JPH07313199A - Detection of target substance in specimen - Google Patents

Abstract

PURPOSE:To detect a target substance in high sensitivity without causing B/F separation by reacting a specimen with plural reagents containing pyrylium(- analog) compound constituting a reaction system varying by a mutual action interposing the target substance and determining the change of the reaction system. CONSTITUTION:A target substance in a specimen is detected in high sensitivity without causing B/F separation by reacting a specimen with two kinds of reagents containing a pyrylium compound, a pyrylium-analog compound, etc. constituting a reaction system varying by a mutual action interposing the target substance and expressed by formula I {the ring is a heterocycllc group expressed by formula II (X is 0, S<2> Se or Te), pyrylium ring or pyrylium-analog ring; R<1> and R<2> each is H, a halogen, a sulfonate, amino, styryl, nitro, OH, COOH, cyano, a (substituted)alkyl, a (substituted)aryl, etc.; R<3> is A [A is a (substituted) aryl or CH=R<5> (R<5> is a heterocyclic group, a cycloalkyl, an aromatic ring, etc.)] or L-A (L is CH=CH, an arylene, an ankylene, etc.)} and determining the change of the reaction system.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to the use of a plurality of reagents capable of forming a reaction system in the presence of a target substance as a detection target, which undergoes a change based on an interaction mediated by the target substance. The present invention relates to a method for detecting, for example, a method useful for detecting and identifying a desired base sequence of a nucleic acid (DNA or RNA) of virus, microorganism, animal or plant, human or the like, or detecting presence or absence of mutation in various base sequences.

[0002]

2. Description of the Related Art A large number of various mutant genes have been found out by the development of nucleic acid analysis techniques, and various genetic diseases based on gene mutations are being elucidated. Among them, it was revealed that some of the bases in the gene were partially deleted and some of the bases had point mutations, which caused mutations in the protein and caused various symptoms. Is coming. At present, these genetic diseases are
After the symptom appears, it is usually found by an enzyme assay or an immunological method using an antibody.
From the viewpoint of early treatment, it has been pointed out that it is important to detect the presence or absence of a mutation in a gene early before a serious symptom appears.

DNA diagnosis is not only used for human genes, but can also be used for identifying infected bacteria.

Conventionally, a method of identifying bacterial species based on similarity based on the morphological and biochemical properties of the isolated bacteria has been used. In this method, there are problems that it takes a long time for culturing, the determination of the properties is different depending on the test method, and the identification result is different depending on which property is emphasized.

Therefore, in recent years, especially in the field of detection and identification of causative bacteria in bacterial infections, DNA-DNA
Attempts have been made to use the hybridization method or the DNA-RNA hybridization method.
This method extracts a nucleic acid (DNA or RNA) from bacteria, focuses on a specific portion of the nucleic acid derived from the bacterium, and a base sequence having a high homology with the base sequence of the portion is detected in the target test nucleic acid sample. It is a method of determining whether or not the problem bacteria are present in the sample by examining the presence or absence of the bacterium in the sample by a hybridization method.

[0006] In recent years, the PCR method has also been used as a new method for detecting a specific sequence of a nucleic acid. This method selects a specific sequence in a target nucleic acid as a detection target, prepares a primer necessary for amplification of this specific sequence, performs PCR using the target nucleic acid as a template, and detects the presence or absence of the amplified specific sequence. As a result, the target nucleic acid can be detected. The detection sensitivity of nucleic acids is improved by using the PCR method, and is used for detecting nucleic acids in various fields in place of the nucleic acid detection method using the hybridization method.

However, in the PCR method, when the specific base sequence to be investigated is clear, the presence or absence of the specific sequence can be investigated for the first time by selecting the optimum primer for the amplification. Often, a sequence is formed that binds to the target nucleic acid or is otherwise not amplified. In addition, a gene abnormality such as a deficiency in a target nucleic acid can be obtained by analyzing its length, but it can be obtained from an abnormality such as a point mutation in which the length does not change. Has the drawback of not knowing.

Therefore, the hybridization method is not completely replaced by the PCR method,
A simple gene detection method by the hybridization method is also required.

In the hybridization reaction, the probe DNA and the target DNA form a hybrid by hydrogen bonding at their complementary sequence portions. In the hybridization reaction, it is necessary to optimally select the reaction temperature and ionic strength in order to form a hybrid accurately between complementary sequences. That is, if the temperature is too high, the probe cannot bind to the nucleic acid having a complementary sequence, and if the temperature is too low, the probe nonspecifically binds to the nucleic acid. Furthermore, for more accuracy, lower the salt concentration of the solution or raise the temperature of the solution to remove unstable hydrogen bonds and wash away nonspecifically bound probes or mismatched probes. Is important. Therefore, a considerable amount of trial and error is required to set appropriate reaction conditions and washing conditions.

In the gene diagnosis, the hybridization reaction and the washing conditions are required to be more accurate in removing the mismatch at the single base pair level.

The hybridization reaction has conventionally been carried out with the target nucleic acid immobilized on a carrier such as nitrocellulose. However, since this method is extremely complicated in operation, development of a simple method such as hybridization in a solution has been expected. The biggest problem in the conventional method of not immobilizing nucleic acid is how to distinguish the probe bound to the target nucleic acid from the excess probe not bound (B / F separation).
There is. Further, also in this case, similarly to the above-mentioned hybridization reaction using immobilized nucleic acid, setting of appropriate reaction conditions and washing conditions for removing nonspecific adsorption of probe and mismatch is an important issue.

As a method for detecting a hybrid of a target nucleic acid and a probe without performing B / F separation, some detection methods using a fluorescence depolarization method have been proposed (JP-A-2-295496). Japanese Patent Laid-Open No. 2-759
58, etc.). In these methods, a fluorescent-labeled single-stranded DNA probe is brought into contact with DNA in an analysis sample to form double-stranded DNA, and fluorescence polarization before double-strand formation and fluorescence polarization after double-strand formation are performed. Is a method for detecting whether or not a base sequence corresponding to the base sequence of the probe is present in the DNA in the sample by measuring the mutation. In this method, the principle of detection is that the fluorescent substance bound to the single-stranded probe becomes difficult to move due to the double-stranded structure and fluorescence anisotropy increases.

However, in these methods, the sample contains contaminants such as proteins, which is the probe DNA.
Non-specifically adsorbing onto the syrup causes an increase in the background for detection of hybrids, and therefore a complicated work of completely removing these contaminants in advance is required. Further, in the case of other solution systems, the pseudo-hybrid body due to nonspecific adsorption of probe DNA and base mismatch requires an operation to remove it in advance. Furthermore, although it is true that B / F separation is not necessary, it is necessary for measuring the mutation of fluorescence that the probe concentration is approximately the same as the target DNA concentration.

As a hybrid detection method using energy transfer, Gardullo et al. Have proposed three methods (Proc. Natl. Acad.
Sci. USA, 85 , 8790-8794).
Each of these uses fluorescein, acridine orange as an energy donor, and rhodamine as an energy acceptor. (I) 5'-oligonucleotide labeled with fluorescein and 5'-rhodamine-complementary oligonucleotide complementary thereto Hybridization of (ii) 5'with fluorescein-labeled oligonucleotides respectively complementary DNAs, (iii) 5'-rhodamine-labeled oligonucleotides with complementary DNAs in the presence of acridine orange Hybridization.
This method is a phenomenon in which excited state energy of a donor is transferred to an adjacent acceptor when the excitation light and fluorescence of an adjacent fluorescent chromophore are overlapped, and as a result, the lifetime of the donor is reduced, Quenching of the fluorescence of the donor and enhancement of the fluorescence intensity of the acceptor are observed. It can be said that it is an epoch-making method in that it is possible to omit a series of complicated operations at once in that it can judge the presence or absence of hybridization as a solution, but the sensitivity is several orders of magnitude lower than that of the existing hybridase and it can be put to practical use. Therefore, improvements in fluorophores and breakthroughs in detection systems were desired.

Further, recently, when two kinds of dyes were mixed in a double strand of DNA in a free state, DN was generated between these dyes.
There is a report that charge transfer through A was detected (J. Ame. Chem. Sco., 1992, 11).
4 , 3656-3660). When a fluorescent electron donor (ethidium bromide or acridine orange) is irradiated with light having a wavelength corresponding to each excitation wavelength, another dye (electron acceptor: N, N-dim) is irradiated.
Ethyl-2,7-diazapyreniumdi
In the presence of chlorode), its fluorescence intensity decreases, and since both dyes are considered to be intercalators, electrons flow from the electron donor to the electron acceptor through the double helix of DNA. It is that there is. However, the degree of the change is very small, and the sensitivity is not sufficient to detect the hybrid.

[0016]

The conventional hybridization method in which a target nucleic acid is immobilized is, of course, B /
Even in a method such as fluorescence depolarization that does not require F separation, complicated processing is required to prevent nonspecific adsorption or mismatch of the probe or to remove the generated nonspecific adsorption or mismatch. Often there is. Moreover, since the optimum conditions for these operations differ depending on the length of the probe or each base sequence, it is necessary to examine and set the conditions in each case. In particular, the position of the mismatched base on the probe is also an important factor affecting the stability of the hybrid,
Depending on the position, it may not be possible to remove the mismatched hybrid, so it is necessary to set the conditions of the hybridization reaction according to each case, considering the possibility of mismatch, which requires more complicated work. .

Although a detection system in a solution utilizing charge transfer or energy transfer is a simple method as compared with a system for immobilizing a target nucleic acid or a system requiring B / F separation, its sensitivity is very high. It is too low to be put to practical use. Furthermore, the dye used in the conventional example has fluorescence even in a free state, and even if a change in fluorescence intensity is detected, it is an energy donor and an energy acceptor (or an electron donor and an electron acceptor). It is difficult to determine whether it is a DNA-mediated interaction with DNA, mere quenching by a solution, or an effect due to the presence of impurities.

The present invention has been made in view of the problems in the prior art as described above, does not require B / F separation in the detection of hybrids, has a simpler process, and has good measurement sensitivity. Another object of the present invention is to provide a method suitable for detecting a target nucleic acid capable of obtaining Another object of the present invention is to provide a method suitable for detecting a target nucleic acid, which can accurately detect only a desired hybrid even when there is a mismatched hybrid.

The present invention further provides a detection system useful for detecting various substances utilizing an immunoreaction such as an immunoassay.

[0020]

The method for detecting a target substance according to the present invention comprises reacting a sample with at least two kinds of reagents capable of forming a reaction system which causes a change based on an interaction mediated by the target substance. In the method for detecting a target substance in the sample by measuring a change based on the interaction that occurs when the target substance is present in the sample, at least one of the reagents constituting the reaction system is a pyrylium compound and a pyrylium analogue. It is characterized by being selected from compounds.

The interaction used in the present invention means an interaction caused by two substances via a reaction system containing a target substance. As this interaction, for example, charge transfer (charge transfer) or energy transfer (energy transfer) can be used. In the case of charge transfer, one of the two interacting substances is an electron donor and the other is an electron acceptor. In the case of energy transfer, one is an energy donor and the other is an energy acceptor. In the present invention, a reagent group containing two substances that cause such an interaction is used to cause the interaction in the presence of the target substance, and the resulting change is measured to detect the target substance. In some cases, a pyrylium compound or a pyrylium-like compound (hereinafter collectively referred to as a pyrylium-based compound) is used as at least one of the reagents constituting the reaction system for performing this interaction.

Further, the interaction between the reagents in the present invention is accompanied by the formation of these complexes during the reaction of the primary and secondary reactants with the substance to be detected in the immunoreaction such as immunoassay. Then, the reaction system using the above-mentioned reagents can be used for detecting an immune reaction. For example,
In the immunoassay, one of the primary reactant and the secondary reactant is labeled with a reagent that is an energy donor or an electron donor, and the other is labeled with a complementary acceptor (eg, energy acceptor or electron donor). Are labeled with a reagent such as an acceptor), these immune reactions occur, and energy transfer and charge transfer due to the proximity of the donor and acceptor resulting from the binding of the primary and secondary reactants. The migration produces a detectable signal, and the presence or absence of an immune response can be detected by detecting this signal. A pyrylium compound can be used as at least one of the labeling reagents.

The present invention will be described below by taking the case of detecting a nucleic acid double helix structure as a target substance as a representative example, but the present invention is not limited to this as long as it can be detected by the same action.

FIGS. 1 and 2 schematically show a model of a reaction system which causes an interaction used in the present invention, taking the case of charge transfer as an example. The one shown in FIG. 1 is the simplest reaction system composed of two reagents 1 and 2 that interact with a target substance 3 (nucleic acid double-helix structure). In this reaction system, the reagent 1 which serves as an electron donor. Through the target substance 3 to the reagent 2 serving as an electron acceptor in the arrow direction. In the reaction system of FIG. 2, in addition to reagents 4 and 5 that cause interaction, reagents 6 and 7 that mediate charge transfer in the reaction path are further added, and charges are transferred in the arrow direction. Taking the illustrated case as an example, a pyrylium compound is used for at least one of the reagents 1, 2, 4 to 7. The number of reagents is at least two, but the number can be set in consideration of the operation efficiency and detection sensitivity in detection.
As the reagents 6 and 7, those functioning as so-called mediators or sensitizers can be used. In addition, the reaction mediation is performed between the respective reaction substances in the reaction path shown in FIG. 2, for example, between the reagents 4 and 6, or between the reagent 6 and the target substance 3.
The above substances may be further involved as necessary. Therefore, the pyrylium compound is used as at least one of the reagents constituting the reaction pathway of the interaction, and one having a function depending on the role in the reaction system to be used is selected, for example, an electron donor, an electron acceptor. It is used as a body, mediator or sensitizer.

Reagents used for the purpose of directly reacting with the double helix structure, for example, Reagents 1, 2, 6, and 7 in FIGS. 1 and 2 have the property as an intercalator that specifically binds to the double helix structure. Those having are preferred. As the reagent in this case, for example, by reacting with a substance such as a nucleic acid that constitutes a double-helix structure to cause a chemical change or structural mutation on its own or on the component side of the nucleic acid,
Alternatively, a substance that reacts with a third substance added to the reaction system in the presence of a double helix structure to cause a change and thereby causes a change based on a detectable interaction can be used.

As the direct reaction, for example, a reaction which causes a charge transfer or an energy transfer between a component of a nucleic acid having a double helix structure and a reagent substance can be used. When charge transfer is used, the reagent substance used is a substance that can be inserted into the double-helix structure in a specific manner to serve as an electron donor or an electron acceptor with respect to the components of the nucleic acid.

Reactions based on this charge transfer include so-called through space and through.
The case of gh bond is considered. In the former case, for example, the electron donor and the electron acceptor interact through the stacking of nucleobase pairs, and the proximity of the electron donor and the electron acceptor accompanying the change to the double helix structure. Cases based on effects are included. The latter (thou
In gh bond), the transfer of charges including the base, the phosphate moiety or the sugar moiety constituting the nucleic acid is considered. In any case, the form is not limited as long as the direct reaction is derived from the formation of a double helix.

In the case of via stacking between nucleobase pairs, the distance between the electron donor and the electron acceptor (for example, the reagents 1 and 2 in FIG. 1) placed at a position capable of reacting with the double helix structure essentially interacts. When the electrons are far enough apart, the electrons emitted from the electron donor are transferred to the adjacent nucleobase pairs one after another through the electron cloud spreading over the nucleobase pairs.
Eventually, the electrons reach the electron acceptor. On the contrary, a mechanism is also established in which the electron acceptor pulls out an electron from the nucleic acid base pair, which is carried out in a chained manner, and finally the electron is taken from the electron donor. In other words, the mediator in charge transfer is nucleic acid base pair.

On the other hand, in the case of being based on the proximity effect of the electron donor and the electron acceptor, the distance between the electron donor and the electron acceptor is close enough to allow the interaction between them due to the formation of the double helix structure. That is the case. For example, even if both an electron donor and an electron acceptor are present, when they are in a single-stranded state where the distance between these compounds is sufficiently large in solution, they do not interact with each other, The formation of a double helix structure can be controlled by the interaction of the electron donor and the electron acceptor when they hybridize with the target nucleic acid to form a double helix structure and the electron donor and the electron acceptor are brought into close proximity to each other. It can be detected depending on whether an action has occurred. When charge transfer between the double-helix structure and the reagent substance is difficult to occur, a mediator that mediates charge transfer, or a substance called a sensitizer, is interposed between them as described above. May be.

As described above, the reagent used at the position where it reacts with the double helix structure needs to be placed at a position where it can react with the double helix structure to cause these reactions.
As a method of arranging such a reagent at a position where it can react with a double helix structure, when it enters between nucleobase pairs like an intercalator, when it is embedded in the groove of a double helix structure, It can be used when it is arranged so as to be close to the heavy spiral structure. In either case, it is essential for the present invention that the reagent be specifically located in the double helix structure of the hybrid.

Among these, the intercalator is
Most advantageous when utilizing charge transfer via stacking. So the intercalator is generally
It is a planar compound having an electron spread, and is oriented in a position parallel to the nucleic acid base pairs at the same distance as the distance between the nucleic acid base pairs on the extension line of the stacking of the nucleic acid base pairs. For example, if an intercalator is used as an electron donor and an electron acceptor is placed on the opposite side of the double helix structure, the electron released from the electron donor is sent to an adjacent nucleobase pair, which then Via the electron cloud of nucleobase pairs,
It can flow in a straight line towards the electron acceptor. Alternatively, on the contrary, if an intercalator is used as an electron acceptor and the electron acceptor is arranged on the opposite side across the double helix, an electron is drawn from an adjacent nucleic acid base pair by an electron hole on the electron acceptor, In some cases, this electron withdrawal occurs between other nucleic acid base pairs one after another, and finally the electron is withdrawn from the electron donor, and charge transfer is performed. Considering these points, in the case of charge transfer through stacking, at least one of an electron donor and an electron acceptor is preferably an intercalator, and when both are intercalators, further, It is more preferable because the charge transfer efficiency can be increased. Intercalators are known to stabilize the double-helix structure and increase its melting temperature, and the fact that electron donors and electron acceptors are intercalators stabilizes the hybrid between the probe and target nucleic acid. It is also advantageous in that it is made into a material.

In the present invention, the target substance is detected by measuring the change based on the interaction via the target substance. This change differs depending on the type of reaction system that causes an interaction and is composed of the target substance and the reagent used for its detection, and the method of detecting the change also differs depending on the configuration of the reaction system. For example, when reagents that interact with each other (for example, reagents 1, 2, 4, and 5 in FIGS. 1 and 2) that interact with each other based on charge transfer are used, the interaction of these reagents through the double helix structure is used. Can be regarded as a change on the electron acceptor side, and can be measured by a means suitable for the change. For example, like a charge transfer absorption band, it can be considered as the appearance or change of a new absorption spectrum. Further, a system in which the solution is colored or discolored as a result of charge transfer can be directly observed by the eye, and is more effective as a simple system. A light emitting system such as fluorescence or phosphorescence can also be used. In this case, it is possible to use a reaction in which fluorescence or phosphorescence is newly generated or a reaction in which a light-emitting substance disappears as a result of an interaction. In addition, a method in which an electron acceptor chemically changes to another substance as a result of electron transfer and is detected can also be used. At this time, it is also possible to add a third substance to the changed substance afterwards and cause chemiluminescence due to a chemical reaction between the two substances for detection. When a protein such as an enzyme or an antibody is used as the third substance, a bioluminescence detection method can be used. Moreover, when the target substance itself causes a detectable change, this can be detected.

As a detection method, the reaction may be detected by a change on the electron donor side in addition to the change on the electron acceptor. And, basically, most of the methods used in the case of electron acceptors can be directly applied. When a fluorescent substance is used as an electron donor, it is possible to detect a direct change such as the disappearance of fluorescence by using a substance in which the quantum yield of fluorescence decreases with the charge transfer. Can be used in combination with several reactions for visualization.

As described above, the change based on the interaction detected in the present invention may be a change in each reagent or target substance constituting the reaction system causing the interaction, and the change may be directly or directly. The measurement may be performed indirectly using another detection reagent.

On the other hand, in the present invention, the electron donor may be activated by light to emit electrons to start charge transfer, and further, as the third substance, the electron donor may be stimulated to emit electrons. There may be a substance that produces
Further, the electron acceptor may be activated and the electron may be extracted from the electron donor by being activated. And
As the initiator, in addition to light, as in the case of the electron donor,
It can be any initiator.

Further, as described above, in addition to the electron donor and the electron acceptor, as a third substance, a mediator that mediates charge transfer or a substance called a sensitizer intervenes. Good. Then, these substances may interact with the double helix to promote charge transfer to the electron donor and electron acceptor which are not directly bound to the double helix.

In the case of detecting a target nucleic acid by hybridization with a probe nucleic acid, a change due to the interaction may be, for example, a pyrylium compound or a compound other than the pyrylium compound which constitutes the reagent before and after formation of a desired hybrid, Alternatively, it is measured and detected as a change in the chemical structure or property of the constituent component of the nucleic acid that is the target substance, a change in the electronic state, or a change in the signal derived from the substance changed by this interaction.

Examples of the form using the electron donor and the electron acceptor include the following. (I) Both of them are present in the reaction system when the double helix structure is formed by the hybridization of the probe and the target nucleic acid. (Ii) Both of them are added to the reaction system after the double helix structure is formed by the hybridization of the probe and the target nucleic acid. (Iii) One of these is allowed to exist in the reaction system when the double helix structure is formed by the hybridization of the probe and the target nucleic acid, and then the other is added.

When utilizing energy transfer as an interaction, the reagent contains at least a set of energy donor and energy acceptor. The interaction between the two includes, for example, a change in the chemical structure of the energy acceptor, the energy donor, or the third substance capable of interacting with these substances before and after the formation of the hybrid, a change in the electronic state, or, It is detected as a change in the signal derived from the changed substance. For example, the hybrid can be detected by decreasing the fluorescence intensity of the energy donor and increasing the fluorescence intensity of the energy acceptor. In such a case, the energy donor and the energy acceptor enter the double-stranded portion of the hybrid by a mode such as intercalation, and the two are closer than in the solution,
It is considered that the interaction becomes possible and energy transfer occurs.

As the mode using the energy donor and the energy acceptor, the same modes (i), (ii) and (iii) as those for the electron donor and the electron acceptor can be used as they are. Light is mainly used as the energy transfer initiator.

When the method of the present invention is used for detection of a target nucleic acid by hybridization, the length of the probe is such that good hybridization with the target nucleic acid is possible and a stable double helix structure is obtained. It is appropriately selected in each case. An electron donor, an electron acceptor, an energy donor, or a reagent as an energy acceptor (eg, reagents 1, 2, 6, and 7 in FIGS. 1 and 2) is necessary for direct interaction with a nucleobase. Any length is possible as long as a stable double helix structure is possible, and the length is, for example, 8 bases or more, preferably 12 bases.
More than base.

However, in addition to the probe length, the base sequence itself, the salt concentration and the ionic strength of the reaction system greatly influence the stabilization of the double helix structure. G-C base pairs are A-
Due to the higher number of hydrogen bonds than T base pairs, a more stable double helix structure is formed in sequences with a high GC. Also, K
When the molar concentration of Cl is increased from 0.01M to 1M, D
The melting point temperature of NA is said to rise by 30 ° C. The presence of the intercalator also greatly contributes to stability. Therefore, a probe having a length of less than 8 bases can be used by appropriately using these stabilizing factors.

The properties of the electron donor, the electron acceptor, the energy donor, and the energy acceptor are also factors to be considered in selecting the probe. There is no guarantee that these reagent substances will enter the same sequence in any sequence of the double helix. Those who prefer G-C base pairs and AT
The properties differ depending on each reagent such as those that prefer base pairs. Therefore, in addition to the stability of the double helix composed of the probe and the target nucleic acid,
It is important to consider the properties of such reagents.

The change based on the interaction between the electron donor and the electron acceptor is preferably irreversible. That is, if the electron donor or the electron acceptor used for detection changes irreversibly, the change can be accumulated and detected, which is advantageous in terms of sensitivity.

As the pyrylium compound used in the present invention, for example, a compound represented by the following general formula [I] can be used.

[0046]

[Chemical 3] In the above general formula [I],

[0047]

[Chemical 4] Represents a heterocycle, and X is O, S, Se or Te. Examples of the heterocycle include 5- and 6-membered rings such as a pyrylium ring and a pyrylium-like ring.

R ¹ and R ² are each independently a hydrogen atom, halogen atom, sulfonate group, amino group, styryl group, nitro group, hydroxyl group, carboxyl group, cyano group, substituted or unsubstituted lower alkyl group, substituted Alternatively, it represents an unsubstituted aryl group, a substituted or unsubstituted lower aralkyl group, or a substituted or unsubstituted cycloalkyl group.

R³Is -A or -LA. L
Is -L¹-, -L²-L³-Or-L ^Four-L^Five-L⁶-In
Yes, L¹~ L⁶Are each independently-(CH = CH)
-, Divalent derived from a substituted or unsubstituted aryl group
Group, a substituted or unsubstituted lower alkylene group or -C
H = R^Four-(R^FourRepresents a ring structure having an oxo group)
Forget 2 derived from a substituted or unsubstituted aryl group
Examples of the valent group include a phenylene group and the like.
Yes, it can be attached at any of the ortho, meta, or para
May be The lower alkylene group has a carbon number of 1 to
To list 4 straight or branched alkylene groups
And a substituent thereof is, for example, represented by -LA.
Groups may be mentioned. Ring structure with oxo group
A heterocyclic ring, an aromatic ring, an aliphatic ring, etc.
Examples thereof include those having a so group.

As -L-, the following general formula [II],
The groups represented by [III], [IV], [V] or [VI] can be mentioned as preferable ones.

[0051]

[Chemical 5] (In the above general formula [II], Z represents a hydrogen atom or a substituted or unsubstituted lower alkyl group, and n is 0, 1 or 2
Is. In addition, as the substituent when Z is an alkyl group, the group defined by the above-mentioned -LA can be mentioned, for example.

[0052]

[Chemical 6] (In the above general formula [III], n is 0, 1 or 2, and Φ represents a substituted or unsubstituted o-, m- or p-phenylene group.)

[0053]

[Chemical 7] (In the above general formula [IV], Φ is a substituted or unsubstituted o
Represents a-, m- or p-phenylene group. )

[0054]

[Chemical 8]

[0055]

[Chemical 9] As the substituent of the phenylene group in the above general formula, those exemplified above can be mentioned.

A in R ³ of the general formula [I] is a substituted or unsubstituted aryl group, —CH═R ⁵ (R ⁵ is a substituted or unsubstituted heterocycle, a substituted or unsubstituted cycloalkyl group or a substituted or unsubstituted Represents an unsubstituted aromatic ring). As the heterocycle of R ⁵ ,

[0057]

[Chemical 10] (M and N each independently represent an oxygen atom, a sulfur atom or a nitrogen atom, and Y ⁻ represents an anion) and the like.
For example, a substituted or unsubstituted aryl group etc. can be mentioned. The substituted or unsubstituted cycloalkyl group may be saturated or unsaturated, for example,

[0058]

[Chemical 11] Groups derived from those capable of forming a resonance system such as Moreover, an azulene ring can be mentioned as a substituted or unsubstituted aromatic ring. Examples of the substituent of these groups include a lower alkyl group and a substituted or unsubstituted aryl group.

The hydrogen atom bonded to the carbon atom to which R ¹ , R ² and R ^{3 of the} pyrylium ring containing X or its analogous ring is not bonded is a halogen atom, a sulfonate group, an amino group, a styryl group or a nitro group. It may be substituted with a group, a hydrosil group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted aryl group or a substituted or unsubstituted lower aralkyl group.

Y ⁻ represents an anion, and examples of the anion include BF ₄ ⁻ , perchlorate ion, HO ₃ SCH ₂ CO
O ⁻ , or halogen ion such as chlorine ion, bromine ion, iodine ion, or fluorine ion, or a compound having an anion function such as aliphatic hydrocarbon or aromatic sulfonate, and further Zn, Ni, Cu, Pt , C
Examples thereof include complex ions of transition metals such as o and Pd.

When the group further substituted by the various substituents mentioned above is a halogen atom, examples of the halogen atom include Cl, Br and I. The lower alkyl group may be linear or branched, and the carbon number thereof is preferably about 1 to 4.

Among the compounds of general formula [I], those in which the heterocycle containing X is substituted with two or more substituted or unsubstituted aryl groups are preferred. For example, when the heterocycle containing X is a 6-membered ring, such compounds include (1) the 2-position and 4-position of the 6-membered ring containing X are substituted with a substituted or unsubstituted aryl group, and the 3-position, 5 that one of the position and 6-position is substituted by R ^3, (2) 3-position and 5-position of the 6-membered ring is substituted with a substituted or unsubstituted aryl group, 2-position, 4-position及6-position of One of which is substituted with R ³ , (3) the 2- and 6-positions of the 6-membered ring are substituted with a substituted or unsubstituted aryl group, and any of the 3-, 4- and 5-positions is R ³ And the like, and the like. Introducing a substituted or unsubstituted aryl group at such a position is
When this pyrylium compound is used for detecting the double helix structure of a nucleic acid, it is preferable for obtaining good properties as an intercalator for a nucleic acid base pair. Furthermore, it is more preferable that the heterocycle containing X is substituted with two or more of the substituted or unsubstituted aryl groups so that these substitution positions are not adjacent to each other.

One or both of at least two reagents capable of forming a reaction system which causes a change based on the interaction mediated by the target substance can be selected from the compounds of the general formula [I] and utilized. In the detection of nucleic acid, the compound of the general formula [I] is preferably one which is inserted into the double helix structure of the nucleic acid as an intercalator.

In the case of detecting the change due to charge transfer, for example, the compound having the electron-donating group among the compounds of the general formula [I] can be used as the electron donor. Examples of such a compound include those having an aryl group substituted with a lower alkyl group (lower alkylamino group) or the like as an aryl group constituting a substituent of the pyrylium ring or the pyrylium-like ring. As the lower alkylamino group, those in which a dimethylamino group, a diethylamino group and the like are substituted at the para position are preferable. This substituted aryl group may be directly substituted on the pyrylium ring or the pyrylium-like ring, or may be substituted with, for example, a lower alkyl group to form a lower aralkyl group. An example of the compound that can be used as the electron donor is shown in Table 1 below. Among these compounds, 2-methyl-4,6-bis- (4
Preferable one is -N, N-dimethylaminophenyl) pyrylium salt or 2-methyl-4,6-bis- (4-N, N-dimethylaminophenyl) thiopyrylium salt.

Examples of the compound that can serve as an electron acceptor with respect to the compounds shown in Table 1 include the compounds shown in Table 2 below.

However, the compounds in Table 1 are not defined as electron donors and the compounds in Table 2 are not defined as electron acceptors. The electron donor and the electron acceptor are relatively determined by their respective redox potentials, and cannot be uniquely determined. Therefore, for example, it is quite possible that both an electron donor and an electron acceptor are selected from the compound group of Table 1, or both an electron donor and an electron acceptor are selected from the compound group of Table 2. Furthermore, a set of an electron donor and an electron acceptor is formed by combining a pyrylium compound having the properties of the compound shown in Table 1 and another reagent, and an electron donor is prepared by combining a pyrylium compound having the properties of Table 2 and another reagent. It is also possible to set a set of and electron acceptors.

In the case of energy transfer, it is most important that the energy levels of the energy donor and energy acceptor are close. As a result of both being inserted into a target substance, for example, a nucleic acid double helix structure, it is considered that the proximity of the energy donor and the energy acceptor causes energy transfer. For example, among the pyrylium compounds shown in Table 1, the fluorescence intensity increases when the compound is inserted into a double-stranded nucleic acid,
Those whose excited state is considered to be stable can be used as an energy donor. It is preferable to select an energy acceptor suitable for each fluorescence energy level. Also in the case of energy transfer, as in the case of charge transfer, both the energy donor and the energy acceptor may be selected from the pyrylium compounds, or a set of the pyrilium compound and the reagent substance other than the pyrylium compound may be selected. it can.

When a substance other than a pyrylium-based compound is used as a reagent, it can be used without particular limitation as long as the target detection of the present invention can be carried out. 4,4-dimethyloxazolidine-N-oxyl (DOXXL: 4,4-Dimethyloxazolidine-N-oxyl) as a labeling agent,
2,2,5,5-Tetramethylpyrrolidine-N-oxyl (PROXL: 2,2,5,5-Tetramethylpyrrolidine-No
xyl), 2,2,6,6-tetramethylpiperidine-N
-Oxyl (2,2,6,6-Tetramethylpiperidine-N-oxyl)
And derivatives thereof. Also,
Riboflavin, N, N-Dimethyl-2,7-diazapyrenium ion (N, N-Dimethyl-2,7-diazapyrenium io
n) and their derivatives are also available.

Examples of compounds other than the pyrylium compound that can be used as the electron donor include, for example, acridine, anthracene, pyrene, as a fluorescent intercalator,
Ethidium bromide, proflavin, porphyrin,
Thiazole orange dimer (TOTO), oxazole yellow (YOYO), 4,6-diamino-2-phenylindole dihydrochloride (DAPI: 4,
6-Diamino-2-phenylindole dihydrochloride), propidium iodide (PI) and the like.

Further, cyanine, azulene, dansyl, fluorescein, which are used as general reagents,
Fluorescent dyes such as eosin, rhodamine and their derivatives can also be used.

Compounds other than the above-mentioned pyrylium compounds are not defined as electron donors, electron acceptors, etc., but the redox potential and the like are determined from the relative relationship with other reagents used at the same time. It can be appropriately used as an electron donor, an electron acceptor, a mediator, a sensitizer, etc.

[0072]

The present invention will be described in more detail with reference to the following Reference Examples and Examples. Reference Example 1 100 ml of acetic anhydride and 30 ml of concentrated sulfuric acid were mixed while being cooled, and the obtained mixed liquid was heated for 3 hours while being kept at 80 ° C. in a water bath. 20 ml of acetic anhydride, p
-Dimethylaminoacetophenone (30 ml) was added at room temperature, the temperature was then raised to 45 ° C, and the mixture was stirred for 24 hours for reaction. To this reaction solution, an equal amount of ethanol was added, cooled, and an aqueous potassium iodide solution was further added to precipitate crude crystals. The crude crystals were collected by filtration and recrystallized from a mixed system of ethanol / ether (volume ratio, 1: 4) to give 2
Green crystals of -methyl-4,6-bis- (4-N, N-dimethylaminophenyl) pyrylium iodide (Compound 1 in Table 1 (where Y is I)) were obtained. Analysis result of the obtained compound 1 (Y: I) Melting point: 254-257 ° C. UV / visible (CH ₃ CN ε × 10 ⁻⁴ ) λ max: 444
nm (2.43), 550 nm (8.24) NMR ( ¹ H, DMSO) δ ppm: 8.3737 (1 H,
s), 8.2729 (1H, d, J = 9.0 Hz),
8.1795 (1H, d, J = 9.0Hz), 7.88
64 (1H, s), 6.9117 (4H, t, J _AB = J
_BC = 9.77), 3.1829 (6H, s), 3.13
40 (6H, s), 2.6809 (3H, s) FAB mass m / z 333 IR (KBr) νcm ^-1 : 1645, 1610 (s
h), 1580 (s), 1490 (s), 1270, 1
200,1160 Further, 2-methyl-4,6-as described above except that an aqueous solution of perchloric acid is used instead of the aqueous solution of potassium iodide.
A perchlorate salt of bis- (4-N, N-dimethylaminophenyl) pyrylium (Compound 1 (Y: CIO ₄ )) was obtained.

Reference Example 2 20 g of sodium sulfide nonahydrate was dissolved in deionized water to make the total amount 50 ml. After 7 g of sodium hydrogen carbonate was added to and dissolved in this solution, 50 ml of ethanol was further added under ice cooling, and the mixture was stirred at room temperature for 30 minutes. Precipitated sodium carbonate was filtered off, washed with 25 ml of ethanol, and the filtrate and the washing solution were combined to obtain about 125 ml of a solution of sodium hydrosulfide in water and ethanol.

Next, 2-methyl-4,6 obtained in Reference Example 1
-Bis- (4-N, N-dimethylaminophenyl) pyrylium iodide 0.92 g was added to 20 ml DMS.
5 ml of the previously prepared water / ethanol solution of sodium hydrosulfide was added to the resulting solution, and the mixture was stirred at room temperature for 5 minutes. After stirring, 0.75 ml of hydroiodic acid was added, and the mixture was further stirred for 5 minutes. Then, after performing dichloromethane extraction and silica gel column purification according to a conventional method, recrystallization was performed with an ethanol / ether mixed solution (volume ratio, 1: 4) to obtain 0.7 g of 2-methyl-4,6-bis-. (4-
Crystals of N, N-dimethylaminophenyl) thiopyrylium iodide (Compound 2 in Table 1 (wherein Y is I)) were obtained. Analysis result of the obtained compound 2 (Y: I) Melting point: 246 to 248 ° C. UV / visible (CH ₃ CN ε × 10 ⁻⁴ ) λ max: 495
nm (2.50), 587 nm (4.95) NMR ( ¹ H, DMSO) δ ppm: 8.5679 (1 H,
s), 8.4323 (1H, s), 8.2436 (2)
H, d, J = 9.27 Hz), 7.9786 (2H,
d, J = 9.28), 6.8959 (4H, t, J _AB =
J _BC = 9.28), 3.1756 (6H, s), 3.1.
157 (6H, s), 2.8323 (3H, s) FAB mass m / z 349 IR (KBr) νcm ⁻¹ : 1600 (s), 1560
(S), 1460 (s), 1430 (s), 1370
(S), 1260 (s), 1160 (s) Furthermore, 2-methyl-4,6-bis- was prepared in the same manner as above except that an aqueous solution of perchloric acid was used instead of hydroiodic acid.
A perchlorate of (4-N, N-dimethylaminophenyl) thiopyrylium (Compound 2 (Y: CIO ₄ )) was obtained. Reference Example 3 Compounds 3 to 55 shown in Table 1 were prepared. In Table 1, Φ is a p-phenylene group:

[0075]

[Chemical 12] Or represents a phenyl group.

[0076]

[Table 1]

[0077]

[Table 2]

[0078]

[Table 3]

[0079]

[Table 4]

[0080]

[Table 5]

[0081]

[Table 6]

[0082]

[Table 7]

[0083]

[Table 8]

[0084]

[Table 9]

[0085]

[Table 10]

[0086]

[Table 11]

[0087]

[Table 12]

[0088]

[Table 13]

[0089]

[Table 14]

[0090]

[Table 15]

[0091]

[Table 16]

[0092]

[Table 17]

[0093]

[Table 18]

[0094]

[Table 19] In addition, these compounds were synthesized by the following known methods. The specific reaction procedure was in accordance with a conventional method.

Compound 7 is the compound described in “New Methods” by W. Foerst et al.
of Preparative Organic Chemistry ", Acad. Press (1
964) and the compound [i]

[0096]

[Chemical 13] After synthesizing

[0097]

[Chemical 14] The compound obtained by reacting with (p-N, N-dimethylaminobenzaldehyde) was further reacted with a desired anion. Compound 17 is the same as compound [i]

[0098]

[Chemical 15] The compound obtained by reacting with (p-diethylaminostyrylbenzaldehyde) was further reacted with a desired anion. In addition, compound [ii] is reacted with sodium hydrosulfide to give compound [ii]

[0099]

[Chemical 16] After obtaining, the compound [ii] was synthesized in the same manner as the compounds 7 and 17 to synthesize the compounds 8 and 18.

R. Wizinger et al., Helv. Chim. Acta, 39 , 2
According to the method described in 17 (1956), the route shown below from acetophenone and acetaldehyde

[0101]

[Chemical 17] Via compound [iii]

[0102]

[Chemical 18] Was synthesized. Using this compound [iii] as a raw material, p-
The compound obtained from the reaction with dimethylaminobenzaldehyde is further reacted with a desired anion to give compound 5.
Compound 15 was similarly prepared from the reaction with p-diethylaminostyrylbenzaldehyde, and Compound 9 was similarly prepared from the reaction with p-dimethylaminocinnamic aldehyde.

[0103]

[Chemical 19] Compound 11 was obtained in the same manner from the reaction with. Alternatively, the compound [iv] can be obtained by reacting the compound [iii] with sodium hydrosulfide.

[0104]

[Chemical 20] Compounds 6, 16, 10 and 12 were obtained in the same manner as in the case of the synthesis of Compounds 5, 15, 9 and 11 except that the compound [iv] was used instead of the compound [iii]. It was

Furthermore, except that the starting acetaldehyde was replaced with p-dimethylaminobenzaldehyde, the cation moiety of the compound 3 was obtained in the same manner as in the synthesis of the compound [iii], and this was reacted with sodium sulfide. The compound thus obtained was further reacted with a desired anion to obtain a compound 4. Further, in the same manner, the compound [v] was obtained from p-methylbenzaldeh and acetophenone.

[0106]

[Chemical 21] And then reacting it with sodium hydrosulfide to give compound [vi]

[0107]

[Chemical formula 22] Got Furthermore, the compounds [v] and [vi] are each
Compound 13 obtained by reacting with dimethylaminobenzaldehyde and further reacting the obtained compound with a desired anion.
And 14 were obtained.

The compounds 19, 20 and 21 are prepared by combining the compound [i] or [ii] with the cation moiety of the compound 1 or 2.

[0109]

[Chemical formula 23] Was obtained by further reacting the obtained compound with a desired anion. Compounds 22, 23 and 24 are compounds [i] or [ii] and the cation moiety of compound 1 or 2,

[0110]

[Chemical formula 24] Was obtained by further reacting the obtained compound with a desired anion. Compounds 25 and 26 are compounds [i] or [ii]

[0111]

[Chemical 25] Was obtained by further reacting the obtained compound with a desired anion. Compounds 27, 28 and 29 are obtained by reacting compound [i] or [ii] and the cation moiety of compound 1 or 2 with triethoxymethane [HC (OC ₂ H ₅ ) ₃ ] It was synthesized by reacting the desired anion. Compounds 30, 31 and 32
Is compound [iii] or [iv], dimethylamino derivative of compound [iii] and [iv] synthesized from p-dimethylaminoacetophenone by the same method as [iii] and [iv], and triethoxymethane. The reaction was performed, and the obtained compound was further reacted with a desired anion to synthesize the compound.

Compounds 33 to 55 were respectively synthesized by the following reactions. Synthesis of compound 33

[0113]

[Chemical formula 26] Synthesis of compound 34

[0114]

[Chemical 27] Synthesis of compound 35

[0115]

[Chemical 28] Synthesis of compound 36

[0116]

[Chemical 29] Synthesis of compound 37

[0117]

[Chemical 30] Synthesis of compound 38

[0118]

[Chemical 31] Synthesis of compound 39

[0119]

[Chemical 32] Compound 40 is used as a raw material in the synthesis of Compound 36.

[0120]

[Chemical 33] From

[0121]

[Chemical 34] It was changed to and synthesized.

Compound 41 was used as a starting material in the synthesis of Compound 37.

[0123]

[Chemical 35] From

[0124]

[Chemical 36] It was changed to and synthesized.

Compound 42 was used as a starting material in the synthesis of Compound 38.

[0126]

[Chemical 37] From

[0127]

[Chemical 38] It was changed to and synthesized.

Compound 43 was used as a starting material in the synthesis of Compound 39.

[0129]

[Chemical Formula 39] From

[0130]

[Chemical 40] It was changed to and synthesized. Synthesis of compound 44

[0131]

[Chemical 41] Synthesis of compound 45

[0132]

[Chemical 42] Synthesis of compound 46

[0133]

[Chemical 43] Synthesis of compound 47

[0134]

[Chemical 44] Compound 48 is used as a raw material in the synthesis of Compound 44.

[0135]

[Chemical formula 45] From

[0136]

[Chemical formula 46] It was changed to and synthesized.

Compound 49 was used as a starting material in the synthesis of Compound 45.

[0138]

[Chemical 47] From

[0139]

[Chemical 48] It was changed to and synthesized.

Compound 50 was used as a starting material in the synthesis of Compound 46.

[0141]

[Chemical 49] From

[0142]

[Chemical 50] It was changed to and synthesized.

Compound 51 was used as a starting material in the synthesis of compound 47.

[0144]

[Chemical 51] From

[0145]

[Chemical 52] It was changed to and synthesized. Synthesis of compound 52

[0146]

[Chemical 53] Synthesis of compound 53

[0147]

[Chemical 54] Synthesis of compound 54

[0148]

[Chemical 55] Synthesis of compound 55

[0149]

[Chemical 56] Further, various compounds for introducing a substituent were variously changed to obtain the compounds shown in Table 2.

[0150]

[Table 20]

[0151]

[Table 21]

[0152]

[Table 22]

[0153]

[Table 23]

[0154]

[Table 24]

[0155]

[Table 25]

[0156]

[Table 26]

[0157]

[Table 27]

[0158]

[Table 28]

[0159]

[Table 29]

[0160]

[Table 30]

[0161]

[Table 31]

[0162]

[Table 32]

[0163]

[Table 33]

[0164]

[Table 34]

[0165]

[Table 35]

[0166]

[Table 36] Example 1 (Detection of DNA Hybrid with Two Pyrylium Dyes) [1] Synthesis of 2-Methyl-4,6-bis- (4-N, N-dimethylaminophenyl) pyrylium perchlorate (Compound a) Anhydrous 100 ml of acetic acid and 30 ml of concentrated sulfuric acid were mixed while being cooled, and the obtained mixed liquid was heated for 3 hours while being kept at 80 ° C. in a water bath. 20 ml of acetic anhydride, p
-Dimethylaminoacetophenone (30 ml) was added at room temperature, the temperature was then raised to 45 ° C, and the mixture was stirred for 24 hours for reaction. An equal amount of ethanol was added to this reaction solution, which was cooled, and a 70% aqueous solution of perchloric acid was further added to precipitate crude crystals. The crude crystals were recovered by filtration, and ethanol /
Recrystallization from a mixed system of ether (volume ratio, 1: 4) gave 2-methyl-4,6-bis- (4-N, N-dimethylaminophenyl) pyrylium perchlorate (compound a).

[2] Synthesis of 4-Methyl-2,6-diphenylpyrylium perchlorate A. T. Balaban et al., Tetrahedron,
20 , 119 (1964), 4-methyl-2,6-diphenylpyrylium perchlorate (compound b) was synthesized.

[3] Synthesis of Oligonucleotide A 20-mer oligonucleotide having a base sequence partially complementary to M13mp18 DNA (single strand) as a target DNA was synthesized by using 381A DNA automatic synthesizer manufactured by ABI. . The 5'-terminal dimethoxytrityl group was removed on an automatic synthesizer. The base sequence is as follows.

5'-GTTGTAAAAACGACGGC
CAGT-3 ′ (SEQ ID NO: 1) [4] Formation reaction of hybrid of probe and M13mp18DNA 0.2 μM of the oligonucleotide probe prepared in the above [3] and M13mp18DNA (Takara Shuzo)
0.2 μM of 1 mM phosphate buffer (pH 7.0) / 1
A probe / target DNA hybrid was prepared by heating to 80 ° C. in 45 mM NaCl / 5 mM KCl, and then gradually cooling to room temperature.

[5] Characteristics of Two Pyrylium Dyes In order to know the interaction between compound a and compound b with nucleic acid, each compound was added to a commercially available DNA solution and its characteristics were examined. Although compound a itself is non-fluorescent in an aqueous solution, it is 640 nm (580 n in a DNA solution).
It had a strong fluorescence upon excitation with m). In addition, the absorption peak is a fluorescent intercalator that shifts to a long wavelength of about 40 nm when DNA is added. Compound b is
Contrary to compound a, there is strong fluorescence in the free state, but DN
The fluorescence disappeared in the coexistence of A.

[6] Detection of Hybrid by Charge Transfer Using Two Pyrylium Dyes Compound a was added to the hybrid of the probe prepared in the above [4] and M13mp18 at a final concentration of 5 μM, Furthermore, various concentrations of compound b were added. This solution was irradiated with visible light of 580 nm, which is the absorption wavelength of compound a, and its fluorescence was observed with IMUC-7000 manufactured by Otsuka Electronics Co., Ltd. As a result, the fluorescence at 640 nm observed before adding the compound b disappeared, and a new fluorescence appeared at 450 nm instead. It is considered that this is because the charge transfer via the double helix structure of the nucleic acid occurred between the compound a and the compound b inserted in the hybrid, and the fluorescence appeared at 450 nm. Further, the fluorescence at 450 nm was maximum when the compound b was 5 μM.

Example 2 (Detection of Mismatch) A probe / target DNA hybrid was prepared in the same manner as in Example 1 except that the oligonucleotide having the following base sequence was used as a probe.

5'-GTTGTAAAAGGACGGGC
CAGT-3 ′ (SEQ ID NO: 2) This base sequence is obtained by converting the 10th C from the 5′-end of the probe base sequence used in Example 1 to G, and is likely to mismatch with M13mp18DNA. It was designed for.

[0174] As in Example 1, 2 was added to this hybrid body.
Pyrylium dyes of various types were added, and the increase in fluorescence and the disappearance of fluorescence were observed using 580 nm light as excitation light. Example 1
Unlike the above, no change was observed in the intensity of the fluorescence of both compound a and compound b. This result is considered to be because the interaction between these dyes did not occur in the mismatched hybrid.

Example 3 (D with Two Pyrylium Dyes)
Detection of NA hybrid) A hybrid of probe / target DNA was prepared in the same manner as in Example 1. To this, compound a was added, and then 2,6-bis (N, N-dimethylaminophenyl) -4-phenylpyrylium iodide (compound c) was added.

The interaction between compound c and DNA was examined beforehand. As a result, the compound c had an absorption around 600 nm, and like the compound a, a shift of the absorption spectrum was observed with the addition of DNA (640 nm), which seems to be an intercalator, but its fluorescence was small even in the DNA solution.

When these two kinds of pyrylium dyes were added to the probe / target DNA hybrid and irradiated with light having an excitation wavelength of 580 nm of compound a, it was originally observed in the presence of DNA when compound c was absent. The strong absorption of the compound a disappeared, and a new fluorescence appeared at 720 nm instead. It is considered that this is because energy transfer occurred from compound a to compound c. Experiments with various amounts of compound c showed that the decrease in the fluorescence of the donor and the increase in the fluorescence of the acceptor were most effective at the same molar number.

Example 4 (Detection of hybrid body by labeled antibody and charge transfer) [1] Preparation of labeled antibody (1) Synthesis of 4-methyl-2,6-diphenylpyrylium perchlorate A. Preparation of labeled antibody T. Balaban et al., Tetrahedron,
20 , 119 (1964), 4-methyl-2,6-diphenylpyrylium perchlorate (compound b) was synthesized.

(2) Labeling of antibody 4.3 g of compound b, 2 g of carboxybenzaldehyde,
After refluxing a mixed solution of 1 drop of 70% perchloric acid and 10 ml of acetic acid for 1 hour, the precipitated precipitate was collected and recrystallized from the above ethanol / ether mixed system to give 4- (4-carboxystyryl) -2, 6-Diphenylpyrylium perchlorate (compound d) was obtained.

170 mg of compound d was added to 5 ml of dry DM
Dissolve in F and add 50 μl of dry pyridine. DSC
After adding 128 mg of (disuccinimidyl carbonate), the mixture was stirred at room temperature in the dark for 20 hours. 150 ml of diethyl ether was added to the reaction mixture, and the deposited precipitates were collected, washed with diethyl ether and dried. The obtained active ester compound [compound e] was reacted with the amino group of the antibody to prepare a labeled antibody.

[2] Detection with FITC-labeled secondary antibody The antigen was reacted with the primary antibody prepared in the above [2] by a conventional method, and then a FITC-labeled anti-mouse IgG antibody (manufactured by Sigma) was used as a secondary antibody. ) Was reacted.

This sample was irradiated with visible light of 480 nm, and its fluorescence was observed with IMUC-7000 manufactured by Otsuka Electronics Co., Ltd. As a result, the fluorescence of FITC at 520 nm observed before reacting the labeled antibody disappeared, and instead of 45
New fluorescence appeared at 0 nm. It is considered that this is because the reaction between the primary antibody and the secondary antibody resulted in charge transfer between the compound b on the labeled antibody and FITC.

[0183]

According to the detection method of the present invention, it is possible to detect a target substance with higher accuracy. For example, when a hybrid substance obtained by hybridization using a probe is used as a target substance, it has an advantage that B / F separation is not necessary. As a result, many operations such as removal of excessive probe, which was inconvenient in the conventional method, complicated treatment for removing non-specific adsorption, and examination of the conditions, became unnecessary. Furthermore, by selecting the reagent substance so that the signal change is observed only in the correct hybrid, even if a mismatch occurs in the reaction system, the hybrid that forms the correct double helix structure. It becomes possible to detect only.

Furthermore, when a pyrylium compound is used as an electron donor in the method of the present invention, a compound which has almost no fluorescence when released and whose fluorescence property is greatly changed when bound to a double helix structure of a nucleic acid may be used. For example, the S / N ratio at the time of detection can be increased. In addition, the pyrylium-based compound is characterized by being inserted more specifically in a double helix structure than in other condensed ring type intercalators, and is less likely to be inserted in a partial double-stranded structure of single-stranded DNA. As a result, by using a pyrylium compound as at least one of the at least two kinds of reagents, it is possible to exclude a signal derived from a pseudo hybrid having a partial double-stranded structure.

[0185]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid, synthetic DNA Sequence characteristics: M13mp18 DNA detection probe sequence Sequence GTTGTAAAAC GACGGCCAGT 20

SEQ ID NO: 2 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid, Synthetic DNA Sequence characteristics: M13mp18 DNA mismatch probe Sequence Sequence GTTGTAAAAG GACGGCCAGT 20

[Brief description of drawings]

FIG. 1 is a diagram showing a model of a reaction system that interacts with a target substance used in the detection method of the present invention.

FIG. 2 is a diagram showing a model of a reaction system that interacts with a target substance used in the detection method of the present invention.

[Explanation of symbols]

 1, 2, 4, 5, 6, 7 Reagent 3 Target substance

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C07D 409/04 309 C12N 15/09

Claims (31)

[Claims] 1. At least two kinds of reagents capable of forming a reaction system that causes a change based on an interaction mediated by a target substance, are reacted with a sample, and the interaction occurs when the target substance is present in the sample. In the method of detecting a target substance in the sample by measuring a change based on
A method for detecting a target substance, wherein at least one of the reagents constituting the reaction system is selected from a pyrylium compound and a pyrylium analogue compound. 2. The detection method according to claim 1, wherein the interaction is due to charge transfer. 3. A reagent comprising the pyrylium compound or pyrylium-like compound is an electron donor, an electron acceptor,
The detection method according to claim 2, which acts as a mediator or a sensitizer. 4. The detection method according to claim 1, wherein the interaction is based on energy transfer. 5. The pyrylium compound and pyrylium analogue compound are represented by the following general formula [I]: (In the above general formula [I], Represents a heterocycle, X is O, S, Se or Te, represents a pyrylium ring or a pyrylium-like ring, and R ¹ and R ² are each independently a hydrogen atom, a halogen atom, a sulfonate group, an amino group, R represents a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted lower aralkyl group or a substituted or unsubstituted cycloalkyl group, R ³ is -A or -L-a, L ^{is, -L 1 -, - L 2} -L 3 - or -L ⁴ -L ⁵ -L ⁶ -
And L ^{1 to} L ⁶ are each independently-(CH = C
H) -, 2-valent group derived from a substituted or unsubstituted aryl group, a substituted or unsubstituted lower alkylene group, or -CH = R ⁴ - (R ⁴ represents a ring structure having an oxo group)
Represents a substituted or unsubstituted aryl group, -CH = R
⁵ (R ⁵ represents a substituted or unsubstituted heterocycle, a substituted or unsubstituted cycloalkyl group or a substituted or unsubstituted aromatic ring), and R ¹ , R ^{2 of} the pyrylium ring containing X or a ring similar thereto,
The hydrogen atom bonded to the carbon atom to which R ³ is not bonded is a halogen atom, a sulfonate group, an amino group, a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, It may be substituted with a substituted or unsubstituted aryl group or a substituted or unsubstituted lower aralkyl group, and Y ⁻ represents an anion. The detection method according to claim 1, which is a compound represented by the formula (1). 6. The detection method according to claim 1, wherein the change caused by the interaction is optically detectable. 7. The detection method according to claim 1, wherein the change based on the interaction is chemically detectable. 8. The detection method according to claim 1, wherein the change based on the interaction is initiated by light irradiation. 9. The detection method according to claim 1, wherein the change based on the interaction is irreversible. 10. A sample is reacted with at least two kinds of reagents capable of forming a reaction system that produces a detectable change due to an interaction mediated by a double helix structure of a nucleic acid, In the method of detecting a double helix structure in the sample by measuring the detectable change that occurs when present, at least one of the reagents that make up the reaction system is selected from pyrylium compounds and pyrylium analog compounds. A method for detecting a double helix structure of a nucleic acid, which is characterized in that 11. The detection method according to claim 10, wherein the interaction is due to charge transfer. 12. The detection method according to claim 11, wherein the reagent comprising the pyrylium compound or the pyrylium-like compound acts as an electron donor, an electron acceptor, a mediator or a sensitizer. 13. The detection method according to claim 11, wherein the charge transfer occurs via stacking of base pairs forming a double helix structure. 14. The charge transfer according to a proximity effect between a double helix structure and an electron donor or an electron acceptor.
Alternatively, the detection method according to 12 above. 15. The detection method according to claim 10, wherein the interaction is based on energy transfer. 16. The detection method according to claim 15, wherein the energy transfer occurs via stacking of base pairs forming a double helix structure. 17. The detection method according to claim 15, wherein the energy transfer is due to a proximity effect of a double helix structure and an energy donor and an energy acceptor. 18. The detection method according to claim 10, wherein the change based on the interaction is initiated by light irradiation. 19. The detection method according to claim 10, wherein the change based on the interaction is irreversible. 20. The detection method according to claim 10, wherein the pyrylium compound and the pyrylium analog compound are compounds represented by the general formula [I] according to claim 5. 21. The detection method according to claim 10, wherein the double helix structure is a hybrid body obtained by reacting a target nucleic acid with a probe nucleic acid. 22. The presence or absence of an immune reaction is detected by measuring the production of a complex obtained by reacting a first reaction product and a second reaction product capable of producing an immune reaction with a substance to be detected in this order. In the method, at least two reagents capable of forming a reaction system in which each of the first and second reactants causes a change based on interaction mediated by a complex obtained when these reactants react with each other. And a pyrylium compound or a pyrylium-like compound is used as at least one of the reagents constituting the reaction system. 23. The detection method according to claim 22, wherein the pyrylium compound or pyrylium analog compound is a compound represented by the general formula [I] according to claim 5. 24. The detection method according to claim 22 or 23, wherein the interaction between the at least two reagents is based on charge transfer between the reagents. 25. The detection method according to claim 22, wherein the interaction between the at least two kinds of reagents is based on energy transfer between these reagents. 26. The detection method according to claim 24, wherein the reaction system that causes a change based on the interaction includes at least an electron donor and an electron acceptor. 27. The detection method according to claim 24 or 26, wherein the charge transfer is due to a proximity effect between an electron donor and an electron acceptor accompanying the reaction between the first reactant and the second reactant of the immune reaction. 28. The detection method according to claim 25, wherein the energy transfer is due to a proximity effect between an energy donor and an energy acceptor accompanying the reaction between the first reactant and the second reactant of the immune reaction. 29. The detection method according to claim 23, wherein the change due to the interaction is optically detectable. 30. The detection method according to claim 23, wherein the interaction is initiated by light irradiation. 31. The detection method according to claim 23, wherein the interaction is irreversible.