JP2000290278A - Double-stranded dna fragment having electroconductivity and water-soluble fullerene derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new water-soluble fullerene derivative having water solubility and capable of imparting electroconductivity to a double-stranded DNA fragment. SOLUTION: This water-soluble fullerene derivative is represented by formula I (R1 and R3 are each a 2-10C heterocyclic group or the like containing 1-4 heteroatoms selected from N, S and P; R2 is H, a 2-7C acyl or the like; X is a counteranion of a cationic group), e.g. a compound represented by formula II. The derivative represented by formula I can be obtained in good yield by carrying out an alkylation, etc., of a precursor of the water-soluble fullerene derivative, e.g. N-methyl-2-(4'-N-pyridyl)-3,4-fulleropyridine with an alkyl triflate, etc., in a solvent such as chlorobenzene or fluorobenzene. The water-soluble fullerene derivative represented by formula I can be brought into contact with a double-stranded DNA fragment to thereby afford the double-stranded DNA fragment having electroconductivity. The resultant double-stranded DNA fragment can be utilized for quantitative determination of a substrate such as glutathione or a cytochrome.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a double-stranded DNA fragment imparted with conductivity, and a novel water-soluble fullerene derivative.

[0002]

2. Description of the Related Art In gene analysis in the fields of biology, medicine and the like, a hybridization method is generally used as a method for detecting DNA having a specific sequence. The detection is performed by a fluorescence method, a luminescence method, an RI (radioisotope) method, an electrochemical method, or the like. The electrochemical method is superior to a fluorescent method or the like in that it does not require a dedicated measuring device for measurement and does not cause fading due to excitation light.

[0003] As an electrochemical method, an electrochemically active embroidery type intercalator (an intercalator generally means a molecule inserted in parallel between DNA base pairs), which is one of the electrochemically active molecules, is used. In general, a method of contacting DNA and measuring the electrochemical characteristics of a complex in which the intercalator has bound to the DNA is used. For example, JP-A-9
Japanese Patent No. 288080 discloses an electrode provided with an output terminal, in which an electrode on which probe DNA is immobilized and a sample DNA are reacted in the presence of a ferrocene naphthalenediimide derivative to form the probe DNA and the sample DNA. By measuring the amount of current flowing through the naphthalenediimide derivative bound to the hybrid DNA,
A method for detecting the complementarity of A is disclosed.

In a complex of a ferrocene-modified naphthalenediimide derivative and a hybrid DNA, the two ferrocene portions of the derivative are closely aligned in the main groove and the minor groove of the hybrid DNA, respectively. It can be considered as a pseudopolyferrocene polymer (a pseudopolymer of ferrocene molecules) with a matrix as a support. When a potential is applied to the polyferrocene polymer by any electrochemical method, the transfer of electrons between divalent iron ions and trivalent iron ions results in the polyferrocene polymer Changes to ferrocene polymer. That is, the hybrid DNA has a conductive D
It can be considered that it has changed to NA.

[0005] However, it is difficult to impart conductivity to the DNA by bringing the electrochemically active molecule into contact with hybrid DNA, DNA obtained from a biological sample, or the like to form a complex of the DNA and the electrochemically active molecule. Does not necessarily require the use of a threaded intercalator such as a ferrocene-modified naphthalenediimide derivative as an electrochemically active molecule. As long as it has a property of binding to DNA, various electrochemically active molecules (hereinafter, molecules having such properties are referred to as “DNA-binding electrochemically active molecules”) are considered to be usable. It is important to develop a novel DNA-binding electrochemically active molecule that is superior in stability, cost, and the like. In addition, such novel molecules are expected to have electrochemical properties different from conventional naphthalenediimide derivatives and their use.

[0006]

SUMMARY OF THE INVENTION The present invention provides a novel DN
The present invention relates to an A-binding electrochemically active molecule and a conductive double-stranded DNA fragment produced using the electrochemically active molecule.

[0007]

Means for Solving the Problems By the researchers of the present invention,
The following general formula (1):

[0008]

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[Wherein R ¹ and R ³ are each independently a heterocyclic group having 2 to 10 carbon atoms containing 1 to 4 heteroatoms selected from the group consisting of N, S and P; , A hydrocarbon group having 1 to 12 carbon atoms containing 1 to 4 heteroatoms selected from the group consisting of N, S and P in the chain formula, wherein 1 is selected from the group consisting of N, S and P
Any one of the ~ 4 heteroatoms has 1 carbon atom
A cationic group having a group selected from the group consisting of an alkyl group having 1 to 6 carbon atoms, a vinyl group which may be substituted with an alkyl group having 1 to 6 carbon atoms, and a benzyl group; Atom, 1 to 6 carbon atoms
Alkyl group, and represents a atom or group selected from the group consisting of an alkoxy group having 1 to 6 carbon atoms in the; however, of R ¹ and R ^3, at least one represents the cationic group; R ² Represents an atom or a group selected from the group consisting of a hydrogen atom, an acyl group having 2 to 7 carbon atoms, and-(CH ₂ ) _n -R ⁴ , wherein R ⁴ is a hydrogen atom, a carbon atom having 1 carbon atom. To 6 alkyl groups, 1 to 6 carbon atoms
And an alkoxy group having 1 to 6 carbon atoms as a substituent
The number of ring-forming carbon atoms which may have 1 to 5 alkyl groups or 1 to 6 alkoxy groups having 1 to 6 carbon atoms is 6 to
X represents an atom or a group selected from the group consisting of 12 aryl groups, and n represents an integer of 1 to 3;
^- it is a water-soluble fullerene derivative represented by a counter anion of the cationic group, the double-stranded DNA fragment having conductivity comprising contacting the DNA fragments of double strand that can solve the aforementioned problems found.

In a preferred embodiment of the conductive double-stranded DNA fragment of the present invention, R ¹ represents a cationic pyridinyl group having a methyl group at a nitrogen atom, R ² represents a methyl group, and R ³ represents a methyl group. , A hydrogen atom and X ⁻ represents a trifluoromethanesulfonyl anion.

Further, the following general formula (1):

[0012]

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Wherein R ¹ and R ³ each independently represent a heterocyclic group having 2 to 10 carbon atoms containing 1 to 4 heteroatoms selected from the group consisting of N, S and P; , A hydrocarbon group having 1 to 12 carbon atoms containing 1 to 4 heteroatoms selected from the group consisting of N, S and P in the chain formula, wherein 1 is selected from the group consisting of N, S and P
Any one of the ~ 4 heteroatoms has 1 carbon atom
A cationic group having a group selected from the group consisting of an alkyl group having 1 to 6 carbon atoms, a vinyl group which may be substituted with an alkyl group having 1 to 6 carbon atoms, and a benzyl group; Atom, 1 to 6 carbon atoms
Alkyl group, and represents a atom or group selected from the group consisting of an alkoxy group having 1 to 6 carbon atoms in the; however, of R ¹ and R ^3, at least one represents the cationic group; R ² Represents an atom or a group selected from the group consisting of a hydrogen atom, an acyl group having 2 to 7 carbon atoms, and-(CH ₂ ) _n -R ⁴ , wherein R ⁴ is a hydrogen atom, a carbon atom having 1 carbon atom. To 6 alkyl groups, 1 to 6 carbon atoms
And an alkoxy group having 1 to 6 carbon atoms as a substituent
The number of ring-forming carbon atoms which may have 1 to 5 alkyl groups or 1 to 6 alkoxy groups having 1 to 6 carbon atoms is 6 to
X represents an atom or a group selected from the group consisting of 12 aryl groups, and n represents an integer of 1 to 3;
^- is a water-soluble fullerene derivative represented by a counter anion of the cationic groups, also water-soluble fullerene derivative represented by the double-stranded DNA fragment having conductivity comprising contacting the DNA fragments of double-stranded The above problem can be solved.

In a preferred embodiment of the water-soluble fullerene derivative of the present invention, R ¹ represents a cationic pyridinyl group having a methyl group at a nitrogen atom, R ² represents a methyl group, and R ³ represents
But represents a hydrogen atom, and wherein X ^- is a water-soluble fullerene derivative, characterized in that represent a trifluoromethanesulfonyl anion.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION The conductive double-stranded D of the present invention
When the water-soluble fullerene derivative of the present invention is brought into contact with the double-stranded DNA fragment, the NA fragment binds the fullerene derivative in the groove (hydrophobic region) of the DNA, and imparts conductivity due to the cationic nature of the fullerene derivative. It is obtained by doing. Although the fullerene derivative of the present invention is one of the DNA-binding electrochemically active molecules, it is considered that the fullerene derivative enters the groove of the DNA fragment and binds to the DNA fragment. Can be considered.
Confirmation of the conductive double-stranded DNA fragment of the present invention is a method of electrochemically measuring a mixed solution of a double-stranded DNA fragment and a fullerene derivative, applying the mixed solution to an electrode,
A method of electrochemically measuring the electrode, a method of applying a solution containing a double-stranded DNA fragment to the electrode, and then contacting a solution containing a fullerene derivative to electrochemically measure the electrode, containing the fullerene derivative After applying the solution to the electrode, any method such as a method of contacting a solution containing a double-stranded DNA fragment and electrochemically measuring the electrode can be used, but the double-stranded DNA fragment and the fullerene derivative can be used. It is particularly preferable to use a method of applying a mixed solution of the above to an electrode and electrochemically measuring the electrode.

Any electrode can be used as long as it can fix a double-stranded DNA fragment, and it is preferable to use glassy carbon, gold, carbon, or the like, and it is particularly preferable to use glassy carbon.

As the double-stranded DNA fragment, a double-stranded DNA fragment prepared in advance or a hybrid DNA prepared on an electrode can be used. As the previously prepared double-stranded DNA fragment, DNA fragment extracted from a biological sample, DNA prepared by genetic manipulation, etc. is cut with a restriction enzyme, and then purified by electrophoretic separation or the like.
Preferably, an NA fragment is used. Alternatively, a DNA fragment obtained by using a single-stranded synthetic orthonucleotide and a single-stranded synthetic oligonucleotide that is complementary to the nucleotide to form a double strand may be used. Hybrid DN
A is preferably a DNA fragment obtained by bringing a single-stranded sample DNA fragment into contact with a probe DNA obtained by immobilizing a single-stranded DNA fragment on an electrode. The degree of duplex formation is not particularly limited. The double-stranded DNA fragment is 1 to 1
It is preferably used at a concentration in the range of 00 picomolar,
It is particularly preferred to use a concentration in the range from 0 to 50 picomoles.

The water-soluble fullerene derivative of the present invention will be described below.

Fullerene (C ₆₀ ) is a molecule discovered in 1970, in which 60 carbon atoms are bonded to a truncated icosahedral soccer ball shape obtained by cutting off the apex of a regular icosahedron. For the derivatives of fullerenes, see the literature (M. Pr.
ato, et al. , Tetrahedron, 5
2, 5221 (1996)), the synthesis of various compounds is described, but all of these compounds are hydrophobic, and no hydrophilic compounds have been known so far. N-methyl-2- (4'-N-pyridyl) having a structure similar to the water-soluble fullerene derivative of the present invention and represented by the following formula:
-3,4-Fullopyrrolidine is also hydrophobic.

[0020]

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As a method for solubilizing the above hydrophobic fullerene derivative in water, an example using a polymer, cyclodextrin, or the like is known. The fullerene derivative of the present invention is the first water-soluble compound in that it has a water solubility of ^-5 M level.

The water-soluble fullerene derivative of the present invention has the following general formula (1):

[0023]

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## EQU1 ##

In the above formula, R ¹ and R ³ are each independently selected from the group consisting of a cationic group, a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, and an alkoxy group having 1 to 6 carbon atoms. Represents an atom or group. However, of R ¹ and R ^3, at least one representative of the cationic group, either one of R ¹ and R ³ represent the cationic group, the other is a hydrogen atom, a methyl group, ethyl Represents an atom or group selected from the group consisting of an alkyl group having 1 to 6 carbon atoms such as a group, a propyl group and an isopropyl group, and an alkoxy group having 1 to 6 carbon atoms such as a methoxy group, an ethoxy group and a propoxy group. Is particularly preferred.

The cationic group has 1 to 4 heteroatoms selected from the group consisting of N, S and P and has 2 carbon atoms.
A heterocyclic group having 1 to 10 carbon atoms or a hydrocarbon group having 1 to 12 carbon atoms containing 1 to 4 heteroatoms selected from the group consisting of N, S and P in a chain portion, wherein N, S and P
Any one of 1 to 4 heteroatoms selected from the group consisting of an alkyl group having 1 to 6 carbon atoms, a vinyl group which may be substituted with an alkyl group having 1 to 6 carbon atoms, And a cationic group having a group selected from the group consisting of benzyl and benzyl.

The heterocyclic ring constituting the heterocyclic group having 2 to 10 carbon atoms is preferably a heterocyclic ring having 3 or 9 carbon atoms containing 1 or 2 S or N, and more preferably thiophene, Pyrrole, thiazole, imidazole, pyridine, pyrimidine, pyrazine, 2-pyrroline, pyrrolidine, 2-imidazoline, imidazolidin,
Piperidine, piperazine, thiolane, 1,3-thiazole, indole, benzo [b] thiophene, quinoline,
It is isoquinoline, quinazoline or quinoxaline, particularly preferably pyridine.

The above-mentioned hydrocarbon group having 1 to 12 carbon atoms is preferably a hydrocarbon group having 1 to 6 carbon atoms such as methyl group, ethyl group, propyl group, butyl group, isobutyl group and tert-butyl group. And a hydrocarbon group containing one or two S or N in the chain structure part of the hydrocarbon group, more preferably a methylamino group, a dimethylamino group, a methylethylamino group, a methylthio group , An ethylthio group, a methylthiomethylene group or an ethylthiomethylene group, particularly preferably a methylamino group or a methylthio group.

The group of S or N is preferably an alkyl group having 1 to 6 carbon atoms such as methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, tert-butyl group, vinyl group, It is a group selected from the group consisting of a vinyl group and a benzyl group substituted with the alkyl group having 1 to 6 carbon atoms, more preferably a methyl group and an ethyl group, and particularly preferably a methyl group.

Therefore, the cationic group is preferably
N-methylpyridyl cation, -methylimidazolyl cation, N-methylpyrimidyl cation, N-methylthiazolyl cation, N-methyl-2-pyrrolinyl cation, N-methylpyrrolidinyl cation, N-methylpiperidi Nyl cation, S-thiofinyl cation or S-thiolanyl cation, particularly preferably N-
It is a methylpyridyl cation. Further, among R ¹ and R ^3, the atom or group represented by the other group other than the cationic group is preferably a hydrogen atom or a methyl group, and particularly preferably a hydrogen atom.

R ² represents an atom or a group selected from the group consisting of a hydrogen atom, an acyl group having 2 to 7 carbon atoms such as an acetyl group, and-(CH ₂ ) _n -R ⁴ . Where R ⁴
Is a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms such as a methyloxy group, an ethyloxy group, a propylopoxy group, and an alkyl group having 1 to 6 carbon atoms as a substituent or 1 to 6 carbon atoms
Represents a group selected from the group consisting of aryl groups having 6 to 12 ring carbon atoms, such as a phenyl group and a naphthyl group which may have 1 to 5 alkoxy groups. n is 1 to
Represents an integer of 3, preferably 1. R ⁴ is preferably a hydrogen atom, a methyl group or a phenyl group, and particularly preferably a hydrogen atom. Therefore, R ² is preferably a methyl group, an acetyl group or a benzyl group, and particularly preferably a methyl group.

[0032] X ^- represents a counter anion of the cationic group. X ^- is preferably a trifluoromethanesulfonyl anion, a methylsulfate anion, a hexafluorophosphate anion, a perchlorate anion, a fluoroanion, a chloroanion or a bromoanion, and particularly preferably a trifluoromethanesulfonyl anion.

Representative water-soluble fullerene derivatives 1a, 1b and 1c of the present invention are shown in the following formula in order.

[0034]

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[0035]

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[0036]

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The precursor of the novel water-soluble fullerene derivative of the present invention represented by the general formula (1), for example, N-methyl-2- (4'-N-pyridyl) -3,4-fulleropyrrolidine Are known methods (M. Prato, et a).
l. , Tetrahedron, 52, 5221 (19
96) etc.). By subjecting this precursor to a reaction such as alkylation using an alkyl triflate or the like in a solvent such as chlorobenzene, fluorobenzene, benzene, toluene, dimethylformamide (DMF), dimethylsulfoxide (DMSO), acetone, or dichloromethane, The water-soluble fullerene derivative of the present invention can be obtained with high yield. As the solvent,
It is preferable to use chlorobenzene, fluorobenzene, benzene or toluene, and it is particularly preferable to use chlorobenzene. As the alkyl triflate, it is preferable to use methyl triflate, ethyl triflate, vinyl triflate or benzyl triflate, and it is particularly preferable to use methyl triflate.

The water-soluble fullerene derivative is 0.1 to 1 m
M is preferably used at a concentration in the range of 0.1 to 0.1.
It is particularly preferred to use a concentration in the range of 5 mM.

The water-soluble fullerene derivative of the present invention is a ferrocene-containing naphthalenediimide derivative (JP-A-9-288).
080 and 57th Analytical Chemistry Symposium, p.
137-138 (1996)), which is one of the electrochemically active molecules, but the former is capable of performing a redox reaction by taking in electrons (which makes the whole molecule cationic). In contrast, the latter performs the reaction by emitting electrons.

The binding of the water-soluble fullerene derivative to the double-stranded DNA fragment depends on the electrostatic interaction between the charge (anion) of the phosphate group of the DNA fragment and the charge (cation) of the fullerene derivative, and the binding of the DNA fragment. It is thought to be due to the hydrophobic interaction of the fullerene derivative in the groove, but the binding mode differs depending on the structure of the fullerene derivative. For example, when the fullerene derivative is compound 1a described above, the ratio of the charge of the fullerene derivative to the charge of the phosphate group of the DNA fragment is 2, and in the case of compounds 1b and 1c, the ratio is Each is 1. From this, it is presumed that one molecule of the compound 1a is bonded to two molecules of the phosphate group, and one molecule of the compound 1b or 1c is bonded to one molecule of the phosphate group. The above can be easily understood from the molecular structures of these compounds. That is, in compound 1a, fullerene (fullerene molecule itself having no functional group)
Is shorter than that of the compound 1b or 1c as compared with the compound 1b or 1c, so that the hydrophobic interaction is weakened. As a result, bonding occurs mainly by electrostatic interaction. It is considered that both of these two interactions greatly contribute to the binding. As described above, although the binding mode differs depending on the structure of the fullerene derivative, when the fullerene derivative binds to the DNA fragment, it remains in the state of being inserted into the groove of the DNA fragment regardless of the structure of the fullerene derivative.

The formation of the complex between the above DNA fragment and the water-soluble fullerene derivative is confirmed by measuring its UV / VIS (ultraviolet / visible spectroscopy) and confirming the pale color effect (color reduction effect) that appears in the maximum absorption peak. Can be. This light-color effect becomes constant when the charge (anion) of the phosphate group of the DNA fragment and the charge (cation) of the fullerene derivative are balanced (charge ratio is 1).

The electrochemical characteristics of the composite can be determined by any electrochemical method that can measure the current flowing through the electrode while changing the potential. As the method, it is preferable to use cyclic voltammetry (CV), linear sweep voltammetry (LSV), differential pulse voltammetry, potentiostat, and the like, and it is particularly preferable to use cyclic voltammetry.

The actual measurement is preferably performed by immersing a DNA film obtained by applying a solution containing a fullerene derivative and a DNA fragment to an electrode in an organic solvent containing a salt, not in an electrolyte solution. . This is because, in an electrolyte solution containing potassium chloride or the like, it is difficult to obtain a CV having a plurality of peaks corresponding to each other, which indicates the presence of a plurality of oxidation-reduction processes. The conductive double-stranded DNA fragment of the present invention is the first example whose properties can be measured by CV. The complex of the double-stranded DNA fragment and compound 1a has three redox processes. Is revealed by its CV measurement (the presence of three pairs of peaks).

As the organic solvent for measurement, it is preferable to use a benzene-based solvent such as chlorobenzene, fluorobenzene, benzene, and toluene, and it is particularly preferable to use chlorobenzene.

Any salt can be used as long as it can be dissolved in the above-mentioned organic solvent. Tetramethylammonium ion, tetraethylammonium ion,
Cations such as tetrapropylammonium ion, tetrabutylammonium ion, ammonium ion, potassium ion and sodium ion; halogen ions such as tetrafluoroborate ion, chloride ion and fluorine ion; and anions such as perchlorate ion It is preferable to use a salt obtained by a combination of: tetrabutylammonium tetrafluoroborate (TBAF)
It is particularly preferred to use Salt is 0.12-0.9
It is preferably used at a concentration in the range of 0M,
It is particularly preferred to use a concentration in the range of 0.50M.

Literatures discussing the electrochemical behavior of a complex of DNA and a metal compound in terms of changes in the value of redox potential (MT Carter, M. Rodriguez,
and A. J. Bard, J.A. Am. Chem. So
c. , 111, 8901 (1989)), it is possible to deduce the interaction relating to the formation of the conductive double-stranded DNA fragment of the present invention with reference to this document (see below). Example).

The conductive double-stranded DNA fragment of the present invention may be used as follows. When a DNA film is prepared by applying a mixed solution obtained by further adding a specific oxidoreductase to a solution containing a double-stranded DNA fragment having conductivity to an electrode, the substrate corresponding to the oxidoreductase is quantified. be able to. In the present invention, since the complex of the double-stranded DNA fragment and the fullerene derivative has a negative oxidation-reduction potential, it is possible to use an oxidoreductase that can cope with this. Examples of oxidoreductases having such properties include glutathione reductase, cytochrome reductase, and the like, and the corresponding glutathione, cytochrome, and the like can be quantified.

[0048]

EXAMPLES Production Example 1 N-methyl-2- (2'-N-
Synthesis of methylpyridyl) -3,4-fulleropyrrolidine (Compound 1a) 2-pyridinecarboxaldehyde (75 mg, 0.7
0 mmol), N-methylglycine (31 mg, 0.3
90 mL of o-chlorobenzene was added to 5 mmol) and fullerene (100 g, 0.14 mmol), and the mixture was refluxed for 3 hours. After confirming the disappearance of the raw materials, o-chlorobenzene is distilled off under reduced pressure, and the obtained residue is purified by flash column chromatography (toluene, hexane / ethyl acetate = 2/1; volume ratio). By that
N-methyl-2- (2'-pyridyl) -3,4-fullaropyrrolidine (45 mg) as a yellow oily substance was obtained as a crude product. Yield 38% (62% calculated excluding the recovered fullerene). This crude product (45 mg) was dissolved in toluene (100 mL), methyl trifluoromethanesulfonate (6 μL, 0.05 mmol) was added, and the mixture was refluxed for 6 hours, treated in the same manner as above, and then purified. This gave 38 mg of the title compound represented by the following formula. Yield 75%. ^{1 H-NMR (270MHz, DMSO} -d 6) δ; 3.28 (3H, s) 4.60,5.15 ( each 1H, d, J = 9.7Hz) 4.69 (3H, s) 6 .28 (1H, s) 8.08 to 8.20 (1H, m) 8.74 (1H, dd, J = 8.0, 7.7 Hz) 8.84 (1H, d, J = 7.6 Hz) 9.10 (1H, d, J = 5.9 Hz) MASS: FAB 868 (M-TfO) ⁺ , 720
(C ₆₀ ) ⁺ .

[0049]

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[Production Example 2] N-methyl-2- (3'-N
-Methylpyridyl) -3,4-fulleropyrrolidine (Compound 1b) 3-pyridinecarboxaldehyde (75 mg, 0.7
0 mmol) in the same manner as in Production Example 1 except that N
-Methyl-2- (3'-pyridyl) -3,4-fulleropyrrolidine (53 mg) was obtained. Yield 44% (calculated excluding recovered fullerene, 71%). Subsequently, 58 mg of the title compound represented by the following formula was obtained in the same manner as described above except that the mixture was refluxed in chlorobenzene for 4 hours. Yield 92%. ^{1 H-NMR (270MHz, DMSO} -d 6) δ; 2.76 (3H, s) 4.43 (3H, s) 4.42,5.15 ( each 1H, d, J = 10.1Hz) 5 .43 (1H, s) 8.25 (1H, dd, J = 8.0, 6.3 Hz) 9.00 to 9.10 (2H, m) 9.53 (1H, d, J = 7.6 Hz) ) MASS: FAB 868 (M-TfO) ⁺ , 720
(C ₆₀ ) ⁺ .

[0051]

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[Production Example 3] N-methyl-2- (4'-N
-Methylpyridyl) -3,4-fulleropyrrolidine (Compound 1c) Reference (M. Prato, et al., Tetrahe)
dr., 52, 5221 (1996)).
-Methyl-2- (4'-pyridyl) -3,4-fullaropyrrolidine was obtained, and then in the same manner as in Production Example 1, 70 mg of the title compound represented by the following formula was obtained. Yield 92%. ^{1 H-NMR (270MHz, DMSO} -d 6) δ; 2.76 (3H, s) 4.35 (3H, s) 4.44,5.16 ( each 1H, d, J = 10.1Hz) 5 .55 (1H, s) 8.59 (2H, d, J = 6.3 Hz) 9.04 (2H, d, J = 6.3 Hz) MASS: FAB 868 (M-TfO) ⁺ , 720
(C ₆₀ ) ⁺ .

[0053]

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Example 1 Confirmation of Conductivity of DNA (1) Measurement of Concentration of Fullerene Derivative (Compound 1a) Compound 1a (10 mg) of Production Example 1 was distilled water (10 m
L) and vigorously shaken under light shielding for 24 hours to prepare a saturated aqueous solution of compound 1a. This is 150
After centrifugation at 00 rpm for 10 minutes, the supernatant (3 mL) was dried under reduced pressure, an appropriate amount of the dried product was dissolved in chlorobenzene, and the absorption spectrum was measured by UV / VIS (ultraviolet / visible spectroscopy). As a result, maximum absorption was observed at 209, 267 and 335 nm, respectively. 33
The molar extinction coefficient in water at 5 nm is equal to the molar extinction coefficient in chlorobenzene at 320 nm (ε = 3.6 × 1
Assuming that the concentration is the same as 0 ⁴ cm ^-1 · M ^-1 ), the concentration of the saturated aqueous solution of the compound 1a is 10 ^-5 M.

(2) Confirmation of formation of complex of compound 1a and DNA From a saturated aqueous solution of compound 1a described above, a 5 μM aqueous solution was obtained, and sonicated calf thymus DNA was added thereto. The absorption spectrum was measured. As a result, compared to the case where the DNA was not added, 335 nm
A decrease in the absorption peak by about 5% (light color effect) was observed in the absorption peak of the above, confirming that a complex of the compound 1a and DNA was formed. In addition, the absorption peaks at 209 and 267 nm both overlapped with the absorption peak of DNA, and the light color effect could not be observed.

(3) Measurement of Electrochemical Characteristics Compound 1a containing calf thymus DNA (10 pmol) was placed on the surface of a glassy carbon electrode having an area of 2.5 mm ² (0.
4 mM) chlorobenzene solution (10 mL),
It was left in the air to prepare a DNA film. Then
The DNA film (working electrode), platinum electrode (counter electrode) and silver / silver chloride reference electrode are immersed in a chlorobenzene solution containing 0.15 M tetrabutylammonium tetrafluoroborate (TBAF) to form three electrodes, Cyclic voltammography (CV) was measured at a scan speed of 100 mV / sec (FIG. 1).

From FIG. 1, three pairs of oxidation-reduction potentials (E^{0 / 1-},
E^{1- / 2-}And E^{3- / 4-}The peak indicating the presence of
Was called. The response potential values showing these peaks are
Average potential value between peak potential and cathode peak potential
(Table 1). In the table, E ^{0 / 1-}, E^{1- / 2-}And E
^{3- / 4-}Are the first, second, and fourth redox processes, respectively.
The average potential value is shown.

Comparative Example 1 A 0.15 M TBAF chlorobenzene solution containing compound 1a (0.4 mM) was placed in a 2.5 mm ² area of a glassy carbon electrode (working electrode), a platinum electrode (counter electrode) and A silver chloride reference electrode was immersed to form three electrodes, and its CV was measured at a scan speed of 100 mV / sec (FIG. 2).

FIG. 2 shows that four pairs of oxidation-reduction potentials (E ^{0 / 1−} ,
E ^{1- / 2-} , E ^{2- / 3-} and E ^{3- / 4-} ). The response potential values showing these peaks were determined as the average potential value of the anode peak potential and the cathode peak potential (Table 1 below). In the table, E ^{2- / 3-}
The average potential value in the third oxidation-reduction process is shown.

Example 2 Compound 1 represented by Production Example 2
The average potential value was determined in the same manner as in Example 1 except that b was used (Table 1 below).

[Comparative Example 2] Compound 1 represented by Production Example 2
The average potential value was determined in the same manner as in Comparative Example 1 except that b was used (Table 1 below).

Example 3 Compound 1 represented by Production Example 3
The average potential value was determined in the same manner as in Example 1 except that c was used (Table 1 below).

[Comparative Example 3] Compound 1 represented by Production Example 3
The average potential value was determined in the same manner as in Comparative Example 1 except that c was used (Table 1 below).

[0064]

[Table 1] Table 1 {Average potential value (mV)} ──────────────────────────── E ^{0 / 1-} E ^{1- / 2-} E ^{2- / 3-} E ^{3- / 4 -} ──────────────────────────────────── example 1 -920 -1,278 - -1,930 Comparative example 1 −808 −1239 −1615 −1941 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥ Example 2 −830 −1409 − 1863-2130 Comparative Example 2 -927 -1411 -1835 -2122 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥ Example 3-829-1313--2010 Comparative Example 3 -846 -1258 -15 6 -1927 ────────────────────────────────────

Table 2 below shows the average potential value of Example 1 (Example 2 or 3) in each oxidation-reduction process and Comparative Example 1 (Example 2 or 3 in Comparative Example 2). Alternatively, the differences (ΔE values) from the average potential value of 3) are shown as A, B, and C, respectively.

[0066]

[Table 2] Table 2 Difference in average potential value (mV) ────────────────────────── △ E ^{0 / 1-} △ E ^{1- / 2-} △ E ^{2- / 3-} △ E ^{3- / 4-} ───────────────────────────────── A-112-39-11B B979-28-8 C 17 -55--83 ─────────────────────────────────

Considering that E ^{0 / 1−} is a criterion for judging the interaction related to the formation of a complex between DNA and a water-soluble fullerene derivative represented by compound 1a, etc., Difference in average potential value in the first redox process (過程 E ^{0 / 1-} )
Thus, the average potential value of the complex of compound 1a and DNA is
When the compound 1a is changed to 1b or 1c while the compound is changed to the negative side (ΔE ^{0 / 1−} : −112 mV) as compared with the average potential value of the compound 1a alone, Also changes to the positive side (97, 17m respectively)
V). Therefore, the formation of the former complex requires
It is considered that the electrostatic interaction mainly contributes, and the formation of the latter complex is not only due to the electrostatic interaction but also to the hydrophobic interaction.

[0068]

The fullerene derivative of the present invention is a novel and first water-soluble compound. In addition, conductivity can be imparted to the double-stranded DNA fragment using the fullerene derivative of the present invention. Furthermore, the double-stranded DNA fragment having conductivity is used for the quantification of substrates such as glutathione and cytochrome by causing a redox enzyme that exchanges negative electrons such as glutathione reductase and cytochrome reductase to be present in the measurement system. can do.

[Brief description of the drawings]

FIG. 1 shows a DN obtained by applying a solution containing DNA and a fullerene derivative (compound 1a) to a glassy carbon electrode.
It is a cyclic voltammography of A film.

FIG. 2 is a cyclic voltammogram obtained when a solution containing only a fullerene derivative (compound 1a) is brought into contact with a glassy carbon electrode.

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Tomohiko Tsuge 2-10-12 Fujisaki, Sawara-ku, Fukuoka Fukuoka F-term (reference) 4B024 AA11 CA01 HA11 HA12 4B063 QA01 QQ79 QR02 QR32 QR41 QR55 QR81 QR84 QS36 QS39 QS40 QX05 4C063 AA01 BB01 DD08 EE01

Claims (4)

[Claims] 1. The following general formula (1): [Wherein, R ¹ and R ³ each independently represent a heterocyclic group having 2 to 10 carbon atoms containing 1 to 4 heteroatoms selected from the group consisting of N, S and P, or N, S and A hydrocarbon group having 1 to 12 carbon atoms containing 1 to 4 heteroatoms selected from the group consisting of P in the chain formula portion, and having 1 to 4 hetero atoms selected from the group consisting of N, S and P; Any one of the atoms has a group selected from the group consisting of an alkyl group having 1 to 6 carbon atoms, a vinyl group which may be substituted with an alkyl group having 1 to 6 carbon atoms, and a benzyl group. Represents an atom or a group selected from the group consisting of a cationic group, a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, and an alkoxy group having 1 to 6 carbon atoms; ¹ and R ³
At least one represents said cationic group; R ² is
A hydrogen atom, an acyl group having 2 to 7 carbon atoms, and-(C
H _{2) n} -R represents an atom or a group selected from the group consisting of ^4, wherein R ⁴ is a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, and a substituted As a group, the number of ring-constituting carbon atoms which may have 1 to 5 alkyl groups having 1 to 6 carbon atoms or 1 to 5 alkoxy groups having 1 to 6 carbon atoms is selected from the group consisting of 6 to 12 aryl groups. N represents an integer of 1 to 3; and X ⁻ represents a counter anion of the cationic group.] The water-soluble fullerene derivative represented by the formula A conductive double-stranded DNA fragment that is brought into contact. 2. R ¹ represents a cationic pyridinyl group having a methyl group at a nitrogen atom, R ² represents a methyl group, R ³ represents a hydrogen atom, and X ⁻ represents a trifluoromethanesulfonyl anion. 2. The double-stranded DNA fragment according to claim 1, wherein 3. The following general formula (1): [Wherein, R ¹ and R ³ each independently represent a heterocyclic group having 2 to 10 carbon atoms containing 1 to 4 heteroatoms selected from the group consisting of N, S and P, or N, S and A hydrocarbon group having 1 to 12 carbon atoms containing 1 to 4 heteroatoms selected from the group consisting of P in the chain formula portion, and having 1 to 4 hetero atoms selected from the group consisting of N, S and P; Any one of the atoms has a group selected from the group consisting of an alkyl group having 1 to 6 carbon atoms, a vinyl group which may be substituted with an alkyl group having 1 to 6 carbon atoms, and a benzyl group. Represents an atom or a group selected from the group consisting of a cationic group, a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, and an alkoxy group having 1 to 6 carbon atoms; ¹ and R ³
At least one represents said cationic group; R ² is
A hydrogen atom, an acyl group having 2 to 7 carbon atoms, and-(C
H ₂ ) represents an atom or group selected from the group consisting of _n- R ⁴ , wherein R ⁴ is a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, and a substituent As a group, the number of ring-constituting carbon atoms which may have 1 to 5 alkyl groups having 1 to 6 carbon atoms or 1 to 5 alkoxy groups having 1 to 6 carbon atoms is selected from the group consisting of 6 to 12 aryl groups. represents an atom or group, n represents represents an integer of 1 to 3; and, X ^- is a water-soluble fullerene derivative represented by a counter anion of the cationic group. 4. R ¹ represents a cationic pyridinyl group having a methyl group at a nitrogen atom, R ² represents a methyl group, R ³ represents a hydrogen atom, and X ⁻ represents a trifluoromethanesulfonyl anion. The water-soluble fullerene derivative according to claim 3, wherein