilize the DNA double helix structure when introduced into a DNA strand. It can be widely used as a probe or a probe for investigating a protein or a local polar environment in a cell.

SEQ ID NO: 1 synthetic DNA
SEQ ID NO: 2: Synthetic DNA
Sequence number 3: Synthetic DNA
Sequence number 4: Synthetic DNA
Sequence number 5: Synthetic DNA
Sequence number 6: Synthetic DNA
SEQ ID NO: 7: synthetic DNA

Claims (10)

The following formula (I) or (II):
[Wherein, ring A ¹ and ring A ² are naphthylene groups ,
X ¹ and X ² are each independently the same or different and are a carbon-carbon double bond (—C═C—) or a carbon-carbon triple bond (—C≡C—),
Y ¹ and Y ² are independent of each other, and are the same or different and are each a C _{1 to} C ₁₀ acyl group, a C _{1 to} C ₁₀ alkoxycarbonyl group, a carboxylic acid amide group (—C (═O) —NR ^a R ^b R ^a and R ^b are each independently the same or different and are a hydrogen atom or a C _{1 to} C ₁₀ alkyl group.), A cyano group, a halogen atom, a nitro group, and a substituent. An amino group (represented by —NR ^a R ^b , where R ^a and R ^b are each independently the same or different and are a hydrogen atom or a C ₁ -C ₁₀ alkyl group), a hydroxyl group, and a thiol group. And a substituent selected from the group consisting of C _{1 to} C ₁₀ alkyl groups,
m and n are each independently the same or different and are 0, 1 or 2,
R ¹ and R ² are each independently of one another, identical or different, Ru hydrogen atom or a hydroxyl group der. ]
A deazapurine nucleoside derivative represented by: The deazapurine nucleoside derivative according to claim 1, wherein R ¹ and R ² are hydrogen atoms. The substituents Y ¹ and Y ² are independent of each other and are the same or different, and are a C ₁ -C ₁₀ acyl group, a C ₁ -C ₁₀ alkoxycarbonyl group, a carboxylic acid amide group (—R—C (═O). -NR ^a R ^b , and R ^a and R ^b are each independently the same or different and are a C _{1 to} C ₁₀ alkyl group.), A cyano group, a halogen atom, a nitro group, and a thiol group The deazapurine nucleoside derivative according to claim 1 or 2 , which is selected from the group. The substituents Y ¹ and Y ² is a cyano group, deazapurine nucleoside derivative according to any one of claims 1-3. The deazapurine nucleoside derivative according to any one of claims 1 to 4, which is represented by any one of the following formulas I (a) and II (a).
[Wherein, Y ¹ and Y ² are as defined above. ] The deazapurine nucleoside derivative according to any one of claims 1 to 5 , wherein the fluorescence emission wavelength changes in response to a change in polar environment. The following formula (III) or (IV):
[Wherein, ring A ¹ and ring A ² are naphthylene groups ,
X ¹ and X ² are each independently the same or different and are a carbon-carbon double bond (—C═C—) or a carbon-carbon triple bond (—C≡C—),
Y ¹ and Y ² are independent of each other, and are the same or different and are each a C _{1 to} C ₁₀ acyl group, a C _{1 to} C ₁₀ alkoxycarbonyl group, a carboxylic acid amide group (—C (═O) —NR ^a R ^b R ^a and R ^b are each independently the same or different and are a hydrogen atom or a C _{1 to} C ₁₀ alkyl group.), A cyano group, a halogen atom, a nitro group, and a substituent. An amino group (represented by —NR ^a R ^b , where R ^a and R ^b are each independently the same or different and are a hydrogen atom or a C ₁ -C ₁₀ alkyl group), a hydroxyl group, and a thiol group. And a substituent selected from the group consisting of C _{1 to} C ₁₀ alkyl groups,
m and n are each independently the same or different and are 0, 1 or 2,
R ¹ and R ² are each independently the same or different and are a hydrogen atom or a hydroxyl group,
s and t are each independently of one another, identical or different, Ru 2 or 3 der. ]
A deazapurine nucleotide derivative represented by: The deazapurine nucleotide derivative according to claim 7 , wherein R ¹ and R ² are hydrogen atoms. A polynucleotide derivative obtained by substituting at least one nucleotide in the polynucleotide with the deazapurine nucleotide derivative according to claim 7 or 8 . A probe comprising the polynucleotide derivative according to claim 9 .