U.S. patent application number 09/733365 was filed with the patent office on 2002-01-31 for threading intercalator having oxidation-reduction activity.
This patent application is currently assigned to Fuji Photo Film Co., Ltd.. Invention is credited to Makino, Yoshihiko, Takagi, Makoto, Takahashi, Kazunobu, Takenaka, Shigeori, Yamashita, Kenichi.
Application Number | 20020012917 09/733365 |
Document ID | / |
Family ID | 18402729 |
Filed Date | 2002-01-31 |
United States Patent
Application |
20020012917 |
Kind Code |
A1 |
Makino, Yoshihiko ; et
al. |
January 31, 2002 |
Threading intercalator having oxidation-reduction activity
Abstract
A compound of the following formula: Ea-La-X-Lb-Eb in which each
of Ea and Eb independently is a group having oxidation-reduction
activity and having a conjugated system in its group; X is a
divalent cyclic group; and each of La and Lb independently is a
group which does not form a conjugated system in combination with
the conjugated system of each of Ea and Eb and at least one of
which has a site imparting water solubility to the compound or a
site that is convertible into a site imparting water solubility to
the compound, is favorably employable as an electroconductive
threading intercalator in an electrochemical method for detecting
complementary DNA fragments.
Inventors: |
Makino, Yoshihiko; (Saitama,
JP) ; Takahashi, Kazunobu; (Kanagawa, JP) ;
Takagi, Makoto; (Fukuoka, JP) ; Takenaka,
Shigeori; (Fukuoka, JP) ; Yamashita, Kenichi;
(Fukuoka, JP) |
Correspondence
Address: |
REED SMITH LLP
375 PARK AVENUE
NEW YORK
NY
10152
US
|
Assignee: |
Fuji Photo Film Co., Ltd.
|
Family ID: |
18402729 |
Appl. No.: |
09/733365 |
Filed: |
December 8, 2000 |
Current U.S.
Class: |
435/6.11 ;
546/108; 546/257; 556/142; 564/443; 568/10 |
Current CPC
Class: |
C07F 17/02 20130101 |
Class at
Publication: |
435/6 ; 546/108;
546/257; 556/142; 564/443; 568/10 |
International
Class: |
C12Q 001/68; C07F
009/02; C07D 41/14; C07F 017/02 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 8, 1999 |
JP |
11-349284 |
Claims
What is claimed is:
1. A compound having the formula (1):Ea-La-X-Lb-Eb (1)in which each
of Ea and Eb independently is a group having oxidation-reduction
activity and having a conjugated system in its group; X is a
divalent cyclic group; and each of La and Lb independently is a
group which does not form a conjugated system in combination with
the conjugated system of each of Ea and Eb and at least one of
which has a site imparting water solubility to the compound or a
site that is convertible into a site imparting water solubility to
the compound.
2. The compound of claim 1, wherein Ea is the same as Eb, and La is
the same as Lb.
3. The compound of claim 1, wherein a main chain of La-X-Lb
contains 10 to 100 atoms, which are counted along the shortest
connection route from Ea to Eb.
4. The compound of claim 3, wherein the main chain contains 15 to
70 atoms.
5. The compound of claim 1, wherein each of Ea and Eb independently
a group having oxidation-reduction activity which is selected from
the group consisting of a metallocene moiety, a 2,2'-bipyridine
complex moiety, a cyclobutadiene moiety, a cyclopentadiene moiety,
a 1,10-phenanthroline moiety, a triphenylphosphine moiety, a
cathecol amine moiety, and a biologen moiety, any moiety possibly
having one or more substituents.
6. A compound according to claim 1, which is represented by the
formula (2):Ea-L1a-L2a-X-L2b-L1b-Eb (2)in which each of Ea and Eb
independently is a group having oxidation-reduction activity and
having a conjugated system in its group; X is a divalent cyclic
group; each of L1a and L1b independently is a group which does not
form a conjugated system in combination with the conjugated system
of each of Ea and Eb; and each of L2a and L2b independently
contains a linking group having a site imparting water solubility
to the compound or a site that is convertible into a site imparting
water solubility to the compound.
7. The compound of claim 6, wherein each of Ea and Eb independently
a group having oxidation-reduction activity which is selected from
the group consisting of a metallocene moiety, a 2,2'-bipyridine
complex moiety, a cyclobutadiene moiety, a cyclopentadiene moiety,
a 1,10-phenanthroline moiety, a triphenylphosphine moiety, a
cathecol amine moiety, and a biologen moiety, any moiety possibly
having one or more substituents.
8. The compound of claim 6, wherein each of L1a and L1b
independently is a hydrocarbyl group which may have one or more
substituents.
9. The compound of claim 6, wherein each of L1a and L1b
independently is an alkylene group having 1 to 6 carbon atoms or an
alkenylene group having 2 to 6 carbon atoms, each group possibly
having one or more substituents.
10. The compound of claim 6, wherein each of L2a and L2b
independently is a linking group containing an atomic element other
than carbon element.
11. The compound of claim 10, wherein each of L2a and L2b
independently is a linking group containing N, O, or S.
12. The compound of claim 11, wherein each of L2a and L2b
independently contains a linking group selected from the group
consisting of an amino bonding, an ester bonding, an ether bonding,
a thioether bonding, a diimide bonding, a thiodiimide bonding, a
thioamide bonding, an imino bonding, a carbonyl bonding, a
thiocarbonyl bonding, and 1,4-piperazinyl bonding, any bonding
possibly having one or more substituents.
13. The compound of claim 11, wherein each of L2a and L2b
independently contains --NHCO-- or --CONH--.
14. The compound of claim 6, wherein Ea is the same as Eb, L1a is
the same as L1b, and L2a is the same as L2b.
15. A threading intercalator having oxidation-reduction activity
which is represented by the formula (1):Ea-La-X-Lb-Eb (1)in which
each of Ea and Eb independently is a group having
oxidation-reduction activity and having a conjugated system in its
group; X is a divalent cyclic group; and each of La and Lb
independently is a group which does not form a conjugated system in
combination with the conjugated system of each of Ea and Eb and at
least one of which has a site imparting water solubility to the
compound or a site that is convertible into a site imparting water
solubility to the compound.
16. The compound of claim 15, wherein Ea is the same as Eb, and La
is the same as Lb.
17. The compound of claim 15, wherein a main chain of La-X-Lb
contains 10 to 100 atoms, which are counted along the shortest
connection route from Ea to Eb.
18. An electrochemical method for detection of oligonucleotide
samples or polynucleotide samples which employs the threading
intercalator of claim 15.
19. A process for electrochemically detecting oligonucleotide
samples or polynucleotide samples complementary to a group of probe
molecules of nucleotide derivatives or their analogues fixed onto
an electrode substrate, which comprises the steps of: bringing the
group of probe molecules into contact with oligonucleotide samples
or polynucleotide samples in an aqueous medium in the presence of a
threading intercalator according to claim 15 so as to for by
hybridization a complex of the group of probe molecules and the
oligonucleotide samples or polynucleotide samples in which the
threading intercalator is intercalated; and detecting an electric
current produced by applying an electric potential to the electrode
substrate.
20. The process of claim 19, in which the electric potential
applied to the electrode substrate is in the range of 100 to 400
mV.
21. A process for electrochemically detecting oligonucleotide
samples or polynucleotide samples complementary to a group of probe
molecules of nucleotide derivatives or their analogues fixed onto
an electrode substrate, which comprises the steps of: bringing the
group of probe molecules into contact with oligonucleotide samples
or polynucleotide samples in an aqueous medium so as to form by
hybridization a complex of the group of probe molecules and the
oligonucleotide samples or polynucleotide samples; bringing a
threading intercalator according to claim 15 in contact with the
formed complex so as to intercalate the intercalator into the
complex; and detecting an electric current produced by applying an
electric potential to the electrode substrate.
22. The process of claim 20, in which the electric potential
applied to the electrode substrate is in the range of 100 to 400
mV.
23. A kit for electrochemically detecting oligonucleotide samples
or polynucleotide samples complementary to a group of probe
molecules of nucleotide derivatives or their analogues fixed onto
an electrode substrate, which comprises an electrode substrate
having a group of probe molecules of nucleotide derivatives or
their analogues fixed to its substrate, and a threading
intercalator according to claim 15.
24. The kit of claim 23, in which the nucleotide derivatives and
their analogues are oligonucleotides, polynucleotides, or peptide
nucleic acids.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a compound which is
favorably employable as an electrochemically active threading
intercalator in a procedure of analyzing oligonucleotides or
polynucleotides such as DNA fragments.
BACKGROUND OF THE INVENTION
[0002] In the gene analysis in the fields of biochemistry and
clinical test, the detection of a DNA or its fragment having a
specific base sequence is performed by way of a hybridization
method, particularly Southern hybridization method (Southern
blotting method). Southern hybridization is performed using a
radioisotope (RI) label. Tne conventional analytical methods using
radioisotope label such as Southern hybridization method are
disadvantageous in that they need troublesome radioisotopes.
[0003] A Southern hybridization method using a fluorescent label in
place of a radioisotope label is also known. This method is
superior to the method using RI in safety and rapidness. Therefore,
DM chips comprising a substrate such as a slide glass or a silicone
plate and a great number of oligonucleotide or polynucleotide
molecules fixed onto the substrate are now commercially available
for the use in the fluorescence detection systems. However, the
fluorescence detection system has other disadvantageous features,
that is, the fluorescent label is gradually faded out under
irradiation of stimulating rays; a specifically designed
fluorescence-measuring apparatus should be installed; and an amount
of a fluorescent label is restricted because internal quenching
takes place.
[0004] Recently, a new system for detection of DNA fragments which
utilizes an electrode sensor onto which a group of probes
comprising oligonucleotide molecules or polynucleotide molecules
are fixed has been proposed in Japanese Patent Provisional
Publication No. H9-288080 and Preprint of 57th Conference of
Analytical Chemistry, pp. 137-138 (1996) In this system, an
electrode which has an output terminal and further has he probe
molecules fixed onto its surface is brought into contact with a DNA
sample in an aqueous medium in the presence of a threading
intercalator, and an electric current produced by applying an
electric voltage between the electrode and another electrode
introduced in the aqueous medium is measured.
[0005] As the threading intercalator, an electroconductive
ferrocene-containing compound having oxidation-reduction activity
(redox activity) which has the following chemical structure and can
be specifically bonded to a hybrid or hybridized DNA is known:
1
[0006] The above-mentioned electrochemical detection system
utilizing an electrode sensor is advantageous in easily detecting
the hybrid DNA structure on real-time basis. No fading-out takes
place.
[0007] The above-illustrated conventional electroconductive
threading intercalator has a structure comprising a core portion of
a naphthalene-diimide cyclic group, a pair of linker porions each
of which is attached to each of the two ends of the core portion,
and a pair of electroconductive ferrocene moieties each of which is
attached to other end of each linker. The ferrocene moiety has an
oxidation-reduction activity and a conjugated system in which
electrons freely move.
[0008] In the procedures for detecting DNA fragments complementary
to the probe molecules fixed on the electrode, the amount of
electric current produced by the application of electric potential
to the electrode essentially depends on the nature of an
electroconductive threading intercalator, though in part depends on
the natures of probe molecules and DNA fragment samples, and the
ionic concentration of the buffer solution employed in the
detection procedure. In the use of the conventional threading
intercalator of the above-mentioned formula, a peak electric
current is observed when an electric potential in the range of
approx. 450 to 620 mV is applied. Therefore, in the detection
procedures utilizing the conventional threading intercalator, an
electric potential of approx. 450 mV or higher should be
applied
[0009] The electric potential of approx. 450 mV or higher is
relative high for current detection devices. Accordingly, the cost
for producing the detection devices for the electrochemical
analysis of DNA fragments is relatively high. Moreover, if the
probe molecules are attached to the electrode surface by weak
bonding such as electrostatic bonding, the probe molecules are apt
to be released from the electrode when a high electric potential is
applied to the electrode. The release of the probe molecules from
the electrode adversely effect to the detection sensitivity and
detection accuracy. Particularly, the easy release of the probe
molecules from the electrode adversely effect when the DNA chip is
repeatedly employed in the detection procedures after the
temporarily fixed DN fragment samples and threading intercalator
are removed.
[0010] Accordingly, it is an object of the invention to provide an
electroconductive threading intercalator which is favorably
employable in the electrochemical method for detecting
polynucleotide samples or oligonucleotide samples (such as DNA
fragments) by means of a DNA chip comprising an electrode and probe
molecules (such as nucleotide derivatives or their analogues).
[0011] Specifically, it is an object of the invention to provide an
electroconductive threading intercalator which is capable of
working in the electrochemical detection procedure at a low
electric potential applied to the electrode.
SUMMARY OF THE INVENTION
[0012] The present invention resides in a compound having the
formula (1):
Ea-La-X-Lb-Eb (1)
[0013] in which each of Ea and Eb independently is a group having
oxidation-reduction activity and having a conjugated system in its
group; X is a divalent cyclic group; and each of La and Lb
independently is a group which does not form a conjugated system in
combination with the conjugated system of each of Ea and Eb and at
least one of which has a site imparting water solubility to the
compound or a site that is convertible into a site imparting water
solubility to the compound.
[0014] In the above-mentioned formula, it is preferred that Ea is
the same as Eb, and La is the same as Lb. The main chain of La-X-Lb
preferably contains 10 to 100 atoms, more preferably 15 to 70, most
preferably 20 to 50, which are counted along the shortest
connection route from Ea to Eb. For the sake of reference, the main
chain of the aforementioned conventional threading intercalator has
32 carbon atoms.
[0015] The compound of the formula (1) preferably has the following
formula (2):
Ea-L1a-L2a-X-L2b-L1b-Eb (2)
[0016] in which each of Ea and Eb independently is a group having
oxidation-reduction activity and having a conjugated system in its
group; X is a divalent cyclic group; each of L1a and L1b
independently is a group which does not form a conjugated system in
combination with the conjugated system of each of Ea and Eb; and
each of L2a and L2b independently contains a linking group having a
site imparting water solubility to the compound or a site that is
convertible into a site imparting water solubility to the
compound.
[0017] It is preferred that each of Ea and Eb of the formulas (1)
and (2) independently a group having oxidation-reduction activity
which is selected from the group consisting of a metallocene
moiety, a 2,2'-bipyridine complex moiety, a cyclobutadiene moiety,
a cyclopentadiene moiety, a 1,10-phenanthroline moiety, a
triphenylphosphine moiety, a cathecol amine moiety, and a biologen
moiety. Any of these moieties may have one or more
substituents.
[0018] In the formula (2), it is preferred that each of L1a and L1b
independently is a hydrocarbyl group which may have one or more
substituents. The hydrocarbyl group preferably has 1 to 6 carbon
atoms in its main chain. More specifically, it is preferred that
each of L1a and L1b independently is an alkylene group having 1 to
6 carbon atoms or an alkenylene group having 2 to 6 carbon atoms.
Each group may have one or more substituents.
[0019] In the formula (2), it is preferred that each of L2a and L2b
independently is a linking group containing an atomic element other
than carbon element. It is particularly preferred that each of L2a
and L2b independently is a linking group containing N, O, or S.
Specifically, it is preferred that each of L2a and L2b
independently contains a linking group selected from the group
consisting of an amino bonding, an ester bonding, an ether bonding,
a thioether bonding, a diimide bonding, a thiodiimide bonding, a
thioaride bonding, an imino bonding, a carbonyl bonding, a
thiocarbonyl bonding, and 1,4-piperazinyl bonding, any bonding
possibly having one or more substituents. Most preferred is that
each of L2a and L2b independently contains --NHCO-- or
--CONH--.
[0020] In the formula (2), it is preferred that Ea is the same as
Eb, L1a is the same as L1b, and L2a is the same as L2b.
[0021] The invention also resides in an electroconductive threading
intercalator having oxidation-reduction activity which is
represented by the aforementioned formula (1) or (2).
[0022] The invention further resides in an electrochemical method
for detection of oligonucleotide samples or polynucleotide samples
which employs the above-mentioned threading intercalator of the
invention.
[0023] The invention furthermore resides in a process for
electrochemically detecting oligonucleotide samples or
polynucleotide samples complementary to a group of probe molecules
of nucleotide derivatives or their analogues fixed onto an
electrode substrate, which comprises the steps of:
[0024] bringing the group of probe molecules into contact with
oligonucleotide samples or polynucleotide samples in an aqueous
medium in the presence of a threading intercalator according to
claim 15 so as to form by hybridization a complex of the group of
probe molecules and the oligonucleotide samples or polynucleotide
samples in which the threading intercalator is intercalated;
and
[0025] detecting an electric current produced by applying an
electric potential to the electrode substrate.
[0026] The invention furthermore resides in a process for
electrochemically detecting oligonucleotide samples or
polynucleotide samples complementary to a group of probe molecules
of nucleotide derivatives or their analogues fixed onto an
electrode substrate, which comprises the steps of:
[0027] bringing the group of probe molecules into contact with
oligonucleotide samples or polynucleotide samples in an aqueous
medium so as to form by hybridization a complex of the group of
probe molecules and the oligonucleotide samples or polynucleotide
samples;
[0028] bringing a threading intercalator according to claim 15 in
contact with the formed complex so as to intercalate the
intercalator into the complex; and
[0029] detecting an electric current produced by applying an
electric potential to the electrode substrate.
[0030] In the above-mentioned processes, it is advantageous that
the electric potential applied to the electrode substrate is in the
range of 100 to 400 mV.
[0031] The invention furthermore resides in a kit for
electrochemically detecting oligonucleotide samples or
polynucleotide samples complementary to a group of probe molecules
of nucleotide derivatives or their analogues fixed onto an
electrode substrate, which comprises an electrode substrate having
a group of probe molecules of nucleotide derivatives or their
analogues fixed to its substrate, and an electroconductive
threading intercalator of the invention. It is preferred that the
nucleotide derivatives and their analogues are oligonucleotides,
polynucleotides, or peptide nucleic acids.
DETAILED DESCRIPTION OF THE INVENTION
[0032] The present inventors have assumed that the high electric
potential is required in the electrochemical detection of DNA
fragment samples using the conventional electroconductive threading
intercalator because the conjugated system present in the
electroconductive moiety such as a ferrocene moiety extends to the
.pi. electron-bonding group of the amide group (i.e., --NHCO--), so
that the electron density of electrons moving in the conjugated
system of the ferrocene moiety decreases. Based on the assumption,
the inventors have synthesized a new electroconductive threading
intercalator in which the conjugated system of the
electroconductive ferrocene moiety is present independently of the
.pi. electron-bonding group of the amide group, and studied the
function of the newly synthesized intercalator in the
electrochemical detection of DNA fragment samples. It has been
confirmed that the newly synthesized electroconductive intercalator
gives a peak electric current at an expected low electric potential
in the electrochemical detection.
[0033] The present invention has been made upon the above-mentioned
discovery.
[0034] As described hereinbefore, the compounds which function as
electroconductive threading intercalators in the electrochemical
detection of DNA fragment samples have the formula (1),
particularly the formula (2):
Ea-La-X-Lb-Eb (1)
Ea-L1a-L2a-X-L2b-L1b-Eb (2)
[0035] In the formulas (1) and (2), X represents a divalent cyclic
group which may have one or more substituents
[0036] The divalent cyclic group preferably is a plane cyclic
group. Examples of the divalent cyclic groups include a naphthalene
diimide group having two bonding sites at its two nitrogen atoms,
an anthracene group having two bonding sites at 2- and 6-positions
or 1- and 5- positions (preferably 2- and 6-positions), an
anthraquinone group having two bonding sites in the same manner as
in the anthracene group, a fluorene group having two bonding sites
at 2- and 6-positions, a biphenylene group having two bonding sites
at 2- and 6-positions, a phenantholene group having two bonding
sites at 2- and 7-positions, and a pyrene group having two bonding
sites at 2- and 7-positions. Preferred is a naphthalene diimide
group having two bondings at the nitrogen atoms. The substituent
can be a halogen atom (e.g., F, Cl, or Br), or an alkyl group
having 1 to 6 carbon atoms such as methyl, ethyl, or n-propyl.
[0037] In the formula (1), each of La and Lb independently is a
group which does not form a conjugated system in combination with
the conjugated system of each of Ea and Eb and at least one of
which has a site imparting water solubility to the compound or a
site that is convertible into a site imparting water solubility to
the compound. The site that is convertible into a site imparting
water solubility to the compound means such site that it can be
converted into a site imparting water solubility to the compound,
for instance, by contact with an aqueous acidic solution such as an
aqueous sulfuric acid. For instance, an imino group having a methyl
substituent can be converted into a site having a sulfate group by
contact with sulfuric acid. Thus formed site having a sulfate group
imparts to the compound a necessary water solubility. The site can
have an electric charge.
[0038] The water solubility is required for the compound in the
case that the compound functions in an aqueous medium as the
threading intercalator.
[0039] Each of La and Lb preferably has a hydrocarbyl group (which
may have one or more substituents) on the side adjacent to Ea and
Eb, respectively. The hydrocarbyl group corresponds to L1a and L1b
of the formula (2) and further has a group having atomic elements
other than carbon atoms on the side adjacent to X. The latter group
corresponds to L2a and L2b of the formula (2). Accordingly, La and
Lb are preferably represented by -L1a-L2a- and -L1b-L2b-,
respectively.
[0040] Each of L1a and L1b preferably is an alkylene group having 1
to 6 carbon atoms or an alkenylene group having 2 to 6 carbon
atoms, provided that each group may have one or more substituents.
Each of L2a and L2b preferably is a linking group containing N, O
or S.
[0041] Examples of the substituents for L1a and L1b include
hydroxyl, halogen, carboxyl, amino, cyano, nitro, formyl,
formylamino, alkyl having 1 to 6 carbon atoms, alkylamino having 1
to 6 carbon atoms, halogenated alkyl having 1 to 6 carbon atoms,
cycloalkylamono having 5 to 7 carbon atoms, dialkylamino having 2
to 12 carbon atoms, aryl having 6 to 12 carbon atoms, aralkyl
having 7 to 18 carbon atoms which contains alkyl of 1-6 carbon
atoms, aralkylamino having 7 to 18 carbon atoms which contains
alkyl of 1-6 carbon atoms, alkanoyl having 2 to 7 carbon atoms,
alkanoylamino having 2 to 7 carbon atoms, N-alkanoyl-N-alkylamino
having 3 to 10 carbon atoms, aminocarbonyl, alkoxycarbonyl having 2
to 7 carbon atoms, heterocyclic ring having 2 to 10 carbo atoms
which has 1 to 4 hetero atoms such as S, N, or O, and aryl having 6
to 12 carbon atoms in its ring structure which may have 1 to 5
substituents such as alkyl of 1-6 carbon atoms, alkoxy of 1-6
carbon atoms, or halogen. The number of the substituents preferably
is in the range of 1 to 12, more preferably 1 to 3, when the main
chain is an alkylene group having 1 to 6 carbon atoms. The number
of the substituents preferably is in the range of 1 to 10,
preferably 1 to 3, when the main chain is an alkenylene group
having 2 to 6 carbon atoms.
[0042] Each of L2a and L2b preferably is a linking group containing
one or more groups such as an amino bonding, an ester bonding, an
ether bonding, a thioether bonding, a diimide bonding, a
thiodiimide bonding, a thioamide bonding, an imino bonding, a
carbonyl bonding, a thiocarbonyl bonding, or 1,4-piperazinyl
bonding, any bonding possibly having one or more substituents. Each
of L2a and L2b preferably contains --NHCO-- or --CONH--.
[0043] Examples of the substituents for L2a and L2b include alkyl
having 1 to 3 carbon atoms (e.g., methyl or ethyl), acyl having 2
to 4 carbon atoms (e.g., acetyl), aryl having 6 to 20 carbon atoms
(e.g., phenyl or naphthyl), and aralkyl having 7 to 23 carbon atoms
which has alkyl of 1-3 carbon atoms (e-g., benzyl).
[0044] When L2a or 12b contains an imino bonding, the imino bonding
preferably contains a methyl substituent Accordingly, each of L2a
and L2b preferably is N-methyl-di(n-propylenyl)imino or
1,4-di(n-propylenyl)piper- azinyl. Most preferred is
N-methyl-di(n-propylenyl)imino.
[0045] Each of Ea and Eb has oxidation-reduction activity so that
each has electroconductivity. It is preferred that each of Ea and
Eb independently is a metallocene moiety, a 2,2'-bipyridine complex
moiety, a cyclobutadiene moiety, a cyclopentadiene moiety, a
1,10-phenanthroline moiety, a triphenylphosphine moiety, a cathecol
amine moiety, and a biologen moiety. Any moieties may have one or
more substituents. Preferred are ferrocene moieties which may have
one or more substituents. Examples of the substituted ferrocene
moieties are illustrated below. 2
[0046] In the above-illustrated substituted ferrocene moieties, the
substituent may be present in other positions on the
cyclopentadienyl group.
[0047] The compound of the invention which is favorably employed as
an electroconductive threading intercalator can be prepared by a
process similar to the process described in the aforementioned
Japanese Patent Provisional Publication No. H9-288080.
[0048] Alternatively, the compound of the invention can be
efficiently synthesized from a known diamine compound in accordance
with the following synthesis route: 3
[0049] The above-illustrated synthesis route comprises three
reactions <a>, <b>, and <c>. Although the core
portion (i.e., naphthalene diimide structure) can have linkers
differing from each other which is synthesized two different
diamine compounds, the synthesis procedures are described for its
representative compound having a symmetric structure.
[0050] The reactions participating in the synthesis route are
explained below.
[0051] The compound (4), namely, A--NH--R--NH.sub.2, can be
synthesized from a known diamine (5) by the known method described
in Green T. W., P.G.M., Protective Groups in Organic Synthesis (2nd
Edition, Wiley, New York, 1991, 315-345, 349-359). "A" preferably
is acyl having 2 to 5 carbon atoms, alkoxycarbonyl having 2 to 5
carbon atoms, benzoyl which may have one or more substituents, or
benzyloxycarbonyl which also may have one or more substituents.
Preferred are acetyl, t-butylcarbonyl (i.e., pivaloyl),
t-butoxycarbonyl, or benzyloxycarbonyl possibly having one or more
substituents. Examples of the substituents possibly attached to the
benzoyl or benzyloxycarbonyl include halogen atoms, hydroxyl, alkyl
having 1 to 6 carbon atoms, and alkoxy having 1 to 6 carbon atoms.
The number of the substituents preferably is in the range of 1 to
5. Most preferred is 1.
[0052] The protected amine (4) can be generally obtained by the
reaction of the diamine compound (5) with an acid halide or an acid
anhydride having a moiety of A. Preferred examples of the acid
halides and acid anhydrides are reactive reagents having a
releasable group such as benzotriazol-1-yl-oxy (OBt),
succinimidyloxy (OSu), or 3-thiazolidine-2-thion. Preferred reagent
is 3-benzyloxy-carbonyl-1,3-thi- azoline-2-thion. The diamine (5)
is employed in an excessive amount as compared with the
above-mentioned reactive reagent, such as 3 to 10 molar times.
[0053] The reaction can be performed in the presence of an organic
base or an inorganic base. Examples of the organic bases include
pyridine, triethylamine, and diisopropylethylamine. Examples of the
inorganic bases include sodium hydroxide, potassium hydroxide,
sodium hydrogen carbonate, sodium carbonate, and potassium
carbonate. The base is preferably employed in a molar amount of 0.1
to excessive amount, more preferably 1 to 10 molar amounts, per one
molar amount of the reactive reagent such as an acid halide.
[0054] The reaction is preferably carried out in a solvent, but the
reaction can be carrier out in the absence of a solvent. The
solvent should dissolve all or a portion of the reactive compounds
and the reaction products and does not participate in the reaction
Examples of the solvents include alcohols (e.g., methanol, ethanol,
isopropyl alcohol, and ethylene glycol), amides (e.g.,
dimethylformamide, dimethylacetamide, acetamide, and
N-methylpyrrolidone), nitrites (e.g., acetonitrile and
n-butylonitrile), ethers (e.g., ethylene glycol monomethyl ether,
ethylene glycol monoethyl ether, tetrahydrofuran, and dioxane),
dimethylsulfoxide, sulforane, and water. The solvents can be
employed in combination.
[0055] The reaction can be performed under chilling conditions or
heated conditions. Generally, the reaction is performed at a
temperature in the range of -50.degree. C. to 150.degree. C.,
preferably -10.degree. C. to 100.degree. C.
[0056] The compound (3), that is, A--NH--R--NHCO--Q--E, can be
prepared by condensing the protective amine compound (4) with
MO.sub.2C--Q--E or M.sup.1OC--Q--E - - - Reaction <a>. M is
hydrogen, alkali metal (e.g., Na, K), and an imide having a bonding
site at the nitrogen atom, such as succinimide, phthalimide, or
glutalimide. M.sup.1 is an active group such as halogen,
--SO.sub.2Cl, or a group corresponding to the below-mentioned
reaction intermediate with the condensation reagent.
[0057] The reaction <a>is a condensation reaction between an
amino group and a carboxyl group. The condensation reaction can be
carried out in the manner described in Larock R. C., Comprehensive
Organic Transformations (VCH, New York, 1989, 972-97) The
condensation reaction is preferably carried out using a
condensating agent. The condensating agent preferably is
1,3-dicyclohexylcarbodiimide or
1-ethyl-3-(3'-dimethylaminopropyl)carbodiinide. The reaction can be
preferably performed in the presence of a solvent. The solvent
should dissolve whole or a portion of the reactive compounds and
the reaction products and should no participate in the reaction.
Examples of the employable solvents include, in addition to the
solvents described for the aforementioned reaction <a>,
halogen atom-containing solvents (e.g., dichloromethane,
chloroform, and 1,2-dichloroethane) and esters (e.g., acetate
esters). Tne organic solvents can be employed in combination. Water
and a mixture of water and the organic solvent also can be
favorably employed. In the reaction, the carboxylic acid (6) is
preferably employed in an excessive amount compared with the
compound (4) in 1 to 2 molar amounts. To the compound (4) is
preferably added the compound of M.sup.1OC--Q--E in an excessive
amount such as 1 to 3 molar amounts.
[0058] When 1,3-dicyclohexylcarbodiimide or
1-ethyl-3-(3'-dimethylaminopro- pyl)carbodiimide is employed as the
condensating agent, an acidic or basic additive can be used in
combination. Preferred acidic additives are N-hydroxysuccinimide,
N-hydroxybenzotriazole, and 3,4-dihydroxy-4-oxo-1,2-
,3-benzotriazine. Preferred basic additives are tertiary amines
(e-g., triethylamine), pyridine, and 4-dimethylaminopyridine. The
additive can be employed in an excessive amount such as 0.1 mole or
extremely excessive amount, preferably 1 to 3 molar amount, per one
mole of the condensating agent. The reaction can be carried out
under chilling conditions or heated conditions. Generally, the
reaction is performed at a temperature in the range of -50.degree.
C. to 150.degree. C., preferably -10.degree. C. to 100.degree. C.,
more preferably 0 to 50.degree. C.
[0059] The reaction <b>is performed for removal of the
protective group of the amino group. The reaction for removing the
protective group can be performed under conditions which ate
selected depending upon the nature of the protective group A.
Generally, the reaction conditions described in the aforementioned
Protective Groups in Organic Synthesis are employed. Preferred is a
method using iodotrimethylsilane. The reaction is preferably
performed in a solvent, but can be conducted in the absence of a
solvent. The solvent should dissolve whole or a portion of the
reactive compounds and the reaction products, and should not
participate in the reaction. Examples of the solvents include
halogenated solvents (e.g., dichloromethane and dichloroethane),
nitriles (e.g., acetonitrile and n-butylonitrile), ethers (e.g.,
tetrahydrofuran and dioxane), aromatic solvents (e.g. toluene and
benzene), and their mixtures. In the reaction, an excessive amount
of iodotrimethylsilane [excessive compared with the compound (3)],
such as 1 to 10 molar amount, is preferably employed. The reaction
can be carried out under chilling conditions or heated conditions.
Generally, the reaction is performed at a temperature in the range
of -50.degree. C. to 150.degree. C., preferably -10.degree. C. to
100.degree. C., more preferably 0 to 50.degree. C.
[0060] The reaction <c>is a reaction for condensating under
dehydration the compound (2) with the naphthalenediimide core (7)
to form side chains, so as to give the compound (1). The
naphthalenediimide can be favorably prepared from
1,4,5,8-naphthalene tetracarboxylic acid dianhydride. The reaction
can be performed in a solvent which should dissolve whole or a
portion of the reactive compounds and the reaction products and
should not participate in the reaction. Most of the aforementioned
solvents are employable. In the reaction, two or more equivalent
moles of the compound is preferably employed for one mole of
1,4,5,8-naphthalene tetracarboxylic dianhydride. Some solvents can
be employed in an amount of less than the two equivalent moles. The
reaction can be carried out under chilling conditions or heated
conditions. Preferably, the reaction is performed at a temperature
in the range of 0.degree. C. to the reflux temperature of the
employed solvent.
[0061] The electrochemical processes for detecting nucleic acid
samples according to the invention are further described below.
[0062] The process of the invention for electrochemically detecting
oligonucleotide samples or polynucleotide samples complementary to
a group of probe molecules of nucleotide derivatives or their
analogues fixed onto an electrode substrate, of the invention
comprises the steps of:
[0063] bringing the group of probe molecules into contact with
oligonucleotide samples or polynucleotide samples in an aqueous
medium in the presence of a threading intercalator of the invention
so as to form by hybridization a complex of the group of probe
molecules and the oligonucleotide samples or polynucleotide samples
in which the threading intercalator is intercalated; and
[0064] detecting an electric current produced by applying an
electric potential to the electrode substrate.
[0065] Alternatively, the process of the invention comprises the
steps of:
[0066] bringing the group of probe molecules into contact with
oligonucleotide samples or polynucleotide samples in an aqueous
medium so as to form by hybridization a complex of the group of
probe molecules and the oligonucleotide samples or polynucleotide
samples;
[0067] bringing a threading intercalator of the invention in
contact with the formed complex so as to intercalate the
intercalator into the complex; and
[0068] detecting an electric current produced by applying an
electric potential to the electrode substrate.
[0069] The electrode employed for fixing probe molecules such
should receive the probe molecules for fixing them onto the surface
of the electrode. Preferred materials of the electrode are gold,
glassy carbon, and carbon. In the practical use of the detection
system of the invention, a number of electrodes are preferably
combined to form one analytical chip.
[0070] The probe molecule which is a single stranded DNA fragment
can be obtained from DNA or its fragment which is obtained by
extraction from a living body, cleavage by restriction enzyme,
separation by electrophoresis, and denaturation by heat-treatment
or alkaline-treatment. The single stranded oligonucleotide can be
chemically synthesized. In any case, it is preferred that the
single stranded probe oligonucleotide such as DNA fragment for the
probe molecules is previously analyzed for base sequencing
according to the known methods.
[0071] The probe molecule is then fixed onto an electrode. The
fixation method is already known. For instance, a thiol group is
attached to 5'- or 3'-terminal (5'-terminal is preferred) of the
probe molecule, such as, oligonucleotide or polynucleotide, and the
attached thiol coordinates a gold atoms of the electrode. The
method for incorporating a thiol group to the DNA is described in
M. Maeda et al., Chem. Lett., 1805-1806 (1994) and A. Connolly,
Nucleic Acids Res., 13, 4484 (1985).
[0072] In the fixation process, the probe molecule having thiol
terminal is dropped onto the gold electrode, and then the desired
probe molecule is fixed on the electrode after allowing it to stand
for a few hours at a low temperature.
[0073] In the use of a glassy carbon electrode, the electrode is
oxidized by potassium permanganate to produce carboxyl groups on
the surface of the electrode. On the surface having carboxyl groups
is dropped the probe molecule having thiol terminal, so that an
amide bonding is formed to fix the probe molecule onto the surface
of the glassy carbon electrode. Details of this method are
described in K. M. Millan et al., Analytical Chemistry, 65,
2317-2323 (1993).
[0074] The hybridization is carried out in the presence of the
electroconductive threading intercalator of the invention, which is
preferably used in a concentration of several nM to several mM. The
intercalator can accelerate the hybridization between the probe
oligonucleotide and a sample DNA fragment and per se inserts into
the complex structure of the hybridized DNA so that the hybridized
DNA is stabilized. Thus produced complex of the intercalator and
the hybrid DM can be understood as a polymer having on its side a
number of ferrocene moieties. Thus aligned ferrocene moieties serve
to assist the electron transfer between the electrode on which the
probe molecules are fixed and a counter electrode which is placed
in an aqueous solution in which the detection procedures are
performed.
[0075] The fixation of the DNA fragment sample to the probe
molecule of the electrode can be detected by applying an electric
potential to the electrode of DNA chip. In the detection, a counter
electrode is employed.
[0076] There are no specific limitations with respect to the
electric potential applied to the electrode. However, since the
hybride structure having the electroconductive threading
intercalator of the invention gives a peak electric current even
when a low electric potential such as 400 mV or lower is applied.
Accordingly, it is advantageous to employ a electric potential in
the range of 100 to 400 mV, particularly 200 to 400 mV for applying
to the electrode of the DNA chip when the electrochemical detection
procedure is performed.
[0077] The threading intercalator of the invention can be also
favorably employable for detecting DNA fragment samples which are
partly complementary to the probe molecules. Such fragment samples
are generally referred to as "mis-match fragment". The detection of
the mis-match fragment can be performed by comparing the strength
of the peak current obtained in the detection of the possibly
mis-matched DNA fragment with the strength of the corresponding
peak current obtained in the detection of a fully complementary DNA
fragment (i.e., full-match fragment).
[0078] The present invention is further described by the following
examples.
Preparation of Threading Intercalator of Invention
[0079] Preparation of
N,N'-bis(7-ferrocene-acetamido-4-methyl-4-azaheptyl)- naphthalene
imide 4
[0080] (1) Preparation of
N-1-benzyloxycarbonyl-1,7-diamino-4-methyl-azahe- ptane
[0081] In dichloromethane (400 mL) was dissolved
di(3-aminopropyl)-N-methy- lamine (73.0 g, 500 mmol.) To the
resulting solution was dropwise added a solution of
3-benzyloxycarbonyl-1,3-thiazolidine-2-thione (12.8 g, 50 mmol.,
Synthesis, 1990, 27) in dichloromethane (100 mL). The mixture was
stirred for 3 hours at room temperature. The resulting precipitate
was removed by filtration. To the filtrate were added ethyl acetate
and water. The aqueous mixture was then extracted twice with ethyl
acetate. The ethyl acetate portion was combined and washed
successively with water and saturated aqueous sodium chloride
solution. The washed ethyl acetate portion was then subjected to
extraction with two portions of 1 N aqueous hydrochloric acid. The
obtained aqueous portions were combined and washed with ethyl
acetate. To the aqueous portion was added 6 N aqueous sodium
hydroxide solution under chilling to adjust the aqueous portion at
pH 9-10. The alkaline solution was extracted with ethyl acetate.
The ethyl acetate portion was washed with saturated aqueous sodium
chloride solution, dried over anhydrous sodium sulfate, and placed
under reduced pressure to distill the solvent out, so as to obtain
9.4 g of the desired product, yield 66%.
[0082] .sup.1H-NMR (300 MHz, CDCl.sub.3) .delta.: 1.58-1.72 (4H,
m), 2.20 (3H, s), 2.35-2.45 (4H, m), 2.64 (2H, t), 3.23-3.32 (2H,
m), 5.15 (2H, s), 7.22-7.45 (5H, m).
[0083] MS:FAB 280 (M.sup.++1) (matrix: m-nitrobenzene)
[0084] (2) Preparation of
N-1-benzyloxycarbonyl-1-amino-7-ferrocene-acetam-
ido-4-methyl-4-azaheptane
[0085] The N-1-benzyloxycarbonyl-1,7-diamino-4-methyl-azaheptane
(3.0 g, 11 mmol.) obtained in (1) above was dissolved in
dichloromethane (30 mL). To the resulting solution were added
ferrocene-acetic acid (2.7 g, 11 mmol.), pyridine (2 mL) and ethyl
N,N'-dimethylaminopropylcarbodiimide (2.3 g, 12 mmol.). The mixture
was then stirred for 3 hours at room temperature. To the reaction
mixture was added an aqueous ammonium chloride solution. The
mixture was extracted twice with ethyl acetate, and the ethyl
acetate portions were combined. The ethyl acetate portion was
washed with saturated aqueous sodium chloride solution and placed
under reduced pressure to distill the solvent off. The residual
brown oil was processed by column chromatography (column: alumina,
eluent:chloroform/methanol=20/1). The obtained crystalline product
was washed with a mixture of hexane and ethyl acetate to give 2.6 g
of the desired product (yield: 91%) as an orange-colored
crystalline product.
[0086] .sup.1H-NMR (300 MHz, CDCl.sub.3) .delta.: 1.50-1.72 (4H,
m), 2.08 (3H, s), 2.20-2.33 (4H, m), 3.15-3.30 (4H, m), 3.34 (2H,
s), 4.15 (5H, s), 4.16 (4H, s), 5.15 (2H, s), 5.54 (1H, bs), 6.44
(1H, bs), 7.32-7.48 (5H, m).
[0087] (3) Preparation of
1-amino-7-ferrocene-acetamido-4-methyl-4-azahept- ane
[0088] In acetonitrile (30 mL) was dissolved the
N-1-benzyloxycarbonyl-1-a-
mino-7-ferrocene-acetamido-4-methyl-4-azaheptane (1.6 g, 3.0 mol.)
obtained in (2) above. The mixture was stirred at room temperature,
and to this stirred mixture was dropwise added trimethylsilane
iodide (1.25 mL, 8.8 nmol.). After 5 minutes, 1 N aqueous
hydrochloric acid and ethyl acetate were added to the reaction
mixture. The reaction mixture was then extracted three times with 1
N aqueous hydrochloric acid. The aqueous portion was washed with
ethyl acetate, and then chilled with ice. To the chilled aqueous
portion was added 2N aqueous potassium hydroxide solution to adjust
the aqueous solution to pH 10. The alkaline aqueous solution was
extracted twice with chloroform. The chloroform portion was washed
with saturated aqueous sodium chloride solution, and placed under
reduced pressure to distill the solvent off, to give 1.0 g of the
desired product (yield 70%) as a brown crystalline product.
[0089] .sup.1H-NMR (300 MHz, CDCl.sub.3) .delta.: 1.48-1.62 (4H,
m), 2.09 (3H, s), 2.25-2.35 (4H, m), 2.71 (2H, t), 3.22-3.33 (2H,
m), 3.35 (2H, s), 4.1-4.21 (9H, m), 6.75 (1H, bs).
[0090] (4) Preparation of
N,N'-bis(7-ferrocene-acetamido-4-methyl-4-azahep- tyl)naphthalene
diimide
[0091] In tetrahydrofuran (50 mL) was dissolved the
1-amino-7-ferrocene-acetamido-4-methyl-4-azaheptane (0.95 g, 2.5
mmol.) obtained in (3) above. The mixture was stirred at room
temperature. To the stirred mixture was added
1,4,5,8-tetracarboxylic acid naphthalene dianhydride (0.3 g, 1.1
mmol.). The mixture was then refluxed for 7 hours. The reaction
mixture was filtered and washed with chloroform. The organic
portions were combined and placed under reduced pressure. The
resulting residue was processed by column chromatography (column:
alumina, eluent: chloroform/methanol=15/1). The obtained
crystalline product was washed with ethyl acetate to give 0.32 g of
the desired product (yield: 30%) as a brown crystalline
product.
[0092] .sup.1H-NMR (300 MHz, CDCl.sub.3) .delta.: 1.56-1.70 (8H,
m), 1.78-1.92 (4H, m), 2.12 (6H, s), 2.33-2.46 (8H, m), 3.30-3.42
(4H, m), 3.36 (4H, s), 4.13 (10H, s), 4.20 (8H, s), 6.85 (2H, bs),
8.80 (4H, s).
[0093] MS:FAB 975 (M+H) (matrix: m-nitrobenzene)
EXAMPLE 1
Detection of Hybrid DNA Fragment
[0094] (1) Manufacture of electrochemical analytical element
[0095] On a gold electrode (surface area: 2.25 mm.sup.2) was
spotted 2 .mu.L of an aqueous solution containing 100
picomol./.mu.L of T.sub.20 (thymine 20-mers having an aminohexyl
group at its 5'-terminal) The spotted solution was allowed to stand
for one hour at room temperature, and the unfixed T.sub.20 was
washed out, and dried, to give an electrochemical analytical
element. The preparation of T.sub.20 and its fixation were carried
out in the manner described in the aforementioned Japanese Patent
Provisional Publication No. H9-288080.
[0096] (2) Preparation of ferocene-labeled oligonucleotide
[0097] Adenine 20-mers (dA.sub.20) sample was prepared in the
mannter as described in the above-mentioned Publication, and
employed as a DNA fragment sample.
[0098] (3) Detection of hybrid DNA fragment
[0099] On the analytical element prepared in (1) above was spotted
2 .mu.L of a Tris buffer (10 mM, pH 7.5) containing the dA.sub.20
obtained in (2) above. The analytical element was then kept at
25.degree. C. for 20 minutes for performing incubation. The
incubated element was washed with an aqueous solution of 0.1 M
sodium dihydrogen phosphate-disodium hydrogen phosphate (pH 7.0) to
remove the unfixed dA.sub.20.
[0100] Thus treated element was placed in 0.1 M potassium
chloride-0.1 M acetic acid buffer (pH 5.6) containing 50 .mu.M of
the threading intercalator prepared in the aforementioned
preparation example, and subjected to differential pulse
voltammetry (DVP) in the applied voltage range of 100 to 700 mV,
pulse oscillation 50 mV, pulse width 50 ms, and a scanning rate 100
mV/sec. A responsive electric current as a peak value at 260 mV was
detected.
[0101] For obtaining control current, the same procedures except
for employing the intercalator were repeated.
[0102] The responsive electric current obtained at 260 mV using the
intercalator of the invention is as high as 36%, as compared with
the control current.
Comparison Example 1
Detection of Hybrid DNA981 Fragment
[0103] The same procedures as in Example 1 were repeated except for
employing the conventional intercalator described in the
aforementioned Japanese Patent Provisional Publication H9-288080.
The responsive current as a peak value was detected at 460 mV.
[0104] The responsive electric current obtained at 460 mV using the
conventional intercalator is as high as 38%, as compared with the
control current.
[0105] The results of Example 1 and Comparison Example 1 indicate
that the threading intercalator of the invention gives at 260 mV a
peak current strength which is almost equal to the peak current
strength at 460 mV obtained in the use of the conventional
threading intercalator.
EXAMPLE 2
Detection of Hybrid DNA Having Mis-match Structure
[0106] (1) Manufacture of electrochemical analytical element
[0107] The procedures of Example 1-(1) were repeated except for
using dT.sub.19G.sub.1 (corresponding to mis-match oligonucleotide)
to manufacture an analytical element.
[0108] (2) Detection of hybrid DNA having mis-match structure
[0109] The procedures of Example 1-(3) and the procedures of
Comparison Example 1 were repeated except for using the analytical
element manufactured in (1) above, to give a peak electric current
of 36% increased from the control value at 260 mV in the use of the
intercalator of the invention, and a peak electric current of 20%
increased from the control value at 260 mV in the use of the
conventional intercalator.
* * * * *