JP6709999B2 - Method for detecting compound, probe, condensate and cytosine - Google Patents

Description

The present invention relates to a compound, a probe, a condensate, and a method for detecting cytosine.

Fluorescent compounds whose fluorescence emission wavelengths change depending on the difference in the polar environment around them are useful as various probes because the difference in the polar environment can be detected by the change in color. As an example of such a compound, a fluorescent nucleoside derivative is known, and it is expected to be used as a gene detection probe or a probe for investigating a local polar environment in a protein or a cell.

For example, a fluorescent nucleoside derivative is introduced, a fluorescent polynucleotide having a base site derived from this derivative, when the base sequence of the target gene is recognized, the nucleic acid base to be identified therein and the base site The fluorescence emission wavelength changes with the formation of base pairs and changes in the polar environment. Such a fluorescent polynucleotide is useful as a gene detection probe.

On the other hand, the present inventors have developed a fluorescent nucleoside derivative whose fluorescence emission wavelength and fluorescence emission intensity change with changes in the pH of the peripheral region, in which the base is uracil (that is, a fluorescent uridine derivative). It has been confirmed that it is useful as a probe for detecting a change in pH (see Patent Document 1).

JP, 2005-221769, A

However, Patent Document 1 does not specifically disclose the use of a fluorescent nucleoside derivative to detect a specific nucleobase in a polynucleotide by utilizing a change in fluorescence emission wavelength. Such a fluorescent nucleoside derivative capable of detecting a specific nucleic acid base by such a change in the fluorescence emission wavelength depending on the pH of the peripheral region is extremely useful for producing a probe.

The present invention has been made in view of the above circumstances, utilizing a change in fluorescence emission wavelength with a change in pH of the peripheral region, a probe composed of a polynucleotide capable of detecting a specific nucleic acid base, and the probe. It is an object of the present invention to provide a novel compound for production, a condensate using the compound, and a method for detecting a nucleobase using the probe.

In order to solve the above problems, the present invention employs the following configurations.
[1]. A compound represented by the following general formula (1).

（式中、Ｒ^１は水素原子又は水酸基であり；Ｚは下記一般式（１）−１１又は（１）−１２で表される基である。）

(In the formula, R ¹ is a hydrogen atom or a hydroxyl group; Z is a group represented by the following general formula (1)-11 or (1)-12.)

（式中、Ｘ^１１及びＸ^１２は水素原子、シアノ基、アミノ基、モノアルキルアミノ基又はジアルキルアミノ基であり；Ｒ^１１及びＲ^１２はアルキル基又はハロゲン原子であり；ｎ_１１及びｎ_１２は０〜５の整数であり、ｎ_１１が２以上の整数である場合、複数個のＲ^１１は互いに同一でも異なっていてもよく、ｎ_１２が２以上の整数である場合、複数個のＲ^１２は互いに同一でも異なっていてもよく；符号＊を付した結合は、Ｚの結合先との間で形成されている。）

(In the formula, X ¹¹ and X ¹² are a hydrogen atom, a cyano group, an amino group, a monoalkylamino group or a dialkylamino group; R ¹¹ and R ¹² are an alkyl group or a halogen atom; and n ₁₁ and n ₁₂ are When n ₁₁ is an integer of 2 or more, a plurality of R ¹¹ may be the same or different, and when n ₁₂ is an integer of 2 or more, a plurality of R ^{12 are} May be the same or different from each other; the bond marked with * is formed between the bond to Z and the target.)

[2]. The compound according to [1], wherein X ¹¹ and X ¹² are cyano groups.
[3]. The compound according to [1] or [2], wherein R ¹ is a hydrogen atom.
[4]. Z is a group represented by the general formula (1)-11, R ¹¹ is an alkyl group having 1 to 5 carbon atoms or a halogen atom, and n ₁₁ is an integer of 0 to 2, The compound according to any one of 1] to [3].
[5]. A compound represented by the following general formula (2).

（式中、Ｒ^１は水素原子又は水酸基であり；Ｚは下記一般式（１）−１１又は（１）−１２で表される基であり；ｍは１〜３の整数である。）

(In the formula, R ¹ is a hydrogen atom or a hydroxyl group; Z is a group represented by the following general formula (1)-11 or (1)-12; and m is an integer of 1 to 3.)

（式中、Ｘ^１１及びＸ^１２は水素原子、シアノ基、アミノ基、モノアルキルアミノ基又はジアルキルアミノ基であり；Ｒ^１１及びＲ^１２はアルキル基又はハロゲン原子であり；ｎ_１１及びｎ_１２は０〜５の整数であり、ｎ_１１が２以上の整数である場合、複数個のＲ^１１は互いに同一でも異なっていてもよく、ｎ_１２が２以上の整数である場合、複数個のＲ^１２は互いに同一でも異なっていてもよく；符号＊を付した結合は、Ｚの結合先との間で形成されている。）

(In the formula, X ¹¹ and X ¹² are a hydrogen atom, a cyano group, an amino group, a monoalkylamino group or a dialkylamino group; R ¹¹ and R ¹² are an alkyl group or a halogen atom; and n ₁₁ and n ₁₂ are When n ₁₁ is an integer of 2 or more, a plurality of R ¹¹ may be the same or different, and when n ₁₂ is an integer of 2 or more, a plurality of R ^{12 are} May be the same or different from each other; the bond marked with * is formed between the bond to Z and the target.)

[6]. The compound according to [5], wherein X ¹¹ and X ¹² are cyano groups.
[7]. The compound according to [5] or [6], wherein R ¹ is a hydrogen atom.
[8]. Z is a group represented by the general formula (1)-11, R ¹¹ is an alkyl group having 1 to 5 carbon atoms or a halogen atom, and n ₁₁ is an integer of 0 to 2, 5] to the compound according to any one of [7].
[9]. A compound represented by the following general formula (11b).

（式中、Ｘ^１１は水素原子、シアノ基、アミノ基、モノアルキルアミノ基又はジアルキルアミノ基であり；Ｒ^１１はアルキル基又はハロゲン原子であり；ｎ_１１は０〜５の整数であり、ｎ_１１が２以上の整数である場合、複数個のＲ^１１は互いに同一でも異なっていてもよい。）

(In the formula, X ¹¹ is a hydrogen atom, a cyano group, an amino group, a monoalkylamino group or a dialkylamino group; R ¹¹ is an alkyl group or a halogen atom; n ₁₁ is an integer of 0 to 5; _{When 11} is an integer of 2 or more, a plurality of R ¹¹ may be the same or different from each other.)

[10]. A compound represented by the following general formula (12b).

（式中、Ｘ^１２は水素原子、シアノ基、アミノ基、モノアルキルアミノ基又はジアルキルアミノ基であり；Ｒ^１２はアルキル基又はハロゲン原子であり；ｎ_１２は０〜５の整数であり、ｎ_１２が２以上の整数である場合、複数個のＲ^１２は互いに同一でも異なっていてもよい。）

(In the formula, X ¹² is a hydrogen atom, a cyano group, an amino group, a monoalkylamino group or a dialkylamino group; R ¹² is an alkyl group or a halogen atom; n ₁₂ is an integer of 0 to 5; _{When 12} is an integer of 2 or more, a plurality of R ¹² may be the same or different from each other.)

[11]. A compound represented by the following general formula (1a).

（式中、Ｒ^１０は水素原子、水酸基、トリアルキルシリルオキシ基又はアルキルジアリールシリルオキシ基であり；Ｚは下記一般式（１）−１１又は（１）−１２で表される基であり；Ｒ^２及びＲ^３は、それぞれ独立にトリアルキルシリル基又はアルキルジアリールシリル基である。）

(In the formula, R ¹⁰ is a hydrogen atom, a hydroxyl group, a trialkylsilyloxy group or an alkyldiarylsilyloxy group; Z is a group represented by the following general formula (1)-11 or (1)-12; R ² and R ³ are each independently a trialkylsilyl group or an alkyldiarylsilyl group.)

（式中、Ｘ^１１及びＸ^１２は水素原子、シアノ基、アミノ基、モノアルキルアミノ基又はジアルキルアミノ基であり；Ｒ^１１及びＲ^１２はアルキル基又はハロゲン原子であり；ｎ_１１及びｎ_１２は０〜５の整数であり、ｎ_１１が２以上の整数である場合、複数個のＲ^１１は互いに同一でも異なっていてもよく、ｎ_１２が２以上の整数である場合、複数個のＲ^１２は互いに同一でも異なっていてもよく；符号＊を付した結合は、Ｚの結合先との間で形成されている。）

(In the formula, X ¹¹ and X ¹² are a hydrogen atom, a cyano group, an amino group, a monoalkylamino group or a dialkylamino group; R ¹¹ and R ¹² are an alkyl group or a halogen atom; and n ₁₁ and n ₁₂ are When n ₁₁ is an integer of 2 or more, a plurality of R ¹¹ may be the same or different, and when n ₁₂ is an integer of 2 or more, a plurality of R ^{12 are} May be the same or different from each other; the bond marked with * is formed between the bond to Z and the target.)

[12]. A probe comprising the compound according to any one of [1] to [4].
[13]. A condensate of the compound according to any one of [5] to [8] and the compound or another compound other than the compound.
[14]. The condensate according to [13], wherein the other compound is a nucleotide and the condensate is a polynucleotide.
[15]. A probe comprising the condensate according to [13] or [14].

[16]. A step of hybridizing a polynucleotide having cytosine with the probe described in [15] under acidic conditions to obtain a hybridized product, and measuring the fluorescence of the hybridized product and the probe under acidic conditions. And a step of detecting cytosine in the polynucleotide by detecting a change in emission wavelength of the fluorescence based on the presence or absence of cytosine.
[17]. The step of hybridizing the polynucleotide having cytosine with the probe described in [15] under a plurality of different pH conditions to obtain a hybridized product, and the hybridization under a plurality of different pH conditions. A step of measuring the fluorescence of the product and the probe; and a step of detecting cytosine in the polynucleotide by detecting a change in the emission wavelength of the fluorescence based on the difference in the pH, the detection of cytosine Method.

According to the present invention, utilizing a change in fluorescence emission wavelength with a change in pH of the peripheral region, a probe comprising a polynucleotide capable of detecting a specific nucleobase, a novel compound for producing the probe, Provided are a condensate using a compound and a method for detecting a nucleobase using the probe.

It is a graph which shows the measurement result of (a) ultraviolet absorption spectrum, (b) fluorescence spectrum, and (c) excitation spectrum in Test Example 1. 5 is a graph showing the measurement results of (a) ultraviolet absorption spectrum and (b) fluorescence spectrum in Test Example 2. 5 is a graph showing the measurement results of (a) ultraviolet absorption spectrum, (b) fluorescence spectrum, and (c) excitation spectrum in Test Example 3. 9 is a graph showing (a) ultraviolet absorption spectrum, (b) fluorescence spectrum, (c) excitation spectrum measurement results, and (d) thermal melting curve in Example 5. 3 is a graph showing (a) ultraviolet absorption spectrum, (b) fluorescence spectrum and (c) excitation spectrum measurement results, and (d) thermal melting curve in Comparative Example 1. 5 is a graph showing the measurement results of (a) ultraviolet absorption spectrum, (b) fluorescence spectrum, and (c) excitation spectrum, and (d) thermal melting curve in Comparative Example 2. It is a graph which shows the measurement result of (a) ultraviolet absorption spectrum, (b) fluorescence spectrum, and (c) excitation spectrum in Reference Example 1, and (d) thermal melting curve. 5 is a graph showing the measurement results of (a) ultraviolet absorption spectrum and (b) fluorescence spectrum in Test Example 4. 9 is a graph showing the measurement results of (a) ultraviolet absorption spectrum and (b) fluorescence spectrum, and (c) thermal melting curve in Example 7. It is a graph which shows the measurement result of (a) ultraviolet absorption spectrum and (b) fluorescence spectrum in Reference Example 2, and (c) thermal melting curve.

Hereinafter, the present invention will be described in detail.
In the present specification, the unit of concentration “M” means “mol/L”.
The abbreviations used in the chemical formulas have the following meanings.
Et: ethyl group _(CH 3 CH 2)
Ph: phenyl group _{(C 6 H} 5)
Ac: acetyl group (CH ₃ CO)

<<Compound (1)>>
The compound of one embodiment of the present invention is represented by the following general formula (1) (in the present specification, it may be referred to as “compound (1)”).

（式中、Ｒ^１は水素原子又は水酸基であり；Ｚは下記一般式（１）−１１又は（１）−１２で表される基である。）

(In the formula, R ¹ is a hydrogen atom or a hydroxyl group; Z is a group represented by the following general formula (1)-11 or (1)-12.)

（式中、Ｘ^１１及びＸ^１２は水素原子、シアノ基、アミノ基、モノアルキルアミノ基又はジアルキルアミノ基であり；Ｒ^１１及びＲ^１２はアルキル基又はハロゲン原子であり；ｎ_１１及びｎ_１２は０〜５の整数であり、ｎ_１１が２以上の整数である場合、複数個のＲ^１１は互いに同一でも異なっていてもよく、ｎ_１２が２以上の整数である場合、複数個のＲ^１２は互いに同一でも異なっていてもよく；符号＊を付した結合は、Ｚの結合先との間で形成されている。）

(In the formula, X ¹¹ and X ¹² are a hydrogen atom, a cyano group, an amino group, a monoalkylamino group or a dialkylamino group; R ¹¹ and R ¹² are an alkyl group or a halogen atom; and n ₁₁ and n ₁₂ are When n ₁₁ is an integer of 2 or more, a plurality of R ¹¹ may be the same or different, and when n ₁₂ is an integer of 2 or more, a plurality of R ^{12 are} May be the same or different from each other; the bond marked with * is formed between the bond to Z and the target.)

The compound (1) is a nucleoside derivative, and as a base, 3-deazaadenine is a ring skeleton condensed with a naphthalene ring skeleton at the carbon atoms at the 2-position and 3-position thereof, that is, a benzimidazoquinoline skeleton (hereinafter referred to as “BIQ skeleton” (Sometimes abbreviated).
The compound (1) is a fluorescent compound.

In the present specification, the term “derivative” refers to a compound in which one or more hydrogen atoms of the original compound are replaced with a group (substituent) other than a hydrogen atom, or one or more carbon atoms of the original compound. Represents a single group or a group which is substituted with another group (substituent) together with a hydrogen atom bonded to this carbon atom.

In the general formula (1), R ¹ is a hydrogen atom or a hydroxyl group. That is, the compound (1) is a deoxyribonucleoside derivative when R ¹ is a hydrogen atom (—H), and a ribonucleoside derivative when R ¹ is a hydroxyl group (—OH).
R ¹ is preferably a hydrogen atom.

In the general formula (1), Z corresponds to a base and is a group represented by the general formula (1)-11 or (1)-12.
That is, the compound (1) is a compound represented by the following general formula (1)-1 (hereinafter sometimes abbreviated as “compound (1)-1”), and a general formula (1)-2 below. It is classified into the represented compounds (hereinafter sometimes abbreviated as “compound (1)-2”). The compound (1)-1 and the compound (1)-2 are structural isomers.

（式中、Ｒ^１、Ｘ^１１、Ｘ^１２、Ｒ^１１、Ｒ^１２、ｎ_１１及びｎ_１２は、いずれも上記と同じである。）

(In the formula, R ¹ , X ¹¹ , X ¹² , R ¹¹ , R ¹² , n ₁₁ and n ₁₂ are the same as above.)

In the group represented by the general formula (1)-11 and the group represented by the general formula (1)-12, the bond marked with * constitutes an imidazole ring skeleton in Z which is a base. It is formed between one nitrogen atom and the carbon atom adjacent to the carbon atom to which R ¹ is bonded in the sugar to which Z is bonded.

In formulas (1)-11 and (1)-12, R ¹¹ and R ¹² are alkyl groups or halogen atoms.
The alkyl group for R ¹¹ and R ¹² may be linear, branched or cyclic, and when cyclic, may be monocyclic or polycyclic. The alkyl group preferably has 1 to 12 carbon atoms, more preferably 1 to 10 carbon atoms, further preferably 1 to 8 carbon atoms, and particularly preferably 1 to 5 carbon atoms. Most preferably, it is -3.

The linear or branched alkyl group preferably has 1 to 12 carbon atoms, and examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, and an n-butyl group. Group, isobutyl group, sec-butyl group, tert-butyl group, n-pentyl group, isopentyl group, neopentyl group, tert-pentyl group, 1-methylbutyl group, n-hexyl group, 2-methylpentyl group, 3-methyl Pentyl group, 2,2-dimethylbutyl group, 2,3-dimethylbutyl group, n-heptyl group, 2-methylhexyl group, 3-methylhexyl group, 2,2-dimethylpentyl group, 2,3-dimethylpentyl group Group, 2,4-dimethylpentyl group, 3,3-dimethylpentyl group, 3-ethylpentyl group, 2,2,3-trimethylbutyl group, n-octyl group, isooctyl group, 2-ethylhexyl group, nonyl group, Examples thereof include a decyl group, an undecyl group and a dodecyl group.
The linear or branched alkyl group preferably has 1 to 10 carbon atoms, more preferably 1 to 8 carbon atoms, particularly preferably 1 to 5 carbon atoms, and 1 to 3 carbon atoms. Most preferably.

The cyclic alkyl group preferably has 3 to 12 carbon atoms, and examples of the alkyl group include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, a cyclooctyl group and a cyclononyl group. , A cyclodecyl group, a norbornyl group, an isobornyl group, a 1-adamantyl group, a 2-adamantyl group, a tricyclodecyl group, and the like, and one or more hydrogen atoms of these cyclic alkyl groups are linear or branched. Examples thereof include those substituted with a chain or cyclic alkyl group. Here, examples of the linear, branched or cyclic alkyl group substituting the hydrogen atom include those described above as examples of the alkyl group for R ¹¹ and R ¹² .
The cyclic alkyl group preferably has 3 to 10 carbon atoms, more preferably 3 to 8 carbon atoms, and particularly preferably 5 to 7 carbon atoms.
The cyclic alkyl group is preferably monocyclic.

The alkyl group for R ¹¹ and R ¹² is preferably linear or branched.

Examples of the halogen atom in R ¹¹ and R ¹² include a fluorine atom, a chlorine atom, a bromine atom and an iodine atom, and the halogen atom is preferably a chlorine atom, a bromine atom or an iodine atom.

R ¹¹ and R ¹² are preferably an alkyl group having 1 to 5 carbon atoms or a halogen atom.

In the Formula (1) -11 and (1) _{-12, n 11} and _{n 12} is an integer from 0 to 5.
n ₁₁ is the bonding number of ^{R 11} in the compound (1) -1 (more specifically BIQ _{skeleton), n 12} is, ^{R 12} in the compound (1) -2 (more specifically BIQ backbone) Is the number of bonds of.
When n ₁₁ is an integer of 1 to 5, in the compound (1) (more specifically, the compound (1)-1), R ¹¹ is a naphthalene skeleton to which X ¹¹ is bonded in the BIQ skeleton. It is bonded to any of the five constituent carbon atoms.
Similarly, when n ₁₂ is an integer of 1 to 5, in the compound (1) (more specifically, the compound (1)-2), R ¹² is bonded to X ^{12 in the} BIQ skeleton. It is bonded to any of the five carbon atoms forming the naphthalene skeleton.

In the compound (1), when n ₁₁ is an integer of 2 or more, a plurality of R ¹¹ may be the same or different from each other. That is, R ^{11 s} may all be the same, all may be different, or only some may be the same. The combination of the plurality of R ¹¹ is not particularly limited.
Similarly, in the compound (1), when n ₁₂ is an integer of 2 or more, a plurality of R ¹² may be the same or different from each other. That is, R ^{12 s} may all be the same, all may be different, or only some may be the same. The combination of these plural R ¹² is not particularly limited.

n ₁₁ and n ₁₂ are preferably an integer of 0 to 3, more preferably an integer of 0 to 2, and particularly preferably 0 or 1.

In general formulas (1)-11 and (1)-12, X ¹¹ and X ¹² are a hydrogen atom, a cyano group (—CN), an amino group (—NH ₂ ), a monoalkylamino group or a dialkylamino group.
The one alkyl group bonded to the nitrogen atom of the monoalkylamino group in X ¹¹ and X ¹² may be linear, branched or cyclic, and such an alkyl group is R ^{The same} as the alkyl group in ¹¹ and R ¹² can be mentioned.

The two alkyl groups bonded to the nitrogen atom of the dialkylamino group in X ¹¹ and X ¹² may be linear, branched or cyclic, and such an alkyl group may be R ¹¹ And the same as the above alkyl group for R ¹² .
The two alkyl groups in the dialkylamino group may be the same or different from each other.

X ¹¹ and X ¹² are preferably hydrogen atoms or cyano groups, and more preferably cyano groups.

When Z is a group represented by the general formula (1)-11, preferable examples of the compound (1), that is, the compound (1)-1, include, for example, R ¹¹ being an alkyl group having 1 to 5 carbon atoms. Or a halogen atom, wherein n ₁₁ is an integer of 0 to 2 and the like.
More preferred compound (1)-1 is, for example, X ¹¹ is a cyano group, R ¹¹ is an alkyl group having 1 to 5 carbon atoms or a halogen atom, and n ₁₁ is an integer of 0 to 2. There are certain things.
More preferred compound (1)-1 is, for example, R ¹ is a hydrogen atom, R ¹¹ is an alkyl group having 1 to 5 carbon atoms or a halogen atom, and n ₁₁ is an integer of 0 to 2. Some are also included.
More preferred compound (1)-1 is, for example, R ¹ is a hydrogen atom, X ¹¹ is a cyano group, R ¹¹ is an alkyl group having 1 to 5 carbon atoms or a halogen atom, and n is Examples thereof include those in which ₁₁ is an integer of 0 to 2.

When Z is a group represented by the general formula (1)-12, preferable examples of the compound (1), that is, the compound (1)-2, include, for example, R ¹² having an alkyl group having 1 to 5 carbon atoms. Or a halogen atom, wherein n ₁₂ is an integer of 0 to 2 and the like.
More preferred compound (1)-2 is, for example, X ¹² is a cyano group, R ¹² is an alkyl group having 1 to 5 carbon atoms or a halogen atom, and n ₁₂ is an integer of 0 to 2. There are certain things.
More preferred compound (1)-2 is, for example, R ¹ is a hydrogen atom, R ¹² is an alkyl group having 1 to 5 carbon atoms or a halogen atom, and n ₁₂ is an integer of 0 to 2. Some are also included.
More preferred compound (1)-2 is, for example, R ¹ is a hydrogen atom, X ¹² is a cyano group, R ¹² is an alkyl group having 1 to 5 carbon atoms or a halogen atom, and Those in which ₁₂ is an integer of 0 to 2 and the like can be mentioned.

Among the compounds (1), preferred as the compound (1)-1 is, for example, a compound represented by the following formula (1A)-101 (hereinafter, may be abbreviated as “compound (1A)-101”). ), a compound represented by the following formula (1B)-101 (hereinafter sometimes abbreviated as “compound (1B)-101”), and the like.
Among the compounds (1), preferred as the compound (1)-2 is, for example, a compound represented by the following formula (1A)-201 (hereinafter, may be abbreviated as “compound (1A)-201”). ), a compound represented by the following formula (1B)-201 (hereinafter sometimes abbreviated as “compound (1B)-201”), and the like.
Both the compound (1A)-101 and the compound (1A)-201 are deoxyribonucleoside derivatives, and in this specification, the compound (1) which is a deoxyribonucleoside derivative may be referred to as “compound (1A)”. .. In addition, both the compound (1B)-101 and the compound (1B)-201 are ribonucleoside derivatives, and in the present specification, the compound (1) which is a ribonucleoside derivative is referred to as “compound (1B)”. There is.
Note that these compounds are merely examples of the preferred compound (1), and the preferred compound (1) is not limited thereto.

The compound (1) is preferably one in which Z is a group represented by the general formula (1)-11, that is, the compound (1)-1.

The compound (1) is a fluorescent compound as described above, and emits fluorescence by irradiating with light (excitation light) having a wavelength in an appropriate range corresponding to the type of the BIQ skeleton.
Furthermore, the compound (1) is not only a fluorescent compound, but also has the property that the fluorescence emission wavelength changes with the change of pH in the peripheral region of the compound (1). In the present specification, “the fluorescence emission wavelength changes” means that “the fluorescence emission wavelength of the main peak of fluorescence emission intensity in the fluorescence spectrum changes”, unless otherwise specified. It is preferable that the fluorescence emission wavelength changes when the emission intensity becomes maximum."

As shown in the following formulas (I-1) and (I-2), the compound (1) focuses on the nitrogen atom constituting the quinoline skeleton, that is, the 3-deazaadenine skeleton in the BIQ skeleton. In such a case, it is presumed that a proton (hydrogen ion, H ⁺ ) is bonded to the nitrogen atom at the N1 position constituting this skeleton to be protonated. Since the fluorescence emission wavelength of the compound (1) thus protonated is different from that of the compound (1) which is not protonated, the compound (1) emits fluorescence as the pH of the peripheral region thereof changes. It is assumed that the emission wavelength changes.

（式中、Ｘ^１１、Ｘ^１２、Ｒ^１１、Ｒ^１２、ｎ_１１及びｎ_１２は、いずれも上記と同じである。）

(In the formula, X ¹¹ , X ¹² , R ¹¹ , R ¹² , n ₁₁ and n ₁₂ are all the same as above.)

As is well known, an adenine base in a nucleoside usually forms a base pair with a thymine base in a nucleoside by hydrogen bonding, as shown in the following formula (II).
On the other hand, as shown in the following formula (III-1) and the following formula (III-2), the BIQ skeleton corresponding to the base site of the compound (1) is also in the nucleoside in the nucleoside in a non-protonated state. It is speculated that a base pair can be formed by hydrogen bonding with thymine base. However, in the protonated state, the BIQ skeleton highly preferentially forms a base pair with the cytosine base rather than the thymine base in the nucleoside. The base pair at this time is presumed to be formed as shown in the following formula (IV-1) and the following formula (IV-2).
As described above, in the compound (1), the presence or absence of the formation of a base pair with the cytosine base of the BIQ skeleton changes depending on the pH of the peripheral region.

（式中、Ｘ^１１、Ｘ^１２、Ｒ^１１、Ｒ^１２、ｎ_１１及びｎ_１２は、いずれも上記と同じである。）

(In the formula, X ¹¹ , X ¹² , R ¹¹ , R ¹² , n ₁₁ and n ₁₂ are all the same as above.)

That is, in the compound (1), the presence or absence of protonation of the BIQ skeleton changes depending on the change in the pH of the peripheral region, and the presence or absence of base pair formation with the cytosine base of the BIQ skeleton changes depending on the presence or absence of this protonation. However, at this time, the fluorescence emission wavelength changes. The compound (1) having such properties is suitable for use as a probe, as described later.

In addition, the compound (1) is one in which not only the fluorescence emission wavelength but also the fluorescence emission intensity is changed by the change in pH of the peripheral region by adjusting the molecular structure such as adjusting the structure of the BIQ skeleton. Becomes Usually, the fluorescence emission intensity of compound (1) can be easily adjusted by adjusting the structure of the BIQ skeleton. In the present specification, “change in fluorescence emission intensity” means “the maximum value of fluorescence emission intensity changes” unless otherwise specified.
As described above, the compound (1) whose fluorescence emission wavelength and fluorescence emission intensity change according to the change of pH in the peripheral region is particularly suitable for use as a probe.

In addition to the change in the pH of the peripheral region, the compound (1) may change the fluorescence emission wavelength depending on, for example, the type of solvent in which it is dissolved.

<<Production Method of Compound (1)>>
Compound (1) can be produced, for example, by subjecting a nucleoside derivative as a raw material to a known reaction to form a BIQ skeleton depending on the type of Z. More specifically, it is as follows.

<Method for producing compound (1)-1>
In the compound (1), the compound (1)-1 includes, for example, a compound represented by the following general formula (1c) (hereinafter, may be abbreviated as “compound (1c)”) and the following general formula (1c). A compound represented by the following general formula (11a) (hereinafter referred to as "compound (11a)") by reacting with a compound represented by 11b) (hereinafter sometimes abbreviated as "compound (11b)"). May be abbreviated) (hereinafter, may be abbreviated as "compound (11a) production step"), and a step of obtaining compound (1)-1 from compound (11a) (hereinafter, "compound (11 1)-1 manufacturing process” may be abbreviated).
Hereinafter, each step will be described in detail.

（式中、Ｒ^１０は水素原子、水酸基、トリアルキルシリルオキシ基又はアルキルジアリールシリルオキシ基であり；Ｒ^２及びＲ^３は、それぞれ独立にトリアルキルシリル基又はアルキルジアリールシリル基であり；Ｇ^１はヨウ素原子、臭素原子、塩素原子又はパーフルオロアルカンスルホニルオキシ基であり；Ｒ^１、Ｘ^１１、Ｒ^１１及びｎ_１１は、いずれも上記と同じである。）

(In the formula, R ¹⁰ is a hydrogen atom, a hydroxyl group, a trialkylsilyloxy group or an alkyldiarylsilyloxy group; R ² and R ³ are each independently a trialkylsilyl group or an alkyldiarylsilyl group; G ¹ Is an iodine atom, a bromine atom, a chlorine atom or a perfluoroalkanesulfonyloxy group; R ¹ , X ¹¹ , R ¹¹ and n ₁₁ are all the same as the above.)

[Compound (11a) Production Process]
In the step of producing the compound (11a), the compound (1c) is reacted with the compound (11b) to obtain the compound (11a).
The above reaction for obtaining the compound (11a) is a known cross coupling reaction.

(Compound (1c))
The compound (1c) is a known compound.
In the compound (1c), R ¹⁰ is a hydrogen atom, a hydroxyl group, a trialkylsilyloxy group or an alkyldiarylsilyloxy group.
Examples of the trialkylsilyloxy group in R ¹⁰ include a trimethylsilyloxy group ((CH ₃ ) ₃ SiO—), a triethylsilyloxy group ((CH ₃ CH ₂ ) ₃ SiO—), and a tert-butyldimethylsilyloxy group ( _{(CH 3) 3 C (CH} 3) 2 SiO-), triisopropyl silyl group _{(((CH 3) 2 CH} ) 3 SiO-) , and the like.
Examples of the alkyldiarylsilyloxy group for R ¹⁰ include a tert-butyldiphenylsilyloxy group ((CH ₃ ) ₃ C(C ₆ H ₅ ) ₂ SiO—) and the like.

In the compound (1c), R ² and R ³ are each independently a trialkylsilyl group or an alkyldiarylsilyl group.
Examples of the trialkylsilyl group in R ² and R ³ include a trimethylsilyl group ((CH ₃ ) ₃ Si-), a triethylsilyl group ((CH ₃ CH ₂ ) ₃ Si-), and a tert-butyldimethylsilyl group (( _{CH 3) 3 C (CH 3} ) 2 Si-), triisopropylsilyl group _{(((CH 3) 2 CH} ) 3 Si-) , and the like.
The alkyldiarylsilyl group in R ² and ^{R 3,} for example, such as tert- butyldiphenylsilyl group _{((CH 3) 3 C (} C 6 H 5) 2 Si-) and the like.

In the compound (1c), G ¹ is an iodine atom (-I), a bromine atom (-Br), a chlorine atom (-Cl) or a perfluoroalkanesulfonyloxy group.
Examples of the perfluoroalkanesulfonyloxy group in G ¹ include a trifluoromethanesulfonyloxy group (CF ₃ —S(═O) ₂ —O—) and a nonafluorobutanesulfonyloxy group (C ₄ F ₉ —S(═O). ) _2- O-) and the like.

(Compound (11b))
The compound (11b) is a novel compound. The method for producing the compound (11b) will be described later.
In the compound ^(11b), X ^{11, R 11} and _{n 11} are the same as ^{X 11, R 11} and _{n 11} in Formula (1) -11.

(Compound (11a))
The compound (11a) is a new compound.
In the compound ^{(11a), R 10, R} 2 and ^{R 3} are the same as ^R 10, ^{R 2} and ^{R 3} in the compound ^{(1c), X 11, R} 11 and _{n 11} are X in the compound (11b) ¹¹ , the same as R ¹¹ and n ₁₁ .
The compound (11a) is a compound in which the hydroxyl group of the target compound (1)-1 is protected by a protecting group.

In the step of producing the compound (11a), for example, an organic solvent such as N,N-dimethylformamide (hereinafter sometimes abbreviated as “DMF”) or an aqueous solvent such as a mixed solvent of DMF and water is used as a reaction solvent. It is preferable to use.
The said solvent may be used individually by 1 type, and may use 2 or more types together, and when 2 or more types are used together, those combination and ratio can be selected arbitrarily.

In the compound (11a) production process, for example, tetrakis(triphenylphosphine)palladium(0)(Pd(P(C ₆ H ₅ ) ₃ ) ₄ ), dichlorobis(triphenylphosphine)palladium(II)(PdCl ₂ ( P _{(C 6} H _{5) 3)} 2), bis (tri -tert- butylphosphine) palladium (0) _(Pd (P _((CH 3) _{3 C),} such as _{3) 2),} using palladium catalysis Is preferably performed.
As the palladium catalyst, one kind may be used alone, two kinds or more may be used in combination, and when two kinds or more are used in combination, a combination and a ratio thereof may be arbitrarily selected. However, it is usually sufficient to use one palladium catalyst alone.
In the compound (11a) production step, the amount of the palladium catalyst used is, for example, preferably 0.01 to 0.3 times, and more preferably 0.01 to 0.1 times the molar amount of the compound (1c) used. More preferably, it is an amount.

In the compound (11a) production step, the amount of the compound (11b) used is, for example, preferably 1 to 3 times, and more preferably 1 to 2 times the molar amount of the compound (1c) used. preferable.

In the compound (11a) production step, it is preferable to carry out the reaction using a base.
Examples of the base include inorganic bases such as sodium hydroxide and potassium hydroxide.
The above bases may be used alone or in combination of two or more, and when two or more are used in combination, their combination and ratio can be arbitrarily selected.
In the step of producing the compound (11a), the amount of the base used is, for example, preferably 1 to 6 times, and more preferably 1 to 4 times the molar amount of the compound (1c) used.

In the compound (11a) production step, the reaction temperature is, for example, preferably 70 to 110°C, and more preferably 80 to 100°C.
In the step of producing the compound (11a), the reaction time is, for example, preferably 1 to 12 hours, more preferably 3 to 9 hours.

In the compound (11a) production step, it is preferable to carry out the reaction in an atmosphere of an inert gas.
Examples of the inert gas include argon gas, helium gas, and nitrogen gas.

In the production process of compound (11a), after the reaction is completed, the compound (11a) may be taken out by post-treatment as necessary by a known method. That is, if necessary, any of post-treatment operations such as filtration, washing, extraction, pH adjustment, dehydration, and concentration may be performed alone or in combination of two or more types to perform concentration, crystallization, reprecipitation, column chromatography. The compound (11a) may be taken out by the above method. In addition, the compound (11a) taken out may be further subjected to operations such as crystallization, reprecipitation, column chromatography, extraction, stirring and washing of crystals with a solvent, or any combination of two or more thereof as necessary. It may be purified by performing it more than once.
In the production process of compound (11a), after completion of the reaction, compound (11a) may be used in the next step without being taken out, but from the viewpoint of improving the yield of compound (1)-1, which is the target, It is preferable to take out the compound (11a) by the method described above.

[Compound (1)-1 production process]
In the compound (1)-1 production step, compound (1)-1 is obtained from compound (11a).
The reaction for obtaining the compound (1)-1 is a known deprotection reaction. That is, in this step, R ² and R ³ (trialkylsilyl group or alkyldiarylsilyl group) are removed to form a hydroxyl group. Also, when R ¹⁰ is a trialkylsilyloxy group or an alkyldiarylsilyloxy group, the trialkylsilyl group or alkyldiarylsilyl group) is similarly removed to form a hydroxyl group.
The deprotection reaction can be performed, for example, under acidic conditions or by adding a fluorine atom-containing compound.
In addition, in this step, when R ¹⁰ is a hydrogen atom, a compound (1)-1 in which R ¹ is a hydrogen atom is obtained, and R ¹⁰ is a hydroxyl group, a trialkylsilyloxy group or an alkyldiarylsilyloxy group. Then, a compound (1)-1 in which R ¹ is a hydroxyl group is obtained.

Examples of the acid used for the acidic condition include inorganic acids such as hydrochloric acid; organic acids such as acetic acid and paratoluenesulfonic acid.
In the deprotection reaction, for example, the amount of the acid used is preferably 1 to 2 times the molar amount of the compound (11a) used, the reaction temperature is preferably 10 to 25° C., and the reaction time is It is preferably 1 to 2 hours.

Examples of the fluorine atom-containing compounds include inorganic fluorine atom-containing compounds such as hydrofluoric acid (HF) and cesium fluoride (CsF); organic fluorine atom-containing compounds such as tetrabutylammonium fluoride (TBAF). Be done.
The fluorine atom-containing compound may be used alone or in combination of two or more, and when two or more are used in combination, the combination and ratio thereof may be arbitrarily selected. However, it is usually sufficient to use one type of the fluorine atom-containing compound alone.
In the deprotection reaction, the amount of the fluorine atom-containing compound used is, for example, preferably 2 to 6 times, and more preferably 2 to 4 times the molar amount of the compound (11a) used.
During the deprotection reaction, the reaction temperature is, for example, preferably 15 to 35°C, and more preferably 18 to 30°C.
During the deprotection reaction, the reaction time is, for example, preferably 10 minutes to 5 hours, and more preferably 30 minutes to 3 hours.

In the compound (1)-1 production process, it is preferable to carry out the reaction under an atmosphere of an inert gas.
Examples of the inert gas include those similar to those used in the compound (11a) production step.

In the compound (1)-1 production step, after the reaction is completed, the compound (1)-1 can be extracted in the same manner as in the compound (11a) production step, and the extracted compound (1)-1 can be obtained. Further, it may be purified by the same method.

(Method for producing compound (11b))
The above-mentioned novel compound (11b) is an organic boron compound which is a raw material for the cross coupling reaction, and many other organic boron compounds used for the same purpose are known. It can be manufactured by the method.
For example, as the compound (11b), a step of obtaining the compound (11b) from the compound represented by the following general formula (11b1) (hereinafter sometimes abbreviated as “compound (11b1)”) (hereinafter, referred to as “compound (11b)”). It may be abbreviated as "manufacturing process").
In the compound (11b) production step, for example, as shown below, the compound (11b1) is reacted with 4,4,5,5-tetramethyl-1,3,2-dioxaborolane in the presence of a palladium catalyst. By doing so, the compound (11b) is obtained.

（式中、Ｘ^１１、Ｒ^１１及びｎ_１１は、いずれも上記と同じである。）

(In the formula, X ¹¹ , R ¹¹ and n ₁₁ are all the same as above.)

In the compound (11b) production step, it is preferable to use an organic solvent such as 1,4-dioxane as a reaction solvent.

In the compound (11b1) production process, the amount of 4,4,5,5-tetramethyl-1,3,2-dioxaborolane used is, for example, 1 to 6 times the molar amount of the compound (11b1) used. Is preferred, and more preferably 1 to 4 times the molar amount.

As the palladium catalyst in the step of producing the compound (11b), here, [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (hereinafter, abbreviated as “Pd(dppf)Cl ₂ ”). However, other than Pd(dppf)Cl ₂ may be used.

In the compound (11b1) production step, the amount of the palladium catalyst used is, for example, preferably 0.01 to 0.3 times, and 0.01 to 0.1 times the amount of the compound (11b1) used. More preferably, it is a molar amount.

In the compound (11b1) production step, it is preferable to carry out the reaction using a base.
Among the above bases, examples of the organic base include trialkylamines such as trimethylamine and triethylamine.
The above bases may be used alone or in combination of two or more, and when two or more are used in combination, their combination and ratio can be arbitrarily selected.
In the compound (11b1) production step, the amount of the base used is, for example, preferably 1 to 8 times, and more preferably 1 to 6 times the molar amount of the compound (1c) used.

In the compound (11b) production step, the reaction temperature is, for example, preferably 70 to 110°C, and more preferably 80 to 100°C.
In the compound (11b) production step, the reaction time is, for example, preferably 1 to 12 hours, and more preferably 3 to 9 hours.

In the compound (11b) production step, the reaction is preferably performed in an atmosphere of an inert gas.
Examples of the inert gas include those similar to those used in the compound (11a) production step.

The obtained compound (11b) can be extracted by the same method as in the above-mentioned compound (11a) production step, and the extracted compound (11b) may be further purified by the same method. Further, the obtained compound (11b) may be used in the next step without being taken out after completion of the reaction, but it is preferably taken out from the viewpoint that the yield of the target compound (11a) is improved.

Each compound such as compound (1)-1, compound (11a) and compound (11b) is known, for example, by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), infrared spectroscopy (IR) and the like. The structure can be confirmed by the method of.

<Method for producing compound (1)-2>
Of the compound (1), the compound (1)-2 includes, for example, the compound (1c) and a compound represented by the following general formula (12b) (hereinafter, may be abbreviated as “compound (12b)”). To obtain a compound represented by the following general formula (12a) (hereinafter sometimes abbreviated as “compound (12a)”) (hereinafter abbreviated as “compound (12a) production step”). Can be carried out) and a step of obtaining compound (1)-2 from compound (12a) (hereinafter sometimes abbreviated as “compound (1)-2 production step”).

（式中、Ｒ^１０は水素原子、水酸基、トリアルキルシリルオキシ基又はアルキルジアリールシリルオキシ基であり；Ｒ^２及びＲ^３は、それぞれ独立にトリアルキルシリル基又はアルキルジアリールシリル基であり；Ｇ^１はヨウ素原子、臭素原子、塩素原子又はパーフルオロアルカンスルホニルオキシ基であり；Ｒ^１、Ｘ^１２、Ｒ^１２及びｎ_１２は、いずれも上記と同じである。）

(In the formula, R ¹⁰ is a hydrogen atom, a hydroxyl group, a trialkylsilyloxy group or an alkyldiarylsilyloxy group; R ² and R ³ are each independently a trialkylsilyl group or an alkyldiarylsilyl group; G ¹ Is an iodine atom, a bromine atom, a chlorine atom or a perfluoroalkanesulfonyloxy group; R ¹ , X ¹² , R ¹² and n ₁₂ are all the same as the above.)

[Process for producing compound (12a)]
In the step of producing the compound (12a), the compound (1c) is reacted with the compound (12b) to obtain the compound (12a).
The reaction for obtaining the compound (12a) is a known cross coupling reaction.

(Compound (1c))
The compound (1c) is the same as the compound (1c) used in the above-mentioned compound (11a) production process.

(Compound (12b))
The compound (12b) is a new compound. The method for producing the compound (12b) will be described later.
In the compound ^(12b), X ^{12, R 12} and _{n 12} are the same as ^{X 12, R 12} and _{n 12} in the general formula (1) -12.

(Compound (12a))
The compound (12a) is a new compound.
In the compound ^{(12a), R 10, R} 2 and ^{R 3} are the same as ^R 10, ^{R 2} and ^{R 3} in the compound ^{(1c), X 12, R} 12 and _{n 12} are X in the compound (12b) ¹² , R ¹² and n ₁₂ are the same.
The compound (12a) is a compound in which the hydroxyl group of the target compound (1)-2 is protected by a protecting group.

The compound (12a) production step can be performed by the same method as the above-mentioned compound (11a) production step, except that the compound (12b) is used instead of the compound (11b).

[Compound (1)-2 production process]
In the compound (1)-2 production step, compound (1)-2 is obtained from compound (12a).
The reaction for obtaining the compound (1)-2 is a known deprotection reaction. That is, in this step, R ² and R ³ (trialkylsilyl group or alkyldiarylsilyl group) are removed to form a hydroxyl group. Also, when R ¹⁰ is a trialkylsilyloxy group or an alkyldiarylsilyloxy group, the trialkylsilyl group or alkyldiarylsilyl group) is similarly removed to form a hydroxyl group.
The deprotection reaction can be performed, for example, under acidic conditions or by adding a fluorine atom-containing compound.
In addition, in this step, when R ¹⁰ is a hydrogen atom, a compound (1)-2 in which R ¹ is a hydrogen atom is obtained, and R ¹⁰ is a hydroxyl group, a trialkylsilyloxy group or an alkyldiarylsilyloxy group. Then, a compound (1)-2 in which R ¹ is a hydroxyl group is obtained.

The compound (1)-2 production step can be performed by the same method as the above-mentioned compound (1)-1 production step, except that the compound (12a) is used instead of the compound (11a). For example, after the reaction is completed, the compound (1)-2 can be extracted by the same method as in the above-mentioned compound (11a) production step, and the extracted compound (1)-2 can be further purified by the same method. You may.

(Method for producing compound (12b))
The compound (12b), which is the above-mentioned novel compound, uses a compound represented by the following general formula (12b1) (hereinafter sometimes abbreviated as “compound (12b1)”) instead of the compound (11b1). Other than the above, the compound (11b) can be produced by the same method as the above-mentioned production method.
That is, the compound (12b) can be produced by a production method having a step of obtaining the compound (12b) from the compound (12b1) (hereinafter sometimes abbreviated as “compound (12b) production step”). The respective conditions such as reaction, extraction, purification and the like in the compound (12b) production step can be the same as those in the compound (11b) production step.

（式中、Ｘ^１２、Ｒ^１２及びｎ_１２は、いずれも上記と同じである。）

(In the formula, X ¹² , R ¹² and n ₁₂ are all the same as above.)

The obtained compound (12b) may be used in the next step without being taken out after the completion of the reaction, but it is preferably taken out from the viewpoint of improving the yield of the target compound (12a).

Each compound such as compound (1)-2, compound (12a) and compound (12b) is known, for example, by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), infrared spectroscopy (IR) and the like. The structure can be confirmed by the method of.

(Compound (1a))
As described above, the compound (11a) and the compound (12a) are novel compounds. These compounds can be comprehensively represented by, for example, the following general formula (1a) (in this specification, the compound represented by the following general formula (1a) may be referred to as “compound (1a)”. ).

（式中、Ｒ^１０は水素原子、水酸基、トリアルキルシリルオキシ基又はアルキルジアリールシリルオキシ基であり；Ｚは下記一般式（１）−１１又は（１）−１２で表される基であり；Ｒ^２及びＲ^３は、それぞれ独立にトリアルキルシリル基又はアルキルジアリールシリル基である。）

(In the formula, R ¹⁰ is a hydrogen atom, a hydroxyl group, a trialkylsilyloxy group or an alkyldiarylsilyloxy group; Z is a group represented by the following general formula (1)-11 or (1)-12; R ² and R ³ are each independently a trialkylsilyl group or an alkyldiarylsilyl group.)

（式中、Ｘ^１１及びＸ^１２は水素原子、シアノ基、アミノ基、モノアルキルアミノ基又はジアルキルアミノ基であり；Ｒ^１１及びＲ^１２はアルキル基又はハロゲン原子であり；ｎ_１１及びｎ_１２は０〜５の整数であり、ｎ_１１が２以上の整数である場合、複数個のＲ^１１は互いに同一でも異なっていてもよく、ｎ_１２が２以上の整数である場合、複数個のＲ^１２は互いに同一でも異なっていてもよく；符号＊を付した結合は、Ｚの結合先との間で形成されている。）

(In the formula, X ¹¹ and X ¹² are a hydrogen atom, a cyano group, an amino group, a monoalkylamino group or a dialkylamino group; R ¹¹ and R ¹² are an alkyl group or a halogen atom; and n ₁₁ and n ₁₂ are When n ₁₁ is an integer of 2 or more, a plurality of R ¹¹ may be the same or different, and when n ₁₂ is an integer of 2 or more, a plurality of R ^{12 are} May be the same or different from each other; the bond marked with * is formed between the bond to Z and the target.)

<<Compound (2)>>
The compound of one embodiment of the present invention is represented by the following general formula (2) (may be referred to as “compound (2)” in the present specification).

（式中、Ｒ^１は水素原子又は水酸基であり；Ｚは下記一般式（１）−１１又は（１）−１２で表される基であり；ｍは１〜３の整数である。）

(In the formula, R ¹ is a hydrogen atom or a hydroxyl group; Z is a group represented by the following general formula (1)-11 or (1)-12; and m is an integer of 1 to 3.)

（式中、Ｘ^１１及びＸ^１２は水素原子、シアノ基、アミノ基、モノアルキルアミノ基又はジアルキルアミノ基であり；Ｒ^１１及びＲ^１２はアルキル基又はハロゲン原子であり；ｎ_１１及びｎ_１２は０〜５の整数であり、ｎ_１１が２以上の整数である場合、複数個のＲ^１１は互いに同一でも異なっていてもよく、ｎ_１２が２以上の整数である場合、複数個のＲ^１２は互いに同一でも異なっていてもよく；符号＊を付した結合は、Ｚの結合先との間で形成されている。）

(In the formula, X ¹¹ and X ¹² are a hydrogen atom, a cyano group, an amino group, a monoalkylamino group or a dialkylamino group; R ¹¹ and R ¹² are an alkyl group or a halogen atom; and n ₁₁ and n ₁₂ are When n ₁₁ is an integer of 2 or more, a plurality of R ¹¹ may be the same or different, and when n ₁₂ is an integer of 2 or more, a plurality of R ^{12 are} May be the same or different from each other; the bond marked with * is formed between the bond to Z and the target.)

The compound (2) is a nucleotide derivative having a base containing a BIQ skeleton, and is the 5'-monophosphate, 5'-diphosphate and 5'-triphosphate of the compound (1).
Further, the compound (2) is a fluorescent compound.

The compound (2) has a group represented by the formula “—P(═O)(—O ⁻ )—O—” as a structural unit labeled with a symbol m. May be represented by the formula "-P(=O)(-OH)-O-" due to the addition of a proton (H ⁺ ).

Compound (2) is 5'-position hydroxyl group (-OH) and a methylene group (-CH 2 _-) between, except having the structural unit denoted by reference numeral m above, the same as compound (1) It is a thing. That is, R ¹ and Z (in other words, X ¹¹ , X ¹² , R ¹¹ , R ¹² , n ₁₁ , n ₁₂ and the symbol *) in the compound (2) are the same as R ¹ and Z in the compound (1) (in other words, X). ¹¹ , X ¹² , R ¹¹ , R ¹² , n ₁₁ , n ₁₂ and symbol *).

In general formula (2), m is an integer of 1-3.
That is, the compound (2) is a compound represented by the following general formula (2)-1-1 (hereinafter sometimes abbreviated as "compound (2)-1-1"), the following general formula (2). -1-2 (Compound (2)-1-2 may be abbreviated below), the following general formula (2)-l-3 compound (hereinafter, "compound (2)-1-3" may be abbreviated), and a compound represented by the following general formula (2)-2-1 (hereinafter may be abbreviated as "compound (2)-2-1"). ), a compound represented by the following general formula (2)-2-2 (hereinafter sometimes abbreviated as “compound (2)-2-2”), and the following general formula (2)-2-3 It is classified into the represented compounds (hereinafter sometimes abbreviated as “compound (2)-2-3”). The compound (2)-1-1 and the compound (2)-2-1 are structural isomers. Similarly, compound (2)-1-2 and compound (2)-2-2 are structural isomers, and compound (2)-1-3 and compound (2)-2-3 are structural isomers. Is.

（式中、Ｒ^１、Ｘ^１１、Ｒ^１１及びｎ_１１は、いずれも上記と同じである。）

(In the formula, all of R ¹ , X ¹¹ , R ¹¹ and n ₁₁ are the same as above.)

（式中、Ｒ^１、Ｘ^１２、Ｒ^１２及びｎ_１２は、いずれも上記と同じである。）

(In the formula, R ¹ , X ¹² , R ¹² and n ₁₂ are all the same as above.)

When Z is a group represented by the general formula (1)-11, that is, compound (2)-1-1, compound (2)-1-2 and compound (2)-1-. 3 (hereinafter, these compounds may be collectively referred to as “compound (2)-1”) are preferable, for example, R ¹¹ is an alkyl group having 1 to 5 carbon atoms or a halogen atom, and n ₁₁ is exemplified such as those an integer of 0 to 2.
More preferred compound (2)-1 is, for example, X ¹¹ is a cyano group, R ¹¹ is an alkyl group having 1 to 5 carbon atoms or a halogen atom, and n ₁₁ is an integer of 0 to 2. There are certain things.
More preferred compound (2)-1 is, for example, R ¹ is a hydrogen atom, R ¹¹ is an alkyl group having 1 to 5 carbon atoms or a halogen atom, and n ₁₁ is an integer of 0 to 2. Some are also included.
More preferred compound (2)-1 is, for example, R ¹ is a hydrogen atom, X ¹¹ is a cyano group, R ¹¹ is an alkyl group having 1 to 5 carbon atoms or a halogen atom, and n is Examples thereof include those in which ₁₁ is an integer of 0 to 2.

Compound (2) in the case where Z is a group represented by the above general formula (1)-12, that is, compound (2)-2-1, compound (2)-2-2 and compound (2)-2- 3 (hereinafter, these compounds may be collectively referred to as “compound (2)-2”) are preferable, for example, R ¹² is an alkyl group having 1 to 5 carbon atoms or a halogen atom, and n ₁₂ is exemplified such as those an integer of 0 to 2.
More preferred compound (2)-2 is, for example, X ¹² is a cyano group, R ¹² is an alkyl group having 1 to 5 carbon atoms or a halogen atom, and n ₁₂ is an integer of 0 to 2. There are certain things.
More preferred compound (2)-2 is, for example, R ¹ is a hydrogen atom, R ¹² is an alkyl group having 1 to 5 carbon atoms or a halogen atom, and n ₁₂ is an integer of 0 to 2. Some are also included.
More preferred compound (2)-2 is, for example, R ¹ is a hydrogen atom, X ¹² is a cyano group, R ¹² is an alkyl group having 1 to 5 carbon atoms or a halogen atom, and Those in which ₁₂ is an integer of 0 to 2 and the like can be mentioned.

Among the compounds (2), preferred as the compound (2)-1 are, for example, compounds represented by the following formula (2A)-101-1 and compounds represented by the following formula (2A)-101-2. A compound represented by the following formula (2A)-101-3 (hereinafter, these compounds are referred to as "compound (2A)-101-1", "compound (2A)-101-2", and "compound (2A)-", respectively. 101-3"), a compound represented by the following formula (2B)-101-1, a compound represented by the following formula (2B)-101-2, a compound represented by the following formula (2B)-101-1: Compound represented by 3 (hereinafter, these compounds may be abbreviated as “compound (2B)-101-1”, “compound (2B)-101-2”, and “compound (2B)-101-3”, respectively). There is) etc.

Among the compounds (2), preferred as the compound (2)-2 are, for example, compounds represented by the following formula (2A)-201-1 and compounds represented by the following formula (2A)-201-2. , A compound represented by the following formula (2A)-201-3 (hereinafter, these compounds are referred to as "compound (2A)-201-1", "compound (2A)-201-2", and "compound (2A)-", respectively. Abbreviated as "201-3"), a compound represented by the following formula (2B)-201-1, a compound represented by the following formula (2B)-201-2, a compound represented by the following formula (2B)-201-1: Compound represented by 3 (hereinafter, these compounds may be abbreviated as "compound (2B)-201-1", "compound (2B)-201-2", and "compound (2B)-201-3", respectively. There is) etc.

Compound (2A)-101-1, Compound (2A)-101-2, Compound (2A)-101-3, Compound (2A)-201-1, Compound (2A)-201-2, Compound (2A)- All 201-3 are deoxyribonucleotide derivatives. In the present specification, compound (2) which is a deoxyribonucleotide derivative may be referred to as “compound (2A)”.
In addition, compound (2B)-101-1, compound (2B)-101-2, compound (2B)-101-3, compound (2B)-201-1, compound (2B)-201-2, compound (2B) )-201-3 are all ribonucleotide derivatives. In the present specification, the compound (2) which is a ribonucleotide derivative may be referred to as “compound (2B)”.
Note that these compounds are merely examples of the preferred compound (2), and the preferred compound (2) is not limited thereto.

The compound (2) is preferably one in which Z is a group represented by the general formula (1)-11, that is, the compound (2)-1.

Since the compound (2) has the same BIQ skeleton as the compound (1), it has the same fluorescent property as the compound (1).
That is, the compound (2) is not only a fluorescent compound, but also has the property that the fluorescence emission wavelength changes with the change of pH in the peripheral region of the compound (2).
The BIQ skeleton corresponding to the base site of the compound (2) is usually presumed to form a base pair with the thymine base in the nucleoside when it is not protonated. However, in the protonated state, the BIQ skeleton highly preferentially forms a base pair with the cytosine base rather than the thymine base in the nucleoside.

As described above, in the compound (2), the presence/absence of protonation of the BIQ skeleton changes depending on the change in the pH of the peripheral region, and the presence/absence of protonation causes the formation of a base pair with the cytosine base of the BIQ skeleton. The presence or absence changes, and the fluorescence emission wavelength changes at this time. The compound (2) having such characteristics is suitable for use as a probe, as described later.
Further, the compound (2) changes not only the fluorescence emission wavelength but also the fluorescence emission intensity due to the change in pH of the peripheral region. The compound (2) having such properties is particularly suitable for use as a probe.

In addition to the change in the pH of the peripheral region, the compound (2) may change the fluorescence emission wavelength depending on, for example, the dissolved solvent species.

The compound (2) can be used as various probes. That is, as the novel probe, a probe composed of the compound (2) can be mentioned.
In addition, the compound (2) is useful as a raw material for producing a condensate described below, and the obtained condensate can be used as various probes. The condensate will be described later in detail.

<<Production Method of Compound (2)>>
The compound (2) can be produced, for example, by utilizing a known phosphorylation method depending on the number of m. More specifically, it is as follows.

<Method for producing 5'-monophosphate>
In the compound (2), the 5′-monophosphate (wherein “5′-monophosphate” may be simply referred to herein) when m is 1, is, for example, a compound ( It can be produced by the production method having the step 1) of monophosphorylating a 5'-hydroxyl group (hereinafter, may be abbreviated as "5'-monophosphate production step"). Here, examples of the 5'-monophosphate include compound (2)-1-1 and compound (2)-2-1.

In 5'-monophosphate body manufacturing process, for example, trimethyl phosphite: dissolved (aka trimethyl phosphite, P _{(OCH 3) 3)} solvent compounds such as (1), preferably from -5 to 5 Under the cooling condition of ℃, the compound (1) in this solution was treated with phosphoryl chloride (also known as phosphorus oxychloride, POCl ₃ ) and then with water to give 5% of the compound (1). '- Can hydroxylate the hydroxyl group.

The obtained 5'-monophosphate can be taken out by the same method as in the case of the above-mentioned compound (11a) production step, and the taken 5'-monophosphate can be further purified by the same method. Good.

<Method for producing 5'-diphosphate>
Of the compound (2), the 5′-diphosphate (when it is simply referred to as “5′-diphosphate” in the present specification) when m is 2 includes the compound (2 )-1-2 and compound (2)-2-2.

In the production of the 5'-diphosphate, various known nucleoside 5'-diphosphate production methods can be used. For example, the 5'-monophosphate of compound (1) is dissolved in a solvent such as Tris-hydrochloric acid buffer having a pH of 7.6, and the nucleoside monophosphate is prepared in the presence of magnesium chloride, preferably at 5 to 25°C. By acting a kinase, a 5'-diphosphate is obtained.
The obtained 5'-diphosphate can be taken out by the same method as in the above-mentioned compound (11a) production step, and the taken 5'-diphosphate is further purified by the same method. Good.

<Method for producing 5'-triphosphate>
In the compound (2), a 5'-triphosphate (when it is simply referred to as "5'-triphosphate" in the present specification) when m is 3, is, for example, a compound ( It can be produced by the production method including the step 1) of triphosphorylating a 5'-hydroxyl group (hereinafter, may be abbreviated as "5'-triphosphate production step"). Here, examples of the 5'-triphosphate include compound (2)-1-3 and compound (2)-2-3.

The 5'-triphosphate is, for example, the above-mentioned 5'-monophosphate produced except that tributylammonium pyrophosphate is added to the solution after the action of phosphoryl chloride and before the action of water. It can be produced by performing the 5'-triphosphate production step in the same manner as in the step.

The structure of the compound (2) can be confirmed by known methods such as nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), and infrared spectroscopy (IR).

<< condensate >>
The condensate of the present invention is a condensate of the compound (2) described above and the compound (2) or another compound other than the compound (2).
The condensate of the present invention is useful as various probes, as described later.

The condensate of the present invention may have the same structure as the structure formed by the reaction of the compounds (2) with each other, or the compound (2) and the other compound with elimination of water (that is, dehydration). However, it is not particularly limited.
Among them, the condensate of the present invention is preferably one in which the phosphoric acid group or the 3′-position hydroxyl group of the compound (2) forms an ester bond.

The other compound is not particularly limited as long as it is other than the compound (2).
Preferred the other compounds, for example, 1 hydroxyl groups in the molecule (OH @ -) and phosphate groups (H 2 _PO 4 _-) having 1 or more in total, i.e., no hydroxyl groups in one molecule , Having one or more phosphoric acid groups, having one or more hydroxyl groups in one molecule and not having a phosphoric acid group, and having one or more hydroxyl groups and one or more phosphoric acid groups in one molecule, etc. Is mentioned. In the other compounds, the phosphate group anions (HPO 4 _- ^-) may be formed.

Among these, more preferable other compounds include, for example, nucleosides, nucleoside derivatives, nucleotides, nucleotide derivatives and the like.
It is particularly preferable that the other compound is a nucleotide and the condensate is a polynucleotide.

In the present specification, the term "polynucleotide" means one having a structure in which a plurality of nucleosides are bound to each other via a linking moiety such as phosphate, and usually a plurality of nucleotides are bound to each other. There is no particular limitation on the number of nucleosides that have the above structure. Thus, what is commonly referred to as an "oligonucleotide", such as, for example, having a structure in which 10 or fewer nucleosides or nucleotides are linked, is also referred to herein as a "polynucleotide."

The number of nucleosides constituting the polynucleotide as the condensate is preferably 3 to 25, more preferably 5 to 20, and particularly preferably 7 to 17.

The content of the constituent unit derived from the compound (2) in the condensate is not particularly limited, and for example, all constituent units may be constituent units derived from the compound (2) (that is, the above-mentioned constituent units). The condensate may be a condensate of only the compound (2), or the number of constitutional units derived from the compound (2) may be 1 (that is, the condensate is a compound having a molecular number of 1). It may be a condensate of (2) and the above-mentioned other compound having a molecular number of 1 or more).
Usually, in the condensate, the ratio of the content (the number of moles) of the structural unit derived from the compound (2) to the total amount (the number of moles) of all the structural units is 3 to 100 mol %. Is preferred, and more preferably 5 to 100 mol %.
For example, when the condensate is a polynucleotide, the number of constitutional units derived from the compound (2) constituting the condensate is preferably 1 to 3, and preferably 1 or 2. More preferably, 1 is particularly preferable. Then, when the number of nucleosides constituting the polynucleotide is 3 to 25 as described above, it is preferable that the constitutional unit derived from the compound (2) satisfies such a number of conditions.

In the above-mentioned polynucleotide of the present invention, the fluorescence emission intensity changes according to the change of pH in the peripheral region. Such a polynucleotide of the present invention typically has a tendency that the lower the pH, the higher the degree of protonation and the lower the fluorescence emission intensity.

In addition, the polynucleotide of the present invention forms a duplex with another polynucleotide, and when cytosine in the other polynucleotide faces the BIQ skeleton in the polynucleotide of the present invention, when another base faces Rather, protonation of the BIQ skeleton is induced even under non-acidic conditions. Then, with the protonation of the BIQ skeleton, the fluorescence emission wavelength of the polynucleotide of the present invention changes markedly.

For example, such a polynucleotide of the present invention typically tends to exhibit the following fluorescence emission characteristics. That is, a weakly basic solution (i) containing the polynucleotide of the present invention and further containing other polynucleotide having cytosine and capable of forming a double strand with this polynucleotide, Fluorescence emission intensity in a solution (ii) containing a polynucleotide, containing no other polynucleotide, and having the same pH and concentration of the polynucleotide of the present invention as in the case of the solution (i). Comparing these, the solution (i) has a lower fluorescence emission intensity. Here, the solutions (i) and (ii) are preferably aqueous solutions, and the pH thereof is preferably 7.0 to 9.0. This is because although the solution (i) is weakly basic, the BIQ skeleton in the polynucleotide of the present invention is protonated, whereas in solution (ii), the polyq of the present invention is It is speculated that the BIQ skeleton in the nucleotide is not protonated or the degree of protonation is extremely low.

Moreover, the polynucleotide of the present invention tends to further exhibit the following fluorescence emission characteristics. That is, the acidic solution (iii) containing the polynucleotide of the present invention and the compound (1) of the present invention are contained, and both the pH and the concentration of the BIQ skeleton are the same as the case of the solution (iii). Comparing the fluorescence emission intensity of the solution (iv), the fluorescence emission intensity of the solution (iii) becomes smaller. Here, the solutions (iii) and (iv) are preferably aqueous solutions, and the pH thereof is preferably 4.0 to 5.0.

<Method for producing condensate>
The condensate can be produced by a production method having a step of condensing the compound (2) as a raw material compound with the compound (2) or another compound other than the compound (2).
The condensation of the raw material compounds may be carried out according to a known method depending on the kind of the raw material compounds.

For example, when the condensate is a polynucleotide or a polynucleotide derivative, these condensates can be produced by condensing raw material compounds by applying a known chemical synthesis method without using an enzyme.

The condensate, which is a polynucleotide or a polynucleotide derivative, is subjected to a polymerase chain reaction method (hereinafter abbreviated as “PCR method”) in addition to a known chemical synthesis method without using an enzyme. It can also be manufactured.
When the PCR method is applied in this way, for example, the PCR method may be performed using the above-mentioned 5'-triphosphate compound (2).
Alternatively, the PCR method may be performed using a derivative of the compound (1) instead of the compound (2) which is a 5'-triphosphate. Hereinafter, the method for producing the condensate using the derivative of the compound (1) will be described.

Examples of the derivative of the compound (1) include a compound represented by the following general formula (5) (hereinafter sometimes abbreviated as “compound (5)”).

（式中、Ｒ^１は水素原子又は水酸基であり；Ｚ’は下記一般式（５）−１１又は（５）−１２で表される基である。）

(In the formula, R ¹ is a hydrogen atom or a hydroxyl group; Z′ is a group represented by the following general formula (5)-11 or (5)-12.)

（式中、Ｘ^１１及びＸ^１２は水素原子、シアノ基、アミノ基、モノアルキルアミノ基又はジアルキルアミノ基であり；Ｒ^１１及びＲ^１２はアルキル基又はハロゲン原子であり；ｎ_１１及びｎ_１２は０〜５の整数であり、ｎ_１１が２以上の整数である場合、複数個のＲ^１１は互いに同一でも異なっていてもよく、ｎ_１２が２以上の整数である場合、複数個のＲ^１２は互いに同一でも異なっていてもよく；符号＊を付した結合は、Ｚ’の結合先との間で形成されている。）

(In the formula, X ¹¹ and X ¹² are a hydrogen atom, a cyano group, an amino group, a monoalkylamino group or a dialkylamino group; R ¹¹ and R ¹² are an alkyl group or a halogen atom; and n ₁₁ and n ₁₂ are When n ₁₁ is an integer of 2 or more, a plurality of R ¹¹ may be the same or different, and when n ₁₂ is an integer of 2 or more, a plurality of R ^{12 are} May be the same or different from each other; the bond marked with * is formed between the bond to which Z′ is bonded.)

For the compound (5), for example, a step of obtaining a compound represented by the following general formula (3) (hereinafter sometimes abbreviated as “compound (3)”) from the compound (1) (hereinafter, referred to as “compound (3 ) Production step"), a step of obtaining a compound represented by the following general formula (4) (hereinafter sometimes abbreviated as "compound (4)") from the compound (3) (hereinafter, referred to as "compound (4)") "Compound (4) production step" and a step of obtaining compound (5) from compound (4) (hereinafter sometimes abbreviated as "compound (5) production step") It can be manufactured by the method. Hereinafter, each step will be described.

（式中、Ｒ^１、Ｚ及びＺ’は、いずれも上記と同じである。）

(In the formula, R ¹ , Z and Z′ are the same as above.)

[Compound (3) Production Process]
In the compound (3) production step, for example, the compound (3) is obtained by reacting the compound (1) with N,N-di-n-butylformamide dimethyl acetal.
The reaction conditions in the production process of compound (3) are not particularly limited as long as the reaction proceeds well.

However, in this step, it is preferable to dissolve the compound (1) in a solvent to carry out the reaction.
The solvent is not particularly limited, but preferred examples thereof include hydrocarbons such as benzene, toluene and xylene; tetrahydrofuran (hereinafter sometimes abbreviated as “THF”) and ethers such as diethyl ether; N,N- Examples thereof include amides (compounds having an amide bond) such as dimethylformamide; sulfoxides such as dimethyl sulfoxide; halogenated hydrocarbons such as methylene chloride and chlorobenzene.
The said solvent may be used individually by 1 type, and may use 2 or more types together, and when 2 or more types are used together, those combination and ratio can be selected arbitrarily.

Among the reaction conditions in the compound (3) production step, for example, the reaction temperature is preferably 50 to 90° C., and the reaction time is preferably 0.5 to 10 hours.
The amount of N,N-di-n-butylformamide dimethyl acetal used is preferably 2 to 5 times the molar amount of the compound (1) used.

In the compound (3) production process, it is preferable to carry out the reaction in an atmosphere of an inert gas.
Examples of the inert gas include the same as those used in the above-mentioned compound (11a) production process.

In the compound (3) production step, for example, after the completion of the reaction, the compound (3) can be extracted by the same method as in the above-mentioned compound (11a) production step, and the extracted compound (3) can be further extracted. It may be purified by the same method. The obtained compound (3) may be used in the next step without being taken out after completion of the reaction, but it is preferably taken out from the viewpoint of improving the yield of the target compound (5).

[Production process of compound (4)]
In the compound (4) manufacturing process, for example, by reacting the compound (3) and 4,4'-dimethoxytrityl chloride _{((CH 3 OC 6 H 4} ) 2 C (C 6 H 5) Cl), Compound (4) is obtained.
The reaction conditions in the compound (4) production step are not particularly limited as long as the reaction proceeds well.

However, in this step, it is preferable to carry out the reaction by dissolving the compound (3) in a solvent.
The solvent is not particularly limited, but preferable examples include organic bases such as pyridine. Further, the solvent is preferably an anhydrous solvent which has been subjected to an operation of removing contained water.
The said solvent may be used individually by 1 type, and may use 2 or more types together, and when 2 or more types are used together, those combination and ratio can be selected arbitrarily.

Among the reaction conditions in the compound (4) production step, for example, the reaction temperature is preferably 15 to 35° C., and the reaction time is preferably 0.5 to 8 hours.
The amount of 4,4′-dimethoxytrityl chloride used is preferably 1 to 1.5 times the molar amount of the amount of compound (3) used.

In the compound (4) production step, it is preferable to carry out the reaction under an atmosphere of an inert gas.
Examples of the inert gas include the same as those used in the above-mentioned compound (11a) production process.

In the compound (4) production step, for example, after the completion of the reaction, the compound (4) can be extracted in the same manner as in the above-mentioned compound (11a) production step, and the extracted compound (4) can be further extracted. It may be purified by the same method. Further, the obtained compound (4) may be used in the next step without being taken out after completion of the reaction, but it is preferable to take it out from the viewpoint of improving the yield of the compound (5) which is a target product.

[Compound (5) Production Process]
In the compound (5) manufacturing process, for example, by reacting the compound (4) with 2-cyanoethyl diisopropylchlorophosphoramidite phosphoramidite _{(((CH 3) 2 CH} ) 2 P (OCH 2 CH 2 CN) Cl) , Compound (5) is obtained.
The reaction conditions in the production process of compound (5) are not particularly limited as long as the reaction proceeds well.

However, in this step, it is preferable to carry out the reaction by dissolving the compound (4) in a solvent.
The solvent is not particularly limited, but preferable examples include nitriles such as acetonitrile.
The said solvent may be used individually by 1 type, and may use 2 or more types together, and when 2 or more types are used together, those combination and ratio can be selected arbitrarily.

Among the reaction conditions in the compound (5) production step, for example, the reaction temperature is preferably 15 to 35° C., and the reaction time is preferably 20 minutes to 4 hours.
The amount of 2-cyanoethyldiisopropylchlorophosphoramidite used is preferably 1 to 12 times the molar amount of the amount of the compound (4) used.

Further, in this step, it is preferable to carry out the reaction using a base.
The base is not particularly limited, but an organic base is preferable, and examples of the organic base include trialkylamine such as triethylamine and N,N-diisopropylethylamine.
The above bases may be used alone or in combination of two or more, and when two or more are used in combination, their combination and ratio can be arbitrarily selected.
The amount of the base used is preferably 3 to 12 times the molar amount of the amount of 2-cyanoethyldiisopropylchlorophosphoramidite used.

In the compound (5) production process, it is preferable to carry out the reaction in an atmosphere of an inert gas.
Examples of the inert gas include the same as those used in the above-mentioned compound (11a) production process.

In the compound (5) production step, for example, after the reaction is completed, the compound (5) can be extracted by the same method as in the above-mentioned compound (11a) production step, and the extracted compound (5) can be further extracted. It may be purified by the same method.

Each compound such as compound (5), compound (4), and compound (3) can be prepared by a known method such as nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), infrared spectroscopy (IR), or the like. You can check the structure with.

<<Probe>>
The compound (1) and the condensate described above are useful respectively as various probes.
That is, examples of the probe of the present invention include those consisting of the compound (1) and those consisting of the condensate.
Hereinafter, of the condensates, a probe composed of a polynucleotide will be described in more detail.

The probe of the present invention emits fluorescence even in a single-stranded state, and when hybridized with a polynucleotide, when the nucleotide paired with the compound (1) is adenylic acid, thymidylic acid or guanylic acid, , No clear change in emission wavelength is observed. However, as shown in “Method for detecting cytosine” described below, the nucleotide paired with the above-mentioned compound (1) is cytidylic acid, and under acidic conditions, the emission wavelength is different from that in the case of single strand. It changes clearly.

The number of nucleotides of the probe of the present invention varies depending on the type or length of the target polynucleotide, but is preferably 3 to 25, more preferably 5 to 20, and particularly preferably 7 to 17. ..

When the sample containing the target polynucleotide contains a nuclease such as a cell extract, the nucleotide constituting the probe of the present invention is phosphorothioate DNA or RNA, H-phosphonate DNA or RNA, etc. so that it is difficult to be cleaved by the nuclease. The modified nucleotide of

Upon hybridizing the probe of the present invention with the target polynucleotide, it is preferable to add the probe of the present invention to the sample containing the target polynucleotide in an amount of about 1 nM to 1 mM, particularly about 1 μM to 50 μM. Although the hybridization conditions vary depending on the length of the polynucleotide, the hybridization can be performed at a temperature of, for example, about 0 to 60°C, for example, for about 5 to 60 minutes. The sample to be hybridized may be adjusted to have a pH of about 4.0 to 9.0, for example.

As used herein, the term "hybridize" means that two nucleic acids having complementary base sequences form a hybrid duplex via hydrogen bonds. Examples of such a combination include DNA/DNA, DNA/RNA, RNA/RNA, DNA/PNA, RNA/PNA or PNA/PNA.

The probe of the present invention can be used for the following purposes.
(I) By using a probe complementary to the target polynucleotide, the presence or absence of cytosine at a specific position of the target polynucleotide can be determined. The same method can be used for detecting single nucleotide polymorphisms.
(Ii) In the DNA chip, by fixing or adsorbing the probe of the present invention on the substrate, it can be confirmed whether or not it has a specific base sequence. In particular, the presence or absence of cytosine at a particular position in the target polynucleotide can be determined.
(Iii) When the probe of the present invention is used in place of the antisense polynucleotide, it differs from an ordinary polynucleotide, and therefore, a nucleic acid degrading enzyme (eg, restriction enzyme) or a nucleic acid binding protein (eg, transcription factor, etc.) ) Etc. to the target nucleic acid can be inhibited. Therefore, it can be used as an experimental reagent utilizing these effects.
(Iv) Since the probe of the present invention emits fluorescence itself, it can be used as a fluorescent label for polynucleotides.
(V) In DNA replication, real-time detection of DNA replication is possible by using as a probe complementary to the DNA to be replicated. Similarly, in transcription into RNA, real-time detection of RNA transcription can be performed by using it as a probe complementary to transcribed RNA.
(Vi) Since the probe of the present invention changes the fluorescence emission wavelength by hybridizing with the complementary sequence having cytosine, the redox potential of the probe of the present invention changes due to the change of this wavelength. Therefore, the probe of the present invention is immobilized on an electrode, and a test polynucleotide is allowed to act on this electrode to measure the redox potential, which can be used as a nucleic acid sequence-responsive biosensor.
(Vii) The polynucleotide of the present invention can be used as a fluorescent probe subjected to fluorescence in situ hybridization (FISH).

The DNA chip (or DNA array) described in (ii) above can be obtained by immobilizing or adsorbing the probe of the present invention on a substrate. Immobilization also includes a bond such as a covalent bond.

The spot diameter of the probe of the present invention on the substrate is not particularly limited, but may be, for example, about 50 to 200 μm. The spot pitch is not particularly limited, but can be set to about 100 to 500 μm, for example.

The material of the base material is not particularly limited, and for example, glass, silica, gold or the like can be used. Further, the shape of the base material may be any shape such as a plate shape (substrate shape) or a spherical shape.

When attaching the probe of the present invention to a substrate, one end of the probe of the present invention can be attached to the substrate using a method such as metal-sulfur bonding.

The number of nucleotides of the probe of the present invention, which is immobilized on a DNA chip, varies depending on the type of sample, but may be, for example, about 10 to 200, for example about 50 to 100.

<< Cytosine detection method >>
The probe described above is useful, for example, for detecting cytosine in the polynucleotide to be analyzed.
That is, the method for detecting cytosine of the present invention comprises a step of hybridizing a polynucleotide having cytosine and the probe described above under acidic conditions to obtain a hybridized product, and the hybridized product under acidic conditions. And the step of measuring the fluorescence of the probe, based on the presence or absence of cytosine, by detecting the change in the emission wavelength of the fluorescence, the step of detecting cytosine in the polynucleotide, (hereinafter, It may be abbreviated as "detection method 1").
Further, the method for detecting cytosine of the present invention comprises a step of hybridizing a polynucleotide having cytosine and the above-mentioned probe under a plurality of different pH conditions to obtain a hybridized product, and a plurality of different pH values. Measuring the fluorescence of the hybridized product and the probe under conditions, and detecting the change in the emission wavelength of the fluorescence based on the difference in pH, the step of detecting cytosine in the polynucleotide; , (Hereinafter, may be abbreviated as “detection method 2”).
Hereinafter, each detection method will be described.

<Detection method 1>
[Hybridization process]
In the detection method 1, an acidic solution containing no probe containing cytosine and containing the probe (hereinafter, may be abbreviated as “acidic solution 1-1”), and a polynucleotide containing cytosine, and the probe And an acidic solution (hereinafter, sometimes abbreviated as “acidic solution 1-2”) are used.

The polynucleotide having cytosine in the acidic solution 1-2 is a polynucleotide to be analyzed, and has one or two or more cytosines to be detected as bases. In acidic solution 1-2, the BIQ backbone in the probe, in the protonated state, identifies cytosine in the polynucleotide when the pH of the solution is within a specific range, Form base pairs. Then, the probe and the polynucleotide hybridize to each other to form a stable duplex. On the other hand, when the pH of this solution is in a range different from the above, the BIQ skeleton in the probe does not identify cytosine in the polynucleotide in a protonated state or a non-protonated state. , Does not form base pairs.

On the other hand, the polynucleotide having cytosine in the acidic solution 1-1 means not only the polynucleotide to be analyzed in the acidic solution 1-2 but also all the polynucleotides having cytosine.
Since the acidic solution 1-1 does not contain such a polynucleotide, the BIQ skeleton of the probe in the acidic solution 1-1 also does not form a base pair with cytosine in the polynucleotide.

The cytosine-containing polynucleotide in the acidic solutions 1-1 and 1-2 may contain impurities such as cell extracts and body fluids such as blood, and may have a purity of PCR products, synthetic polynucleotides or the like. It can be expensive.

The pH of the acidic solutions 1-1 and 1-2 may be any pH as long as it is acidic and the polynucleotide having cytosine and the probe can be stably dissolved without being decomposed. For example, pH 4.0 or more and pH 7 The pH may be less than 0.0, for example, pH 4.0 or more and pH 6.0 or less. As the acidic solution used for the acidic solutions 1-1 and 1-2, for example, an acid such as citric acid, lactic acid, phosphoric acid, acetic acid, gluconic acid, succinic acid, or a hydrate thereof is used to adjust the pH to the above. Any buffer solution may be used. The acidic solutions used for the acidic solutions 1-1 and 1-2 may be the same type of acidic solution or different types of acidic solutions. Among them, the same type of acidic solution is preferable because the measurement can be performed as it is in the subsequent fluorescence measurement step.

Hybridization conditions are as described in the above "Probe".

[Fluorescence measurement process]
The fluorescence may be measured at an irradiation wavelength of about 250 to 600 nm according to a conventional method. The fluorescence measurement of the probe in the acidic solution 1-1 and the fluorescence measurement of the hybridized product in the acidic solution 1-2 are preferably performed in the state of being dissolved or suspended in the same kind of acidic solution. Usually, it may be measured in a state of being dissolved or suspended in an acidic solution used for hybridization.

[Cytosine detection process]
The probe in the acidic solution 1-2 clearly shows the fluorescence emission wavelength when the BIQ skeleton forms a base pair with cytosine in a protonated state and when it does not form a base pair. Differently, the fluorescence emission intensity may be different.
Further, the probe in the acidic solution 1-1 does not form a base pair with cytosine and may have a different fluorescence emission wavelength and a different fluorescence emission intensity from the case where it forms a base pair with cytosine. is there.
Therefore, when the fluorescence of the probe is measured in the measuring step for the acidic solution 1-1 and the acidic solution 1-2, the change in the fluorescence emission wavelength is detected based on the presence or absence of cytosine in these acidic solutions. By doing so, cytosine in the polynucleotide can be detected.

<Detection method 2>
[Hybridization process]
In the detection method 2, a plurality of types of solutions (hereinafter, may be abbreviated as “solution 2 group”) containing a polynucleotide having cytosine and the probe and having different pHs are used.
Solution group 2 is a group of two or more solutions in which one or both of the types and contents of the components other than the polynucleotide and the probe are adjusted so that the pH is different from each other. The second solution group usually contains at least one acidic solution as an essential solution.

The polynucleotide having cytosine in the detection method 2 is a polynucleotide to be analyzed, and is the same as the polynucleotide having cytosine in the acidic solution 1-2 described above. Of the two solutions, in acid solution, the BIQ skeleton in the probe identifies cytosine in the polynucleotide in a protonated state when the pH of the solution is within a specific range, It forms a base pair with this cytosine. Then, the probe and the polynucleotide hybridize to each other to form a stable duplex. On the other hand, when the pH of the other solution of the second group of solutions is in a range different from the above, the BIQ skeleton in the probe is in a protonated state or in a non-protonated state. It does not identify cytosines and does not base pair.

The polynucleotide having cytosine in the second solution group is the same as the polynucleotide having cytosine in the detection method 1 described above.

The pH of the plurality of types of solutions having different pHs may be any pH as long as the polynucleotide having cytosine and the probe can be stably dissolved without being decomposed, and for example, it may be pH 4.0 or more and pH 9.0 or less. .. Examples of plural kinds of solutions having different pHs include acids such as citric acid, lactic acid, phosphoric acid, acetic acid, gluconic acid, succinic acid and hydrates thereof, or sodium acetate, trisodium citrate, potassium carbonate, carbonic acid. Use an alkali such as monovalent or divalent salts of sodium hydrogen, monosodium hydrogen phosphate, disodium hydrogen phosphate, dipotassium phosphate, ammonium chloride, or their hydrates to bring the pH to the target value. The buffer solution obtained by adjustment is included.
The pH and composition of the acidic solution, which is an essential solution, are the same as those of the acidic solutions 1-1 and 1-2 in the detection method 1 described above.

Hybridization conditions are as described in the above "Probe".

[Fluorescence measurement process]
The fluorescence measurement step in Detection Method 2 is the same as the fluorescence measurement step in Detection Method 1 described above.

[Cytosine detection process]
In the probe, the fluorescence emission wavelength is clearly different and the fluorescence emission intensity is different when the BIQ skeleton is protonated and forms a base pair with cytosine, and when the base pair is not formed. Sometimes.
Therefore, when the fluorescence of the probe is measured in the measurement step for a plurality of types of solutions having different pHs in the two solution groups, the change in the fluorescence emission wavelength is detected based on the difference in the pH of these solutions. The cytosine in the polynucleotide can be detected.

<<Method for detecting gene mutation>>
The cytosine detection method described above is useful, for example, for detecting a gene mutation.
That is, the method for detecting a gene mutation of the present invention uses the above-described method for detecting cytosine.
Hereinafter, a method for detecting a gene mutation will be described.

In the method for detecting a gene mutation, the presence or absence of a gene mutation can be determined by detecting the change in fluorescence emission wavelength based on the presence or absence of cytosine or the difference in pH using the above detection method 1 or detection method 2.

In the method for detecting a gene mutation of the present invention, as a gene to be detected, a mutation in which at least one base of adenine, guanine or thymine is replaced with cytosine, or a mutation in which at least one base of cytosine is inserted is used. Any gene may be used, and examples thereof include cancer-related genes such as oncogenes and tumor suppressor genes, and other disease-related genes. More specifically, examples thereof include BRCA1 (Breath Cancer Susceptibility gene I) which is a tumor suppressor gene shown in Examples described later.

The BRCA1 gene is a breast cancer susceptibility gene, and it is known that mutation of the BRCA1 gene causes gene instability and eventually causes breast cancer and ovarian cancer. Furthermore, in the cDNA of BRCA1 gene, a mutation in which cytosine is inserted at position 5382 is known to increase the risk of developing breast cancer. Therefore, according to the method for detecting a gene mutation of the present invention, a mutation in which cytosine is inserted at position 5382 of the BRCA1 gene can be easily detected with high sensitivity.

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited to the examples shown below.
In addition, in the following, for example, a compound represented by the general formula (1A)-101 will be referred to as a “compound (1A)-101”, and a reference numeral attached to each compound will be used. The name was confirmed.

[Example 1]
<Production of Compound (11b)>
Compound (11b)-101 was produced as compound (11b) by the route shown below.

[Production of Compound (11b3)-101]
Compound (11b4)-101 (1.6 g, 7.95 mmol) was dissolved in acetic acid (25 mL) under an argon atmosphere. Bromine (20 mmol) was added little by little to the resulting acetic acid solution, and sodium acetate (1.3 g, 16 mmol) was further added, followed by stirring in an oil bath at 50° C. for 2.5 hours. Then, bromine (10 mmol) was added little by little, sodium acetate (0.7 g, 7 mmol) was further added, and the mixture was stirred for 1.5 hours. Then, acetic acid (50 mL), 48% hydrobromic acid (50 mL) and sodium sulfite (12 g, 95 mmol) were added, and the mixture was stirred at 130° C. under reflux overnight. After cooling the obtained reaction liquid to room temperature, the pH was adjusted to 5 with a 10% aqueous sodium hydroxide solution to precipitate black crystals, suction filtration was performed, and the precipitate was taken out by washing with water. .. Since the reaction product was also contained in the filtrate, liquid separation operation was performed on the filtrate with a two-layer system of chloroform and pure water, sodium sulfate was added to the organic layer for dehydration and concentration under reduced pressure to yield white crystals. Got The white crystals and the precipitate taken out by suction filtration were combined and purified by silica gel column chromatography (methanol/chloroform/acetic acid (10/100/1, volume ratio)) to give compound (11b3)-101. Obtained as a white powder (yield 1 g, yield 100%).

[Production of Compound (11b2)-101]
Compound (11b3)-101 (1 g), toluene (15 mL) and thionyl chloride (1.5 mL) were mixed under an argon atmosphere, and the mixture was stirred at 120° C. for 2 hours under reflux. The obtained reaction solution was concentrated under reduced pressure, and while cooling the concentrate with ice water, saturated ammonia water was added to the concentrate and the mixture was stirred for about 30 minutes. The brown crystals precipitated in the reaction solution were taken out by suction filtration. The obtained brown crystals were purified by silica gel column chromatography (methanol/chloroform (1/100, volume ratio)) to obtain compound (11b2)-101 as a skin-colored powder (yield 750 mg, yield 36%).

The analysis results by ¹ H-NMR and ¹³ C-NMR of the obtained compound (11b2)-101 are shown below.
¹ H-NMR(DMSO-d ₆ , 400MHz)σ 5.77(br s, 2H), 7.11(s, 1H), 7.27(br s, 1H), 7.58(d, J = 8.7 Hz, 1H), 7.79 (dd, J = 1.4 Hz, 1H), 7.92(br s, 1H), 8.11(s, 1H), 8.23(d, J = 1.4 Hz, 1H).
¹³ C-NMR(DMSO-d ₆ , 100 MHz)σ 107.3, 112.4, 124.8, 124.9, 125.7, 127.2, 127.6, 132.2, 135.2, 145.1, 167.9.

[Production of Compound (11b1)-101]
Compound (11b2)-101 (700 mg, 0.27 mmol) was suspended in acetonitrile (20 mL) under an argon atmosphere. Phosphoryl chloride (50 μL, 0.5 mmol) was added little by little to the obtained suspension, and the mixture was stirred in an oil bath at 80° C. for 2.5 hours. The obtained reaction solution was slowly poured into ice water (about 50 mL) and then stirred for about 30 minutes. Next, the obtained reaction solution was subjected to a liquid separation operation with a two-layer system of ethyl acetate and an aqueous solution of sodium hydrogen carbonate, the organic layer was dehydrated with sodium sulfate, and then concentrated under reduced pressure. The obtained concentrate was purified by silica gel column chromatography (hexane/ethyl acetate (5/1 to 3/1, volume ratio) to give compound (11b1)-101 as a white powder (yield 300 mg, yield 44 %).

The analysis results by ¹ H-NMR and ¹³ C-NMR of the obtained compound (11b1)-101 are shown below.
¹ H-NMR(CDCl ₃ , 400MHz)σ4.56(br s, 2H), 7.06(s, 1H), 7.47(dd, J = 1.4, 8.2 Hz, 1H), 7.59(d, J = 8.2 Hz, 1H), 7.96(d, J = 1.4 Hz, 1H), 8.00(s, 1H).
¹³ C-NMR(CDCl ₃ , 100MHz) σ105.9, 108.5, 114.0, 119.6, 126.7, 127.1, 132.2, 133.0, 135.5, 144.5.

[Production of Compound (11b)-101]
Under an argon atmosphere, compound (11b1)-101 (100 mg, 0.4 mmol) and [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (17 mg, 20 μmol) were added to 1,4-dioxane. It was dissolved in (2 mL). Trimethylamine (230 μL, 1.7 mmol) was added to the obtained 1,4-dioxane solution, and 4,4,5,5-tetramethyl-1,3,2-dioxaborolane (300 μL, 1.3 mmol) was added dropwise. Then, after confirming that the reaction solution in the flask was foaming and changing from a yellow solution to a reddish purple solution, this solution was stirred overnight in an oil bath at 90°C. The obtained reaction solution was cooled to room temperature and then purified by silica gel column chromatography (hexane/ethyl acetate (10/1, volume ratio) to obtain compound (11b)-101 as a yellow powder (yield 70 mg, yield 58%).

The analysis results by ¹ H-NMR and ¹³ C-NMR of the obtained compound (11b)-101 are shown below.
¹ H-NMR(CDCl ₃ , 400MHz) σ 1.40(s, 12H), 5.12(br s, 2H), 6.83(s, 1H), 7.42(dd, J = 1.4, 8.7 Hz, 1H), 7.51( d, J = 8.7 Hz, 1H), 8.03(d, J = 1.4 Hz, 1H), 8.22(s, 1H).
¹³ C-NMR (CDCl ₃ , 100MHz) σ24.9, 84.3, 104.2, 106.9, 120.2, 125.3, 126.2, 127.9, 135.0, 138.4, 139.8, 152.3.

[Example 2]
<Production of Compound (11a)>
By the procedure shown below, Compound (11Aa)-101 was produced as Compound (11a) among Compound (1a).
Under an argon atmosphere, compound (1Ac)-101 (125 mg, 0.2 mmol), compound (11b)-101 (70 mg, 0.2 mmol), potassium carbonate (80 mg, 0.6 mmol), and tetrakis (triphenylphosphine). A mixed solvent of DMF (3 mL) and water (600 μL) was added to a mixture of palladium (0) (11 mg, 10 μmol) to obtain a suspension. The suspension was stirred overnight in an oil bath at 90° C., cooled to room temperature, and concentrated under reduced pressure. The obtained concentrate was subjected to a liquid separation operation with a two-layer system of ethyl acetate and an aqueous solution of sodium hydrogen carbonate, the organic layer was dehydrated with sodium sulfate, and then concentrated under reduced pressure. The obtained concentrate was purified by silica gel column chromatography (hexane/ethyl acetate (1/1-1/3, volume ratio) to obtain compound (11Aa)-101 as a yellow powder (amount 125 mg, yield 94). %).

The analysis results of the obtained compound (11Aa)-101 by ¹ H-NMR and ¹³ C-NMR are shown below.
¹ H-NMR(DMSO-d ₆ , 400MHz)σ1.12(m, 42H), 2.71(m, 2H), 3.95(m, 2H), 4.33(m, 1H), 4.87(m, 1H), 5.82 (br s, 2H), 6.88(dd, J = 1.8, 8.7 Hz, 1H), 7.55(dd, J = 1.8, 8.7 Hz, 1H), 7.97(d, J = 8.7 Hz, 1H), 8.25(s , 1H), 8.30(complex, 2H), 8.59(s, 1H).
¹³ C-NMR(DMSO-d ₆ ,100MHz)σ 11.9, 12.2, 17.9, 18.1, 42.3, 63.4, 72.8, 77.2, 86.6, 89.2, 107.4, 117.8, 119.7, 120.8, 123.4, 125.3, 127.4, 128.9, 129.6, 131.7, 133.3, 134.9, 138.3, 145.1, 153.0.

[Example 3]
<Production of Compound (1)-1>
According to the procedure shown below, Compound (1A)-101 was produced as Compound (1)-1 among Compound (1).
Compound (11Aa)-101 (125 mg, 0.2 mmol) was dissolved in THF (2 mL) under an argon atmosphere. While stirring the obtained yellow THF solution at room temperature, tetrabutylammonium fluoride (360 μL, 0.4 mmol) was added dropwise thereto, and the mixture was further stirred for 1 hour. After confirming that yellow crystals were precipitated in the reaction solution, acetic acid (360 μL) was added to the reaction solution for neutralization, and the mixture was further stirred for about 10 minutes. After confirming that the amount of the yellow precipitate increased in the reaction solution, suction filtration was performed, and the yellow precipitate was washed with an acetone/ethanol (5/1, volume ratio) mixed solvent at 0° C. to remove the compound (1A )-101 was obtained as a yellow powder (yield 54 mg, 80% yield).

The analysis results by ¹ H-NMR and ¹³ C-NMR of the obtained compound (1A)-101 are shown below.
¹ H-NMR(DMSO-d ₆ , 400MHz)σ2.71(m, 2H), 3.61(m, 1H), 3.66(m, 1H), 4.05(m, 1H), 4.49(m, 1H), 5.07 (dd, J = 5.0, 5.5 Hz, 1H), 5.47(d, J = 4.1 Hz, 1H), 6.87(dd, J = 5.5, 6.0 Hz, 1H), 7.18(br s, 2H), 7.64(dd , J = 1.8, 8.7 Hz, 1H), 8.10(d, J = 8.7 Hz, 1H), 8.67(s, 1H), 8.73(s, 1H), 8.80(s, 1H).
¹³ C-NMR(DMSO-d ₆ ,100MHz)σ 60.6, 69.1, 78.0, 85.8, 87.8, 105.3, 117.4, 119.7, 121.6, 124.8, 126.4, 128.4, 129.5, 130.7, 132.8, 135.4, 139.7, 145.6, 153.7.

[Test Example 1]
<Evaluation of Optical Properties of Compound (1)-1>
[Measurement of UV absorption spectrum]
The compound (1A)-101 obtained above was converted into (S1) 1,4-dioxane, (S2) THF, (S3) ethyl acetate, (S4) DMF, (S5) dimethyl sulfoxide (DMSO), (S6). The solvent was dissolved in 11 kinds of solvents such as acetonitrile, (S7)2-propanol, (S8)ethanol, (S9)methanol, (S10)ethylene glycol, and (S11)glycerol at a concentration of 10 μM, respectively. A solution was prepared.
Next, an ultraviolet absorption spectrum of each of the obtained solutions was measured using an ultraviolet-visible spectrophotometer (“UV-2550” manufactured by Shimadzu Corporation). The results are shown in Fig. 1(a). In the graph of FIG. 1A, the vertical axis represents the absorbance and the horizontal axis represents the wavelength of light (nm).

[Measurement of fluorescence spectrum]
About the 11 kinds of solutions of the compound (1A)-101 obtained above, the fluorescence spectrum was measured using a fluorometer (“RF-5300PC” manufactured by Shimadzu Corporation). The results are shown in Fig. 1(b). In the graph of FIG. 1B, the vertical axis represents the light intensity and the horizontal axis represents the light wavelength (nm).

[Measurement of excitation spectrum]
With respect to 11 kinds of solutions of the compound (1A)-101 obtained above, an excitation spectrum was measured using a fluorometer (“RF-5300PC” manufactured by Shimadzu Corporation). The results are shown in Fig. 1(c). In the graph of FIG. 1C, the vertical axis represents the light intensity and the horizontal axis represents the light wavelength (nm).

As shown in FIG. 1, some solutions of compound (1A)-101 showed similar spectra in all of ultraviolet absorption spectrum, fluorescence spectrum and excitation spectrum, but different spectra depending on the type of solvent. showed that. For example, the fluorescence emission wavelength greatly changed depending on the type of solvent, and when the fluorescence emission wavelength changed, the fluorescence emission intensity tended to change greatly.

[Test Example 2]
<Evaluation of pH Dependence of Optical Properties of Compound (1)-1>
[Measurement of UV absorption spectrum under various pH conditions]
Using 4 types of buffer solutions (b1) to (b4) below, various aqueous solutions of buffer solution/ethanol (9/1, volume ratio) adjusted to pH 4.0 to 9.0 were prepared, and these aqueous solutions were prepared. The above-obtained compound (1A)-101 was dissolved in each of the solutions to a concentration of 10 μM to prepare various solutions having different pH.
(B1): a buffer solution having a total concentration of glycine hydrochloride and sodium chloride of 10 mM and having a pH of 4.0.
(B2): A buffer solution in which the total concentration of acetic acid and sodium acetate is 10 mM, and the pH of the components is adjusted to 4.3 to 6.0 by adjusting the ratio of these components.
(B3): A buffer solution in which the total concentration of sodium monohydrogen phosphate and sodium dihydrogen phosphate is 10 mM, and the pH is adjusted to any of pH 6.3 to 8.0 by adjusting the ratio of these components.
(B4): A buffer solution in which the total concentration of ammonium chloride and ammonia is 10 mM, and the pH is adjusted to any of 8.3 to 9.0 by adjusting the ratio of these components.

Next, with respect to these various solutions having different pHs, an ultraviolet absorption spectrum was measured using an ultraviolet-visible spectrophotometer (“UV-2550” manufactured by Shimadzu Corporation). The results are shown in Fig. 2(a).

[Measurement of fluorescence spectrum under various pH conditions]
Fluorescence spectra of various solutions of the compound (1A)-101 having different pHs obtained above were measured using a fluorometer (“RF-5300PC” manufactured by Shimadzu Corporation). The results are shown in Fig. 2(b).

As shown in FIG. 2, the solution of compound (1A)-101 showed different spectra depending on pH in both the ultraviolet absorption spectrum and the fluorescence spectrum. In particular, the fluorescence spectrum changed greatly, and both the fluorescence emission wavelength and the fluorescence emission intensity greatly changed depending on pH. From this, it was inferred that changes in protonation and deprotonation occurred in the nitrogen atom contained in the BIQ skeleton in the compound (1A)-101.

In addition, among the measurement results of the above-mentioned ultraviolet absorption spectrum, the pKa of the compound (1A)-101 was calculated using the measurement value at the wavelength of 394 nm, and the result was 6.16.
In addition, among the measurement results of the above-mentioned fluorescence spectrum, the pKa of the compound (1A)-101 was calculated using the measurement value at the wavelength of 379 nm, and the result was 6.08.
From these results, the pKa of compound (1A)-101 was considered to be 6.1 to 6.2.

In the compound (1A)-101, the absolute quantum yield was 0.856, and the fluorescence lifetime was 9.6 nanoseconds in the case where the wavelength of the excitation light was 370 nm in the acid system of pH 4.5.
In compound (1A)-101, the absolute quantum yield was 0.852 and the fluorescence lifetime was 5.2 nanoseconds when the wavelength of the excitation light was 370 nm in the pH 8.5 base system. ..

[Example 4]
<Production of condensate (polynucleotide)>
By the procedure shown below, a polynucleotide was produced as a condensate using Compound (1A)-101 as a monomer.

[Production of Compound (3A)-101]
Compound (1A)-101 (42 mg, 0.1 mmol) was dissolved in DMF (2 mL) under an argon atmosphere. N,N-Di-n-butylformamide dimethyl acetal (100 μL) was added to the obtained DMF yellow solution, and the mixture was stirred in an oil bath at 80° C. for 2 hours. The obtained reaction solution was cooled to room temperature and then concentrated under reduced pressure, and the obtained concentrate was purified by silica gel column chromatography (chloroform/methanol/triethylamine (300/10/3 to 100/5/1) to give a compound. (3A)-101 was obtained as a yellow powder (yield 42 mg, yield 72%).

The analysis results of the obtained compound (3A)-101 by ¹ H-NMR and ¹³ C-NMR are shown below.
¹ H-NMR(DMSO-d ₆ , 400MHz)σ 0.95(m, 6H), 1.36(m, 4H), 1.65(m, 4H), 2.71(m, 2H), 3.46(m, 2H), 3.61 -3.67(complex, 4H), 4.05(m, 1H), 4.49(m, 1H), 5.07(m, 1H), 5.49(d, J = 4.8 Hz, 1H), 6.93(t, J = 5.7, 6.0 Hz, 1H), 7.69(m, 1H), 8.20(d, J = 8.8 Hz, 1H), 8.48(s, 1H), 8.68(s, 1H), 8.82(s, 1H), 8.90(s, 1H ), 8.93(s, 1H).
¹³ C-NMR(DMSO-d ₆ ,100MHz)σ13.6, 13.7, 19.1, 19.6, 28.7, 30.5, 40.1, 44.3, 50.7, 60.4, 68.9, 85.6, 87.6, 106.2, 118.0, 119.5, 121.5, 124.3, 124.6, 127.3, 128.8, 131.9, 132.2, 133.9, 135.4, 139.9, 144.2, 156.7, 157.7.

[Production of Compound (4A)-101]
Compound (3A)-101 (50 mg, 9.72×10 ⁻² mmol) was dissolved in pyridine (1.5 mL) under an argon atmosphere. 4,4′-Dimethoxytrityl chloride (40 mg, 0.1 mmol) was added to the obtained pyridine yellow solution, and the mixture was stirred overnight at room temperature. The obtained reaction solution was concentrated under reduced pressure, the obtained concentrate was subjected to a liquid separation operation with a two-layer system of ethyl acetate and an aqueous solution of sodium hydrogen carbonate, the organic layer was dehydrated with sodium sulfate, and then concentrated under reduced pressure. .. The obtained concentrate was purified by silica gel column chromatography (chloroform/methanol/triethylamine (200/1/2, volume ratio) to obtain compound (4A)-101 as a yellow powder (amount 38 mg, yield 53%). ).

The analysis results by ¹ H-NMR and ¹³ C-NMR of the obtained compound (4A)-101 are shown below.
¹ H-NMR(DMSO-d ₆ , 400MHz)σ 0.98(m, 6H), 1.44(m, 4H), 1.74(m, 4H), 2.88(m, 1H), 3.09(dd, J = 10.5 Hz , 1H), 3.21(m, 1H), 3.33(m, J = 4.6, 10.5 Hz, 1H), 3.52(m, 2H), 3.53(s, 3H), 3.59(s, 3H), 3.75(m, 2H), 4.30(m, 1H), 4.89(m, 1H), 6.41(m, 2H), 6.50(m, 2H), 6.90(m, 1H), 7.02-7.15(complex, 9H), 7.56(dd , J = 1.4, 8.7 Hz, 1H), 8.39(s, 1H), 8.45(s, 1H), 8.57(s, 1H), 9.00(s, 1H), 9.11.
¹³ C-NMR(DMSO-d ₆ ,100MHz)σ14.1, 14.3, 20.5, 20.9, 30.1, 32.0, 40.3, 45.9, 52.3, 55.3, 55.3, 63.6, 70.7, 86.2, 86.5, 87.2, 108.0, 113.6( 2C), 113.6(2C), 113.9, 119.5, 120.2, 123.0, 125.4, 125.5, 128.4(2C), 128.7(2C), 128.8, 129.8, 130.5(2C), 130.8(2C), 133.6, 134.0, 135.7, 136.1, 136.4, 136.6, 139.6, 145.8, 146.1, 158.1, 158.9, 159.2, 159.3.

[Production of Compound (5A)-101]
Compound (4A)-101 (37 mg, 0.05 mmol) was dissolved in acetonitrile (1 mL) under an argon atmosphere. Triethylamine (0.5 mL) was added to the obtained acetonitrile solution, 2-cyanoethyldiisopropylchlorophosphoramidite (100 μL) was added dropwise, and the mixture was stirred at room temperature for 1 hr. The obtained reaction liquid is subjected to liquid separation operation with a two-layer system of ethyl acetate and an aqueous solution of sodium hydrogencarbonate, and the organic layer obtained is further separated with a two-layer system of ethyl acetate and an aqueous sodium chloride solution. The operation was performed. Then, the obtained organic layer was dehydrated with sodium sulfate and then concentrated under reduced pressure to obtain compound (5A)-101.

[Production of polynucleotide]
Compound (5A)-101 was dissolved in acetonitrile (600 μL), and the resulting solution and an automatic DNA synthesizer (“DNA Synthesizer 3400” manufactured by Applied Biosystems) were used to automatically synthesize a DNA chain. A polynucleotide having the base sequence shown in SEQ ID NO: 1 (5′-CGCAATNTAACGC-3′ (N represents a base site having a BIQ skeleton in the compound (5A)-101), hereinafter may be abbreviated as “ODN1”. I got).

The obtained ODN1 was further purified by fractionation by high performance liquid chromatography under the following conditions.
Mobile phase: AF buffer/acetonitrile (concentration of acetonitrile increased from 3% to 20% by volume in 45 minutes).
Flow rate: 2.0 mL/min
Column: "Chemcobond 5-ODS-H" manufactured by Chemco Plus (10 mm x 150 mm)

The obtained ODN1 was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and as a result, the mass value of the proton adduct ion [M+H] ⁺ was 4052.02, It almost agreed with the theoretical value of 4052.8. From this, it was confirmed that ODN1 was the target product.

[Test Example 3]
<Evaluation of pH Dependence of Optical Properties of Polynucleotide>
[Measurement of UV absorption spectrum under various pH conditions]
The following three types of buffers (b5) to (b7) adjusted to pH 4.0 to 8.0 were prepared, and the ODN1 obtained above was added to each of these buffers so that the concentration was 5 μM. Various solutions with different pHs were prepared by dissolving.
(B5): A buffer solution in which the total concentration of acetic acid and sodium acetate is 10 mM, and the pH is adjusted to any of pH 4.0 to 6.0 by adjusting the ratio of these components.
(B6): A buffer solution in which the total concentration of sodium monohydrogen phosphate and sodium dihydrogen phosphate is 10 mM, and the pH is adjusted to any of pH 6.3 to 8.0 by adjusting the ratio of these components.
(B7): A buffer solution in which the total concentration of ammonium chloride and ammonia is 10 mM, and the pH is adjusted to any of 8.3 to 9.0 by adjusting the ratio of these components.

Next, with respect to these various solutions having different pHs, an ultraviolet absorption spectrum was measured using an ultraviolet-visible spectrophotometer (“UV-2550” manufactured by Shimadzu Corporation). The results are shown in Fig. 3(a).

[Measurement of fluorescence spectrum under various pH conditions]
The fluorescence spectra of the various solutions of ODN1 having different pHs obtained above were measured using a fluorometer (“RF-5300PC” manufactured by Shimadzu Corporation). The results are shown in Fig. 3(b).

[Measurement of excitation spectrum under each pH condition]
The excitation spectra of the various solutions of ODN1 having different pHs obtained above were measured using a fluorometer (“RF-5300PC” manufactured by Shimadzu Corporation). The results are shown in Fig. 3(c).

As shown in FIG. 3( b ), the solution of ODN1 has a tendency that the fluorescence emission intensity decreases as the pH decreases in the wavelength range of 430 to 480 nm, and the fluorescence emission intensity depends on the pH. changed. This means that in the state where ODN1 does not form a double chain, the BIQ skeleton in ODN1 has a higher degree of protonation as the pH of the solution becomes lower (in other words, as the pH of the solution becomes higher, The degree of deprotonation is increased). However, in the fluorescence spectrum, there was no change in the fluorescence emission wavelength of the main peak with high fluorescence emission intensity, and there was no clear change in the fluorescence emission wavelength.

The fluorescence spectrum shown in FIG. 3B is for a solution of ODN1 having a concentration of 5 μM, and the fluorescence spectrum shown in FIG. 2B above is for a solution of compound (1A)-101 having a concentration of 10 μM. In these solutions, the concentration of the target substance is different. However, among these fluorescence spectra, for example, when the fluorescence spectra at the same pH are compared under acidic conditions such as pH 4.5, the fluorescence emission intensity is maximum when the wavelength is 410 nm or in the vicinity thereof. The maximum value (maximum value of fluorescence emission intensity) is assumed from the fluorescence spectrum shown in FIG. 2(b) in the fluorescence spectrum shown in FIG. 3(b) even when the difference in the concentration of the target substance is taken into consideration. It was clearly smaller than the value. That is, when the fluorescence emission intensity of the solution of compound (1) and that of the polynucleotide of the present invention having the same concentration of BIQ skeleton are compared, the solution of the polynucleotide has a higher fluorescence emission intensity. It was confirmed to be low.

<Detection of cytosine in polynucleotide>
[Example 5]
The above-mentioned three types of buffer solutions (b5) to (b7) adjusted to pH 4.0 to 8.0 were prepared, and the ODN1 obtained above and the nucleotide sequence shown in SEQ ID NO: 2 were prepared in these buffer solutions. (3'-GCGTTACATTGCG-5', which may be abbreviated as "TrichC" hereinafter) having the following: were dissolved. Then, the mixture was heated at 90° C. for 5 minutes and then slowly cooled to room temperature for hybridization to prepare various solutions containing these polynucleotide duplexes and having different pHs. The amounts of ODN1 and TrichC used were adjusted so that the concentration of these polynucleotide duplexes in the solution was 5 μM.

Next, with respect to these various solutions having different pHs, the ultraviolet absorption spectrum, the fluorescence spectrum and the excitation spectrum were measured by the same method as in Test Example 3. The measurement result of the ultraviolet absorption spectrum is shown in FIG. 4(a), the measurement result of the fluorescence spectrum is shown in FIG. 4(b), and the measurement result of the excitation spectrum is shown in FIG. 4(c).

Further, with respect to these various solutions having different pHs, the absorbance was measured while heating, a thermal melting curve was prepared, and the melting temperature of the double chain (hereinafter, sometimes abbreviated as "Tm") was measured. .. The thermal melting curve obtained at this time is shown in FIG. In the graph of FIG. 4D, the vertical axis represents standardized absorbance and the horizontal axis represents heating temperature (°C). Table 1 shows the measured values of Tm.

As shown in FIG. 4B, in the fluorescence spectrum of the solution, the fluorescence emission intensity decreases as the pH decreases, and the degree of protonation increases as the pH of the solution decreases. Was shown. In addition, comparing the fluorescence spectra at the same pH in the region of pH 7.0 to 8.0 and the like in FIG. 4(b) and FIG. 3(b), the figure shows that the concentration of ODN1 is the same. The fluorescence spectrum of 4(b) had a lower fluorescence emission intensity as a whole. This is because the BIQ skeleton in ODN1 is already protonated when ODN1 coexists with TrichC in a weakly basic solution, whereas when ODN1 is present alone. , Showed that the BIQ backbone in ODN1 was either unprotonated or had a significantly lower degree of protonation.
Furthermore, in the fluorescence spectrum of the solution, the fluorescence emission wavelength of the main peak with high fluorescence emission intensity changed as the pH decreased. In particular, the fluorescence emission wavelength when the fluorescence emission intensity becomes maximum is 440 nm or near when the pH is 6.5 or more, whereas it is 415 nm or around when the pH is 6.0 or less. And there was a clear change.
Further, it was found that such fluorescence emission changed to the short wavelength side is strongly quenched due to the influence of the nucleic acid bases present in the periphery. That is, the intensity of the fluorescence emission, the emission wavelength of which was changed by the change of pH, was decreased by the quenching.
These results show that, under acidic conditions such that the pH of the solution is 6.0 or less, the BIQ skeleton in ODN1 is protonated and the facing cytosine in TrichC is identified. It was shown that it was stably forming a base pair.

On the other hand, as shown in Table 1, Tm has the highest value at pH 5.5 and is considerably high even at pH 6.0, and when the pH is 5.5 to 6.0, the duplex is most stable. Was presumed to have been formed.

From the results of Test Example 3 and Example 5, an acidic solution containing no probe containing a cytosine-containing polynucleotide and a probe containing the polynucleotide of the present invention, and a polynucleotide containing cytosine and the polynucleotide of the present invention were used. Probe, for an acidic solution containing, the fluorescence of the probe is measured, from the measurement result, based on the presence or absence of the polynucleotide, by detecting the change in fluorescence emission wavelength, the polynucleotide of the analysis target It was confirmed that the cytosine contained therein could be detected.
Further, from the results of Example 5, the fluorescence of the probe was measured for a plurality of types of solutions containing a polynucleotide having a cytosine and a probe comprising the polynucleotide of the present invention and having different pHs, and the measurement result From this, it was confirmed that cytosine in the polynucleotide to be analyzed can be detected by detecting a change in fluorescence emission wavelength based on a difference in pH of the solution.

Further, containing a probe consisting of the polynucleotide of the present invention, a weak basic solution containing no polynucleotide capable of forming a double strand with the probe, preferably as a polynucleotide, only the probe consisting of the polynucleotide of the present invention Weak basic solution containing, and a probe consisting of the polynucleotide of the present invention, and a polynucleotide capable of forming a double strand with the probe, a weak basic solution containing two kinds of weak basic solution, By using the same concentration of the probe, the fluorescence of the probe is measured for these two types of weakly basic solutions, and the difference in fluorescence emission intensity is detected from the measurement result, whereby It was confirmed that the presence or absence of protonation of the BIQ skeleton can be detected. Here, the weakly basic solution is, for example, a solution having a pH of 7.5 to 8.0.

[Comparative Example 1]
The same method as in Example 5 except that a polynucleotide having the base sequence shown in SEQ ID NO: 3 (3′-GCGTTAAATTGCG-5′, hereinafter sometimes abbreviated as “TrichA”) was used instead of TrichC. Then, the formation of a double chain was tried, and an ultraviolet absorption spectrum, a fluorescence spectrum, an excitation spectrum and Tm were measured. The base sequence of TrichA is the same as the above-mentioned base sequence of TrichC except that the central base is adenine instead of cytosine. The measurement result of the ultraviolet absorption spectrum is shown in FIG. 5(a), the measurement result of the fluorescence spectrum is shown in FIG. 5(b), the measurement result of the excitation spectrum is shown in FIG. 5(c), and the thermal melting curve is shown in FIG. 5(d). , Respectively. Table 1 shows the measured values of Tm.

As shown in FIG. 5B, when TrichA was used, the fluorescence emission intensity decreased as the pH of the solution decreased, and the degree of protonation increased as the pH of the solution decreased. It was showing that it was doing. Further, comparing the fluorescence spectra at the same pH in the region of pH 7.0 to 8.0 and the like in FIG. 5(b) and FIG. 4(b), although the concentration of ODN1 is the same, The fluorescence spectrum of 4(b) had a lower fluorescence emission intensity as a whole. This is because the BIQ skeleton in ODN1 has already been protonated when ODN1 coexists with TrichC in a weakly basic solution, whereas when ODN1 coexists with TrichA. , Showed that the BIQ backbone in ODN1 was either unprotonated or had a significantly lower degree of protonation.
Further, when TrichA was used, no clear change was observed in the fluorescence emission wavelength of the main peak with high fluorescence emission intensity in the fluorescence spectrum of the solution even when the pH was changed. More specifically, the fluorescence emission wavelength at which the fluorescence emission intensity becomes maximum was approximately 440 nm or near regardless of pH. As in the case of Example 5, there is a possibility that the strong quenching action of fluorescence emission on the short wavelength side is also involved in this result.
These results indicate that, in the presence of TrichA, the BIQ skeleton in ODN1 increased the degree of protonation as the pH of the solution became lower, but the adenine facing the TrichA was not affected by the pH of the solution. No distinction was made, indicating that it did not form a base pair with this adenine.

Further, as shown in Table 1, Tm has the highest value at pH 6.5 and is considerably high at pH 7.0 and 7.5, but both are significantly lower than the highest value of Tm in Example 5. It was From this, it was speculated that the duplex with TrichA was significantly less stable than the duplex in Example 5.

[Comparative example 2]
The same method as in Example 5 except that a polynucleotide having the base sequence shown in SEQ ID NO: 4 (3′-GCGTTAGATTGCG-5′, hereinafter sometimes abbreviated as “TrichG”) was used instead of TrichC. Then, the formation of a double chain was tried, and an ultraviolet absorption spectrum, a fluorescence spectrum, an excitation spectrum and Tm were measured. The base sequence of TrichG is the same as the above-mentioned base sequence of TrichC except that the central base is guanine instead of cytosine. The measurement result of the ultraviolet absorption spectrum is shown in FIG. 6(a), the measurement result of the fluorescence spectrum is shown in FIG. 6(b), the measurement result of the excitation spectrum is shown in FIG. 6(c), and the thermal melting curve is shown in FIG. 6(d). , Respectively. Table 1 shows the measured values of Tm.

As shown in FIG. 6B, when TrichG is used, the fluorescence emission intensity decreases as the pH of the solution decreases, and the degree of protonation increases as the pH of the solution decreases. It was showing that it was doing. Further, comparing the fluorescence spectra at the same pH in the region of pH 7.0 to 8.0, etc., between FIG. 6(b) and FIG. 4(b), it is shown that the concentration of ODN1 is the same, The fluorescence spectrum of 4(b) had a lower fluorescence emission intensity as a whole. This is because the BIQ skeleton in ODN1 is already protonated when ODN1 coexists with TrichC in a weakly basic solution, whereas when ODN1 coexists with TrichG. , Showed that the BIQ backbone in ODN1 was either unprotonated or had a significantly lower degree of protonation.
Furthermore, when TrichG was used, no clear change was observed in the fluorescence emission wavelength of the main peak with high fluorescence emission intensity in the fluorescence spectrum of the solution even when the pH was changed. More specifically, the fluorescence emission wavelength at which the fluorescence emission intensity becomes maximum was approximately 440 nm or near regardless of pH. As in the case of Example 5, this result may also be involved in the strong quenching action of fluorescence emission on the short wavelength side.
These results show that, in the presence of TrichG, the BIQ skeleton in ODN1 increased the degree of protonation as the pH of the solution decreased, but the guanine facing TrichG was not affected by the pH of the solution. No distinction was made, indicating that it did not form a base pair with this guanine.

Further, as shown in Table 1, Tm had the highest value at pH 7.0 and was considerably high even at pH 6.5, but both were significantly lower than the highest value of Tm in Example 5. From this, it was speculated that the duplex using TrichG was significantly less stable than the duplex in Example 5.

[Reference Example 1]
The same method as in Example 5 except that a polynucleotide having the base sequence shown in SEQ ID NO: 5 (3′-GCGTTTATTATGCG-5′, hereinafter sometimes abbreviated as “TrichT”) was used instead of TrichC. Then, the formation of a double chain was tried, and an ultraviolet absorption spectrum, a fluorescence spectrum, an excitation spectrum and Tm were measured. The base sequence of TrichT is the same as the above-mentioned base sequence of TrichC except that the central base is thymine instead of cytosine. The measurement result of the ultraviolet absorption spectrum is shown in FIG. 7(a), the measurement result of the fluorescence spectrum is shown in FIG. 7(b), the measurement result of the excitation spectrum is shown in FIG. 7(c), and the thermal melting curve is shown in FIG. 7(d). , Respectively. Table 1 shows the measured values of Tm.

As shown in FIG. 7B, when TrichT is used, the fluorescence emission intensity decreases as the pH of the solution decreases, and the degree of protonation increases as the pH of the solution decreases. It was showing that it was doing. Further, comparing the fluorescence spectra at the same pH in the region of pH 7.0 to 8.0 and the like in FIG. 7(b) and FIG. 4(b), although the concentration of ODN1 is the same, the figure The fluorescence spectrum of 4(b) had a lower fluorescence emission intensity as a whole. This is because the BIQ skeleton in ODN1 is already protonated when ODN1 coexists with TrichC in a weakly basic solution, whereas when ODN1 coexists with TrichT. , Showed that the BIQ backbone in ODN1 was either unprotonated or had a significantly lower degree of protonation.
Furthermore, when TrichT was used, no clear change was observed in the fluorescence emission wavelength of the main peak with high fluorescence emission intensity in the fluorescence spectrum of the solution even when the pH was changed. More specifically, the fluorescence emission wavelength at which the fluorescence emission intensity becomes maximum was approximately 440 nm or near regardless of pH. As in the case of Example 5, this result may also be involved in the strong quenching action of fluorescence emission on the short wavelength side.

Further, as shown in Table 1, Tm had the highest value at pH 7.5, but was clearly lower than the highest value of Tm in Example 5. From this, it was speculated that the duplex using TrichT is more unstable than the duplex in Example 5.
However, the maximum value of Tm in this reference example was obviously higher than the maximum value of Tm in Comparative Examples 1 and 2. It was speculated that this is because, in the state where the BIQ skeleton in ODN1 is not protonated, as in the case of adenine, the facing thymine in TrichT is identified to form a base pair.

[Example 6]
<Production of condensate (polynucleotide)>
A polynucleotide having the nucleotide sequence shown in SEQ ID NO: 6 (5′-GGTACCANTGAAATA-3′(N is the same as in Example 4 except that the reaction order of each monomer during the automatic synthesis of a DNA chain was changed. Compound (5A)-101, which represents a base site having a BIQ skeleton), hereinafter, may be abbreviated as "ODN2").

The obtained ODN2 was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and as a result, the mass value of the proton adduct ion [M+H] ⁺ was 4734.13, It was almost in agreement with the theoretical value of 4734.26. From this, it was confirmed that ODN2 was the target product.

[Test Example 4]
<Evaluation of pH Dependence of Optical Properties of Polynucleotide>
[Measurement of UV absorption spectrum under various pH conditions]
The two types of buffers (b5) to (b6) adjusted to pH 4.5 to 8.0 and the buffer (b7) type adjusted to pH 8.5 to 9.0 are used. The ODN2 obtained above was dissolved in each of these buffers to a concentration of 5 μM to prepare various solutions having different pH.

Next, with respect to these various solutions having different pHs, an ultraviolet absorption spectrum was measured using an ultraviolet-visible spectrophotometer (“UV-2550” manufactured by Shimadzu Corporation). The results are shown in Fig. 8(a).

[Measurement of fluorescence spectrum under various pH conditions]
The fluorescence spectra of the various solutions of ODN2 having different pHs obtained above were measured using a fluorometer (“RF-5300PC” manufactured by Shimadzu Corporation). The results are shown in Fig. 8(b).

As shown in FIG. 8B, the solution of ODN2 shows a tendency that the fluorescence emission intensity decreases as the pH decreases in the wavelength range of 420 to 500 nm, and the fluorescence emission intensity depends on the pH. changed. This means that, in the state where ODN2 does not form a double chain, the BIQ skeleton in ODN2 has a higher degree of protonation as the pH of the solution becomes lower (in other words, as the pH of the solution becomes higher, it becomes higher). The degree of deprotonation is increased). However, in the fluorescence spectrum, there was no change in the fluorescence emission wavelength of the main peak with high fluorescence emission intensity, and there was no clear change in the fluorescence emission wavelength.

The fluorescence spectrum shown in FIG. 8(b) is for a solution of ODN2 having a concentration of 5 μM, and the fluorescence spectrum shown in FIG. 2(b) above is for a solution of compound (1A)-101 having a concentration of 10 μM. In these solutions, the concentration of the target substance is different. However, comparing the fluorescence spectra at the same pH under acidic conditions, such as pH 4.5, among these fluorescence spectra, the fluorescence emission intensity shows that when the wavelength is 440 nm or near in FIG. 8B. It becomes the maximum when the wavelength is 410 nm or in the vicinity thereof in FIG. 2B, but the maximum value (the maximum value of the fluorescence emission intensity) is shown in FIG. The fluorescence spectrum shown in (b) was clearly smaller than the value expected from the fluorescence spectrum shown in FIG. 2(b). That is, as in the case of ODN1 described above, comparing the fluorescence emission intensities of the compound (1) solution and the polynucleotide solution of the present invention having the same BIQ skeleton concentration, It was confirmed that the fluorescence emission intensity was lower in the nucleotide solution.

<Detection of cytosine in polynucleotide>
[Example 7]
The two types of buffers (b5) to (b6) adjusted to pH 4.5 to 8.0 and the buffer (b7) type adjusted to pH 8.5 to 9.0 are used. Prepared in these buffers, the ODN2 obtained above and a polynucleotide having the base sequence shown in SEQ ID NO: 7 (3′-CCATGGTCACTTTAT-5′, hereinafter sometimes abbreviated as “BRCA1-C”) And were dissolved. Then, the mixture was heated at 90° C. for 5 minutes and then slowly cooled to room temperature for hybridization to prepare various solutions containing these polynucleotide duplexes and having different pHs. The amounts of ODN2 and BRCA1-C used were adjusted so that the concentration of these polynucleotide duplexes in the solution was 5 μM.

Next, with respect to these various solutions having different pHs, the ultraviolet absorption spectrum, the fluorescence spectrum and the Tm were measured by the same method as in Test Example 3. The measurement result of the ultraviolet absorption spectrum is shown in FIG. 9(a), the measurement result of the fluorescence spectrum is shown in FIG. 9(b), and the thermal melting curve is shown in FIG. 9(c).

As shown in FIG. 9B, in the fluorescence spectrum of the solution, the fluorescence emission intensity generally decreases as the pH decreases, and the degree of protonation increases as the pH of the solution decreases. It was showing that. Further, comparing the fluorescence spectra at the same pH in the region of pH 7.0 to 9.0 between FIG. 9(b) and FIG. 8(b), although the concentration of ODN2 is the same, The fluorescence spectrum of 9(b) had a lower fluorescence emission intensity as a whole. This was the same result as in the case of ODN1 described above. That is, when ODN2 coexists with BRCA1-C in a weakly basic solution, the BIQ skeleton in ODN2 is already protonated, whereas when ODN2 is present alone. Showed that the BIQ backbone in ODN2 was either unprotonated or had a significantly lower degree of protonation.
Furthermore, in the fluorescence spectrum of the solution, the fluorescence emission wavelength of the main peak with high fluorescence emission intensity changed as the pH decreased. More specifically, when the pH was 7.0 or higher, the main peak was present only at or near 440 nm. On the other hand, when the pH was 6.5 or less, there was a main peak at 390 nm or its vicinity in addition to 440 nm or its vicinity. When the pH was 4.5 and 5.0, a main peak was present at 440 nm or its vicinity and 390 nm or its vicinity, as well as 410 nm or its vicinity. Thus, a clear change in the fluorescence emission wavelength was observed depending on the pH of the solution. Further, as in the case of ODN1 described above, the fluorescence emission changed to the short wavelength side is It was found that the quenching is strong due to the influence of the nucleic acid bases present in. That is, the intensity of the fluorescence emission, the emission wavelength of which was changed by the change of pH, was decreased by the quenching.
These results show that, for example, when the pH of the solution is under 6.5 or less, the BIQ skeleton in ODN2 is protonated and the facing cytosines in BRCA1-C are identified. It was shown that it forms a stable base pair with this cytosine.

In addition, as shown in FIG. 9( c ), the thermal melting curve was a sigmoid curve in any of the solutions, the double chain was formed, and the BIQ skeleton in ODN2 had a pH of the solution of 6.5. It was protonated under the acidic conditions below, and the cytosine that faced in BRCA1-C was identified, and it could be determined that it stably forms a base pair with this cytosine.

[Reference Example 2]
Examples except that a polynucleotide having a base sequence shown in SEQ ID NO: 8 (3′-CCATGGTTACTTTAT-5′, hereinafter sometimes abbreviated as “BRCA1-T”) was used instead of BRCA1-C Double strand formation was tried in the same manner as in 7, and the ultraviolet absorption spectrum, fluorescence spectrum and Tm were measured. The base sequence of BRCA1-T is the same as the base sequence of BRCA1-C described above, except that the central base is thymine instead of cytosine. The measurement result of the ultraviolet absorption spectrum is shown in FIG. 10(a), the measurement result of the fluorescence spectrum is shown in FIG. 10(b), and the thermal melting curve is shown in FIG. 10(c).

As shown in FIG. 10(b), when BRCA1-T was used, the fluorescence emission intensity decreased as the pH of the solution decreased, and the degree of protonation decreased as the pH of the solution decreased. Was increasing. Further, comparing the fluorescence spectra at the same pH in the region of pH 7.0 to 9.0 and the like in FIG. 10(b) and FIG. 9(b), although the concentration of ODN2 is the same, the figure The fluorescence spectrum of 9(b) had a lower fluorescence emission intensity as a whole. This means that when ODN2 coexists with BRCA1-C in a weakly basic solution, the BIQ skeleton in ODN2 has already been protonated, whereas ODN1 coexists with BRCA1-T. In that case, the BIQ backbone in ODN2 was either unprotonated or had a significantly lower degree of protonation.
Further, when BRCA1-T was used, no clear change was observed in the fluorescence emission wavelength of the main peak with high fluorescence emission intensity in the fluorescence spectrum of the solution even when the pH was changed. More specifically, the fluorescence emission wavelength at which the fluorescence emission intensity becomes maximum was approximately 440 nm or near regardless of pH. This result may also involve the strong quenching action of fluorescence emission on the short wavelength side, as in the case of Example 7.

The Tm when the pH was 7.5 was 47.9° C. in this reference example and 49.3° C. in Example 7. Thus, since the Tm was significantly lower in this reference example than in Example 7, the duplex with BRCA1-T was the duplex with BRCA1-C. More likely to be unstable.

INDUSTRIAL APPLICABILITY The present invention can be used for pH sensors, gene detection reagents, gene test kits and the like.

Claims (16)

A compound represented by the following general formula (1).

(In the formula, R ¹ is a hydrogen atom or a hydroxyl group; Z is a group represented by the following general formula (1)-11 .)

(In the formula, X ¹¹ is a hydrogen atom, a cyano group, an amino group, a monoalkylamino group or a dialkylamino group; R ¹¹ is an alkyl group or a halogen atom; n ₁₁ is an integer of 0 to 5; _{When 11} is an integer of 2 or more, a plurality of R ¹¹ may be the same or different from each other ; the bond marked with * is formed between the bond to Z and the bonding target. The compound according to claim 1, wherein X ¹¹ is a cyano group. The compound according to claim 1 or 2, wherein R ¹ is a hydrogen atom. Before Symbol ^{R 11} is an alkyl group or a halogen atom having 1 to 5 carbon atoms, wherein _{n 11} is an integer from 0 to 2 A compound according to any one of claims 1 to 3. A compound represented by the following general formula (2).

(In the formula, R ¹ is a hydrogen atom or a hydroxyl group; Z is a group represented by the following general formula (1)-11 ; and m is an integer of 1 to 3.)

(In the formula, X ¹¹ is a hydrogen atom, a cyano group, an amino group, a monoalkylamino group or a dialkylamino group; R ¹¹ is an alkyl group or a halogen atom; n ₁₁ is an integer of 0 to 5; _{When 11} is an integer of 2 or more, a plurality of R ¹¹ may be the same or different from each other ; the bond marked with * is formed between the bond to Z and the bonding target. The compound according to claim 5, wherein X ¹¹ is a cyano group. The compound according to claim 5 or 6, wherein R ¹ is a hydrogen atom. Before Symbol ^{R 11} is an alkyl group or a halogen atom having 1 to 5 carbon atoms, wherein _{n 11} is an integer from 0 to 2 A compound according to any one of claims 5-7. A compound represented by the following general formula (11b).

(In the formula, X ¹¹ is a hydrogen atom, a cyano group, an amino group, a monoalkylamino group or a dialkylamino group; R ¹¹ is an alkyl group or a halogen atom; n ₁₁ is an integer of 0 to 5; _{When 11} is an integer of 2 or more, a plurality of R ¹¹ may be the same or different from each other.) A compound represented by the following general formula (1a).

(In the formula, R ¹⁰ is a hydrogen atom, a hydroxyl group, a trialkylsilyloxy group or an alkyldiarylsilyloxy group; Z is a group represented by the following general formula (1)-11 ; R ² and R ³ are , Each independently being a trialkylsilyl group or an alkyldiarylsilyl group.)

(In the formula, X ¹¹ is a hydrogen atom, a cyano group, an amino group, a monoalkylamino group or a dialkylamino group; R ¹¹ is an alkyl group or a halogen atom; n ₁₁ is an integer of 0 to 5; _{When 11} is an integer of 2 or more, a plurality of R ¹¹ may be the same or different from each other ; the bond marked with * is formed between the bond to Z and the bonding target. A probe comprising the compound according to any one of claims 1 to 4. A condensate of the compound according to any one of claims 5 to 8 and the compound or another compound other than the compound. The condensate according to claim 12 , wherein the other compound is a nucleotide and the condensate is a polynucleotide. A probe comprising the condensate according to claim 12 or 13 . A step of hybridizing the polynucleotide having cytosine and the probe according to claim 14 under acidic conditions to obtain a hybridized product;
Measuring the fluorescence of the hybridized product and the probe under acidic conditions,
Based on the presence or absence of cytosine, by detecting the change in the emission wavelength of the fluorescence, the step of detecting cytosine in the polynucleotide,
A method for detecting cytosine, comprising: A step of hybridizing the polynucleotide having cytosine and the probe according to claim 14 under a plurality of different pH conditions to obtain a hybridized product;
Measuring fluorescence of the hybridized product and the probe under a plurality of different pH conditions,
A step of detecting cytosine in the polynucleotide by detecting a change in emission wavelength of the fluorescence based on the difference in pH;
A method for detecting cytosine, comprising:

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