JP5747432B2 - Disubstituted cyclodextrin and method for detecting nucleic acid using the same - Google Patents

Description

  The present invention relates to a 2-substituted modified cyclodextrin and a nucleic acid detection method using the same.

In recent years, with the progress of genome sequencing, the whole base sequence of the genome of each organism is being clarified, and a technique for effectively utilizing this result is desired.
Nucleic acid microarrays are technologies that can analyze multiple genes at the same time, and are developed not only as methods for determining nucleotide sequences, but also as methods for efficiently examining gene expression levels and polymorphisms. Technological developments such as identification of biological classification and diagnosis of diseases are being developed.

In general, nucleic acid microarrays used for tailor-made medicine and biological classification or mutant identification need only be able to qualitatively detect a specific base sequence. It has been demanded.
For example, Patent Document 1 discloses a line probe (LiPA: Line Probe Assay, registered trademark of Inodogenetics) method as a nucleic acid microarray used in a method for identifying an HCV (hepatitis C virus) isolate. .
In this assay, nucleic acid probes are immobilized as parallel lines on a polyamide membrane piece.

Nucleic acid detection on such an assay is performed mainly using an RI labeling method, a fluorescence labeling method, an enzyme coloring method, and the like.
Among these methods, the enzyme color development method is particularly effective as it is an inexpensive and simple method.
However, even when this enzyme coloring method is used, it is necessary to modify the primer with biotin, so that there is a problem that the cost increases accordingly.
Furthermore, in the enzyme color development method, it is necessary to act and wash alkaline phosphatase modified with streptavidin and a substrate for streptavidin, and there is a problem that the detection process becomes extremely complicated.

On the other hand, as a highly sensitive and simple detection method, research on an intercalator that acts on the π-electron stacking structure of a hybridized nucleic acid base has been actively conducted (see Non-Patent Document 1).
However, since the intercalator disclosed in Non-Patent Document 1 is hardly soluble in water, it is extremely difficult to prepare a concentration that can act on the hybridized double-stranded nucleic acid. Further, since the storage stability of the reagent is poor, it is not suitable as an industrial method.

As a new technique for solving the above problems, the present inventors have found a mono-substituted modified cyclodextrin that is soluble in water and can be selectively adsorbed to a hybridized double-stranded nucleic acid. This has been reported (see Patent Document 2).
By using this mono-substituted modified dextrin, a difference in fluorescence intensity occurs between the single-stranded DNA and the double-stranded DNA. By detecting this, it becomes possible to discriminate them easily and inexpensively. .
However, the technique of Patent Document 2 is insufficient in that it can completely and accurately discriminate between single-stranded DNA and double-stranded DNA because the fluorescence intensity changes even in the case of single-stranded DNA. Met.

JP 7-503143 A International Publication No. 2007/105786 Pamphlet

Bioorganic & Medical Chemistry Letters, vol. 16, 2005, P154-157

  The present invention has been made in view of such circumstances, and more specifically, a 2-substituted modified cyclodextrin that selectively interacts with a double-stranded DNA, and a single-stranded DNA and a double-stranded DNA with higher accuracy. An object of the present invention is to provide a nucleic acid detection method that can be discriminated.

  As a result of intensive studies to achieve the above object, the inventors of the present invention have obtained a 2-substitution in which two fluorescent chromophores are bonded to cyclodextrin using a specific neutral divalent organic group as a linker. The modified cyclodextrin selectively interacts with the double-stranded DNA, and by using this compound, the single-stranded DNA and the double-stranded DNA can be distinguished with higher accuracy than the conventional method described above. As a result, the present invention has been completed.

（式中、ＣｙＤは、α−、β−またはγ−シクロデキストリン骨格を、Ａ¹およびＡ²は、互いに独立して、エーテル結合またはチオエーテル結合を含んでいてもよい炭素数１〜１０のアルキレン基を、Ｂ¹およびＢ²は、互いに独立して、単結合、−Ｃ（Ｏ）−、−Ｃ（Ｏ）ＮＨ−、−ＮＨＣ（Ｏ）−、−Ｃ（Ｏ）Ｏ−または−ＯＣ（Ｏ）−を、Ｃ¹およびＣ²は、互いに独立して、エーテル結合を含む炭素数１〜２０のアルキレン基を、Ｘ¹およびＸ²は、互いに独立して、ピレン、ナフタレン、ダンシルグリシン、アントラセンおよびローダミンＢから選ばれる少なくとも１種の蛍光性発色団を表す。）
２． 前記蛍光性発色団が、ピレンおよびナフタレンから選ばれる少なくとも１種である１の２置換修飾シクロデキストリン、
３． 式（２）で示される１の２置換修飾シクロデキストリン、

（式中、ＣｙＤ、Ａ¹、Ａ²、Ｂ¹、Ｂ²、Ｃ¹およびＣ²は、前記と同じ意味を表す。）
４． 式（３）で示される３の２置換修飾シクロデキストリン、

（式中、ＣｙＤ、Ｃ¹およびＣ²は、前記と同じ意味を表す。）
５． 式（４）で示される４の２置換修飾シクロデキストリン、

（式中、ｍおよびｎは１〜１０の整数を表し、ｍ＋ｎ≦１９を満足し、ｏおよびｐは１〜１０の整数を表し、ｏ＋ｐ≦１９を満足する。ＣｙＤは、前記と同じ意味を表す。）
６． 式（５）で示される５の２置換修飾シクロデキストリン、

（式中、ＣｙＤは、前記と同じ意味を表す。）
７． 前記ＣｙＤが、γ−シクロデキストリン骨格である１〜６のいずれかの２置換修飾シクロデキストリン、
８． 前記γ−シクロデキストリン骨格を構成する８個のＤ−グルコースの任意の１つをグルコースＡとし、その隣に結合するグルコースから順にグルコースＢ〜Ｈとした場合に、グルコースＡとグルコースＥとがそれぞれ置換されているＡＥ２置換体である７の２置換修飾シクロデキストリン、
９． １〜８のいずれかの２置換修飾シクロデキストリンを用いることを特徴とする核酸検出方法
を提供する。 That is, the present invention
1. A disubstituted modified cyclodextrin characterized by the formula (1)

(Wherein CyD is an α-, β- or γ-cyclodextrin skeleton, and A ¹ and A ² are each independently an alkylene having 1 to 10 carbon atoms which may contain an ether bond or a thioether bond. The groups B ¹ and B ² are independently of each other a single bond, —C (O) —, —C (O) NH—, —NHC (O) —, —C (O) O— or —OC; (O)-, C ¹ and C ² are independently of each other an alkylene group having 1 to 20 carbon atoms containing an ether bond, and X ¹ and X ² are independently of each other of pyrene, naphthalene, dansyl glycine. And represents at least one fluorescent chromophore selected from anthracene and rhodamine B.)
2. 1 disubstituted modified cyclodextrin, wherein the fluorescent chromophore is at least one selected from pyrene and naphthalene;
3. 1 disubstituted modified cyclodextrin of formula (2)

(In the formula, CyD, A ¹ , A ² , B ¹ , B ² , C ¹ and C ² represent the same meaning as described above.)
4). 3 disubstituted modified cyclodextrins of formula (3),

(In the formula, CyD, C ¹ and C ² have the same meaning as described above.)
5. 4 disubstituted modified cyclodextrins of formula (4)

(In the formula, m and n represent an integer of 1 to 10, satisfying m + n ≦ 19, o and p represent an integer of 1 to 10, and satisfy o + p ≦ 19. CyD has the same meaning as described above. Represents.)
6). 5 disubstituted modified cyclodextrins of formula (5)

(In the formula, CyD represents the same meaning as described above.)
7). The bisubstituted modified cyclodextrin according to any one of 1 to 6, wherein the CyD is a γ-cyclodextrin skeleton,
8). When any one of the eight D-glucoses constituting the γ-cyclodextrin skeleton is glucose A, and glucose B to H in order from glucose bound to the glucose A, glucose A and glucose E are respectively 7 disubstituted modified cyclodextrins that are substituted AE2 substituents,
9. Provided is a nucleic acid detection method characterized by using any one of the disubstituted cyclodextrins of 1 to 8.

The disubstituted modified cyclodextrins of the present invention selectively interact with double stranded DNA, and as a result, an increase in fluorescence spectrum is observed.
For this reason, by using the 2-substituted modified cyclodextrin of the present invention, it becomes possible to distinguish between double-stranded DNA and single-stranded DNA more accurately and easily than in the conventional method.
The disubstituted cyclodextrin of the present invention having such characteristics can be suitably used as a label for nucleic acid detection, such as a reagent for a DNA chip.

2 is a diagram showing a fluorescence spectrum of a mixed solution of 2-substituted pyrene-modified γ-CyD and dsDNA obtained in Example 1. FIG. 2 is a diagram showing a fluorescence spectrum of a mixed solution of 2-substituted pyrene-modified γ-CyD and ssDNA obtained in Example 1. FIG. FIG. 3 is a first intercalation estimation diagram of the 2-substituted pyrene-modified γ-CyD obtained in Example 1 into dsDNA. FIG. 2 is a second intercalation estimation diagram of the 2-substituted pyrene-modified γ-CyD obtained in Example 1 into dsDNA. It is a figure which shows CD spectrum at the time of adding sodium adamantanecarboxylate to 2 substituted pyrene modification gamma-CyD obtained in Example 1. FIG.

Hereinafter, the present invention will be described in more detail.
The disubstituted cyclodextrin according to the present invention is represented by the formula (1).

In the formula (1), CyD means an α-, β-, or γ-cyclodextrin skeleton.
Cyclodextrin is a compound in which D-glucose is cyclically linked to 1,4-α-glycoside, and α-cyclodextrin (6 glucoses), β-cyclodextrin (glucose 7) depending on the difference in the number of glucose constituting this. And γ-cyclodextrin (8 glucoses), and any of α-, β-, and γ- can be used for the disubstituted cyclodextrin of the present invention. A dextrin skeleton is preferred.
In addition, in 2-substituted cyclodextrins, there are various positional isomers depending on the bonding position of two substituents on glucose constituting this. For example, in 2-substituted γ-cyclodextrin, as shown below, AB form In the present invention, an AE form in which glucose A and glucose E are substituted, respectively, is preferable.
The binding site of the substituent may be either a primary hydroxyl group or a secondary hydroxyl group of glucose, but a primary hydroxyl group is preferred from the viewpoint of production cost.

A ¹ and A ² each independently represent an alkylene group having 1 to 10 carbon atoms which may contain an ether bond or a thioether bond, and particularly preferably 1 to 5 carbon atoms.
Specific examples thereof include —CH ₂ —O—CH ₂ —, — (CH ₂ ) ₂ —O— (CH ₂ ) ₂ —, — (CH ₂ ) ₃ —O— (CH ₂ ) ₃ —, — ( _{CH 2) 4 -O- (CH 2} ) 4 -, - (CH 2) 5 -O- (CH 2) 5 -, - CH 2 -S-CH 2 -, - (CH 2) 2 -S- ( _{CH 2) 2 -, - (} CH 2) 3 -S- (CH 2) 3 -, - (CH 2) 4 -S- (CH 2) 4 -, - (CH 2) 5 -S- (CH 2 ₅ ) etc. are mentioned.

B ¹ and B ² are each independently a single bond, —C (O) —, —C (O) NH—, —NHC (O) —, —C (O) O— or —OC (O). In particular, —C (O) NH— and —NHC (O) — are preferable.
In the case of a single bond, A ¹ and C ¹ , A ² and C ² are directly bonded.

C ¹ and C ² each independently represent a C 1-20 alkylene group containing an ether bond, preferably a C 2-10 alkylene group containing an ether bond, more preferably an ether group. It is a C2-C6 alkylene group containing a bond.
Specific examples of C ¹ include —CH _{2 —o} (O _2p H _p C) — (where o and p represent an integer of 1 to 10 and satisfy o + p ≦ 19). The thing of 1-5 and o = 1-5 is suitable.
Specific examples of C ² include — (C _n H _2n O) _m —CH ₂ — (m and n represent an integer of 1 to 10, satisfying m + n ≦ 19). The thing of 1-5 and n = 1-5 is suitable.

X ¹ and X ² represent, independently of each other, a fluorescent chromophore. The fluorescent chromophore is not particularly limited as long as it is a fluorescent substituent, but a neutral chromophore is preferred in the present invention.
Specific examples of the chromophore include pyrene, naphthalene, dansyl glycine, anthracene, rhodamine B and the like. Among these, pyrene and naphthalene capable of forming an excimer are preferable, and pyrene is more preferable.
Specific examples of the chromophore including the substitution position include those represented by the following formula, but are not limited thereto.

  Suitable disubstituted dextrins include, but are not limited to, those represented by the following formulas (3) to (5).

（式中、ＣｙＤ、Ｃ¹およびＣ²は、上記と同じ意味を表す。ｍおよびｎは１〜１０の整数を表し、ｍ＋ｎ≦１９を満足し、ｏおよびｐは１〜１０の整数を表し、ｏ＋ｐ≦１９を満足する。）

(In the formula, CyD, C ¹ and C ² represent the same meaning as described above. M and n represent an integer of 1 to 10, m + n ≦ 19 is satisfied, and o and p represent an integer of 1 to 10. , O + p ≦ 19 is satisfied.)

The disubstituted dextrin of the present invention can be produced using a known organic synthesis reaction, and the production method is not limited.
Modification of the dextrin to the primary hydroxyl group can be performed, for example, by tosylating this as shown in the following scheme.

In this case, the usage-amount of p-toluenesulfonyl chloride (TsCl) is about 2-10 equivalent with respect to CyD1 equivalent.
As the reaction solvent, an amine solvent is suitable, and for example, pyridine or the like can be used.
The reaction temperature is about 0 to 50 ° C., and the reaction time is about 0.1 to 10 hours.
After completion of the reaction, a crude product is obtained by neutralization with a base such as sodium hydroxide and precipitation with an organic solvent such as acetone.
The obtained crude product can be purified by reverse phase silica gel column chromatography.

The tosylated CyD can introduce a desired side chain by a substitution reaction utilizing the detachability of the tosyl group.
For example, as shown in the following scheme, CyD substituted with thioacetic acid can be obtained by reacting with mercaptoacetic acid.

In this case, the amount of mercaptoacetic acid used can be about 2 to 10 equivalents relative to 1 equivalent of tosylated CyD.
In the reaction, a base is preferably used, and specific examples thereof include sodium carbonate. The usage-amount of a base can be about 2-20 equivalent with respect to CyD1 equivalent.
The reaction solvent is not particularly limited, but DMF is preferable.
Although reaction temperature is about 0-100 degreeC, about 50-100 degreeC is preferable.
The reaction time is about 1 to 240 hours.
After completion of the reaction, the solvent is distilled off, neutralized with hydrochloric acid or the like, and precipitated with an organic solvent such as acetone to obtain a crude product.
The obtained crude product can be purified by gel filtration or the like.

CyD having the thioacetic acid group can be converted to CyD having a chromophore by condensation with an amine or alcohol having a chromophore to form an amide bond or an ester bond.
For example, as shown in the following scheme, CyD having pyrene as a chromophore by condensation with 8- (1-pyrenemethoxy) -3,6- (dioxa) oct-1-amine to form an amide bond Is obtained.

In this case, the use amount of the amine having a chromophore can be about 2 to 10 equivalents relative to 1 equivalent of CyD, but is preferably about 2 to 5 equivalents.
In the reaction, a known dehydration condensing agent such as N, N′-dicyclohexylcarbodiimide can also be used. The amount of the condensing agent used can be about 2 to 10 equivalents relative to CyD.
The reaction solvent is not particularly limited, but DMF is preferably used.
The reaction temperature is about 0 to 100 ° C.
The reaction time is about 1 to 240 hours.
After completion of the reaction, the solvent is distilled off, and a crude product is obtained by precipitation with an organic solvent such as acetone.
The obtained crude product can be purified by reverse phase silica gel column chromatography.

The method for synthesizing the amine having a chromophore is not particularly limited. For example, the 8- (1-pyrenemethoxy) -3,6- (dioxa) oct-1-amine is 1-pyrene. 8- (1-Pyrenemethoxy) -3,6- (dioxa) oct-1-chloride obtained by reacting methanol with 1,2-bis (2-chloroethoxy) ethane in the presence of a base is phthalimide. And can be produced by a method of reducing it.
In the above explanation, the carboxyl group on the CyD side and the amino group on the chromophore side are reacted, but this may be reversed.
Further, when an alcohol having a chromophore is used, a known esterification method using an acid or base as a catalyst may be used.

As described above, the 2-substituted modified cyclodextrin of the present invention selectively interacts with double-stranded DNA and increases its fluorescence spectrum, so that double-stranded DNA and single-stranded DNA can be distinguished with high accuracy. be able to.
Therefore, the 2-substituted modified dextrin of the present invention can be suitably used as a label for nucleic acid detection such as a DNA chip reagent. For example, an analyte nucleic acid or a nucleic acid probe is immobilized on a substrate to prepare an array. A first step, a second step of preparing a double-stranded nucleic acid by hybridizing a sample nucleic acid and a nucleic acid probe using the array obtained in the first step, and a double strand obtained in the second step The nucleic acid can be suitably used in a nucleic acid detection method including the third step of treating a nucleic acid with the 2-substituted modified cyclodextrin of the present invention.

Here, the sample nucleic acid means a nucleic acid to be detected, and the nucleic acid probe means a nucleic acid that can hybridize with the sample nucleic acid.
As the nucleic acid probe, synthesized DNA or cDNA reversely transcribed from mRNA may be used. DNA synthesis can be performed by an automatic nucleic acid synthesizer.
As long as the base sequence of the nucleic acid probe can hybridize with the sample nucleic acid, a sequence in which several bases are inserted with respect to the completely complementary sequence of the base sequence of the sample nucleic acid, or a few bases in the completely complementary sequence are mutated or missing. Although it may be a lost sequence, in view of detection accuracy, a completely complementary sequence is desirable.

  Since the number of bases of a nucleic acid probe varies depending on the base sequence to be detected, it cannot be defined unconditionally. However, when the purpose is to detect a single base level mutation in genomic DNA extracted from a living body. Is preferably about 10 to 30 bases, particularly 12 to 26 bases.

On the other hand, genomic DNA extracted from biological samples such as blood, saliva and hair can be used as the sample nucleic acid. Examples of the extraction method include a phenol extraction method, a guanidine thiocyanate extraction method, and a vanadyl ribonucleoside complex extraction method.
Further, the genomic DNA may be obtained by amplifying a specific base sequence to be detected by a known method such as a PCR method, a LAMP method, or an ICAN method.

The base material may be any material that can immobilize a sample nucleic acid or a nucleic acid probe (hereinafter also referred to as a nucleic acid), such as polyethylene, polypropylene, polycarbonate, polymethyl methacrylate, polyamide, polystyrene, polyethylene terephthalate, Examples thereof include organic materials such as nitrocellulose; inorganic materials such as glass and silica; metal materials such as gold, silver and copper. Among these, organic materials are preferable because they can be easily molded, and polypropylene, polycarbonate, polymethyl methacrylate, and polyamide are particularly preferable.
The shape of the substrate is also arbitrary, and can be an appropriate shape such as a plate shape, a film shape, or a tube shape.

The technique for immobilizing the nucleic acid on the substrate may be a physical technique or a chemical technique.
Examples of the physical technique include a method of spotting a nucleic acid solution on a base material. Examples of the spotting method include an extrusion method using a dispenser, a suction method using a Coulomb force, and an ink jet method.
In this case, an irrelevant base sequence may be added to the nucleic acid probe, and the immobilization rate to the nucleic acid probe on the substrate may be improved by UV irradiation. Examples of the base sequence unrelated to the nucleic acid probe include polyadenine, polycytosine, polythymine, polyguanine and the like.

Further, the substrate may be surface-treated to improve the nucleic acid immobilization rate.
Surface treatment methods include coating the substrate surface with a polycationic polymer such as polylysine, treating the inorganic substrate surface with a silane coupling agent, and metal substrate with an amino group-containing thiol or disulfide compound. The method of processing the surface etc. are mentioned.

On the other hand, examples of chemical methods include a method of modifying a nucleic acid probe with a functional group capable of covalent bonding with a substrate, a method of modifying both a nucleic acid probe and a substrate with a functional group capable of covalent bonding, and the like. It is done.
Specific examples thereof include (1) a method of modifying a nucleic acid probe with a functional group capable of silane coupling reaction such as trimethoxysilane and triethoxysilane, and (2) a base material with an amino group such as aminoethoxysilane. And a method in which a nucleic acid probe is modified with a carboxyl group.

  In addition, before immobilizing the nucleic acid, the substrate may be plasma-treated for the purpose of increasing the amount of nucleic acid immobilized. This plasma treatment can be performed using corona discharge, arc discharge, glow discharge, or the like.

The sample nucleic acid and the nucleic acid probe are hybridized using the array produced by the above method.
The hybridization method is not particularly limited, and a general method may be used.
Specific examples thereof include a method of immersing an array in which a nucleic acid probe or a sample nucleic acid is immobilized in a sample nucleic acid solution or a nucleic acid probe solution. Hybridization temperature conditions may be appropriately set in consideration of the heat denaturation temperature of the DNA probe used.
Unreacted nucleic acid can be easily removed by washing.

The double-stranded nucleic acid prepared by hybridizing by the above method is treated with the 2-substituted modified cyclodextrin of the present invention, and the change in the fluorescence spectrum is detected.
The treatment method with the 2-substituted modified cyclodextrin is not particularly limited, and the nucleic acid may be added to the solution of the 2-substituted modified cyclodextrin at an appropriate concentration, or vice versa. In this case, the solvent is not particularly limited as long as it can dissolve the 2-substituted modified cyclodextrin and the nucleic acid, but in the present invention, a mixed solvent of DMSO-water can be preferably used. The concentration of DMSO is preferably about 1 to 30% by volume.

By the above treatment, the 2-substituted modified cyclodextrin of the present invention interacts with the double-stranded nucleic acid, and as a result, a change occurs in the detected fluorescence spectrum.
Specifically, as described in detail in Examples below, the interaction between the 2-substituted modified cyclodextrin and the double-stranded nucleic acid increases the intensity of the monomer fluorescence of the fluorescent chromophore possessed by the cyclodextrin, If the fluorescent chromophore is capable of forming an excimer, the intensity of excimer fluorescence increases at the same time, but this phenomenon is not observed for single-stranded nucleic acids.
Due to this phenomenon difference, double-stranded nucleic acid and single-stranded nucleic acid can be detected and identified with high accuracy.

In addition, it is estimated that the said interaction is what the fluorescent chromophore which cyclodextrin has intercalates to double stranded DNA.
In this embodiment of intercalation, an increase in excimer fluorescence is confirmed depending on the substituted fluorescent chromophore, so one fluorescent chromophore intercalates into a double-stranded nucleic acid, and from the outside, Possible to have immobilized a chromophore intercalated with one fluorescent chromophore or inserted between two bases of a double-stranded nucleic acid into two fluorescent chromophores .

EXAMPLES Hereinafter, although a synthesis example and an Example are given and this invention is demonstrated more concretely, this invention is not limited to the following Example. In addition, the analyzers and conditions used in the examples are as follows.
[1] ¹ H NMR and ¹³ C NMR
Equipment: Bruker: BURUKER-SPECTROSPIN 300
Measuring solvent: DMSO-d6
Reference substance: Tetramethylsilane [2] IR
Equipment: PerkinElmer: FT-IR Spectrometer SPECTRUM 2000
[3] FAB-MS
Device: JEOL (JEOL): JMS-700
[4] Elemental analyzer: Yanako Analytical Co., Ltd .: CHN coder [5] Fluorescence spectrometer: Fluorometer Perkin-Elmer LS 40B Fluorescence Spectrophotometer
Cell: Standard square fluorescent cell (2500μL)
Measurement conditions: Start wavelength: 300 nm, End wavelength: 600 nm, Excitation wavelength: 343.0 nm, Excitation slit: 5 nm, Emission slit: 5 nm, Scan speed: 100 nm / min.
[6] CD spectrum device: JASCO (JSCO): J-720 spectrophotometer

[Synthesis Example 1] Synthesis of 8- (1-pyrenemethoxy) -3,6- (dioxa) oct-1-chloride

1-pyrenemethanol (1.03 g, 4.42 mmol) and 1,2-bis (2-chloroethoxy) ethane (4.86 g, 26.1 mmol) were dissolved in 50 mL of dry-THF, and then sodium hydride (1.65 g, 43.2 mmol) was added and allowed to react for 6 hours under heated reflux. The reaction was traced using TLC (n-hexane: ethyl acetate = 1: 1 (v / v), TLC; silica gel 60F ₂₅₄ ).
After completion of the reaction, a small amount of methanol was added to decompose sodium hydride, the solvent was distilled off, water was added, and the mixture was extracted 3 times with chloroform. The organic phase was washed with water, dried over sodium sulfate and filtered. The filtrate was concentrated and then dried under reduced pressure at 60 ° C. overnight to recover 4.45 g of a yellow oily crude product.
The crude product was dissolved in a small amount of chloroform and purified by normal phase silica gel column chromatography. The developing solvent is n-hexane-ethyl acetate, the ratio of ethyl acetate is gradually increased, and the eluate of about 26 to 32 vol% of ethyl acetate is collected and concentrated, and then dried under reduced pressure at 60 ° C. to give a yellow oily form. 0.86 g (yield; 51.1%) of the desired product was obtained.

R _f = 0.71 (n-hexane: ethyl acetate = 1: 1, TLC; silica gel 60F ₂₅₄ )
¹ H NMR (300 MHz, CDCl ₃ )
δ = 3.5-3.8 (12H, m, -OCH ₂ CH ₂ O-), 5.2-5.3 (2H, s, CH ₂ of pyrenemethoxy), 7.9-8.4 (9H, m, aromatic H of pyrene).
¹³ C NMR (75 MHz, CDCl ₃ )
δ = 41-45 (1C, t, -CCl), 67-74 (6C, m, -OCH _2- ), 122-132 (16C, m, aromatic C of pyrene).
IR (KBr) ν 3041.4 (aryl-H), 2865.2 (CH), 1588.6 (C = C), 1108.9 (COC), 682.3 (C-Cl) cm ^-1 .
FAB-MS (m / z); 382.2 [M] ⁺

[Synthesis Example 2] Synthesis of 8- (1-pyrenemethoxy) -3,6- (dioxa) oct-1-amine-phthalimide

8- (1-pyrenemethoxy) -3,6- (dioxa) oct-1-chloride (0.85 g, 2.2 mmol) obtained in Synthesis Example 1 and potassium phthalimide (1.07 g, 5.78 mmol) were used. , And dissolved in 15 mL of dry-DMF and reacted at 90 ° C. for 6 hours. The reaction was traced using TLC (n-hexane: ethyl acetate = 1: 1 (v / v), TLC; silica gel 60F ₂₅₄ ).
After completion of the reaction, the solvent was distilled off, water was added, and the mixture was extracted 3 times with chloroform. The organic phase was washed successively with 0.2M aqueous sodium hydroxide and water, dried over sodium sulfate, and filtered. The filtrate was concentrated and then dried under reduced pressure at 60 ° C. overnight to recover 0.98 g of a yellow oily crude product.
The crude product was dissolved in a small amount of chloroform and purified by normal phase silica gel column chromatography. The developing solvent is n-hexane-ethyl acetate, the ratio of ethyl acetate is gradually increased, and the eluate of about 30 to 55 vol% of ethyl acetate is collected, concentrated and dried under reduced pressure at 60 ° C. 0.45 g (yield; 41.6%) of the product was obtained.

R _f = 0.39 (n-hexane: ethyl acetate = 1: 1, TLC; silica gel 60F ₂₅₄ )
¹ H NMR (300MHz, CDCl ₃ )
δ = 3.6-3.9 (12H, m, -OCH ₂ CH ₂ O-), 5.2-5.3 (2H, s, CH ₂ of pyrenemethoxy), 7.4-7.8 (4H, m, aromatic H of phthalimide), 7.9-8.4 (9H, m, aromatic H of pyrene).
¹³ C NMR (75MHz, CDCl ₃ )
δ = 35-40 (1C, t, -CN), 65-74 (6C, m, -OCH _2- ), 121-135 (22C, m, aromatic C of pyrene and phthalimide), 168-169 (2C, s, C = O).
IR (KBr) ν 3042.3 (aryl-H), 2865.5 (CH), 1772.8 (C = O), 1707.6 (C = O), 1603.4 (C = C), 1106.2 (COC) cm ^-1 .
FAB-MS (m / z); 493.3 [M] ⁺

[Synthesis Example 3] Synthesis of 8- (1-pyrenemethoxy) -3,6- (dioxa) oct-1-amine

8- (1-Pyrenemethoxy) -3,6- (dioxa) oct-1-amine-phthalimide (1.76 g, 3.57 mmol) obtained in Synthesis Example 2 was dissolved in 75 mL of acetone, and then 80% hydrazine Hydrate (75 mL) was added, and the mixture was reacted for 1 hour under heating and reflux. The reaction was traced using TLC (acetone: aqueous ammonia (28%) = 20: 1 (v / v), TLC; silica gel 60F ₂₅₄ ).
After completion of the reaction, the mixture was neutralized with 1M hydrochloric acid, the solvent was distilled off, 1M aqueous sodium hydroxide solution was added, and the mixture was extracted 3 times with chloroform. The organic phase was washed successively with 1M aqueous sodium hydroxide solution and water, then dried over sodium sulfate and filtered. The filtrate was concentrated and then dried under reduced pressure at 60 ° C. overnight to recover 1.06 g (yield: 81.3%) of the desired product as a yellow oil.

R _f = 0.61 [acetone: ammonia _aq (28%) = 20: 1, TLC; silica gel 60F ₂₅₄ ]
¹ H NMR (300 MHz, CDCl ₃ )
δ = 1.5-1.7 (2H, s, NH ₂ ), 2.7-2.9 (2H, t, -CH ₂ N), 3.4-3.8 (10H, m, -CH ₂ OCH _2- ), 5.2-5.3 (2H, s, CH ₂ of pyrenemethoxy), 7.9-8.5 (9H, m, aromatic H of pyrene).
¹³ C NMR (75 MHz, CDCl ₃ )
δ = 39-44 (1C, t, -CN), 67-74 (6C, m, -OCH _2- ), 122-132 (16C, m, aromatic C of pyrene).
IR (KBr) ν 3397.2 (NH), 3040.9 (aryl-H), 1587.5 (C = C), 1090.1 (COC) cm ^-1 .
Calcd. For 3C ₂₃ H ₂₅ NO ₃ · H ₂ O: C; 74.76, H; 7.01, N; 3.79% .; Found: C; 74.89, H; 6.83, N; 3.64%.
FAB-MS (m / z); 364.2 [M + H] ⁺

[Synthesis Example 4] di -6 ^A, 6 ^E -p- toluenesulfonyloxy-gamma-CyD Synthesis of

γ-CyD (5.0960 g, 3.93 mmol) was completely dissolved in 100 mL of pyridine, p-toluenesulfonyl chloride (4.7101 g, 24.7 mmol) was added, and the mixture was reacted at room temperature for 3 hours. The reaction was traced using TLC (1-butanol: ethanol: water = 5: 4: 3 (v / v / v), TLC; silica gel 60F ₂₅₄ ).
After completion of the reaction, the solvent was distilled off to some extent, neutralized with 1M aqueous sodium hydroxide solution, and reprecipitated with 500 mL of acetone. The precipitate was filtered with suction, dried at 30 ° C. under reduced pressure, and 9.7270 g of a crude product of white crystals was recovered.
The crude product was dissolved in 30 mL of dry-DMF, the insoluble material was filtered with Kiriyama funnel (manufactured by Kiriyama Seisakusho), and the filtrate was subjected to reverse phase silica gel column chromatography (Lobar column LiChroprep RP-18, manufactured by Merck, 310 mm × 10 mm). As the developing solvent, methanol-water was used, and the methanol ratio was gradually increased to recover an eluate of 40% methanol. After concentration, acetone was added to crystallize, and the precipitate was filtered by suction and dried under reduced pressure at 30 ° C. to obtain 0.2040 g (yield; 3.23%) of the desired product as white crystals.

R _f = 0.58 (1-butanol: ethanol: water = 5: 4: 3, TLC; silica gel 60F ₂₅₄ )
¹ H NMR (300MHz, DMSO-d ₆ )
δ = 4.5-4.6 (6H, w, C ¹ H of CyD), 5.7-5.9 (16H, s, OH of C ² and C ³ of CyD), 7.4-7.5 (4H, d, aromatic H of benzene), 7.7-7.8 (4H, d, aromatic H of benzene).

[Synthesis Example 5] di -6 ^A, 6 ^E - deoxy -6 ^A, 6 ^E - thioacetate-gamma-CyD Synthesis of

Di -6 obtained in Synthesis Example 4 ^A, 6 ^E -p- toluenesulfonyloxy -γ-CyD (0.2040g, 0.13mmol) , mercaptoacetic acid (0.0623g, 0.66mmol), dry- DMF15mL Then, 3.8 mL of water in which sodium carbonate (0.1467 g, 1.38 mmol) was dissolved was added dropwise and reacted at 70 ° C. for 4 days. The reaction was traced using TLC (1-butanol: ethanol: water = 5: 4: 3 (v / v / v), TLC; silica gel 60F ₂₅₄ ).
After completion of the reaction, the solvent was distilled off, and a small amount of water was added to dissolve sodium carbonate, followed by neutralization with 1M hydrochloric acid. After concentration, it was reprecipitated with 300 mL of acetone. The precipitate was filtered with suction, dried at 60 ° C. under reduced pressure, and 0.3295 g of a white crystalline crude product was recovered.
The crude product was dissolved in a small amount of water and purified with Sephadex G-15 open column (100 cm × 1.8 cm). Elution solvent was water. The target eluate was collected, concentrated, and crystallized by adding acetone. The precipitate was filtered by suction and dried under reduced pressure at 60 ° C. to obtain 0.1266 g (yield; 68.7%) of the target product as white crystals.

R _f = 0.33 (1-BuOH: EtOH: H ₂ O = 5: 4: 3, TLC; silica gel 60F ₂₅₄ )
¹ H NMR (300 MHz, DMSO-d ₆ )
δ = 4.7-5.0 (8H, w, C ¹ H of CyD), 5.5-5.9 (16H, s, OH of C ² and C ³ of CyD).
IR (KBr) ν 3430.4 (OH), 2934.8 (CH), 1635.4 (C = O), 1029.6 (COC) cm ^-1 .

[Example 1] di -6 ^A, 6 ^E - deoxy ^{-6 A, 6 E - [{} 8- (1- pyrene methoxy) -3,6 (dioxa) oct-l-amino} - (thioacetyl)] -Γ-CyD synthesis

Di -6 ^A obtained in Synthesis Example 5, 6 ^E - deoxy -6 ^A, 6 ^E - thioacetate -γ-CyD (0.1479g, 0.10mmol) , 1- hydroxybenzotriazole (1-HOBt, 0 0.0626 g, 0.46 mmol) was dissolved in 15 mL of dry-DMF. The solution was cooled to 0 ° C. under a nitrogen stream, and N, N′-dicyclohexylcarbodiimide (DCC, 0.0879 g, 0.42 mmol) was added. After reacting for 2 hours, 8- (1-pyrenemethoxy) -3,6- (dioxa) oct-1-amine (0.1646 g, 0.45 mmol) obtained in Synthesis Example 3 was added. After 30 minutes, the temperature was gradually returned to room temperature and allowed to react for 1 day, then heated (81.2 ° C.) and reacted for 3 days. The reaction was traced using TLC (1-butanol: ethanol: water = 5: 4: 3, TLC; silica gel 60F ₂₅₄ ).
After completion of the reaction, the solvent was distilled off and reprecipitated with 200 mL of acetone. The precipitate was filtered with suction, dried at 60 ° C. under reduced pressure, and 0.1628 g of a white crystalline crude product was recovered.
The crude product was dissolved in a small amount of dry-DMF and water and purified by reverse-phase silica gel column chromatography (Lobar column LiChroprep RP-18, manufactured by Merck, 310 mm × 10 mm). As a developing solvent, methanol-water was used, and the methanol ratio was gradually increased to recover an eluate of 80 to 100 vol% methanol. After concentration, acetone was added to crystallize. The precipitate was filtered with suction, and dried under reduced pressure at 60 ° C. to obtain 0.0134 g (yield; 6.14%) of the desired product as white crystals.

R _f = 0.56 (1-BuOH: EtOH: H ₂ O = 5: 4: 3, TLC; silica gel 60F ₂₅₄ )
¹ H NMR (300 MHz, DMSO-d ₆ )
δ = 4.5-4.7 (6H, m, OH of C ⁶ of CyD), 4.8-5.0 (8H, d, H of C ¹ of CyD), 5.1-5.3 (2H, s, CH ₂ of pyrenemethoxy), 5.8- 6.2 (16H, s, OH of C ² and C ³ of CyD), 7.9-8.0 (2H, t, H of amide), 8.0-8.5 (18H, m, aromatic H of pyrene).
IR (KBr) ν 3423.5 (OH), 2923.5 (CH), 1655.1 (C = C), 1634.9 (C = O), 1029.8 (CO) cm ^-1 .

[Example 2] Measurement of fluorescence spectrum In all the following procedures, a 10% DMSO aqueous solution was used as a solvent.
<Interaction with dsDNA>
Di -6 ^A obtained in Example 1, 6 ^E - deoxy ^{-6 A, 6 E - [{} 8- (1- pyrene methoxy) -3,6 (dioxa) oct-l-amino} - (thioacetyl )]-Γ-CyD (hereinafter referred to as disubstituted pyrene-modified γ-CyD) was adjusted to a concentration of 3 × 10 ⁻⁷ M.
Thereto, commercially available dsDNA (200 Mer) adjusted to a predetermined concentration with a solvent, the DNA concentration in the fluorescent cell was 0,0.9375 × 10 ⁻¹⁰ , 1.875 × 10 ⁻¹⁰ , 3.75 ×. In addition to 10 ⁻¹⁰ , 7.5 × 10 ⁻¹⁰ , 15 × 10 ⁻¹⁰ , 30 × 10 ⁻¹⁰ , 60 × 10 ⁻¹⁰ , 120 × 10 ⁻¹⁰ , and 250 × 10 ⁻¹⁰ M, the fluorescence spectrum Was measured. The results are shown in FIG.

<Interaction with ssDNA>
The disubstituted pyrene-modified γ-CyD obtained in Example 1 was adjusted to a concentration of 3 × 10 ⁻⁷ M.
Then, commercially available ssDNA (40 Mer) adjusted to a predetermined concentration with a solvent was used in a fluorescent cell, and the DNA concentration was 0,4.6875 × 10 ⁻¹⁰ , 9.375 × 10 ⁻¹⁰ , 18.75. ^{× 10 -10, 37.5 × 10 -10} , 75 × 10 -10, 150 × 10 -10, 300 × 10 -10, 600 × 10 -10, added to a 1200 × 10 ^-10 M, fluorescent The spectrum was measured. The results are shown in FIG.

As shown in FIG. 1, when dsDNA is added to 2-substituted pyrene-modified γ-CyD, it can be seen that the intensities of pyrene monomer fluorescence (377 nm) and excimer fluorescence (479 nm) are increased.
The degree of increase in fluorescence intensity increased rapidly until 7.5 × 10 ⁻¹⁰ M of dsDNA was added, and the fluorescence intensity did not change substantially even when dsDNA was further added thereafter.
On the other hand, as shown in FIG. 2, it can be seen that the fluorescence intensity of the 2-substituted pyrene-modified γ-CyD does not increase even when ssDNA is added.
This is because when dsDNA was added, the pyrene site of the 2-substituted pyrene-modified γ-CyD was inserted between the hydrophobic base pairs of dsDNA and became more stable than in 10% DMSO aqueous solution. Is considered to have increased.

  As described above, an increase in the excimer fluorescence of pyrene was confirmed in the aspect of intercalation of 2-substituted pyrene-modified γ-CyD into dsDNA. As shown in FIG. 3, one pyrene was converted into dsDNA. Intercalating and immobilizing pyrene with the other pyrene intercalating from the outside, or two pyrenes inserted between one base pair as shown in FIG. It is guessed.

Further, sodium adamantanecarboxylate (48 × 10 ⁻⁵ M, 10) easily entrapped in CyD vacancies in 2-substituted pyrene-modified γ-CyD (3.0 × 10 ⁻⁵ M, diluted with 10% DMSO aqueous solution). When the CD spectrum was measured, no significant change was observed in the spectrum as shown in FIG.
This shows that the 2-substituted pyrene-modified γ-CyD is not self-inclusion in a 10% DMSO aqueous solution.
As described above, it can be seen that by using 2-substituted pyrene-modified γ-CyD, dsDNA and ssDNA can be identified with high accuracy and can be suitably used for detection of nucleic acids.

Claims (9)

A disubstituted modified cyclodextrin represented by the formula (1):

(Wherein CyD represents an α-, β- or γ-cyclodextrin skeleton,
A ¹ and A ² each independently represent a C 1-10 alkylene group which may contain an ether bond or a thioether bond,
B ¹ and B ² are each independently a single bond, —C (O) —, —C (O) NH—, —NHC (O) —, —C (O) O— or —OC (O). −
C ¹ and C ² are each independently a C 1-20 alkylene group containing an ether bond,
X ¹ and X ² each independently represent at least one fluorescent chromophore selected from pyrene, naphthalene, dansyl glycine, anthracene and rhodamine B. )   The disubstituted cyclodextrin according to claim 1, wherein the fluorescent chromophore is at least one selected from pyrene and naphthalene. The disubstituted cyclodextrin according to claim 1 represented by the formula (2).

(In the formula, CyD, A ¹ , A ² , B ¹ , B ² , C ¹ and C ² represent the same meaning as described above.) The disubstituted cyclodextrin according to claim 3 represented by formula (3).

(In the formula, CyD, C ¹ and C ² have the same meaning as described above.) The disubstituted cyclodextrin according to claim 4 represented by formula (4).

(In the formula, m and n represent an integer of 1 to 10, satisfying m + n ≦ 19, o and p represent an integer of 1 to 10, and satisfy o + p ≦ 19. CyD has the same meaning as described above. Represents.) The disubstituted cyclodextrin according to claim 5 represented by formula (5).

(In the formula, CyD represents the same meaning as described above.)   The disubstituted cyclodextrin according to any one of claims 1 to 6, wherein the CyD is a γ-cyclodextrin skeleton.   When any one of the eight D-glucoses constituting the γ-cyclodextrin skeleton is glucose A, and glucose B to H in order from glucose bound to the glucose A, glucose A and glucose E are respectively The disubstituted modified cyclodextrin according to claim 7, which is a substituted AE2 substituted product.   A method for detecting a nucleic acid, comprising using the 2-substituted modified cyclodextrin according to any one of claims 1 to 8.

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