JP3078793B2 - Dye having rotaxane structure, labeling agent, and labeling method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel dye having a rotaxane-type structure having cyclodextrin, a labeling agent using the same, and a labeling method using the same.

[0002]

2. Description of the Related Art As a method for detecting various substances or a method for labeling a specific substance in the field of biotechnology including genetic engineering, a method using a dye (in particular, a fluorescent analysis method using a fluorescent dye) is the most widely used analytical method. It is one of. For this purpose, labeling agents with dyes having various properties (particularly fluorescent dyes) have been developed and used.

However, (1) there is a problem that conventionally known dyes or labeling agents using the same are not sufficient in terms of water solubility. Furthermore, (2) there is a problem that there is no labeling agent using a multi-wavelength type dye capable of coping with multicoloring, which has been strongly demanded in recent years, and a labeling method using the same.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide a dye exhibiting excellent water solubility and capable of multicoloring. It is another object of the present invention to provide a labeling agent containing such a dye and a labeling method using the labeling agent.

[0005]

Means for Solving the Problems The present inventors have intensively studied to solve the above-mentioned problems of the prior art, and combined a water-insoluble or poorly water-soluble dye with a water-soluble cyclodextrin. By using a rotaxane structure, it has been found that a multi-wavelength dye excellent in water solubility and capable of coping with multicoloring can be obtained. Further, they have found that a labeling agent containing such a dye having a rotaxane structure can be obtained. Furthermore, a labeling method using the labeling agent was developed.

In particular, when the dye is a fluorescent dye, a multi-wavelength fluorescent dye having excellent water solubility and capable of coping with multicoloring, a labeling agent containing the fluorescent dye, The present inventors have found that a labeling method using this labeling agent is achieved, and have completed the present invention.

That is, the structure of the rotaxane type dye according to the present invention is such that a dye molecule is combined with (1) cyclodextrin and (2) at least one end of a chain group penetrating the cyclodextrin ring. It is based on Therefore, the dye having the cyclodextrin-rotaxane structure has excellent water solubility because of having cyclodextrin. Further, it is possible to have a structure in which dye molecules are linked at both ends of a chain group penetrating the cyclodextrin ring. In this case, not only those of the same kind but also different kinds of dyes are bound. It is also possible.

The dye according to the present invention can be used as a labeling agent for labeling various substances in an aqueous solution by introducing various functional groups or various reactive groups of the dye molecule itself. . The dye according to the present invention is a dye selected from the group consisting of fluorescein, rhodamine, and Cy5.

[0009] More specifically, the dye according to the present invention is a dye characterized in that the dye is bonded to at least one end of a chain group, and the chain group is a rotaxane type penetrating through a cyclodextrin ring. .

The dye according to the present invention is a rotaxane type dye represented by the following formula 1.

[0011]

Embedded image

(Where FL1 and FL2 may be the same or different and represent a dye selected from the group consisting of fluorescein, rhodamine and Cy5, and n is 8 to 1
2 represents an integer, and m represents an integer of 6 to 8. X is OH, C
1, represents any of Br, I, NH2, NCO, and NHCO (CH2) 3CO2H. The dye according to the present invention is a rotaxane type dye represented by the following formula 2.

[0013]

Embedded image

(Here, FL3 represents a dye selected from the group consisting of fluorescein, rhodamine and Cy5, k represents an integer of 6 to 8, and 1 represents an integer of 8 to 12.)

Further, the dye according to the present invention is a rotaxane type dye characterized in that the dye is a dye selected from the group consisting of fluorescein, rhodamine and Cy5.

The labeling agent according to the present invention is a labeling agent characterized by using the dye described above.

Further, a labeling method according to the present invention is a labeling method using the labeling agent described above.

Hereinafter, the present invention will be described in more detail based on embodiments.

[0019]

DESCRIPTION OF THE PREFERRED EMBODIMENTS One basic structure of a rotaxane-type dye according to the present invention is, as schematically shown in FIG. 1 (a), a dye molecule at both terminal portions of a chain group penetrating CD. (Shown as dyes 1 and 2 in the figure).

As shown in FIG. 1 (b), another basic structure is such that a dye molecule (shown as dye 3 in the figure) is attached to one end of a chain group penetrating the CD. ), And a rotaxane structure having a reactive group bonded to the other end.

The dyes 1, 2 and 3 have sufficiently large molecular sizes that they cannot be displaced from the cyclodextrin ring. Further, the chain group penetrates the cyclodextrin ring, but depending on the type of the chain group, relatively molecular movement is possible in the cyclodextrin.

The rotaxane type dye according to the present invention has a large hydrophilic contribution of a cyclodextrin ring even if the dye molecules (dyes 1 to 3 in the figure) are insoluble or hardly soluble. And sufficient water solubility. In addition, it can be extremely stably present under a normal labeling reaction or various subsequent measurement conditions.

Hereinafter, the rotaxane type dye according to the present invention will be described in more detail. 1. Cyclodextrin (hereinafter abbreviated as CD) (1) The type of CD used for the rotaxane-type dye according to the present invention is not particularly limited. It is sufficient that the hydrophobic space inside the CD has a size sufficient to penetrate a chain group described below. Specifically, although it depends on the type of the chain group, in the case of a methylene chain, the number of glucose is preferably 6 or more.

As the CD usable in the present invention, those commercially available as various cyclodextrins can be obtained and used. That is, depending on the number of glucose, α-cyclodextrin, β-cyclodextrin, or γ
-Cyclodextrin is available in high purity. Therefore, these or cyclodextrin derivatives having a structure similar thereto can be suitably used as the CD of the present invention. Specifically, α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin, and glucosyl-α-cyclodextrin having a branched structure, glucosyl-β-cyclodextrin, glucosyl-γ-cyclodextrin, maltosyl-α -Cyclodextrin, maltosyl-β-cyclodextrin, maltosyl-γ-cyclodextrin, or an alkylated cyclodextrin derivative such as 6-O-methyl-α-cyclodextrin,
O-methyl-β-cyclodextrin, 6-O-methyl-γ-cyclodextrin, 2,6-di-O-methyl-
α-cyclodextrin, 2,6-di-O-methyl-β
-Cyclodextrin, 2,6-di-O-methyl-γ-
Cyclodextrin, 2,3,6-tri-O-methyl-
α-cyclodextrin, 2,6-di-O-ethyl-α
-Cyclodextrin, 2,3,6-tri-O-ethyl-α-cyclodextrin, and the corresponding β-
Cyclodextrin and γ-cyclodextrin, etc.
Or a hydroxyalkylated cyclodextrin, 2-hydroxyethyl-α-cyclodextrin, 2-hydroxypropyl-α-cyclodextrin, 3-hydroxypropyl-α-cyclodextrin, 2,3-dihydroxypropyl-α-cyclo Dextrin, 2,3,6-tri-O-acyl (C2-C1
8) -α-cyclodextrin, O-carboxylmethyl-O-ethyl-α-cyclodextrin, α-cyclodextrin sulfate, α-cyclodextrin phosphate, and the like, and β-cyclodextrin and γ-cyclodextrin corresponding thereto. (Kaneto Kamigama, Proceedings of the 12th Cyclodextrin Symposium, 1 (1993)).

Further, cyclodextrin oligomers and polymers having various molecular weights are commercially available, and these can be used.

(2) The number of CDs contained in the rotaxane type dye according to the present invention is not particularly limited. FIGS. 2 (a) to 2 (c) show examples of CDs which can be preferably used in the present invention. The embodiment of (a) in the figure shows that two or more CDs
1 shows an aspect in which a hole penetrates. (B) shows an embodiment in which two or more CDs are bonded. further,
The two or more CDs need not necessarily be of the same type.

When a plurality of CDs are included, an embodiment having a plurality of chain groups (further having dye molecules at both ends) is also possible, and an example thereof is shown in FIG. 2 (c).

(3) Cyclodextrin into which an active group has been introduced As the active group (reactive group or reactive functional group) for labeling a specific substance using the rotaxane type dye according to the present invention, CD Although a hydroxyl group having the same can be preferably used, the present invention further includes those in which a suitable active group is introduced into CD. Specifically, it is easy to convert one or more hydroxyl groups of the CD into a carboxyl group, a halogen group, an amino group, an isocyanate group, or the like.

It is also possible to use the reactive group bonded to one end of a chain group, and an example is shown in FIG. 1 (b).

2. Chain group (1) The chain group of the rotaxane type dye according to the present invention,
There is no particular limitation as long as it can bind a dye molecule to both ends and can penetrate the internal space of the CD. From the size of the CD to be used, the size of the internal space can be estimated based on a molecular model, molecular calculation, and the like, and it is easy to estimate the type and length of a usable chain group from the estimated size. Furthermore, it is preferred that the chain group or the portion other than the both end groups for binding the dye molecule is chemically stable, and the requirement makes it easy to select the type of the chain group. Specifically, methylene chain, polyether chain, polyamine chain, polyester chain, polyamide chain, etc.
Or a chain group containing two or more of them. In particular, it is preferable to use a polymethylene chain whose length can be easily adjusted, which is chemically stable, and whose molecular size is sufficiently small.

(2) The chain group described above is capable of binding a dye molecule to both ends thereof, and specifically, the following combinations are preferably selected. Functional group of dye molecule Chain-end bonding group -------------------------------------- ----------------------------- Carboxyl group Amino group, hydroxyl group Type of dye molecule bonded to both ends of chain-like group It is not necessary that they be the same.

Further, it is easy to select the length of the chain group according to the size of the internal space of the CD. In particular,
In the case of a methylene chain, it is preferably a hexamethylene chain or more for α, β, and γCD. Further, in the case of a chain group other than a methylene chain, the conversion into 1
The length can be selected so as to be equal to or longer than the hexamethylene chain (more preferably equal to or longer than the octamethylene chain) with respect to the penetration of one CD ring. Further, in an embodiment in which two or more CD rings penetrate, it is easy to select the length so as to be equal to or more than the length penetrating each CD ring.

3. Dye molecule The dye molecule used in the rotaxane-type dye according to the present invention is a dye selected from the group consisting of fluorescein-based, rhodamine-based, or Cy5, and labels a specific analyte with the rotaxane-type dye. The presence of the object is determined by measuring the fluorescence of the labeled object. Depending on the mode of using the present rotaxane-type dye, dye molecules having various properties can be selected. For example, various dye molecules used as tracers in vivo are included.

The rotaxane type dye according to the present invention is CD
, The water solubility is improved.

Examples of the fluorescein and rhodamine dyes according to the present invention include carboxyfluorescein (FAM) and carboxytetramethylrhodamine (TAMR).
A) can be mentioned.

In the case of a fluorescent dye molecule, the selection of the excitation wavelength and the fluorescence wavelength is not particularly limited. The same fluorescent dye molecule may be used to increase the fluorescence intensity. Further, when a plurality of different fluorescent dye molecules are used to utilize a plurality of fluorescent light, a combination of the fluorescent dye molecules may be appropriately selected so that the excitation wavelength and the fluorescent wavelength of each fluorescent dye molecule do not overlap. Easy. Furthermore, when utilizing the phenomenon of fluorescence energy transfer between different fluorescent dyes, they may be used as known energy donor fluorescent dye molecules and energy acceptor fluorescent dye molecules. By combining the same energy donor fluorescent dye molecule with a different energy acceptor fluorescent dye molecule, a polychromatic compound having a fluorescence wavelength with the same excitation wavelength is possible. 4. Synthesis Method The method for synthesizing the rotaxane type dye according to the present invention is not particularly limited, and any known organic chemical synthesis means used for synthesizing rotaxanes can be used. Generally, there is a method of (i) mixing each of CD, a dye molecule, and a chain group in an appropriate solvent to form a rotaxane structure, and at the same time, performing a bonding reaction between the dye molecule and the chain group. . According to this method, the solvent is a polar non-aqueous solvent, dimethyl sulfoxide.
(DMSO) and dimethylformamide (DMF) are preferably used. Further, it is easy to optimize conditions such as reaction temperature and time depending on the type of the binding reactive group. There is also a method of (ii) first mixing only CD and a chain group in an appropriate solvent to obtain or isolate an inclusion complex, and then reacting the obtained inclusion complex with a dye molecule. This method is particularly preferred for the purpose of synthesizing the dye according to the present invention having one type of dye.

Hereinafter, a more preferable method of the synthesis method (ii) will be described specifically. The conditions for the synthesis of the rotaxane with the chain group and CD are preferably performed using two phases of water and an organic solvent. Here, as the organic solvent, those which dissolve the chain group and sufficiently separate from water can be used, and examples thereof include ethyl ether. Dissolve (preferably saturate) the linear group in the organic solvent, and
Is dissolved in water (preferably saturated). Said 2
The two phases are stirred vigorously at a suitable temperature, preferably in the range of 30-50 ° C. By such agitation, the clathrate of the chain group and CD is mainly dissolved in the aqueous phase, and thus moves to the aqueous phase. In the aqueous phase, the mixture of the clathrate and CD is present. Become. In this case, when the solubility of the obtained clathrate in water is lower than that of CD itself, the clathrate may precipitate from the aqueous phase and partially precipitate. Particularly when the aqueous phase is saturated with CD, the precipitation often occurs. If necessary, the precipitate can be easily obtained by cooling the aqueous phase.

When the precipitate thus obtained contains substantially no CD other than the clathrate, it can be easily isolated by filtration. In this case, it is possible to remove the mixed chain group by washing the precipitate obtained with ether.

In addition, in addition to the clathrate, C
When D is mixed, appropriate separation means is required, and various chromatographic techniques can be used.
Specifically, a gel filtration method (GPC), which is a method of separating according to the size of a molecule, can be preferably used. Specifically, for the separation of the inclusion complex of α-CD and don and α-CD,
Sephadic G10 can be selected. As for GPC conditions, ordinary conditions can be preferably used. For monitoring the separation, TLC, HPLC, etc. can be used.
In order to minimize the equilibrium in which the chain group deviates from the CD ring,
It is preferable to use water as the elution solvent. By such a separation means, a chain group contained in a small amount is also removed.

A known organic chemical reaction can be generally used for binding a dye molecule to the obtained clathrate.
In this case, different dye molecules can be reactively bonded, and selection of such conditions is easy.

The monitoring of the reaction or the identification and confirmation of the reaction product can be performed by separating and purifying by a usual separation means and then using a usual organic chemical analysis technique. Specifically, monitoring of the progress of the reaction by TLC or HPLC, measurement of an infrared absorption spectrum, a nuclear magnetic resonance absorption spectrum, an absorption spectrum, a fluorescence spectrum, and the like, a mass spectrometric method, and the like can be used.

In order to confirm the presence in a solution, it is preferable to compare the retention times of peaks obtained by HPLC under specific conditions. Alternatively, comparison of nuclear magnetic resonance absorption spectrum, absorption spectrum, fluorescence spectrum, and the like is preferable.

In order to confirm the presence in the solid state, it is preferable to compare absorption peaks in a specific region (for example, 2000 to 500 cm ^-1 ) of the infrared absorption spectrum. Alternatively, comparison of molecular peaks by a mass spectrometry (for example, TOFMS) is preferable. FIG. 3 shows a method for synthesizing another basic structure of the dye of the present invention by reacting the chain group bonded with the dye with CD to form an inclusion body and then reacting the bond group. Indicated. The linking group can be easily selected based on the chemically reactive groups of various substances to be labeled. Specifically, when an amino group of an amino acid, an oligopeptide, a protein or the like can be used for labeling, a succinimidyl group or the like can be mentioned. When a thiol group can be used, a maleimide group, an alkyl halide group and the like (including iodoacetamide) can be mentioned. Other examples include a hydrazino group and a hydrazide group for the carboxyl group and the carbonyl group, and an active ester for the phenolic hydroxyl group.

FIG. 4 shows an example of introducing a reactive group into CD. A reactive group having a terminal carboxyl group can be introduced from a hydroxyl group via an amino group.

6 Labeling Method The rotaxane type dye according to the present invention can have a reactive group for labeling reaction introduced into any of cyclodextrin, a dye molecule, and a chain group. Therefore, it becomes possible to introduce various reactive groups for labeling reaction based on the functional groups of the compound to be labeled. Specific examples include a hydroxyl group of cyclodextrin, or a halide starting from the hydroxyl group, an amino group, an isocyanate group, and the like. Such a reactive group can be generally introduced by a known method. Further, various functional groups can be introduced into the dye molecule. Specifically, carboxyl group, amino group,
Examples include an isocyanate group and an active ester group. Such a reactive group can be generally introduced by a known method. In addition, various functional groups can be introduced into the chain group. Specific examples include a hydroxyl group, a halide, an amino group, a carboxyl group, an active ester group, and an isocyanate group. Such a reactive group can be generally introduced by a known organic synthesis method.

The rotaxane type dye according to the present invention into which the functional group described above has been introduced can be efficiently labeled under mild conditions in an aqueous solution. The selection of such reaction conditions depends on the labeling reaction, but can be easily optimized.

The substance to be labeled by the labeling method with the rotaxane type dye according to the present invention is not particularly limited, and various kinds of biological substances existing in an aqueous solution can be labeled. The substance to be labeled preferably has at least one functional group for reacting with the reagent according to the invention.
However, when the substance to be labeled does not have a functional group for reacting with the reagent according to the present invention, the functional group can be introduced by an appropriate pretreatment. For this purpose, it is easy to utilize generally known chemical reactions.
Furthermore, as described above, it is easy to select a binding group of the labeling reagent according to the present invention depending on the functional group of the substance to be labeled. Specifically, the substances to be labeled include amino acids, sugars, peptides, nucleic acids, proteins and the like. In such a case, the binding reaction with the labeling reagent reaction according to the present invention can be easily selected by a generally known organic chemical technique. For example, in the case of an amino acid or a peptide, an amino group can be used, and a coupling reaction can be easily performed by introducing an isocyanate or an active ester group into a dye molecule or cyclodextrin.

7. Detection method When the rotaxane-type dye according to the present invention is used as a labeling agent, the method for detecting the dye is not particularly limited, and an optimum detection method can be selected according to the dye molecule used. For example, measurement of a fluorescence spectrum and the like can be mentioned. In particular, when the dye molecules used are fluorescent, conventionally known high-sensitivity detection methods and specific detection methods can be applied as described in detail below.

(1) When the rotaxane-type dye according to the present invention has one type of fluorescent dye molecule The substance labeled with the dye according to the present invention emits fluorescence based on the fluorescent dye and detects this fluorescence. This makes it possible to detect the labeled substance. As described above, the dye according to the present invention can bind a plurality of one type of fluorescent dye. In such a case, the obtained fluorescence intensity can be increased by the number of fluorescent molecules, and the detection sensitivity can be improved.

(2) When the dye according to the present invention has two or more types of fluorescent dye molecules In this case, it is only possible to detect the fluorescence spectrum of each of the ordinary fluorescent dye molecules described above. Instead, a detection method based on various interactions between the fluorescent dye molecules becomes possible. For example, there is a method in which two types of fluorescent dye molecules are an energy donor fluorescent dye and an energy acceptor fluorescent dye, and detection is performed using a fluorescent energy transfer phenomenon between them. Further, a method is also possible in which two or more kinds of different fluorescent dye molecules are one energy donor fluorescent dye and another plurality of energy acceptor fluorescent dyes, and detection is performed by utilizing a fluorescent energy transfer phenomenon between them. In this case, excitation of a single energy donor fluorescent dye makes it possible to detect fluorescence emission from a plurality of energy acceptor fluorescent dyes.

[0051]

[0052]

Further, by using a labeling agent containing a plurality of rotaxane-type dyes according to the present invention, multicolor labeling becomes possible. That is, it is possible to separately label a plurality of labels (for example, a plurality of types of proteins).

For example, they have the same energy donor fluorescent dye (for example, dye 1 is common in FIG. 1) and different energy acceptor fluorescent dyes (for example, dyes 2, 3, 4, etc. in FIG. 1). By labeling a corresponding plurality of labeling substances with a plurality of fluorescent labeling agents having, it is possible to monitor each labeling substance present in a sample in which such labeling substances are mixed. is there. At this time, by irradiating the dye 1 with excitation light (single excitation light), the fluorescent wavelength of each energy acceptor fluorescent dye (that is, dyes 2, 3, and
(Fluorescence wavelengths such as 4) can be separately measured. From the above, specifically, it can be used as a dye for DNA sequencing. Utilizing the feature that one kind of excitation light can emit various wavelengths, it is used for DNA sequencing 4
Color fluorescent dyes can be synthesized. In addition, multiple stained cells can be detected efficiently. Whether excitation is performed at a single wavelength or at multiple wavelengths, the range of dye combinations that can be used is expanded by changing the donor dye and the acceptor dye. In addition, since the Stoke shift increases, a clear image with a low background can be expected. Application to a fluoroimmunoassay as an antibody labeling dye is also possible. By preparing an antibody to which a dye having a different fluorescence wavelength is bound according to the substance to be measured, a group of substances to be measured in a sample such as blood can be analyzed at a time. By measuring fluorescence at a single excitation wavelength with a photodiode or CCD at a time, highly sensitive analysis can be performed with an inexpensive device.

In this specification, carboxytetramethylrhodamine is TAMRA, carboxyfluorescein is FAM, and diaminododecane is don. In addition, (TAMRA) 2-don-CD rotaxane (or (TAMRA)-
don- (TAMRA) -CD rotaxane) is TAMRA
And the chain group is “don”, and has a rotaxane structure (FIG. 5 (a)) by cyclodextrin. (TAMRA) -don- (TAMRA) indicates that both fluorescent dyes are TAM
RA, which are linked by a chain group don (FIG. 5 (b)). Also, (FAM) 2-don-CD rotaxane (or
(FAM) -don- (FAM) -CD rotaxane)
M, the chain group is don, and it has a rotaxane structure (FIG. 6 (a)) by cyclodextrin. Further, (FAM) -don- (FAM) indicates that the fluorescent dyes are both FAM, and these are linked by a chain group don (FIG. 6).
(b)). (FAM) -don- (TAMRA) -CD rotaxane has a rotaxane structure with fluorescent dyes of TAMRA and FAM, a chain group of don, and cyclodextrin (FIG. 7 (a)).
It is assumed that (FAM) -don- (TAMRA)
Indicates that the fluorescent dyes are TAMRA and FAM,
It is assumed that they are linked by n (FIG. 7B).

Further, (FAM) -don- (Cy5) -CD rotaxane is
The fluorescent dyes are FAM and Cy5, the chain group is don, and the rotaxane structure is formed by cyclodextrin (Fig. 8 (a)).
It is assumed that (FAM) -don- (Cy5)
It is assumed that the fluorescent dyes are FAM and Cy5, and these are linked by a chain group don (FIG. 8 (b)).

(FAM) -don- (TAMRA) -CDcooh rotaxane is the (FAM) -don-CD- (TAMRA) rotaxane fluorescent dye.
It has a structure in which a reactive group is bonded to CD and has a carboxyl group (FIG. 9).

Hereinafter, the present invention will be described in more detail with reference to examples.

[0059]

EXAMPLES Example 1 Synthesis of (TAMRA) 2-don-CD Rotaxane (Part 1) 100 μl of a dimethyl sulfoxide (DMSO) solution saturated with α-cyclodextrin (hereinafter referred to as α-CD) was added to 3 mg (15 μmol) of 12-diaminododecane (don) was dissolved by stirring at 40 ° C. 5-carboxytetramethylrhodamine-succinimidyl ester (5-TAMRA, SE) dissolved in 50 μl DMF
25 mg (47 μmol) was added, and the mixture was stirred at 40 ° C. overnight. The reaction solution was analyzed by high performance liquid chromatography (hereinafter referred to as HPLC) under the following conditions, and it was confirmed that the retention time of 12.2 minutes was the target substance. Separation and purification were performed by preparative HPLC under the following conditions. After removing the solvent under reduced pressure from the fraction eluted with the target component (TAMRA) 2-don-CD rotaxane, the remaining aqueous solution was freeze-dried to obtain a purple powder (yield 8%). Analytical HPLC equipment and analysis conditions: Equipment: Tosoh HPLC system Detection: UV-visible detection made by Hitachi Ltd. L-7420 546nm Fluorescence detector L-7480 made by Hitachi Ltd. Excitation 546nm, emission 5
90 nm column: AsahipackODP-50 4E 4.6 mm x 250 mm Eluent: 50% water (10 mM ammonium acetate) / 50% methanol (10 mM ammonium acetate) to methanol (10 mM)
Ammonium acetate) 100% gradient Flow rate: 0.6 ml / min Mass spectrometry: Equipment: Shimadzu Laser Ionization Time-of-Flight Mass Spectrometer (MALDI-IV) (hereinafter abbreviated as TOF-MS, and used unless otherwise specified) Matrix: DHBA (gentisic acid) [M + 1] +: 2001 Absorption spectrum (methanol): 543 nm (FIG. 12) Fluorescence spectrum (methanol): excitation 543 nm, fluorescence 578 nm IR spectrum (KBr): FIG. 10 (Example 2) (TAMRA) ) Synthesis of 2-don-CD rotaxane (No. 2) (Synthesis and isolation of clathrate) Don was dissolved in diethyl ether at room temperature to prepare a saturated solution. Similarly, a saturated aqueous solution of α-CD was prepared. 0.5 ml of each saturated solution was placed in a sample tube, stoppered and stirred at 40-50 ° C overnight. After returning to room temperature, only the aqueous phase was separated with a pipette. A white precipitate was formed in the obtained aqueous phase, and the white precipitate was confirmed to be the target clathrate (don and αCD rotaxane) from the analysis results of IR spectrum and the like.

The aqueous phase after the above reaction was washed several times with ether in a sample tube to remove don, and as described below, an attempt was made to separate and purify the clathrate using a gel permeation column (GPC).

That is, the aqueous solution obtained above was applied to a Sephadex G10 GPC column described below, and eluted with water. The first eluted fraction contained the desired clathrate, but also contained α-CD. T
LC (normal phase silica gel, chloroform-methanol 10: 1)
The fractions substantially free of free don were collected while monitoring in step, and the obtained fractions were concentrated. (Synthesis of (TAMRA) 2-don-CD rotaxane) To the aqueous solution of the clathrate obtained above, 5 mg of 5-carboxytetramethylrhodamine succinimidyl ester (5-TAMRA, SE) and 1 ml of DMF solution were added. And stirred at room temperature overnight. The obtained reaction solution is
With the column using Chromatorex NH-DM3050, (TAMRA)
Three compounds, 2-don-CD rotaxane, (TAMRA) -don-CD and α-CD, were separated. The fraction components were concentrated under reduced pressure to remove acetonitrile, and the residue was lyophilized (yield 30%). Separation conditions for inclusion compound and non-inclusion CD: Column: Sephadex G10 10mmx70mm Monitor: TLC (silica gel, developing solvent chloroform:
The inclusion compound is near the origin in methanol 10: 1). (TAMRA) Separation conditions of 2-don-CD rotaxane: Column: Chromatorex NH-DM3050 20 mm x 250 mm (manufactured by Fuji Silysia Chemical) Eluent: 70% acetonitrile Monitor: TLC (HPTLC-NH-F254 manufactured by Merck, developing solvent 7)
Example 3 Synthesis of (TAMRA) -don- (TAMRA) 3 mg (15 μmol) of 1,12-diaminododecane (don) was dissolved in 100 μl of methanol by stirring at 40 ° C. 25 mg (47 μmol) of 5-carboxytetramethylrhodamine-succinimidyl ester (5-TAMRA, SE) dissolved in 50 μl of DMF was added, and the mixture was stirred at 40 ° C. overnight. The obtained reaction solution was separated by HPLC and purified. Target component (TAMRA) -don- (TAMRA)
After the solvent was removed from the fraction eluted under reduced pressure, the remaining aqueous solution was freeze-dried to obtain a purple powder. Analytical HPLC instruments and conditions: Equipment: Tosoh HPLC system Detection: Hitachi UV-visible detector L-7420 546nm Hitachi Fluorescence detector L-7480 Excitation 546nm, emission 590nm Column: AsahipackODP-50 4E 4.6mmx250mm Eluent: Water (10 mM ammonium acetate) 50% / methanol (10 mM ammonium acetate) 50% to methanol (10 mM ammonium acetate) 100% gradient Flow rate: 0.6 ml / min Mass spectrometry: Equipment: Shimadzu laser ionization time-of-flight mass spectrometer (MALDI) -IV) Matrix: DHBA (gentisic acid) [M + 1] +: 1028 Absorption spectrum (methanol): 543 nm (FIG. 12) Fluorescence spectrum (methanol): excitation 543 nm, fluorescence 578 nm IR spectrum (KBr): FIG. 11 (Example 4) ) Synthesis of FAM-don 84.5 mg (420 μmol) of 1,12-diaminododecane (don) was completely dissolved in 1.8 ml of methanol. 5-carboxyfluorescein-succinimidyl ester (5-FAM, SE) 10mg
(20 μmol) dissolved in 400 μl of DMF was added dropwise little by little, and after completion of the addition, the mixture was stirred at 40 ° C. overnight. Analyze the reaction solution by HPLC, confirm that 18.0 minutes is the target substance, and
Separation and purification were performed by HPLC. After removing the solvent under reduced pressure from the fraction eluted with the target component, (FAM) -don, the remaining aqueous solution was lyophilized to give an orange powder (7.6 mg). Analytical HPLC equipment and analytical conditions Equipment: Tosoh HPLC system Detection: Hitachi UV-visible detector L-7420 495nm Hitachi Fluorescence detector L-7480 Excitation 495nm, emission 520nm Column: AsahipackODP-50 4E 4.6mmx250mm Eluent: Water ( Gradient from 50% of 10 mM ammonium acetate) / 50% of methanol (10 mM ammonium acetate) to 100% of methanol (10 mM ammonium acetate) Flow rate: 0.6 ml / min Retention time: FAM-don 18.5 min Preparative HPLC instrument and preparative conditions: Instrument: Tosoh Detection: UV-visible detector UV-8020 495nm Column: AsahipackODP-90 21F 21.4mmx300mm Eluent: 45% water (10mM ammonium acetate) / 55% methanol (10mM ammonium acetate) to water (10mM ammonium acetate) 5 % / Methanol (10 mM ammonium acetate) 95% gradient of methanol (10 mM ammonium acetate) Flow rate: 5 ml / min Mass spectrometry: Matrix: DHBA (gentisic acid) [M + 1 +: 559 absorption spectrum (methanol): 499 nm (FIG. 17) Fluorescence spectrum (methanol): excitation 499 nm, fluorescence 525 nm IR spectrum (KBr): FIG. 13 (Example 5) (FAM) -don- (TAMRA) -CD rotaxane The obtained (FAM) -don7.6mg (13.6μmol) was dissolved in α-cyclodextrin (α-CD) saturated DMSO solution 1.4ml,
Stirred for days and included on CD. 5-carboxytetramethylrhodamine-succinimidyl ester (5-TAMRA, SE) 1
Add 0mg (18.9μmol) dissolved in 400μl DMF,
The mixture was further stirred for one day. The reaction liquid was analyzed by HPLC, and it was confirmed that 6.4 minutes was the target substance and the unreacted TAMRA. Therefore, the reaction liquid was separated and purified by preparative HPLC. Target substance, (FAM) -don
The fraction eluted with-(TAMRA) -CD rotaxane is concentrated, and the target substance and unreacted TAMRA are analyzed by HPLC using a gel filtration column.
And separated. It was confirmed that the retention time of 9.0 minutes was the target substance, and the fraction was purified by preparative HPLC. The solvent was removed from the eluted fraction under reduced pressure, and the remaining aqueous solution was freeze-dried to obtain a purple powder. From the fluorescence spectrum of the obtained substance (Fig. 1
6), maximum absorption wavelength of donor dye (fluorescein)
When excited at (499 nm), the fluorescence of the acceptor dye (rhodamine) was confirmed, indicating that intramolecular fluorescence energy transfer had occurred. Analytical HPLC instruments and conditions: Equipment: Tosoh HPLC system Detection: Hitachi UV-visible detector L-7420 495nm Hitachi Fluorescence detector L-7480 Excitation 495nm, emission 520nm Column: AsahipackODP-50 4E 4.6mmx250mm Eluent: water (10 mM ammonium acetate) 50% / methanol (10 mM ammonium acetate) 50% to methanol (10 mM ammonium acetate) 100% gradient Flow rate: 0.6 ml / min Retention time: (FAM) -don- (TAMRA) -CD rotaxane and TAMRA
6.4 minutes Instrument: Tosoh HPLC System Detection: Hitachi UV-visible detector L-7420 495nm Hitachi Fluorescence detector L-7480 Excitation 495nm, emission 580nm Column: YMC-pack Diol-60 Eluent: water (10mM ammonium acetate) 50 % / Methanol (10 mM ammonium acetate) 50% Flow rate: 1.0 ml / min Retention time: (FAM) -don- (TAMRA) -CD rotaxane 9.0 min Preparative HPLC instrument and conditions: Equipment: Tosoh HPLC System Detection: UV-visible detector UV-8020 546nm Column: AsahipackODP-90 21F 21.4mmx300mm Eluent: water (10mM ammonium acetate) 45% / methanol (10mM ammonium acetate) 55% to water (10mM ammonium acetate) 5% / methanol (10mM acetic acid) Ammonium) 95%
Gradient Flow rate: 5 ml / min Instrument: Tosoh HPLC system Detection: UV-visible detector UV-8020 546 nm Column: YMC-Pack Diol-60 Eluent: water (10 mM ammonium acetate) 50% / methanol (10 mM ammonium acetate) 50% Flow rate: 6 ml / min Mass spectrometry: Matrix: DHBA (gentisic acid) [M + 1] +: 1943 Absorption spectrum (methanol): 499 nm, 548 nm (FIG. 1)
7) Fluorescence spectrum (methanol): excitation 499 nm, fluorescence 573 nm
(FIG. 16) IR spectrum (KBr): FIG. 14 (Example 6) Synthesis of (FAM) -don- (TAMRA) 7.6 mg (13.6 μmol) of (FAM) -don obtained was methanol 400
Dissolve in μl and add 5-carboxytetramethylrhodamine-
10 mg of succinimidyl ester (5-TAMRA, SE) (18.9 μm
ol) dissolved in 400 μl of DMSO, and the mixture was stirred at 40 ° C. for 1 day. The reaction solution was analyzed by HPLC, and it was confirmed that 24.5 minutes was the target substance, and fractionated by preparative HPLC. Target substance
The solvent was removed from the fraction eluted with (FAM) -don- (TAMRA) under reduced pressure, and the remaining aqueous solution was lyophilized to obtain a purple powder. Analytical HPLC equipment and analytical conditions Equipment: Tosoh HPLC system Detection: Hitachi UV-visible detector L-7420 495nm Hitachi Fluorescence detector L-7480 Excitation 495nm, emission 580nm Column: AsahipackODP-50 4E 4.6mmx250mm Eluent: Water ( Gradient from 50% of 10 mM ammonium acetate) / 50% of methanol (10 mM ammonium acetate) to 100% of methanol (10 mM ammonium acetate) Flow rate: 0.6 ml / min Retention time: (FAM) -don- (TAMRA) 24.5 minutes Mass spectrometry: Matrix : DHBA (gentisic acid) [M + 1] +: 970 Absorption spectrum (methanol): 500 nm, 542 nm (FIG. 1)
7) Emission spectrum (methanol): excitation 500nm, emission 569nm
(FIG. 16) IR spectrum (KBr): FIG. 15 (Example 7) Labeling reaction of phenethylamine 100 pmol of (TAMRA) 2-don-CD rotaxane was converted to methanol 20 μm.
1 M phenethylamine methanol solution 25 μl
In addition, 50 μg of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC) and dimethylaminopyridine 500
The mixture was stirred at 40 ° C. for 1 day with μg. The reaction solution was analyzed by HPLC, and it was confirmed that 21.5 minutes was phenethylamine labeled with a rotaxane dye. Analytical HPLC equipment and conditions Equipment: Tosoh HPLC system Detection: Hitachi, Ltd. UV-visible detector L-7420 546nm Hitachi, Ltd. Fluorescence detector L-7480 Excitation 546nm, emission 600nm Column: AsahipackODP-50 4E 4.6mmx250mm Eluent: Water ( Gradient from 50% of 10 mM ammonium acetate / 50% of methanol (10 mM ammonium acetate) to 100% of methanol (10 mM ammonium acetate) Flow rate: 0.6 ml / min Mass spectrometry: Matrix: DHBA (gentisic acid) [M + 1] +: 2205 (implementation Example 8) Synthesis of (FAM) -don- (Cy5) -CD rotaxane (FAM) -don 7.6 mg (13.6 μmol) was dissolved in α-cyclodextrin (α-CD) saturated aqueous solution (400 μl) at 40 ° C. for 2 days Stir and cover with CD. Cy5-OSu (Amersham) 5
mg (6.6 μmol) dissolved in 200 μl of DMF is added,
The mixture was further stirred for one day. The reaction solution was fractionated on a gel filtration column according to molecular weight. The component eluted first is (FAM) -don- (Cy
5) Since it was a mixture of -CD rotaxane and (FAM) -don- (Cy5), the two components were further separated by a reversed-phase column. 5.3
Min, 20.6 / 2 peaks appeared, 5.3 min was (FAM) -don- (Cy5)-
The CD rotaxane, 20.6 minutes, was (FAM) -don- (Cy5). 5.3
The components were fractionated and lyophilized. Analytical HPLC equipment and conditions Equipment: Tosoh HPLC system Detection: Hitachi UV-visible detector L-7420 495nm Hitachi Fluorescence detector L-7480 Excitation 495nm, emission 670nm Column: AsahipackODP-50 4E 4.6mmx250mm Eluent: Water ( Gradient from 50% of 10mM ammonium acetate) / 50% of methanol (10mM ammonium acetate) to 100% of methanol (10mM ammonium acetate) Flow rate: 0.6ml / min Mass spectrometry: Matrix DHBA absorption / fluorescence spectrum measurement 50% in MeOH Excitation wavelength 495nm IR Spectrum analysis (film method) IR Cards Type 61 Polyethylene 19mm Aperture (3M) Analysis result: HPLC: retention time 5.3 minutes TOF-MS: [M + 1] +: 2175 Absorption spectrum: Fig. 20 Fluorescence spectrum: Fig. 21 IR Spectra: FIG. 18 (Example 9) Synthesis of (FAM) -don- (Cy5) 7.6 mg (13.6 μmol) of (FAM) -don was dissolved in 400 μl of DMF, stirred at 40 ° C. for 2 days, and Cy5-OSu (Amersham) 5m
g (6.6 μmol) dissolved in 200 μl of DMF was added, and the mixture was further stirred for 1 day. The reaction solution was fractionated on a gel filtration column according to molecular weight. The component eluted first is (FAM) -don- (Cy5)-
Since it was a mixture of CD rotaxane and (FAM) -don- (Cy5), the two components were further separated on a reversed-phase column. 5.3
Min, 20.6 / 2 peaks appeared, 5.3 min was (FAM) -don- (Cy5)-
The CD rotaxane, 20.6 minutes, was (FAM) -don- (Cy5). 20.
The 6-minute components were fractionated and lyophilized. Analytical HPLC equipment and conditions Equipment: Tosoh HPLC system Detection: Hitachi UV-visible detector L-7420 495nm Hitachi Fluorescence detector L-7480 Excitation 495nm, emission 670nm Column: AsahipackODP-50 4E 4.6mmx250mm Eluent: Water ( Gradient from 50% of 10mM ammonium acetate) / 50% of methanol (10mM ammonium acetate) to 100% of methanol (10mM ammonium acetate) Flow rate: 0.6ml / min Mass spectrometry: Matrix DHBA absorption / fluorescence spectrum measurement 50% in MeOH Excitation wavelength 495nm IR Spectral analysis (film method) IR Cards Type 61 Polyethylene 19mm Aperture (manufactured by 3M) Analysis result: HPLC: retention time 20.6 minutes Mass spectrum: [M + 1] +: 1199 Absorption spectrum: Fig. 20 Fluorescence spectrum: Fig. 21 IR spectrum : FIG. 19 (Example 10) Synthesis of Ts-α-CD Under a nitrogen atmosphere, take 500 ml of pyridine and 62 g (66 mmol) of α-CD, and p-toluenes with stirring at room temperature. In several portions acid hydrochloride 39.4 g (210 mmol), was as it was stirred overnight at room temperature, the solvent was distilled off under reduced pressure to give a syrup of the crude reaction product. The crude reaction product showed four peaks by HPLC analysis, the first peak corresponding to excess p-toluenesulfonic acid and the last peak to unreacted α-CD. This crude reaction product was purified by preparative HPLC. After removing acetonitrile under reduced pressure from the fraction in which the component corresponding to peak 3 corresponding to the main product eluted, the remaining aqueous solution was lyophilized and mono- (6-tosyl-6-deoxy) -α-cyclodextrin (Ts
-α-CD) as a white powder (13.8 g, yield 27%). Analytical HPLC: Apparatus: Shimadzu LC-64 Detection: Differential refraction detector RID-64 Column: Kaseisorb LC-NH2 Super, 6mmφ × 250mm Eluent: 60% acetonitrile Flow rate: 1.0 ml / min Preparative HPLC: Column: 40 mmφ × 1000 mm Filler: Fuji Chemical Co., Ltd. NH-DU 3050 Eluent: 60% acetonitrile Flow rate: 30 ml / min (Example 11) Synthesis of N3-α-CD Ts-α-CD 0.34 g (0.45 mmol), 0.32 g of sodium azide
(4.9 mmol) was dissolved in 10 ml of water, heated to 80 ° C. with stirring, and stirred for 4 hours. After confirming that the spot of Ts-α-CD as a raw material had disappeared, the reaction was terminated. The solvent was distilled off, and N3-α-CD (0.06 g, yield 16%) was obtained by acetone precipitation. TLC analysis conditions TLC: MERCK.HPTLC-Fertigplatten NH2 Solvent: 60% acetonitrile Detection: diphenylamine / aniline / phosphoric acid / acetone
2: 2: 15: 100 Rf value: 0.49 (Example 12) Synthesis of NH2-α-CD 0.50 g (0.52 mmol) of N3-α-CD was dissolved in 20 ml of water, 40 mg of palladium carbon was added, and hydrogen was added at room temperature. Gas was vented for 3 hours. The reaction solution became positive by ninhydrin color development. The catalyst was removed by filtration under reduced pressure, the filtrate was concentrated under reduced pressure, and 0.41 g of NH2-α-CD (80.8% yield) was obtained by acetone precipitation. TLC analysis conditions TLC: MERCK.HPTLC-Fertigplatten NH2 Solvent: 60% acetonitrile Detection: diphenylamine / aniline / phosphoric acid / acetone
2: 2: 15: 100 Rf value: 0.43 TLC: MERCK. Kieselgel 60 F254 Detection: ninhydrin coloring Mass spectrometry conditions Matrix: DHBA (Gentisic acid) [M-1 + Na] +: 994 (Example 13) Glutaric acid monohydrate Synthesis of t-butyl ester 5.0 g of glutaric anhydride (44 mmo) in 70 ml of dichloromethane
l), 5.4 g (44 mmol) of dimethylaminopyridine are dissolved, and 3.3 g of t-butyl alcohol (44 mmo) is cooled to 0 ° C.
l) was added. After stirring at room temperature overnight, most of the methylene chloride was recovered and 100 ml of water was added. The aqueous phase was acidified with citric acid and extracted with ethyl acetate. The organic phase was washed with brine and dried over anhydrous sodium sulfate, and the solvent was distilled off to obtain 3.3 g of t-butyl ester. (Yield: 40
(Example 14) Synthesis of succinimidyl carbonate mono-t-butylglutarate 3.2 g (17 mmol) of monot-butyl glutarate, 2.9 g (25 mmol) of N-hydroxysuccinic acid, 0.2 g of dimethylaminopyridine (1.7 mmol), N-ethyl-N-yu (N, N-dimethyl-3-aminopropyl) -carbodiimide hydrochloride 4.9 g
(26 mmol) was dissolved in 80 ml of dichloromethane and stirred at room temperature overnight. The usual post-treatment was carried out to obtain 2.2 g of the objective white crystals. (Yield: 46%) (Example 15) α-cyclodextrin glutaramide (C
2.17 g (2 mmol) of NH2-α-CD dissolved in 40 ml of DMF was added to 0.68 g (2.4 mmo) of t-butyl glutarate-succinimidyl ester
l) was added and stirred at room temperature for 2 days. The reaction solution is
% Acetonitrile as an eluent, purified by column chromatography on aminopropylated silica gel.
0.31 g was obtained. This was dissolved in 1N hydrochloric acid (5 ml), stirred at room temperature overnight, and then freeze-dried to obtain the desired product (105 mg). (Yield: 1
1.3%) Preparative conditions: Column: 40mmφ × 1000mm Filler: Fuji Chemical Co., Ltd. NH-DU 3050 Eluent: 60% acetonitrile (Example 16) Synthesis of (FAM) -don- (TAMRA) -CDcooh rotaxane ( FAM) -don 7.8mg (13.6μmol) and CDcooh 0.15g (70μmo
l) was stirred overnight at room temperature in 1.2 ml of pH 9.0 phosphate buffer. A solution prepared by dissolving 5 mg (6.6 μmol) of 5-TAMRA, SE in 200 μl of DMF was added, and the mixture was further stirred for 1 day. The reaction solution was fractionated on a gel filtration column according to molecular weight. Ma that the first eluted component is (FAM) -don- (TAMRA) -CDcooh rotaxane
It was confirmed from ss spectrum analysis. The solvent of the eluted component was distilled off and dried to obtain a red powder. Analytical HPLC instruments and conditions Equipment: Tosoh HPLC system Detection: Hitachi UV-visible detector L-7420 495nm Hitachi Fluorescence detector L-7480 Excitation 495nm, emission 580nm Column: AsahipackODP-50 4E 4.6mmx250mm Eluent: Water ( Gradient from 50% of 10 mM ammonium acetate) / 50% of methanol (10 mM ammonium acetate) to 100% of methanol (10 mM ammonium acetate) Flow rate: 0.6 ml / min Mass spectrometry: Matrix DHBA IR spectrum analysis (film method): IR Cards Type 61 P
olyethylene 19mm Aperture (3M) Analysis result: HPLC: retention time 5.3 minutes Mass spectrum: [M + 1] +: 2059 IR spectrum: FIG. 22 (Example 17) Labeling of phenethylamine (FAM) -don- (TAMRA) ) -CDcooh rotaxane 3.0 nmol DMF 90
dissolved in 10 μl of 6 mM phenethylamine DMF solution
(30 nmol: 10 times the amount of the dye) and 6 μl of a 10 mM dicyclohexylcarbodiimide DMF solution were added, and the mixture was stirred at 40 ° C. for 2 days in a dark place. From HPLC, a new peak appeared at 39.3 minutes, and the mass spectrum confirmed that the product was rotaxane-labeled phenethylamine. Analytical HPLC equipment and conditions: Equipment: Tosoh HPLC system Detection: Hitachi UV-visible detector L-7424 546nm Hitachi Fluorescence detector L-7480 Excitation 495nm Emission 580nm Column: Asahipak ODP-50 4E 4.6mm × 250mm Eluent : Water (10 mM ammonium acetate) 20% / methanol (10 mM ammonium acetate) 80% to methanol (10 mM ammonium acetate) 100% gradient Flow rate: 0.6 ml / min Mass spectrometry: matrix DHBA Analysis result: HPLC: retention time 39.3 min TOF- MS: [M] +: 2062 (Example 18) Measurement of fluorescence lifetime of each dye The fluorescence lifetime of FAM used alone as a donor is 9.4 ns, which is the lifetime when energy transfer is not involved. In systems where acceptor dyes are present, B-1 and B-2
The fluorescence lifetime of FAM was about 0.3 ns, and that of C-1 and C-2 was about 0.2 ns, indicating that energy transfer occurred at an efficiency of 95% or more in each case. This can be confirmed from the presence of the rise of the acceptor dye (indicated by (r) in the table). The two components of the acceptor dye are because there are a rising component for excitation by energy transfer and a fluorescence lifetime component of the acceptor dye itself.

Even when A-1 and A-2, B-1 and B-2, and C-1 and C-2 are compared, no problem such as quenching of the acceptor dye due to rotaxane formation is observed. Therefore, by rotaxane-forming, it is possible to obtain a dye having high water solubility and capable of multicoloring without giving a problem to fluorescence emission. ==================== Fluorescence Lifetime (ns) ------------------------- ----------------------------------- FAM-don 9.4 TAMRA-don 5.1 A-1 2.7 A-2 4.2 B-1 (FAM) 0.29 3.1 (TAMRA) 0.07 (r) 3.0 B-2 (FAM) 0.28 2.8 (TAMRA) 0.08 (r) 3.5 C-1 (FAM) 0.21 3.9 (Cy5) 0.14 (r) 2.3 C -2 (FAM) 0.2 3.6 (Cy5) 0.40 (r) 2.4 ==================== (Example 19) Synthesis of TAMRA-don 1,12-diamino 84.5 mg (420 μmol) of dodecane was completely dissolved in 1.8 ml of methanol. 5-carboxytetramethylrhodamine-succinimidyl ester (5-TA
A solution of 10 mg (20 μmol) of MRA, SE in 400 μl of DMF was added dropwise little by little. After the addition, the mixture was stirred at 40 ° C. overnight. The reaction solution was analyzed by HPLC, and it was confirmed that 23.3 minutes was the target substance, and fractionation and purification were performed by preparative HPLC. Target ingredient, TAMR
After removing the solvent from the fraction eluted with A-don under reduced pressure, the remaining aqueous solution was freeze-dried and dried to obtain 8.4 mg of red powder (yield 60%).
%). Analytical HPLC equipment and conditions Equipment: Tosoh HPLC system Detection: Hitachi, Ltd. UV-visible detector L-7420 546nm Hitachi, Ltd. Fluorescence detector L-7480 Excitation 546nm, emission 630nm Column: AsahipackODP-50 4E 4.6mmx250mm Eluent: Water ( Gradient from 50% of 10 mM ammonium acetate) / 50% of methanol (10 mM ammonium acetate) to 100% of methanol (10 mM ammonium acetate) Flow rate: 0.6 ml / min Mass spectrometry condition Sample: Prepare 10 μM 50% methanol solution Matrix: DHBA Analysis result: HPLC: retention time 23.3 minutes TOF-MS: [M + 1] +: 613 (Example 20) Synthesis of TAMRA-don-CD-ML (maleimide) 0.6 ml of 0.05 M αCD aqueous solution, 2.4 ml of 0.05 M αCD DMF solution 10.9 mg of TAMRA-don obtained by the above method (1
7.8 μmol) and stirred in the dark at room temperature for 2 days to allow TAMRA-don to be included in the CD. After inclusion, DMF 200μl
A solution prepared by dissolving 50 mg (178 μmol) of GMBS (N- (4-maleimidobutyryloxy) succinimide) (manufactured by Dojin Kagaku) is added to the mixture, and the mixture is further stirred at room temperature for 1 day. The low molecular weight unreacted substances were removed by HPLC, and lyophilized. Analytical HPLC equipment and conditions Equipment: Tosoh HPLC System Detection: Hitachi, Ltd. UV-visible detector L-7420 546nm Hitachi, Ltd. Fluorescence detector L-7480 Excitation 546nm, emission 630nm Column: YMC-Pack Diol-60 Eluent: water ( 10 mM ammonium acetate) 50% / methanol (10 mM ammonium acetate) 50% Flow rate: 1.0 ml / min Separation result: Retention time: 13.8 minutes (Example 21) Labeling of GSH by TAMRA-don-CD-ML As SH compound, Reducing glutathione (GSH), a low-molecular peptide, was selected and labeled (GSH: Glu-
Cys-Gly) Dissolve 50 nmol of GSH in 25 μl of pH 7.5 phosphate buffer, and add TA
A solution of 100 nmol of MRA-don-CD-ML in 100 μl of 20% acetonitrile was added to the above solution. The mixture was stirred overnight at room temperature in the dark. From HPLC analysis, a new peak appeared at 5.7 minutes, and Mass spectrum analysis of the component confirmed that the component was GSH labeled with a dye. Analytical HPLC equipment and conditions Equipment: Tosoh HPLC system Detection: Hitachi UV-visible detector L-7420 546nm Hitachi Fluorescence detector L-7480 Excitation 546nm, emission 630nm Column: AsahipackODP-50 4E 4.6mmx250mm Eluent: 40% Gradient from acetonitrile (0.1% TFA) to 80% acetonitrile (0.1% TFA) Flow rate: 0.6 ml / min Mass spectrometry condition Sample: Adjust 10 μM 50% acetonitrile solution Matrix: CHCA Analysis result: HPLC: retention time 5.7 min TOF-MS : [M] +: 2059 (Example 22) Bioactive peptide (Gly-Cys-Glu-Tyr-Ty)
r-Lys-Lys) labeled peptide (150 nmol) is dissolved in pH 7.5 phosphate buffer (25 μl), and TAMRA-don-CD-ML (75 μmol)
A solution dissolved in 125 μl of 20% acetonitrile was added to the above solution. The mixture was stirred overnight at room temperature in the dark. From the HPLC analysis, a new peak appeared at 6.6 minutes.
Ass spectrum analysis confirmed that the peptide was a dye-labeled peptide. Analytical HPLC equipment and conditions Equipment: Tosoh HPLC system Detection: Hitachi UV-visible detector L-7420 546nm Hitachi fluorescent detector L-7480 Excitation 546nm, emission 630nm Column: AsahipackODP-50 4E 4.6mmx250mm Eluent: 20% Gradient from acetonitrile (0.1% TFA) to 80% acetonitrile (0.1% TFA) Flow rate: 0.6 ml / min Mass spectrometry conditions Sample: Adjust 10 μM 50% acetonitrile solution Matrix: CHCA Analysis result: HPLC: retention time 6.6 minutes TOF-MS : [M + Matrix] +: 2830 (Example 23) Bioactive peptide (Gly-Cys-Asp-Arg-Va)
l-Tyr-Ile-His-Pro-Phe (C-Ang)) Labeling Dissolve 250 nmol of peptide in 25 μl of pH 7.5 phosphate buffer, and add 125 μmol of TAMRA-don-CD-ML to 20% acetonitrile 12
The solution dissolved in 5 μl was added to the above solution. In a dark place,
Stirred overnight at room temperature. From HPLC analysis, a new peak appeared at 6.0 minutes, and the mass spectrum analysis of the component confirmed that the peptide was a dye-labeled peptide. Analytical HPLC equipment and conditions Equipment: Tosoh HPLC system Detection: Hitachi UV-visible detector L-7420 546nm Hitachi Fluorescence detector L-7480 Excitation 546nm, emission 630nm Column: AsahipackODP-50 4E 4.6mmx250mm Eluent: 40% Gradient from acetonitrile (0.1% TFA) to 80% acetonitrile (0.1% TFA) Flow rate: 0.6 ml / min Mass spectrometry conditions Sample: Adjust 10 μM 50% acetonitrile solution Matrix: CHCA Analysis result: HPLC: Retention time 6.0 min TOF-MS : [M + Matrix] +: 3151 (Example 24) Labeling of BSA Bovine serum albumin (BSA), which is a high molecular protein, was labeled. One BSA molecule combined with one dye molecule
Confirmed by s spectrum and the like.

50 nmol of TAMRA-don-CD-ML was dissolved in a mixed solution of 400 μl of a pH 7.5 phosphate buffer and 50 μl of DMF, and 1% BSA
99 μl of aqueous solution (pH 7.5 phosphate buffer + 5 mM EDTA)
(30 volumes) and left at 4 ° C. overnight. Sephadex G-25
As a result, a high molecular weight component and a low molecular weight component (unbound dye) were separated. HPLC analysis of the fractionated high molecular weight component showed a peak at 7.8 minutes, and mass spectrometry analysis of the component indicated that it was consistent with the molecular weight of the dye-labeled BSA. Analytical HPLC equipment and conditions Equipment: Tosoh HPLC system Detection: Hitachi UV-visible detector L-7420 546nm Hitachi Fluorescence detector L-7480 Excitation 546nm, emission 630nm Column: TSKgel G3000SW _XL Eluent: 50mM phosphate buffer +0.3 M NaCl flow rate: 1.0 ml / min Mass spectrometry conditions Matrix: SA (Sinapinic acid) Analysis result: HPLC: retention time 8.6 min TOF-MS: [M] +: 67315

[0064]

[Sequence List] SEQUENCE LISTING <110> LAboratory of Molecular Biophotonics <120> A Dye having a rotaxane structure, labeling a
gent using the dye, and a method for labeling <160> 1 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220><223> Biologically active peptide <400> 1

[0065]

The rotaxane-type dye according to the present invention has excellent water solubility and is excellent in water solubility because it has a rotaxane structure in which a dye molecule is bonded to both terminal portions of a chain group penetrating a cyclodextrin ring. It is possible to have a plurality of dyes.

Further, various substances can be labeled under aqueous conditions by various labeling reaction bonding groups contained in the dye molecule, CD, or chain group of the rotaxane type dye of the present invention. It becomes possible.

[Brief description of the drawings]

FIG. 1 is a diagram schematically showing two basic structures ((a) and (b)) of a rotaxane-type dye according to the present invention.

2 (a) to 2 (c) are views showing some structures of the rotaxane type according to the present invention.

FIG. 3 is a diagram showing an example of a case where one basic structure of the rotaxane type dye according to the present invention is synthesized.

FIG. 4 is a diagram showing an example of a case where a reactive group is introduced into one CD of the rotaxane-type dye according to the present invention.

FIG. 5 (a) is a view showing the structure of (TAMRA) -don- (TAMRA) -CD rotaxane shown in Examples of the rotaxane type dye according to the present invention. FIG. 5 (b) is a diagram showing the structure of (TAMRA) -don- (TAMRA) shown in Examples of the rotaxane type dye according to the present invention.

FIG. 6 (a) shows the rotaxane type dye according to the present invention.
It is a figure which shows the structure of (FAM) -don- (FAM) -CD rotaxane.
FIG. 6 (b) shows (FAM) -don of the rotaxane type dye according to the present invention.
It is a figure which shows the structure of-(FAM).

FIG. 7 (a) is a diagram showing the structure of (FAM) -don- (TAMRA) -CD rotaxane shown in Examples of the rotaxane type dye according to the present invention. FIG. 7 (b) is a diagram showing the structure of (FAM) -don- (TAMRA) shown in Examples of the rotaxane type dye according to the present invention.

FIG. 8 (a) is a diagram showing the structure of (FAM) -don- (Cy5) -CD rotaxane shown in Examples of the rotaxane type dye according to the present invention. FIG. 8 (b) is a diagram showing the structure of (FAM) -don- (Cy5) shown in Examples of the rotaxane type dye according to the present invention.

FIG. 9 is a diagram showing the structure of (FAM) -don- (TAMRA) -CDcooh rotaxane shown in Examples of the rotaxane type dye according to the present invention.

FIG. 10 is a diagram showing infrared absorption spectra of (TAMRA) -don- (TAMRA) -CD rotaxane and α-CD.

FIG. 11 is a diagram showing an infrared absorption spectrum of (TAMRA) -don- (TAMRA).

FIG. 12 is a diagram showing absorption spectra of (TAMRA) -don- (TAMRA) -CD rotaxane and (TAMRA) -don- (TAMRA). (Concentration 1.0 × 10 ^-6 M)

FIG. 13 is a view showing an infrared absorption spectrum of (FAM) -don.

FIG. 14 is a diagram showing infrared absorption spectra of (FAM) -don- (TAMRA) -CD rotaxane and α-CD.

FIG. 15 is a diagram showing an infrared absorption spectrum of (FAM) -don- (TAMRA).

FIG. 16 shows (FAM) -don- at 499 nm excitation.
It is a figure which shows the fluorescence spectrum of (TAMRA) -CD rotaxane, (FAM) -don- (TAMRA) (concentration 1.0x10 < ^-7 > M).

FIG. 17 shows (FAM) -don- (TAMRA) CD-rotaxane, (FAM) -don- (TAMRA), and (FAM) -don, (TAMRA) -don
FIG. 3 is a view showing an absorption spectrum of the compound in methanol: water (1: 1).

FIG. 18 is a diagram showing an infrared absorption spectrum of (FAM) -don- (Cy5) -CD rotaxane.

FIG. 19 is a diagram showing an infrared absorption spectrum of (FAM) -don- (Cy5).

FIG. 20 shows (FAM) -don- (Cy5) -CD rotaxane,
It is a figure which shows the absorption spectrum in methanol: water (1: 1) of (FAM) -don- (Cy5). (Concentration 1.0 × 10 ^-6 M)

FIG. 21 shows (FAM) -don- (Cy5) -CD rotaxane,
It is a figure which shows the fluorescence spectrum of (FAM) -don- (Cy5) in methanol: water (1: 1).

FIG. 22 is a diagram showing an infrared absorption spectrum of (FAM) -don- (TAMRA) -CDcooh rotaxane.

────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-9-3011893 (JP, A) JP-A-9-216815 (JP, A) Tokuyohei 4-500229 (JP, A) International Publication 98/26287 (WO, A1) Bull. Chem. Soc. Jpn. , 66 [11] (1993), 3382-3386 Bull. Chem. Soc. Jpn. , 57 [6] (1984), 1566-1603 Bull. Chem. Soc. Jpn. , 56 [8] (1983), 2283-2289. Am. Chem. Soc. , 103 [5] (1981), 1303-1304 CHEMISTRY LETTER S, [6] (1993), 1033-1036 Angew. Chem. Int E d. Engl. , 36 [12] (1997), 1310-1313 (58) Fields investigated (Int. Cl. ⁷ , DB name) CA (STN) CAOLD (STN) REGISTRY (STN) JICST file (JOIS)

Claims (4)

(57) [Claims] 1. A rotaxane type dye represented by the following formula 1. Embedded image (Where FL1 and FL2 may be the same or different and represent a dye selected from the group consisting of fluorescein, rhodamine or Cy5, n represents an integer of 8 to 12, and m represents an integer of 6 to 8 X is OH, Cl, Br, I, NH
2, represents any of NCO and NHCO (CH2) 3CO2H. ) 2. A rotaxane type dye represented by the following formula 2: (Where FL3 represents a dye selected from the group consisting of fluorescein, rhodamine or Cy5, and k is 6 to 8
And 1 represents an integer of 8 to 12. ) 3. A labeling agent characterized by using the rotaxane type dye according to claim 1. 4. A labeling method using the labeling agent according to claim 3.