Compound, detection system, and measurement device

Abstract

The present disclosure relates to a technique for detecting molecules to be analyzed etc. by using a compound having a chromophore, and the main object thereof is to enable simultaneous observation of a plurality of factors. As a result of intensive studies, the present inventors have found that with a compound having a plurality of chromophores in a molecule in which Förster resonance energy transfer between the plurality of chromophores is suppressed and the waveform of the spectrum derived from each of the plurality of chromophores is maintained, it is possible to simultaneously observe a plurality of factors to be measured by analyzing the waveform of the spectrum in a specific wavelength region using the compound.

Description

Compounds, detection systems and measurement devices

[0001] The present technology relates to compounds, detection systems and measurement devices.

[0002] BACKGROUND ART Conventionally, a method for detecting a molecule to be analyzed or the like using a compound having a chromophore in the molecule has been known.

[0003] For example, Patent Document 1 below discloses a compound having a FRET donor and a FRET acceptor in the molecule and a method for observing a sample using the compound. However, this technique detects an analyte molecule by increasing a luminescence signal through Förster resonance energy transfer (FRET) and detecting a strong colored or fluorescent peak, but does not simultaneously observe multiple factors by analyzing the waveform of a spectrum.

[0004] Special table 2021-518410 publication

[0005] The present technology is related to a technology for detecting analyte molecules and the like using a compound having a chromophore, and the main object of the present technology is to provide a technology that can observe multiple factors simultaneously.

[0006] As a result of extensive research, the present inventors have discovered that a compound having multiple chromophores in the molecule suppresses Förster resonance energy transfer between the multiple chromophores, thereby maintaining the spectral waveforms derived from each of the multiple chromophores, and thereby enabling multiple factors of the measurement target to be observed simultaneously by analyzing the spectral waveforms in specific wavelength regions using the compound.

[0007] That is, the present technology provides a compound for analyzing the waveform of a spectrum in a specific wavelength region, which has a structure of the following formula (1): (1) n is an integer of 1 or more, m is an integer of 0 or more, and when n is an integer of 2 or more, the two or more m's may be the same or different from each other, M1 and M2 are chromophores having different chemical structures and not undergoing Förster resonance energy transfer with each other, and when n is an integer of 2 or more, the two or more M2's may be the same or different from each other, L1, L2, and L3 are single bonds or divalent groups, and L1 and L2 or L1 and L3 may be bonded to each other to form a ring, and the two or more L1, L2, and L3 may be the same or different from each other, L4 is a single bond or divalent group, and when m is an integer of 2 or more, the two or more L4's may be the same or different from each other, R1 is H or a monovalent group, and the two or more R1's may be the same or different from each other, R2 and R3 are each H, a monovalent group, or Q, R4 and R5 are each H or a monovalent group, or R4 and R5 are one unsaturated substituent, and when there are two or more R4 and R5, the two or more R4 and R5 may be the same or different from each other, and Q is a reactive group that can form a bond with an analyte molecule, a targeting moiety, a solid support, or a complementary reactive group Q', and when there are two or more Q, the two or more Q' may be the same or different from each other. The compound of the present technology may have a structure of the following formula (1A): (1A) z is an integer of 1 or more, and when m1 or m3 is an integer of 2 or more, the two or more z's may be the same or different from each other, m1, m2, and m3 are integers of 0 or more, and when n is an integer of 2 or more, the two or more m1, m2, and m3 may be the same or different from each other. The compound of the present technology may have a structure of the following formula (1B). (1B) z is an integer of 1 or more, and when m is an integer of 2 or more, the two or more z's may be the same or different from each other. The compound of the present technology may have a structure represented by the following formula (1C): (1C) z is an integer of 1 or more, and when m is an integer of 2 or more, the two or more z's may be the same or different from each other, x1 and x2 are integers of 0 or more, x3 and x4 are integers of 0 or more, and when n is an integer of 2 or more, the two or more x3's and x4's may be the same or different from each other. The compound of the present technology may have a structure represented by the following formula (1D). (1D) m2 is an integer of 0 or greater, and when n is an integer of 2 or greater, the two or more m2s may be the same or different from each other. In the compound of the present technology, for at least one L1, the L1 may be a linker containing a triazolyl functional group. In the compound of the present technology, for at least one L1-M (the M may be M1 or M2), the L1-M may have the following structure: L1a and L1b are a single bond or a divalent group. In the compound of the present technology, for at least one of L1-M (the M may be M1 or M2), the L1-M may have the following structure: L and L are a single bond or a divalent group. In the compound of the present technology, for at least one L, the structure of the L may include one or more structures selected from the following: a, b, and c are integers of 1 or more, and when a, b, and c are 2 or more, the two or more a, b, and c may be the same or different from each other. In the compounds of the present technology, the distance between M1 and M2 and the distance between different M2s are preferably 2.25 nm or more, more preferably 3.25 nm or more. In the compounds of the present technology, it is preferable that M1 and M2, or M1 and two or more different M2s, have different absorption or emission spectra. In the compounds of the present technology, the efficiency of Förster resonance energy transfer (FRET efficiency) from M1 or M2 to another M2 is preferably 99% or less, more preferably 50% or less. In the compounds of the present technology, it is preferable that M1 or M2, or both, contain aryl or heteroaryl, or a combination thereof in the structure. In the compounds of the present technology, M1 or M2, or both, may have a fused polycyclic structure containing at least four fused rings. In the compounds of the present technology, M1 or M2, or both, are selected from the group consisting of dimethylaminostilbene, quinacridone, fluorophenyl-dimethyl-BODIPY, his-fluorophenyl-BODIPY, acridine, terylene, sexiphenyl, porphyrin, benzopyrene, (fluorophenyl-dimethyl-difluorobora-diaza-indacene)phenyl, (bis-fluorophenyl-difluorobora-diaza-indacene)phenyl, quaterphenyl, bi-benzothiazole, ter-benzothiazole, bi-naphthyl, bi-anthracyl, squaraine, squarylium, 9,10-ethylenediamine, ethylenediamine, ethylenediamine-1, ethylenediamine-2, ethylenediamine-3, ethylenediamine-4, ethylenediamine-5, ethylenediamine-6, ethylenediamine-7, ethylenediamine-8, ethylenediamine-9, ethylenediamine-10, ethylenediamine-11, ethylenediamine-12, ethylenediamine-13, ethylenediamine-14, ethylenediamine-15, ethylenediamine-15, ethylenediamine-16, ethylenediamine-17, ethylenediamine-18, ethylenediamine-19, ethylenediamine-21, ethylenediamine-22, ethylenediamine-23, ethylenediamine-24, ethylenediamine-25, ethylenediamine-25, ethylenediamine-25, ethylenediamine-26, ethylenediamine-27, ethylenediamine-28, ethylenediamine-29, ethylenediamine-30, ethylenediamine-31, ethylenediamine-32, ethylenediamine-33, ethylenediamine-34, ethylenediamine-35, ethylenediamine-35, ethylenediamine-35, ethylenediamine-35, ethylenediamine-36, ethylenediamine-37, ethylenedi The compound may be one or more structures selected from the group consisting of thionylanthracene, ter-naphthyl moiety, p-terphenyl, perylene, azobenzene, phenazine, phenanthroline, acridine, thioxanthrene, chrysene, rubrene, coronene, cyanine, perylene imide, perylene amide, coumarin dye, resorufin dye, dipyrromethene boron difluoride dye, ruthenium bipyridyl dye, thiazole orange dye, polymethine, N-aryl-1,8-naphthalimide dye, boron-dipyrromethene, rhodamine, cyanine, pyrene, perylene, perylene monoimide, and 6-FAM, or derivatives thereof.In the compounds of the present technology, M1 or M2, or both, may be one or more structures selected from the following or derivatives thereof: In the compounds of the present technology, R2 or R3, or both, may be Q.

[0008] Next, the present technology provides a detection system that detects the distribution of a target in a measurement object by analyzing the waveform of a spectrum in a specific wavelength region using the compound of the present technology. In the detection system of the present technology, the measurement object may be a cell. In the detection system of the present technology, the distribution of a target in the measurement object may be detected by flow cytometry. Furthermore, the present technology provides a measurement device that employs the detection system of the present technology.

[0009] FIG. 1 is an image of the spectrum of a compound of the present technology. FIG. 2 is an image showing the concept of the detection system of the present technology. FIG. 3 is an image showing the concept of a detection system according to the prior art. FIG. 4 is an image of the absorption spectrum of a compound of the present technology having two different chromophores. FIG. 5 is an image of the fluorescence spectrum of a compound of the present technology having two different chromophores. FIG. 6 is an image of the absorption spectrum of a compound of the present technology having three different chromophores. FIG. 7 is a diagram schematically showing the overall configuration of a biological sample analyzer. FIG. 8 is a graph showing the calculated relationship between the distance between chromophores in a compound and FRET efficiency. FIG. 9 is a graph showing the calculated relationship between the distance between chromophores and FRET efficiency for each J value. FIG. 10 is a graph showing the calculated relationship between the distance between chromophores and the change in the fluorescence spectrum of each chromophore.

[0010] Preferred embodiments of the present technology will be described below. However, the embodiments shown below are examples of typical embodiments of the present technology, and the present technology is not limited to only the preferred embodiments below and can be freely modified within the scope of the present technology.

[0011] <Compound> The compound according to the present technology is a compound for analyzing the waveform of a spectrum in a specific wavelength region, and has a structure of the following formula (1). (1)

[0012] The above n is an integer of 1 or more, and can be any integer selected within a range that can be synthesized, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc. There is no particular upper limit to n, but it can be any integer selected, for example, from 30 or less, 20 or less, 15 or less, 10 or less, etc.

[0013] The above m is an integer of 0 or more, and can be any integer selected within a range that can be combined, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc. From the viewpoint of suppressing the efficiency of Förster resonance energy transfer (FRET efficiency) from M1 or M2 (described later) to another M2 and maintaining the waveform of the spectrum derived from M1 and M2 (or each M2 when there are two or more M2), the distance between M1 and M2 and the distance between different M2s can be increased by increasing the value of m or by increasing the length of L1 or L2 (described later).

[0014] Here, the "distance" refers to the distance in the three-dimensional structure of the compound, not the distance when the compound is linear.

[0015] The upper limit of m is not particularly limited, and can be selected arbitrarily, for example, from 30 or less, 20 or less, 15 or less, 10 or less, etc. When n is an integer of 2 or more, the 2 or more m's may be the same or different from each other.

[0016] M1 and M2 are chromophores having different chemical structures and not undergoing Förster resonance energy transfer with each other. When n is an integer of 2 or more, two or more M2s may be the same or different from each other.

[0017] It is preferable that M1 and M2, or M1 and two or more different M2, have different absorption or emission spectra. That is, M1 and M2, or M1 and two or more different M2, have different spectral waveforms. In the present technology, by using such M1 and M2 as chromophores, Förster resonance energy transfer between the chromophores is suppressed and the spectral waveforms derived from each of the multiple chromophores are maintained, thereby making it possible to simultaneously observe multiple factors. In the present technology, "spectrum" includes both absorption spectra and emission spectra such as fluorescence spectra.

[0018] Furthermore, in the present technology, depending on the purpose and application of use, the compound of the present technology may be measured using a single light source, or may be measured using multiple light sources emitting light of different wavelengths.

[0019] In the present technology, the efficiency of Förster resonance energy transfer (FRET efficiency) from M1 or M2 to another M2 is related to the detection capability of a measurement device employed in the detection system of the present technology, and the lower the efficiency, the better. Depending on the detection capability of the measurement device employed in the detection system of the present technology, the efficiency can be adjusted to, for example, 99% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, etc.

[0020] The FRET efficiency can be calculated using the following formulas (1) to (3).

[0021]

[0022]

[0023]

[0024] As shown in the above formula, in the present technology, by increasing the distance between M1 and M2 and the distance between mutually different M2, it is possible to suppress the efficiency of Förster resonance energy transfer (FRET efficiency) from M1 or M2 to the other M2. The FRET efficiency also depends on the Förster distance of the chromophore used, but for example, by setting the distance between M1 and M2 and the distance between mutually different M2 to 2.25 nm or more, more preferably 3.00 nm or more, even more preferably 3.25 nm or more, even more preferably 3.50 nm or more, and particularly preferably 4.00 nm or more, the FRET efficiency can be adjusted to any of the above-mentioned ranges, such as the above-mentioned 99% or less, in accordance with the measurement conditions for the spectral waveform in a specific wavelength region.

[0025] Here, the distance between the chromophores refers to the distance between the chromophores in the ground state of the compound of the present technology, which is the state of the compound with the lowest energy level.

[0026] In this technology, the specific wavelength range can be set to any wavelength range that matches the absorption or emission spectrum of the chromophore contained in the compound of this technology. This technology does not focus on individual peaks in the spectrum, but instead focuses on the overall waveform shape of the spectrum measured in the specific wavelength range and analyzes the overall waveform shape of the spectrum. This makes it possible to simultaneously observe multiple factors by utilizing spectral information that has not previously been the subject of analysis.

[0027] The distance between M1 and M2 and the distance between different M2s can be adjusted by increasing the value of m as described above or by increasing the length of L1 or L2, which will be described later.

[0028] M1 and M2 that can be used in the present technology preferably contain an aryl or heteroaryl, or a combination thereof, in the structure, and particularly preferably have a fused polycyclic structure containing at least four fused rings.

[0029] Examples of M1 and M2 that can be used in the present technology include dimethylaminostilbene, quinacridone, fluorophenyl-dimethyl-BODIPY, his-fluorophenyl-BODIPY, acridine, terylene, sexiphenyl, porphyrin, benzopyrene, (fluorophenyl-dimethyl-difluorobora-diaza-indacene)phenyl, (bis-fluorophenyl-difluorobora-diaza-indacene)phenyl, quaterphenyl, bi-benzothiazole, ter-benzothiazole, bi-naphthyl, bi-anthracyl, squaraine, squarylium, 9,10-ethynylanthracene, ternary One or more structures selected from a butyl moiety, p-terphenyl, perylene, azobenzene, phenazine, phenanthroline, acridine, thioxanthrene, chrysene, rubrene, coronene, cyanine, perylene imide, perylene amide, coumarin dye, resorufin dye, dipyrromethene boron difluoride dye, ruthenium bipyridyl dye, thiazole orange dye, polymethine, N-aryl-1,8-naphthalimide dye, boron-dipyrromethene, rhodamine, cyanine, pyrene, perylene, perylene monoimide, and 6-FAM, or derivatives thereof can be suitably used, but the present invention is not limited to these examples.

[0030] The term "derivative" refers to a structure in which some atoms in the structure have been changed by substitution or the like.

[0031] As more preferred examples of M1 and M2 that can be used in the present technology, for example, one or more structures selected from the following or derivatives thereof can be suitably used.

[0032]

[0033] In the structural formulas of M1 and M2 shown above, R is H or a monovalent group, and two or more R may be the same or different from each other. Furthermore, the above M1 and M2 may have a substituent at any position. Furthermore, the above M1 and M2 may be substituted with L1 at any position in the structure.

[0034] The above-described M1 and M2 or derivatives thereof may have the following structures, for example.

[0035]

[0036]

[0037] L1, L2, and L3 are each a single bond or a divalent group, and L1 and L2 or L1 and L3 may be bonded to each other to form a ring. Two or more L1, L2, and L3 may be the same or different from each other.

[0038] When L1 and L2 or L1 and L3 are bonded to each other to form a ring, the structure of the formed ring is not particularly limited. For example, when L1 and L3 are bonded to each other to form a ring, the following structure is considered, but is not limited thereto.

[0039]

[0040] In the above formula, L1' and L3' are modifications of L1 and L3, and are single bonds or divalent groups like L1 and L3. The other elements in the formula are the same as those defined in formula (1).

[0041] As described above, the distance between M1 and M2 and the distance between different M2s can be increased by increasing the value of m or the length of L1 or L2.

[0042] L4 is a single bond or a divalent group, and when m is an integer of 2 or more, two or more L4s may be the same or different from one another.

[0043] In the compound of the present technology, for at least one L1, the structure of the L1 may include one or more structures selected from the following:

[0044] In this specification, the term "comprise" means that the target structure is included as an element, and the target structure is allowed to include other elements besides the element. In the above example, L1 may include a structure other than the structure below, for example, a triazolyl functional group, etc., which will be described later.

[0045]

[0046] In the formula, a, b, and c are integers of 1 or more, and can be selected from any integer within a range that allows synthesis, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc. There are no particular upper limits for a, b, and c, but they can be selected from any values, such as 30 or less, 20 or less, 15 or less, or 10 or less. Furthermore, when there are two or more a, b, and c, the two or more a, b, and c may be the same or different from each other.

[0047] In the compounds of the present technology, for at least one L1, the L1 may be a linker containing a triazolyl functional group.

[0048] In the case where L1 contains a triazolyl functional group, for example, L1-M (where M may be M1 or M2) may have the following structure:

[0049]

[0050]

[0051] L1a and L1b in the above formula are modified versions of L1, and are a single bond or a divalent group, just like L1.

[0052] In some embodiments, L1a and L1b may, for example, independently have the following structures:

[0053]

[0054] The ion implantation device may have one of the following:

[0055] R1 is H or a monovalent group, and two or more R1's may be the same or different from each other.

[0056] R2 and R3 are each H, a monovalent group, or Q as described below.

[0057] R4 and R5 are each H or a monovalent group, or R4 and R5 are a single unsaturated substituent. When there are two or more R4s and R5s, the two or more R4s and R5s may be the same or different from each other.

[0058] The Q is a reactive group that can form a bond with an analyte molecule, a targeting moiety, a solid support, or a complementary reactive group Q'. These may further have one or more substituents as needed, as long as the desired physical properties are not significantly impaired. In addition, when there are two or more Qs in the compound of the present technology, the two or more Qs may be the same or different from each other.

[0059] When Q is a moiety containing a reactive group capable of forming a bond with a complementary reactive group Q′, Q and Q′ can be bonded to form a conjugate in which the compound of the present technology is bound to, for example, an antibody.

[0060] Here, a "reactive group" is a moiety that can react with a second reactive group (a "complementary reactive group") to form one or more bonds, for example, by substitution, oxidation, reduction, addition, or cycloaddition reaction, etc. Exemplary reactive groups are provided in Table 1 below and include, for example, nucleophilic groups, electrophilic groups, dienes, dienophiles, aldehydes, oximes, hydrazones, alkynes, amines, azides, acyl azides, acyl halides, nitriles, nitrones, sulfhydryls, disulfides, sulfonyl halides, isothiocyanates, imidoesters, activated esters, ketones, α,β-unsaturated carbonyls, alkenes, maleimides, α-haloimides, epoxides, aziridines, tetrazines, tetrazoles, phosphines, biotin, thiirane, and the like.

[0061] In the above case, Q and Q' are not particularly limited as long as they are a combination that can form a bond through interaction, and an example is a combination of biotin and avidin.

[0062] In the present technology, by making one of Q and Q' avidin or an avidin derivative and the other biotin or a biotin derivative, a conjugate between the compound of the present technology and an antibody or the like can be suitably formed by avidin-biotin bonding.

[0063] In the compounds of the present technology, examples of the analyte molecule that can be Q include "biomolecules."

[0064] "Biomolecule" refers to any of a variety of biological substances, including nucleic acids, carbohydrates, amino acids, polypeptides, glycoproteins, hormones, aptamers, and mixtures thereof. More specifically, the term is intended to include, but is not limited to, RNA, DNA, oligonucleotides, modified or derivatized nucleotides, enzymes, receptors, prions, receptor ligands (including hormones), and the like.

[0065] In the compounds of the present technology, the "solid support" which can be Q refers to any solid substrate known in the art for solid-phase support of molecules, such as microparticles, cells, etc.

[0066] As used herein, "microparticle" refers to any of a number of small particles useful for binding to the compounds of the invention, including, but not limited to, glass beads, magnetic beads, polymeric beads, non-polymeric beads, etc. In certain embodiments, the microparticle comprises a polystyrene bead.

[0067] "Solid support residue" refers to a functional group that remains attached to a molecule when the molecule is cleaved from the solid support. Solid support residues are known in the art and can be readily obtained based on the structure of the solid support and the group linking the molecule to the solid support.

[0068] In the compounds of the present technology, a "targeting moiety," which can be Q, is a moiety that selectively binds to or associates with a specific target (e.g., an analyte molecule). "Selectively" binds or associates means that the targeting moiety preferentially associates with or binds to the desired target compared to other targets. In some embodiments, the compounds disclosed herein include a linkage to a targeting moiety for the purpose of selectively binding or associating the compound with the analyte of interest (i.e., the target of the targeting moiety), thereby enabling detection of the analyte. Exemplary targeting moieties include, but are not limited to, antibodies, antigens, nucleic acid sequences, enzymes, proteins, cell surface receptor antagonists, and the like. In some embodiments, the targeting moiety is a moiety that selectively binds to or associates with a target feature on or in a cell (e.g., a target feature on the cell membrane or other cellular structure), such as an antibody, thereby enabling detection of the cell of interest. Small molecules that selectively bind to or associate with the desired analyte are also contemplated as targeting moieties in certain embodiments. Those skilled in the art will recognize other analytes and corresponding targeting moieties that are useful in various embodiments.

[0069] Examples of Q that can be used in the compounds of the present technology are shown in Table 1 below. Note that Table 1 shows examples of Q, and Q is not limited to these.

[0070]

[0071]

[0072]

[0073]

[0074] In this specification, the term "compound" includes stereoisomers, tautomers, etc. The compounds of the present technology may also exist as salts.

[0075] As described above, the compound of the present technology can suppress Förster resonance energy transfer from M1 or M2 to another M2, thereby maintaining the spectral waveforms originating from the chromophores of M1 and M2, respectively. Therefore, by analyzing the spectral waveforms in specific wavelength regions using the compound of the present technology, multiple factors to be measured can be observed simultaneously.

[0076] In the present technology, the "monovalent group" is not particularly limited as long as it is a group having one binding site, and suitable examples include monovalent groups such as OH, SH, alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkyl ether, alkoxyalkyl ether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether, thiophosphoalkyl ether, monovalent carbocyclic groups such as aryl or cycloalkyl, and heterocyclic groups such as heteroaryl or heterocycloalkyl. These may further have one or more substituents as necessary, as long as the desired physical properties are not significantly impaired.

[0077] In the compounds of the present technology, any of R1 to R5 may be, for example, a monovalent group having the following structure:

[0078]

[0079] In the above formula, m" and n" are integers of 1 or more, and any integer can be selected within the range in which synthesis is possible, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc. There are no particular upper limits for m" and n", but they can be selected arbitrarily, for example, from 30 or less, 20 or less, 15 or less, 10 or less, etc. When there are two or more m"s and n"s, the two or more m"s and n"s may be the same or different from each other.

[0080] Re is H, an electron pair, or a counter ion.

[0081] L″ may be the aforementioned Re, Q, a nucleoside, a monovalent group containing a nitrogen-containing ring, or a monovalent group containing the structure of formula (1).

[0082] In the compounds of the present technology, any of R1 to R5 may be, for example, a monovalent group selected from the following:

[0083]

[0084] In the present technology, the "divalent group" is not particularly limited as long as it is a group having two binding sites, and for example, divalent groups such as alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, or heteroatom linker, arylene, heteroarylene, etc. can be suitably used. These may further have one or more substituents as necessary, as long as the desired physical properties are not significantly impaired.

[0085] In the present technology, the term "substituent" refers to an atom or atomic group other than a hydrogen atom that is introduced by replacing a hydrogen atom that constitutes a hydrocarbon having a target chemical structure.

[0086] "Alkyl" refers to a straight or branched hydrocarbon chain group consisting of carbon and hydrogen atoms, containing no unsaturation, e.g., 1 to 12 carbon atoms (C1-C12 alkyl), 1 to 8 carbon atoms (C1-C8 alkyl), or 1 to 6 carbon atoms (C1-C6 alkyl), attached to the remainder of the molecule by a single bond (e.g., methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, etc.). Unless otherwise specifically stated in the specification, the hydrogen atoms comprising an alkyl group may optionally be substituted.

[0087] "Alkylene" or "alkylene chain" refers to a straight or branched divalent hydrocarbon chain, consisting of carbon and hydrogen, containing no unsaturation, and having 1 to 12 carbon atoms (e.g., methylene, ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene, propynylene, n-butynylene, etc.), that connects the rest of the molecule to the radical group. The alkylene chain is attached to the rest of the molecule through a single bond and to the radical group through a single bond. The points of attachment of the alkylene chain to the rest of the molecule or to the radical group can be through one carbon or any two carbons within the chain. Unless stated otherwise specifically in the specification, alkylene can have optional substituents.

[0088] "Alkenylene" or "alkenylene chain" refers to a straight or branched divalent hydrocarbon chain, consisting of carbon and hydrogen, containing at least one carbon-carbon double bond, e.g., 2 to 12 carbon atoms, that connects the rest of the molecule to a radical group (e.g., ethenylene, propenylene, n-butenylene, etc.). The alkenylene chain is attached to the rest of the molecule through a single bond and to the radical group through a double bond or a single bond. The points of attachment of the alkenylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless stated otherwise specifically in the specification, alkenylene is optionally substituted.

[0089] "Alkynylene" or "alkynylene chain" refers to a straight or branched divalent hydrocarbon chain, consisting of carbon and hydrogen, containing at least one carbon-carbon triple bond, e.g., 2 to 12 carbon atoms, (e.g., ethenylene, propenylene, n-butenylene, etc.), that connects the rest of the molecule to a radical group. The alkynylene chain is attached to the rest of the molecule through a single bond and to the radical group through a double bond or a single bond. The points of attachment of the alkynylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless stated otherwise specifically in the specification, alkynylene can optionally have substituents.

[0090] "Alkyl ether" refers to any alkyl group, as defined above, in which at least one carbon-carbon bond is replaced with a carbon-oxygen bond. The carbon-oxygen bond can be terminal (i.e., C-O-H) (as in an alkoxy group) or the carbon-oxygen bond can be internal (i.e., C-O-C). An alkyl ether contains at least one carbon-oxygen bond, but can contain more than one. For example, polyethylene glycol (PEG) is included within the meaning of an alkyl ether. Unless otherwise specifically stated herein, alkyl ether groups can optionally have substituents.

[0091] "Alkoxy" refers to a group of the formula -ORa, where Ra is an alkyl group as defined above, for example, containing 1 to 12 carbon atoms. Unless stated otherwise specifically in the specification, an alkoxy group may optionally bear a substituent.

[0092] "Alkoxyalkyl ether" refers to a group of the formula -ORaRb, where Ra is an alkylene group as defined above, for example, containing 1 to 12 carbon atoms, and Rb is an alkyl ether group as defined herein. Unless stated otherwise specifically in the specification, an alkoxyalkyl ether group may optionally bear a substituent.

[0093] "Heteroalkyl" refers to an alkyl group (as defined above) that includes at least one heteroatom (e.g., N, O, P, or S) within or at the terminus of the alkyl group. Unless otherwise specifically stated in the specification, heteroalkyl groups can have optional substituents.

[0094] "Heteroalkoxy" refers to a radical of the formula -ORa, where Ra is a heteroalkyl radical as defined above, e.g., containing 1 to 12 carbon atoms. Unless stated otherwise specifically in the specification, a heteroalkoxy radical may optionally bear substituents.

[0095] "Heteroalkylene" refers to an alkylene group, as defined above, containing at least one heteroatom (e.g., N, O, P, or S) within the alkylene chain or at the terminus of the alkylene chain. Unless stated otherwise specifically in the specification, heteroalkylene groups can have optional substituents.

[0096] Exemplary heteroalkylene groups include ethylene oxide (e.g., polyethylene oxide) and the "C" linking groups illustrated below: Includes:

[0097] Multimers of the above C-linkers are included in the various embodiments of heteroalkylene linkers.

[0098] "Heteroalkenylene" is a heteroalkylene (as defined above) containing at least one carbon-carbon double bond. Unless stated otherwise specifically in the specification, a heteroalkenylene group may optionally bear substituents.

[0099] "Heteroalkynylene" means a heteroalkylene (as defined above) containing at least one carbon-carbon triple bond. Unless stated otherwise specifically in the specification, a heteroalkynylene group may optionally bear substituents.

[0100] A "heteroatom linker" refers to a linker group consisting of one or more heteroatoms. Exemplary heteroatom linkers contain one or more heteroatoms selected from the group consisting of O, N, P, and S (e.g., linkers having the formula -P(O-)(=O)O- or -OP(O-)(=O)O-, as well as multimers and combinations thereof). Unless otherwise specifically stated herein, heteroatom linkers may optionally have substituents.

[0101] "Phosphate" refers to the group -OP(=O)(Ra)Rb, where Ra is OH, O-, or ORc; and Rb is OH, O-, ORc, a thiophosphate group as described below, or a further phosphate group, where Rc is a counterion (e.g., Na+, etc.). Unless otherwise specifically stated herein, the phosphate group may have optional substituents.

[0102] "Phosphoalkyl" refers to the group -OP(=O)(Ra)Rb, where Ra is OH, O-, or ORc; and Rb is -Oalkyl, where Rc is a counterion (e.g., Na+, etc.). Unless otherwise specifically stated herein, phosphoalkyl groups may optionally have substituents.

[0103] "Phosphoalkyl ether" refers to the group -OP(=O)(Ra)Rb, where Ra is OH, O-, or ORc; and Rb is an -O alkyl ether, where Rc is a counterion (e.g., Na+, etc.). Unless otherwise specifically stated herein, phosphoalkyl ether groups may optionally have substituents.

[0104] "Thiophosphate" refers to the group -OP(=Ra)(Rb)Rc, where Ra is O or S; Rb is OH, O-, S-, ORd, or SRd; and Rc is OH, SH, O-, S-, ORd, SRd, a phosphate group, or an additional thiophosphate group, where Rd is a counterion (e.g., Na+), and with the proviso that: i) Ra is S; ii) Rb is S- or SRd; iii) Rc is SH, S-, or SRd; or iv) a combination of i), ii), and / or iii). Unless specifically stated otherwise herein, thiophosphate groups can optionally bear substituents.

[0105] "Thiophosphoalkyl" refers to the group -OP(=Ra)(Rb)Rc, where Ra is O or S; Rb is OH, O-, S-, ORd, or SRd; and Rc is -Oalkyl, where Rd is a counterion (e.g., Na+), and with the proviso that: i) Ra is S; ii) Rb is S- or SRd; or iii) Ra is S and Rb is S- or SRd. Unless stated otherwise specifically in the specification, thiophosphoalkyl groups may bear optional substituents.

[0106] "Thiophosphoalkyl ether" refers to the group -OP(=Ra)(Rb)Rc, where Ra is O or S, Rb is OH, O-, S-, ORd, or SRd; and Rc is an -O alkyl ether, where Rd is a counterion (e.g., Na+), and with the proviso that: i) Ra is S, ii) Rb is S- or SRd, or iii) Ra is S and Rb is S- or SRd. Unless otherwise specifically stated in the specification, thiophosphoalkyl ether groups may bear optional substituents.

[0107] "Carbocyclic" refers to a stable 3- to 18-membered aromatic or non-aromatic ring containing, for example, 3 to 18 carbon atoms. Unless stated otherwise specifically in the specification, a carbocyclic ring can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system which can include fused or bridged ring systems and can be partially or fully unsaturated. Non-aromatic carbocyclyl radicals include cycloalkyl, while aromatic carbocyclyl radicals include aryl. Unless stated otherwise specifically in the specification, a carbocyclic group can optionally bear substituents.

[0108] "Cycloalkyl" refers to a stable non-aromatic monocyclic or polycyclic carbocyclic ring, for example, having 3 to 15 carbon atoms, preferably 3 to 10 carbon atoms, saturated or unsaturated, and attached to the remainder of the molecule by a single bond, which may include fused or bridged ring systems. Monocyclic cycloalkyls include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic cycloalkyls include, for example, adamantyl, norbornyl, decalinyl, 7,7-dimethyl-bicyclo-[2.2.1]heptanyl, and the like. Unless otherwise specifically stated in the specification, cycloalkyl groups may optionally have substituents.

[0109] "Aryl" refers to a ring system containing at least one carbocyclic aromatic ring. In some embodiments, an aryl contains 6 to 18 carbon atoms. The aryl ring can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which can include fused or bridged ring systems. Aryl includes, but is not limited to, aryl derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene. Unless stated otherwise specifically in the specification, aryl groups can optionally bear substituents.

[0110] "Heterocyclic" refers, for example, to a stable 3- to 18-membered aromatic or non-aromatic ring containing from 1 to 12 carbon atoms and from 1 to 6 heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. Unless otherwise specifically stated herein, the heterocyclic ring can include fused or bridged ring systems, the nitrogen, carbon, or sulfur atoms in the heterocyclic ring can be optionally oxidized; the nitrogen atom can be optionally quaternized; and the heterocyclic ring can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system which can be partially or fully unsaturated.

[0111] Examples of aromatic heterocyclic rings are listed below in the definition of heteroaryl below (i.e., heteroaryl is a subset of heterocyclic). Examples of non-aromatic heterocyclic rings include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4- Piperidonyl, pyrrolidinyl, pyrazolidinyl, pyrazolopyrimidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trioxanyl, trithianyl, triazinanyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. Unless stated otherwise specifically in the specification, heterocyclic groups may optionally bear substituents.

[0112] "Heteroaryl" refers, for example, to a 5-14 membered ring system containing 1-13 carbon atoms, 1-6 heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur, and at least one aromatic ring. For purposes of certain embodiments of this invention, the heteroaryl radical can include fused or bridged ring systems; the nitrogen, carbon, or sulfur atoms in the heteroaryl radical can be optionally oxidized; and the nitrogen atom can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which can be optionally quaternized.Examples include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzthiazolyl, benzoindolyl, benzodioxolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl). , benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, benzoxazolinonyl, benzimidazolethionyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1-oxidopyridinyl, 1- Oxidopyrimidinyl, 1-oxidopyrazinyl, 1-oxidopyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, pteridinonyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyridinonyl, pyrazinyl, pyrimidinyl, pyrimidinonyl , pyridazinyl, pyrrolyl, pyrido[2,3-d]pyrimidinonyl, quinazolinyl, quinazolinonyl, quinoxalinyl, quinoxalinonyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, thieno[3,2-d]pyrimidin-4-onyl, thieno[2,3-d]pyrimidin-4-onyl, triazolyl, tetrazolyl, triazinyl, and thiophenyl (i.e., thienyl). Unless stated otherwise specifically in the specification, heteroaryl groups may optionally bear substituents.

[0113] "Heterocycloalkyl" refers to a cycloalkyl group, as defined above, containing at least one heteroatom (e.g., N, O, P, or S) within the cycloalkyl chain or at the terminus of the cycloalkyl chain. Unless stated otherwise specifically in the specification, heterocycloalkyl groups may optionally bear substituents.

[0114] "Arylene" refers to a divalent ring system containing at least one carbocyclic aromatic ring that links the rest of the molecule to a radical group. Unless stated otherwise specifically in the specification, an arylene group can optionally bear substituents.

[0115] "Heteroarylene" refers to an arylene group, as defined above, containing at least one heteroatom (e.g., N, O, P, or S) within or at the terminus of the arylene group. Unless otherwise specifically stated in the specification, heteroarylene groups may optionally bear substituents.

[0116] "Fused" refers to a ring system containing at least two rings, wherein the two rings share at least one common ring atom, e.g., two common ring atoms. When the fused ring is a heterocyclyl ring or a heteroaryl ring, the common ring atom can be carbon or nitrogen. Fused rings include bicyclic, tricyclic, tetracyclic, etc.

[0117] "Conjugation" refers to the overlap of one p orbital with another p orbital across an intervening sigma bond. Conjugation can occur in cyclic or acyclic compounds. "Degree of conjugation" refers to the overlap of at least one p orbital with another p orbital across an intervening sigma bond. For example, 1,3-butadiene has a degree of conjugation of one, while benzene and other aromatic compounds typically have multiple degrees of conjugation. Fluorescent and colored compounds typically contain at least one degree of conjugation.

[0118] The compound of the present technology may have a structure of the following formula (1A) as a modification of the structure of formula (1).

[0119] (1A)

[0120] z is an integer of 1 or more, and can be any integer selected within a range that allows synthesis, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc. There is no particular upper limit to z, and it can be any integer selected, for example, from 30 or less, 20 or less, 15 or less, 10 or less, etc. When m1 or m3 is an integer of 2 or more, the two or more z's may be the same or different from each other.

[0121] m1, m2, and m3 are integers of 0 or more, and can be selected from any integer within a range that allows synthesis, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc. There are no particular upper limits for m1, m2, and m3, and they can be selected from any values, such as 30 or less, 20 or less, 15 or less, or 10 or less. When n is an integer of 2 or more, the two or more m1, m2, and m3 may be the same or different from each other.

[0122] In formula (1A), the other elements are the same as those defined in formula (1).

[0123] The compound of the present technology may have a structure of the following formula (1B) as a modification of the structure of formula (1).

[0124] (1B)

[0125] z has the same definition as in formula (1A). When m is an integer of 2 or more, two or more z's may be the same or different from each other.

[0126] In formula (1B), the other elements are the same as those defined in formula (1).

[0127] The compound of the present technology may have a structure of the following formula (1C) as a modification of the structure of formula (1).

[0128] (1C)

[0129] z has the same definition as in formula (1A). When m is an integer of 2 or more, two or more z's may be the same or different from each other.

[0130] x1 and x2 are integers of 0 or more, and can be selected from any integer within a range that can be synthesized, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc. There are no particular upper limits for x1 and x2, but they can be selected from any values, such as 30 or less, 20 or less, 15 or less, 10 or less, etc.

[0131] x3 and x4 are integers of 0 or more, and can be selected from any integer within a range that can be synthesized, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc. There are no particular upper limits for x3 and x4, and they can be selected from any values, such as 30 or less, 20 or less, 15 or less, or 10 or less. When n is an integer of 2 or more, the two or more x3 and x4 may be the same or different from each other.

[0132] In formula (1C), the other elements are the same as those defined in formula (1).

[0133] The compound of the present technology may have a structure of the following formula (1D) as a modification of the structure of formula (1).

[0134] (1D)

[0135] m2 has the same definition as in formula (1A). When n is an integer of 2 or more, two or more m2's may be the same or different from one another.

[0136] In formula (1D), the other elements are defined the same as in formula (1).

[0137] <Synthesis of the compounds of the present technology> The following reaction scheme shows exemplary methods for preparing the compounds of the present technology. Those skilled in the art can prepare these compounds by similar methods or by combining other methods known to those skilled in the art. Those skilled in the art can prepare compounds of the present technology that are not specifically exemplified herein by using appropriate starting components in a manner similar to that described below, and modifying synthesis parameters as necessary.

[0138] Generally, the starting components can be obtained from sources such as Sigma-Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, or can be synthesized according to sources known to those skilled in the art (see, for example, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th Edition (Wiley, December 2000)), or can be prepared as described herein.

[0139] In particular, methods of preparing embodiments of the compounds disclosed herein can be found in, for example, PCT Publication Nos. WO2015 / 027176, WO2016 / 138461, WO2016 / 183185, WO2017 / 173348, WO2017 / 173355, WO2017 / 177065, WO2017 / 196954, WO2017 / 214165, and WO2018 / 022925, each of which is incorporated by reference in its entirety.

[0140] Reaction Scheme I

[0141] Reaction Scheme I illustrates an exemplary method for preparing intermediates useful for preparing compounds of the present technology. Referring to Reaction Scheme 1, compounds of structure a can be purchased or prepared by methods well known to those skilled in the art. Reaction with M-X (where X is a halogen, such as bromo) under art-known Suzuki coupling conditions yields compounds of structure b. Compounds of structure b can be used for preparing compounds of the present technology as described below.

[0142] In the formula of the above structure a, L is a monovalent group having at least one double structure, and becomes a divalent group by reaction with the above M-X. The other elements in the formula are defined as in formula (1).

[0143] [Reaction Scheme II]

[0144] Reaction Scheme II illustrates an alternative method for preparing intermediates useful in preparing compounds of the present technology. Referring to Reaction Scheme II, a compound of structure c (which can be purchased or prepared by well-known techniques) is reacted with M-G' to provide a compound of structure d, where G and G' represent functional groups with complementary reactivity (i.e., functional groups that react to form a covalent bond). G' can be pendant to M or can be part of the structural backbone of M. G and G' can be any number of functional groups described herein, such as an alkyne and azide, respectively, an amine and activated ester, respectively, or an amine and isothiocyanate, respectively.

[0145] In the above formula, L1a is a single bond or a divalent group, and the other elements in the formula are the same as those defined in formula (1).

[0146] The compound of the present technology can be synthesized under well-known automated DNA synthesis conditions with the following structure (e): (hereinafter "click" reaction)

[0147] DNA synthesis methods are well known in the art. Briefly, two alcohol groups (e.g., R2 and R3 in intermediates b or d above) are functionalized with a dimethoxytrityl (DMT) group and a 2-cyanoethyl-N,N-diisopropylaminophosphoramidite group, respectively. The phosphoramidite group is typically coupled to the alcohol group in the presence of an activator such as tetrazole, followed by oxidation of the phosphorus atom with iodine. The dimethoxytrityl group can be removed with an acid (e.g., chloroacetic acid) to reveal a free alcohol, which can be reacted with the phosphoramidite group. The 2-cyanoethyl group can be removed after oligomerization by treatment with aqueous ammonia.

[0148] The preparation of phosphoramidites used in the above oligomerization methods is also well known in the art. For example, a primary alcohol (e.g., R3) can be protected as a DMT group by reaction with DMT-Cl. A secondary alcohol (e.g., R2) is then functionalized as a phosphoramidite by reaction with an appropriate reagent, such as 2-cyanoethyl N,N-diisopropylchlorophosphoramidite. Methods for the preparation of phosphoramidites and their oligomerization are well known in the art and are described in more detail in the Examples.

[0149] The compounds of the present technology are prepared by oligomerization of intermediates b or d and e according to the well-known phosphoramidite chemistry described above. The desired number of m and n repeat units are incorporated into the molecule by repeating the phosphoramidite coupling a desired number of times.

[0150] In various other embodiments, compounds useful for preparing the compounds of the present technology are provided. These compounds can be prepared as described above in the form of monomers, dimers, and / or oligomers, and then an M moiety can be covalently attached to the compound via any number of synthetic methodologies (e.g., the "click" reaction described above) to form the compounds of the present technology.

[0151] Alternatively, the compound of the present technology can be prepared by subjecting the compound of structure c to the above-mentioned "click" reaction to form an intermediate shown in the following formula (2), and then reacting the intermediate with the above-mentioned M-G' (where M may be M1 or M2).

[0152] In the following formula (2) relating to the intermediate, G (which may be G1 or G2 in the following formula) and G' defined in Structure C are functional groups having complementary reactivity (i.e., functional groups that react to form a covalent bond). The other elements in the formula are the same as those defined in formula (1).

[0153] As a modification of the structure of formula (2), the structure of formula (2) can take the modified structures corresponding to formulas (1A) to (1D), which are modifications of the structure of formula (1), similarly to formula (1).

[0154] (2)

[0155] <Detection System> As described above, the compound of the present technology can suppress Förster resonance energy transfer from M1 or M2 to other M2, thereby maintaining the spectral waveforms derived from the respective chromophores of M1 and M2. This allows multiple factors of the measurement target to be observed simultaneously. Therefore, a detection system for detecting the distribution of a target in a measurement target can be suitably constructed by analyzing the spectral waveform of a specific wavelength region using the compound of the present technology.

[0156] In this specification, the term "detection system" is not limited to a method for detecting the distribution of a target, but also includes a detection kit for realizing the method, a measuring device equipped with a system for detecting the distribution of a target, and the like.

[0157] Examples of measurement targets of the detection system of the present technology include fine particles, cells, and the like.

[0158] The compounds of the present technology are used in detection systems for labeling purposes, making them useful in a wide variety of analytical applications (e.g., biochemical and biomedical applications) that require determining the presence, location, or amount of a specific "target" analyte (e.g., a biomolecule) in a measurement target. Thus, in another aspect, the detection system of the present technology may be a method for detecting a biomolecule, comprising: (a) providing a biological system with a detectable biomolecule that includes a compound of the present technology linked to the biomolecule; and (b) detecting the biomolecule based on its visible properties.

[0159] Here, "detecting a biomolecule" means detecting the biomolecule of interest directly or indirectly using an optical-based detection device (including, but not limited to, an absorption spectrophotometer, a transmission light microscope, a digital camera, and a scanner).

[0160] In addition, in the detection system of this technology, a densitometer can be used to quantify the amount of detectable biomolecules present. For example, the relative amounts of the biomolecules in two samples can be determined by measuring their relative optical density. If the stoichiometry of dye molecules per biomolecule is known and the extinction coefficient of the dye molecule is known, the absolute concentration of the biomolecule can also be determined from the optical density measurement. As used herein, the term "biological system" refers to any solution or mixture that contains one or more biomolecules in addition to a detectable biomolecule. Non-limiting examples of such biological systems include cells, cell extracts, tissue samples, electrophoresis gels, assay mixtures, and hybridization reaction mixtures.

[0161] In the detection system of the present technology, from the viewpoint of suitably labeling a target in a measurement subject, it is preferable that R2 or R3, or both, of the compound of the present technology used in the detection system of the present technology be Q.

[0162] The detection system of the present technology will be described below with reference to the drawings.

[0163] Figure 1 shows an image of the spectrum of the compound of the present technology. Dye A and dye B in <1A> in Figure 1 correspond to M1 and M2, which are chromophores in formula (1).

[0164] <1A> in Figure 1 indicates that a compound of the present technology is synthesized using dye A and dye B. In the synthesized compound of formula (1), dye A constitutes M1, and dye B constitutes M2.

[0165] <1B> in Figure 1 is a graph of the spectrum of the above compound. The graphs of "Dye A" and "Dye B" in <1B> are spectra of the dye A and dye B compounds related to M1 and M2 alone. On the other hand, the graph of "Dye A + Dye B" is the spectrum of the compound of the present technology.

[0166] In the compound of the present technology, M1 and M2 do not undergo Förster resonance energy transfer with each other, and therefore, as shown in graph <1B>, the spectral waveform of the compound maintains the spectral waveforms derived from dye A and dye B.

[0167] Next, Figure 2 is an image diagram showing the concept of the detection system of the present technology. <2A> in Figure 2 indicates that a specific analyte, such as a target biomolecule in a cell to be measured, is bound to a compound of the present technology by any method.

[0168] <2B> in Figure 2 is a graph of the spectrum obtained when cells bound to the compound of the present technology are measured using the detection system of the present technology. As described above, this graph maintains the spectral waveforms derived from dye A and dye B, allowing multiple dyes to be used to distinguish multiple targets in the cells being measured. Therefore, by analyzing the spectral waveforms in specific wavelength regions, multiple factors corresponding to the type of dye can be simultaneously observed. For example, in the example of Figure 2, two factors can be simultaneously observed in a single measurement.

[0169] In contrast, Figure 3 is an image diagram showing the concept of a detection system according to the prior art. <3A> and <3C> in Figure 3 show that a specific analyte, such as a target biomolecule in a cell, and a dye compound or the like are bound to the cell by any method.

[0170] <3B> and <3C> in Figure 3 are graphs of spectra obtained when cells bound to the above-mentioned conventional compound are measured using the detection system of the present technology. In the detection system of the conventional technology, the presence of a target in the cells to be measured can be confirmed by detecting the spectral peak of a dye compound or the like. Therefore, in principle, measurements are performed according to the number of factors to be observed. That is, in this example, two measurements are required to observe two factors, as shown in Figure 3.

[0171] 4 is an image diagram of the absorption spectrum of a compound of the present technology having two different chromophores. The graphs of "Dye A" and "Dye B" in <4B> are the absorption spectra of the dye A and dye B compounds related to M1 and M2 alone. On the other hand, the graph of "Dye A + B" is the absorption spectrum of the compound of the present technology.

[0172] As shown in <4B>, the absorption spectrum of the compound of the present technology maintains the waveform of the absorption spectrum derived from dye A or dye B.

[0173] 5 is an image of the fluorescence spectrum of a compound of the present technology having two different chromophores. The graphs of "Dye A" and "Dye B" in <5B> are the absorption and fluorescence spectra of the dye A and dye B compounds related to M1 and M2 alone. On the other hand, the graph of "Dye A + B" is the absorption and fluorescence spectrum of the compound of the present technology.

[0174] As shown in <5B>, the waveform of the absorption spectrum derived from dye A or dye B is also maintained in the fluorescence spectrum of the compound of the present technology, and therefore, it can be confirmed from Figures 4 and 5 that FRET between the two chromophores is suppressed in the compound of the present technology. As described above, by using the compound of the present technology having such a spectrum and analyzing the waveform of the spectrum in a specific wavelength region, two targets in a cell to be measured can be distinguished by the dyes associated with the two chromophores, and two factors can be observed simultaneously.

[0175] 6 is an image diagram of the absorption spectrum of a compound of the present technology having three chromophores. The graphs of "Dye A," "Dye B," and "Dye C" in <6B> are absorption spectra of the compounds M1, M2, and dye A, dye B, and dye C associated with M2 different from M1 alone. On the other hand, the graph of "Dye A+B+C" is the absorption spectrum of the compound of the present technology.

[0176] In the compound of the present technology, the waveform of the absorption spectrum derived from dye A or dye B is maintained in the absorption spectrum of the compound of the present technology, and FRET between chromophores can be suppressed. Therefore, by using the compound and analyzing the waveform of the spectrum in a specific wavelength region, three targets in a cell to be measured can be distinguished by dyes associated with three chromophores, and three factors can be observed simultaneously.

[0177] As in the example shown above, by synthesizing a compound of the present technology with an even greater number of chromophores and using the compound to analyze the waveform of a spectrum in a specific wavelength region, it is possible to distinguish multiple targets in the cells to be measured using dyes associated with multiple chromophores and observe multiple factors simultaneously.

[0178] Furthermore, in the detection system of the present technology, the compound of the present technology can be used in any manner, similar to conventional dye compounds, to detect the distribution of a target in the measurement subject by flow cytometry.

[0179] <Measuring Device> The detection system according to the present technology can also be suitably employed in measuring devices such as a biological sample analyzing device (a flow cytometer (including a microparticle analyzing device (cell analyzer) and a microparticle sorting device (cell sorter)) that includes a flow cell having a measurement flow path, a microscope, a plate reader, etc.

[0180] An example of a biological sample analyzer that can be equipped with a detection system according to the present technology will be described in more detail with reference to FIG.

[0181] An example configuration of a biological sample analyzer according to the present disclosure is shown in Figure 7. The biological sample analyzer 6100 shown in Figure 7 includes a light irradiation unit 6101 that irradiates light onto a biological sample S flowing through a flow path C, a detection unit 6102 that detects light generated by irradiating the biological sample S with light, and an information processing unit 6103 that processes information related to the light detected by the detection unit. Examples of the biological sample analyzer 6100 include a flow cytometer and an imaging cytometer. The biological sample analyzer 6100 may also include a fractionation unit 6104 that separates specific biological particles P from within the biological sample. An example of a biological sample analyzer 6100 that includes the fractionation unit is a cell sorter.

[0182] (Biological Sample) The biological sample S may be a liquid sample containing biological particles. The biological particles may be, for example, cells or non-cellular biological particles. The cells may be living cells, and more specific examples include blood cells such as red blood cells and white blood cells, and reproductive cells such as sperm and fertilized eggs. The cells may be directly collected from a specimen such as whole blood, or may be cultured cells obtained after culturing. Examples of the non-cellular biological particles include extracellular vesicles, particularly exosomes and microvesicles. The biological particles may be labeled with one or more labeling substances (e.g., dyes (particularly fluorescent dyes) and fluorescent dye-labeled antibodies). Note that the biological sample analyzer of the present disclosure may also analyze particles other than biological particles, such as beads for calibration purposes.

[0183] (Flow Channel) The flow channel C is configured to allow the biological sample S to flow. In particular, the flow channel C can be configured to form a flow in which biological particles contained in the biological sample are aligned in a substantially straight line. The flow channel structure including the flow channel C may be designed to form a laminar flow. In particular, the flow channel structure is designed to form a laminar flow in which the flow of the biological sample (sample flow) is surrounded by the flow of sheath liquid. The design of the flow channel structure may be appropriately selected by those skilled in the art, and a known design may be adopted. The flow channel C may be formed in a flow channel structure such as a microchip (a chip having flow channels on the order of micrometers) or a flow cell. The width of the flow channel C may be 1 mm or less, particularly 10 μm or more and 1 mm or less. The flow channel C and the flow channel structure including it may be formed from a material such as plastic or glass.

[0184] The biological sample analyzer of the present disclosure is configured so that light from light irradiation unit 6101 is irradiated onto the biological sample flowing within flow path C, and particularly onto biological particles within the biological sample. The biological sample analyzer of the present disclosure may be configured so that the interrogation point of light on the biological sample is within the flow path structure in which flow path C is formed, or so that the interrogation point of light is outside the flow path structure. An example of the former is a configuration in which the light is irradiated onto flow path C within a microchip or flow cell. In the latter, the light may be irradiated onto biological particles after they have left the flow path structure (particularly its nozzle portion), and an example of this is a jet-in-air flow cytometer.

[0185] (Light Irradiation Unit) The light irradiation unit 6101 includes a light source unit that emits light and a light-guiding optical system that guides the light to an irradiation point. The light source unit includes one or more light sources. The type of light source is, for example, a laser light source or an LED. The wavelength of the light emitted from each light source may be any of ultraviolet light, visible light, and infrared light. The light-guiding optical system includes optical components such as a beam splitter group, a mirror group, or an optical fiber. The light-guiding optical system may also include a lens group for focusing light, such as an objective lens. There may be one or more irradiation points where the light intersects with the biological sample. The light irradiation unit 6101 may be configured to focus light irradiated from one or more different light sources onto one irradiation point.

[0186] (Detection Unit) The detection unit 6102 includes at least one photodetector that detects light generated by irradiating the bioparticles with light. The detected light is, for example, fluorescence or scattered light (e.g., one or more of forward scattered light, back scattered light, and side scattered light). Each photodetector includes one or more light-receiving elements, for example, a photodetector array. Each photodetector may include one or more PMTs (photomultiplier tubes) and / or photodiodes such as APDs and MPPCs as light-receiving elements. The photodetector includes, for example, a PMT array in which multiple PMTs are arranged in a one-dimensional direction. The detection unit 6102 may also include an imaging element such as a CCD or CMOS. The detection unit 6102 can acquire images of the bioparticles (e.g., bright-field images, dark-field images, and fluorescence images) using the imaging element.

[0187] The detection unit 6102 includes a detection optical system that allows light of a predetermined detection wavelength to reach a corresponding photodetector. The detection optical system includes a spectroscopic unit such as a prism or a diffraction grating, or a wavelength separation unit such as a dichroic mirror or an optical filter. The detection optical system is configured to, for example, disperse light generated by irradiating bioparticles with light, and detect the dispersed light using a plurality of photodetectors, the number of which is greater than the number of fluorescent dyes with which the bioparticles are labeled. A flow cytometer that includes such a detection optical system is called a spectral flow cytometer. Furthermore, the detection optical system is configured to, for example, separate light corresponding to the fluorescent wavelength range of a specific fluorescent dye from the light generated by irradiating bioparticles with light, and detect the separated light using a corresponding photodetector.

[0188] The detection unit 6102 may also include a signal processing unit that converts the electrical signal obtained by the photodetector into a digital signal. The signal processing unit may include an A / D converter as a device that performs the conversion. The digital signal obtained by the conversion by the signal processing unit may be transmitted to the information processing unit 6103. The digital signal may be handled by the information processing unit 6103 as data related to light (hereinafter also referred to as "light data"). The light data may be light data including, for example, fluorescent light data. More specifically, the light data may be light intensity data, and the light intensity may be light intensity data of light including fluorescent light (which may include feature quantities such as area, height, and width).

[0189] (Information Processing Unit) The information processing unit 6103 includes, for example, a processing unit that processes various data (e.g., optical data) and a storage unit that stores various data. When the processing unit acquires optical data corresponding to a fluorescent dye from the detection unit 6102, the processing unit may perform fluorescence spillover correction (compensation processing) on ​​the light intensity data. Furthermore, in the case of a spectral flow cytometer, the processing unit performs fluorescence separation processing on the optical data to acquire light intensity data corresponding to the fluorescent dye. The fluorescence separation processing may be performed, for example, according to the unmixing method described in Japanese Patent Application Laid-Open No. 2011-232259. When the detection unit 6102 includes an image sensor, the processing unit may acquire morphological information of bioparticles based on images acquired by the image sensor. The storage unit may be configured to store the acquired optical data. The storage unit may further be configured to store spectral reference data used in the unmixing processing.

[0190] If the biological sample analyzer 6100 includes a fractionating unit 6104 (described below), the information processing unit 6103 can determine whether to fractionate bioparticles based on the optical data and / or morphological information. The information processing unit 6103 can then control the fractionating unit 6104 based on the result of this determination, allowing the fractionating unit 6104 to fractionate the bioparticles.

[0191] The information processing unit 6103 may be configured to output various data (e.g., optical data and images). For example, the information processing unit 6103 may output various data (e.g., two-dimensional plots, spectral plots, etc.) generated based on the optical data. The information processing unit 6103 may also be configured to accept input of various data, such as accepting gating processing on a plot by a user. The information processing unit 6103 may include an output unit (e.g., a display, etc.) or an input unit (e.g., a keyboard, etc.) for executing the output or input.

[0192] The information processing unit 6103 may be configured as a general-purpose computer, for example, as an information processing device including a CPU, RAM, and ROM. The information processing unit 6103 may be included in a housing that includes the light irradiation unit 6101 and the detection unit 6102, or may be located outside the housing. Furthermore, various processes or functions performed by the information processing unit 6103 may be realized by a server computer or a cloud connected via a network.

[0193] (Sorting unit) The sorting unit 6104 sorts the bioparticles according to the determination result by the information processing unit 6103. The sorting method may be a method of generating droplets containing bioparticles by vibration, applying an electric charge to the droplets to be sorted, and controlling the direction of travel of the droplets using electrodes. The sorting method may also be a method of controlling the direction of travel of the bioparticles within the flow channel structure to perform sorting. The flow channel structure is provided with, for example, a control mechanism using pressure (spray or suction) or electric charge. An example of such a flow channel structure is a chip (for example, the chip described in JP 2020-76736 A) having a flow channel structure in which a flow channel C branches downstream into a recovery flow channel and a waste flow channel, and specific bioparticles are recovered into the recovery flow channel.

[0194] The present technology will be described in detail below based on specific examples, but the present technology is not limited to the contents of the examples shown below.

[0195] <Investigation of the distance between chromophores> For the dye compounds capable of forming the chromophores shown in Table 1, data on the absorption, fluorescence spectrum, maximum molar extinction coefficient, and fluorescence quantum yield of the dye compounds were obtained from papers, websites of the dye compound manufacturers (Thermo Fisher, AAT Bioquest websites), and actual measurements.

[0196] The normalized donor emission spectrum was calculated from the above data of the donor fluorescence spectrum and the acceptor absorption spectrum, and the J value was calculated using the above-mentioned formula (3) based on the molar extinction coefficient of the acceptor, etc. The calculated J values ​​are shown in Table 1.

[0197] For each of the dye compounds, the Förster distance (R0; the distance at which the FRET efficiency is 50%) was calculated using the above-mentioned formula (2) from the J value, the fluorescence quantum yield Q0 of the donor, the refractive index n of the medium, the mutual molecular orientation k, etc. The calculated R0 is shown in Table 1.

[0198] From the above R0, the FRET efficiency at each inter-dye distance was calculated using the aforementioned mathematical formula (3), and the distance required to achieve each FRET efficiency was calculated. The results are shown as a graph in Figure 8. Furthermore, based on the graph in Figure 8, Table 1 shows the distance at which the FRET efficiency is 50% and the distance at which the FRET efficiency is less than 99%.

[0199]

[0200] From the above results, it can be confirmed that even if the J value is large, FRET can be suppressed if the distance between chromophores is large.

[0201] 9 is a graph showing the calculated relationship between the distance between chromophores and the FRET efficiency for each J value. From this graph, it can be seen that, assuming J = 1E14, the FRET efficiency can be suppressed to less than 99% by separating the chromophores by 2.25 nm or more. Furthermore, it can be seen that the FRET efficiency can be suppressed to less than 50% by separating the chromophores by 4.90 nm or more. By utilizing this graph, it is possible to design a suitable compound based on the J value and the distance between chromophores, in accordance with the detection capabilities of the measurement device employed in the detection system of this technology and the intended use of the measurement device.

[0202] That is, when J = 1E14, for example, if a measuring device employing the detection system of the present technology can detect, for example, 1% of the original luminescence intensity of the chromophore, the spectral waveform of a specific wavelength region can be suitably analyzed by using a compound with a chromophore distance of 2.25 nm or more. If the measuring device can detect, for example, 50% of the luminescence, the spectral waveform of a specific wavelength region can be suitably analyzed by using a compound with a chromophore distance of 4.90 nm or more. Thus, by using a compound that matches the characteristics of the measuring device, the position of the target in the measurement target can be identified by the label derived from the chromophore.

[0203] <Compounds of the Present Technology> Next, based on the results shown in Figure 9, when the following dyes are designated as M1 and M2 and a compound of the present technology is synthesized, how the fluorescence spectrum changes depending on the distance between M1 and M2 was calculated based on the calculation results of the above-mentioned formulas (1) to (3). The results are shown in Figure 10. The compound of the present technology may have any structure shown in the present specification. Furthermore, the compound may be suitably synthesized, for example, by the method shown in the present specification.

[0204] M1: Rhodamine 101 inner salt (structure shown below)

[0205] M2: Sodium fluorescein (uranine) (structure shown below)

[0206] 10, it can be seen that, when the intensity of the uranine fluorescence spectrum of the compound is a predetermined amount relative to the 100% intensity of the uranine fluorescence spectrum alone, it is possible to suitably analyze the waveform of the spectrum in a specific wavelength region. In particular, it can be seen that by setting the distance between the dyes at 2.25 nm or more, the FRET efficiency can be suppressed to 99% or less, enabling suitable detection of light emission. It can also be seen that by increasing the distance between the dyes, the FRET efficiency can be further suppressed.

[0207] Therefore, by using this compound and focusing on the overall shape of the waveform of the spectrum in a specific wavelength range measured and analyzing the overall shape of the waveform of the spectrum, rather than focusing on individual peaks in the spectrum, it is suggested that, for example, when the measurement target is a cell, two targets in the cell can be distinguished using dyes associated with two chromophores and two factors can be observed simultaneously. Furthermore, although this example shows an example of a compound having two chromophores, when a compound having three or more chromophores is used, targets can be distinguished using dyes associated with the three or more chromophores and three or more factors corresponding to the chromophores can be observed simultaneously.

[0208] The present technology can have the following configurations: [1] A compound for analyzing the waveform of a spectrum in a specific wavelength region, having a structure of the following formula (1): (1) n is an integer of 1 or more, m is an integer of 0 or more, and when n is an integer of 2 or more, the two or more m's may be the same or different from each other, M1 and M2 are chromophores having different chemical structures and not undergoing Förster resonance energy transfer with each other, and when n is an integer of 2 or more, the two or more M2's may be the same or different from each other, L1, L2, and L3 are single bonds or divalent groups, and L1 and L2 or L1 and L3 may be bonded to each other to form a ring, and the two or more L1, L2, and L3 may be the same or different from each other, L4 is a single bond or divalent group, and when m is an integer of 2 or more, the two or more L4's may be the same or different from each other, R1 is H or a monovalent group, and the two or more R1's may be the same or different from each other, R2 and R3 are each H, a monovalent group, or Q, R4 and R5 are each H or a monovalent group, or R4 and R5 are one unsaturated substituent, and when there are two or more R4 and R5, the two or more R4 and R5 may be the same or different from each other, and Q is a reactive group capable of forming a bond with an analyte molecule, a targeting moiety, a solid support, or a complementary reactive group Q', and when there are two or more Q, the two or more Q' may be the same or different from each other. [2] The compound according to [1], having a structure of the following formula (1A): (1A) z is an integer of 1 or more, and when m1 or m3 is an integer of 2 or more, the two or more z's may be the same or different from each other, m1, m2, and m3 are integers of 0 or more, and when n is an integer of 2 or more, the two or more m1's, m2's, and m3's may be the same or different from each other. [3] The compound according to [1], having a structure of the following formula (1B): (1B) z is an integer of 1 or more, and when m is an integer of 2 or more, the two or more z's may be the same or different from each other. [4] The compound according to [1], having a structure of the following formula (1C): (1C) z is an integer of 1 or more, and when m is an integer of 2 or more, the two or more z's may be the same or different from each other, x1 and x2 are integers of 0 or more, x3 and x4 are integers of 0 or more, and when n is an integer of 2 or more, the two or more x3's and x4's may be the same or different from each other. [5] The compound according to [1], having a structure of the following formula (1D): (1D) m2 is an integer of 0 or greater, and when n is an integer of 2 or greater, the two or more m2s may be the same or different from each other. [6] The compound according to any one of [1] to [5], wherein at least one L1 is a linker containing a triazolyl functional group. [7] The compound according to any one of [1] to [6], wherein at least one L1-M (wherein M may be M1 or M2) has the following structure: [8] The compound according to any one of [1] to [6], wherein at least one of L1-M (wherein M may be M1 or M2) has the following structure: L1a and L1b are a single bond or a divalent group. [9] The compound according to any one of [1] to [8], wherein for at least one L1, the structure of the L1 contains one or more structures selected from the following: a, b, and c are integers of 1 or more, and when a, b, and c are 2 or more, the two or more a, b, and c may be the same or different from each other.

[10] The compound according to any one of [1] to [9], wherein the distance between M1 and M2 and the distance between the different M2s are 2.25 nm or more.

[11] The compound according to any one of [1] to [9], wherein the distance between M1 and M2 and the distance between the different M2s are 3.25 nm or more.

[12] The compound according to

[10] or

[11] , wherein the distances are the distances when the compound is in the ground state.

[13] The compound according to any one of [1] to

[12] , wherein M1 and M2, or M1 and two or more different M2s, have different absorption or emission spectra.

[14] The compound according to any one of [1] to

[13] , wherein the efficiency of Förster resonance energy transfer (FRET efficiency) from M1 or M2 to other M2 is 99% or less.

[15] The compound according to any one of [1] to

[13] , wherein the efficiency of Förster resonance energy transfer (FRET efficiency) from M1 or M2 to another M2 is 50% or less.

[16] The compound according to any one of [1] to

[15] , wherein M1 or M2, or both, contain an aryl or heteroaryl in the structure, or a combination thereof.

[17] The compound according to any one of [1] to

[16] , wherein M1 or M2, or both, have a fused polycyclic structure containing at least four fused rings.

[18] M1 or M2, or both, are selected from the group consisting of dimethylaminostilbene, quinacridone, fluorophenyl-dimethyl-BODIPY, his-fluorophenyl-BODIPY, acridine, terylene, sexiphenyl, porphyrin, benzopyrene, (fluorophenyl-dimethyl-difluorobora-diaza-indacene)phenyl, (bis-fluorophenyl-difluorobora-diaza-indacene)phenyl, quaterphenyl, bibenzothiazole, ter-benzothiazole, binaphthyl, bianthracyl, squaraine, squarylium, 9,10-ethynylanthracene, ter- The compound according to any one of [1] to

[17] , wherein M1 or M2, or both of M1 and M2, are one or more structures selected from the following: a naphthyl moiety, p-terphenyl, perylene, azobenzene, phenazine, phenanthroline, acridine, thioxanthrene, chrysene, rubrene, coronene, cyanine, perylene imide, perylene amide, coumarin dye, resorufin dye, dipyrrometheneboron difluoride dye, ruthenium bipyridyl dye, thiazole orange dye, polymethine, N-aryl-1,8-naphthalimide dye, boron-dipyrromethene, rhodamine, cyanine, pyrene, perylene, perylene monoimide, or 6-FAM, or a derivative thereof.

[19] The compound according to any one of [1] to

[17] , wherein M1 or M2, or both of M1 and M2, are one or more structures selected from the following:

[20] The compound according to any one of [1] to

[19] , wherein R2 or R3, or both, are Q.

[21] A detection system for detecting the distribution of a target in a measurement object by analyzing the waveform of a spectrum in a specific wavelength region using the compound according to any one of [1] to

[20] .

[22] The detection system according to

[21] , wherein the measurement object is a cell.

[23] The detection system according to

[21] or

[22] , which detects the distribution of a target in the measurement object by flow cytometry.

[24] A measurement device employing the detection system according to any one of

[21] to

[23] .

Claims

1. A compound for analyzing the waveform of a spectrum in a specific wavelength region, having a structure represented by the following formula (1): (1) n is an integer of 1 or more; m is an integer of 0 or more; when n is an integer of 2 or more, the two or more m may be the same or different from each other; M1 and M2 are chromophores having different chemical structures and not transferring Förster resonance energy to each other; when n is an integer of 2 or more, the two or more M2 may be the same or different from each other; L1, L2 and L3 are single bonds or divalent groups; L1 and L2 or L1 and L3 may be bonded to each other to form a ring; two or more L1, L2 and L3 may be the same or different from each other; L4 is a single bond or divalent group; when m is an integer of 2 or more, the two or more L4 may be the same or different from each other; R1 is H or a monovalent group; two or more R1 may be the same or different from each other; R2 and R3 are each H, a monovalent group, or Q; R4 and R5 are each H or a monovalent group, or R4 and R5 are an unsaturated substituent, and when there are two or more R4 and R5, the two or more R4 and R5 may be the same or different from each other; and Q is a reactive group capable of forming a bond with an analyte molecule, a targeting moiety, a solid support, or a complementary reactive group Q', and when there are two or more Q, the two or more Q may be the same or different from each other.

2. The compound of claim 1 having the structure of formula (1A): (1A) z is an integer of 1 or more, and when m1 or m3 is an integer of 2 or more, the two or more z's may be the same or different from each other, and m1, m2, and m3 are integers of 0 or more, and when n is an integer of 2 or more, the two or more m1's, m2's, and m3's may be the same or different from each other.

3. The compound according to claim 1, having the structure of formula (1B): (1B) z is an integer of 1 or more, and when m is an integer of 2 or more, two or more z's may be the same or different from each other.

4. The compound of claim 1 having the structure of formula (1C): (1C) z is an integer of 1 or more, and when m is an integer of 2 or more, the two or more z's may be the same or different from each other, x1 and x2 are integers of 0 or more, x3 and x4 are integers of 0 or more, and when n is an integer of 2 or more, the two or more x3's and x4's may be the same or different from each other.

5. The compound of claim 1 having the structure of formula (1D): (1D) m2 is an integer of 0 or more, and when n is an integer of 2 or more, the 2 or more m2's may be the same or different from each other.

6. The compound of claim 1, wherein for at least one instance of L1, said L1 is a linker that includes a triazolyl functional group.

7. The compound according to claim 1, wherein at least one of L1-M (wherein M may be M1 or M2) has the following structure: L1a and L1b are each a single bond or a divalent group.

8. The compound according to claim 1, wherein at least one of L1-M (wherein M may be M1 or M2) has the following structure: L1a and L1b are each a single bond or a divalent group.

9. The compound according to claim 1, wherein at least one of L1 includes one or more structures selected from the following in the structure of said L1: a, b and c are integers of 1 or more, and when a, b and c are 2 or more, the 2 or more a, b and c may be the same or different from each other.

10. The compound according to claim 1, wherein the distance between M1 and M2 and the distance between M2s different from each other are 2.25 nm or more.

11. The compound according to claim 1, wherein the distance between M1 and M2 and the distance between M2s different from each other are 3.25 nm or more.

12. The compound according to claim 1, wherein M1 and M2, or M1 and two or more mutually different M2, have different absorption or emission spectra.

13. The compound according to claim 1, in which the efficiency of Forster resonance energy transfer (FRET efficiency) from M1 or M2 to another M2 is 99% or less.

14. The compound according to claim 1, wherein the efficiency of Forster resonance energy transfer (FRET efficiency) from M1 or M2 to another M2 is 50% or less.

15. The compound of claim 1, wherein M1 or M2, or both, contain an aryl or heteroaryl in the structure, or a combination thereof.

16. The compound of claim 1, wherein M1 or M2, or both, have a fused polycyclic structure containing at least four fused rings.

17. M1 or M2, or both, are selected from the group consisting of dimethylaminostilbene, quinacridone, fluorophenyl-dimethyl-BODIPY, his-fluorophenyl-BODIPY, acridine, terylene, sexiphenyl, porphyrin, benzopyrene, (fluorophenyl-dimethyl-difluorobora-diaza-indacene)phenyl, (bis-fluorophenyl-difluorobora-diaza-indacene)phenyl, quaterphenyl, bi-benzothiazole, ter-benzothiazole, bi-naphthyl, bi-anthracyl, squaraine, squarylium, 9,10-ethynylanthracene, The compound according to claim 1, which is one or more structures selected from a ter-naphthyl moiety, p-terphenyl, perylene, azobenzene, phenazine, phenanthroline, acridine, thioxanthrene, chrysene, rubrene, coronene, cyanine, perylene imide, perylene amide, coumarin dye, resorufin dye, dipyrromethene boron difluoride dye, ruthenium bipyridyl dye, thiazole orange dye, polymethine, N-aryl-1,8-naphthalimide dye, boron-dipyrromethene, rhodamine, cyanine, pyrene, perylene, perylene monoimide, and 6-FAM, or a derivative thereof.

18. The compound of claim 1, wherein M1 or M2, or both, are one or more structures selected from the following or derivatives thereof:

19. The compound of claim 1, wherein R2 or R3, or both, are Q.

20. A detection system for detecting the distribution of a target in a measurement subject by analyzing the waveform of a spectrum in a specific wavelength region using the compound according to claim 19.

21. The detection system according to claim 20, wherein the measurement object is a cell.

22. The detection system according to claim 20, wherein the distribution of the target in the measurement subject is detected by flow cytometry.

23. A measuring device employing a detection system according to claim 20.

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