Fluorescent compound and fluorescently labeled biological substance using the same

Abstract

A fluorescent compound represented by the following general formula (1) and a fluorescently labeled biological substance using the fluorescent compound. In the above formula, FL represents an n-valent fluorophore moiety, and n is an integer of 1 or more. R 11 ~R 15 represents a hydrogen atom or a substituent. Among them, at least one of the combination of R 11 and R 12 and the combination of R 12 and R 13 is a combination in which one is -SO3 ‑ X + and the other is a substituent. X + represents a hydrogen ion or a metal ion.

Description

TECHNICAL FIELD

[0001] The present application relates to a fluorescent compound and a fluorescently labeled biological substance using the same. BACKGROUND

[0002] In order to observe changes in vivo to various stimuli (disease, environmental changes, etc.), a fluorescently labeled biological substance in which a fluorescent compound (fluorescent dye) is used to label a living body molecule (antibody, etc.) having a binding property to a target detection object substance is used most often.

[0003] For example, in Western blotting (hereinafter, also referred to as WB.) in which a specific protein is detected from a protein mixture, a fluorescent method in which a fluorescently labeled antibody having a binding property to the protein is used to detect the presence or absence or the amount of the specific protein is also used.

[0004] Also, in biological imaging technology in which dynamics and functions of living body molecules, cells, tissues, and the like in a living body are analyzed, living body fluorescence imaging in which a specific site of a living body visualized by fluorescent labeling is observed is used as one of techniques for living body observation.

[0005] In the above-described fluorescent labeling, an organic fluorescent dye can be used generally.

[0006] However, most of the organic dyes such as dipyrrometheneboron complex dyes, cyanine dyes, and rhodamine dyes, which exhibit fluorescence, include an aromatic chromophore having high planarity, and thus interaction between the dyes easily occurs, as a result of which reduction in fluorescence intensity based on self-association between the dyes and the like easily occurs.

[0007] Also, the organic fluorescent dyes have low light resistance, and are deteriorated due to excitation light irradiation, and sometimes, target living body observation cannot be performed sufficiently.

[0008] One of the organic fluorescent dyes, a dipyrrometheneboron complex dye, is known as a fluorescent dye having high quantum yield and exhibiting sharp emission characteristics, and research on the applicability to fluorescent labeling is being conducted.

[0009] For example, in Patent Literature 1, a dipyrrometheneboron complex dye having an aryl group or a heteroaryl group at at least two of positions 2, 4, and 6 of a dipyrromethene skeleton is described, and it is described that, thereby, decomposition of the dye is suppressed by light irradiation, and high light resistance is achieved. Also, in Patent Literature 2, a dipyrrometheneboron complex dye in which at least one alkyl group is bonded to a boron atom, and further, a specific hydrophilic group is present at a specific position of a dipyrromethene skeleton is described, and it is described that, thereby, decomposition of the dye is suppressed by light irradiation, and high water solubility is maintained and high light resistance is achieved.

[0010] PRIOR ART DOCUMENTS

[0011] Patent Literature

[0012] Patent Literature 1: Japanese Patent Application Laid-Open No. 2019-172826

[0013] Patent Literature 2: International Publication No. 2021 / 100814 SUMMARY

[0014] Technical Problem to be Solved by the Invention

[0015] Among pigments used for fluorescent labeling, it is required to have high hydrophilicity, to show excellent fluorescence intensity, and to show excellent light resistance as a compound or a fluorescently labeled biological substance. However, as a result of the present inventors' studies, it was found that the fluorescent pigment described in Patent Literature 1 above does not have sufficient characteristics in light resistance and there is room for improvement, the fluorescent pigment described in Patent Literature 2 above does not have sufficient characteristics in molar absorption coefficient and there is room for improvement in terms of fluorescence intensity, and the fluorescent pigment and the obtained fluorescently labeled biological substance do not have sufficient characteristics in light resistance and there is room for improvement.

[0016] Furthermore, as described in Patent Literature 1 or 2 above or the like, not limited to dipyrrometheneboron complex pigments, it is important to search for a chemical structure of all organic fluorescent pigments having high hydrophilicity, showing excellent fluorescence intensity, and being able to show excellent light resistance as a compound or a labeled biological substance.

[0017] The present invention has an object to provide a fluorescent compound having high water solubility required for fluorescent labeling, high molar absorption coefficient and fluorescence quantum yield, excellent fluorescence intensity (brightness), and being able to show excellent light resistance as a compound or a fluorescently labeled biological substance. Furthermore, the present invention has an object to provide a fluorescently labeled biological substance in which the fluorescent compound is bonded to a biological substance.

[0018] Means for Solving the Technical Problem

[0019] That is, the above-described object of the present invention is solved by the following method.

[0020] 〔1〕

[0021] A fluorescent compound represented by the following general formula (1).

[0022] [Chemical Formula 1]

[0023]

[0024] In the above formula, FL represents a phosphor portion, and n is an integer of 1 or more.

[0025] R 11 ~ R15 Represents a hydrogen atom or a substituent. Where R... 11 With R 12 Combinations and R 12 With R 13 At least one of the combinations is -SO3 - X + And the other is a combination of substituents. X + It represents hydrogen ions or metal ions.

[0026] [2]

[0027] According to the fluorescent compound described in [1], wherein,

[0028] The above R 11 With R 12 Combinations and R 12 With R 13 At least one of the combinations is -SO3 - X + And the other is a combination of hydroxyl, alkoxy, aryloxy, or heteroaryloxy groups.

[0029] [3]

[0030] The fluorescent compound according to [1] or [2] is represented by the following general formula (2).

[0031] [Chemical Formula 2]

[0032]

[0033] In the above formula, R 1 and R 2 Indicates a hydrogen atom, alkyl group, aryl group, or heteroaryl group. FL, R 13 R 14 X + and n and the above FL, R 13 R 14 X + The meanings of "and n" are the same.

[0034] [4]

[0035] According to the fluorescent compound described in [3], wherein,

[0036] The above R 1 and R 2 It can be alkyl, aryl, or heteroaryl.

[0037] [5]

[0038] According to the fluorescent compound described in [3] or [4], wherein,

[0039] The above R1 and R 2 is an alkyl group having an alkylene glycol structure.

[0040] 〔6〕

[0041] The fluorescent compound according to any one of the items [1] to [5], wherein,

[0042] The FL is a structural portion including a dipyrrometheneboron complex dye.

[0043] 〔7〕

[0044] The fluorescent compound according to the item [6], wherein,

[0045] The FL is a structural portion including a dipyrrometheneboron complex dye.

[0046] 〔8〕

[0047] The fluorescent compound according to the item [7], wherein,

[0048] The dipyrrometheneboron complex dye is represented by the following general formula (6).

[0049] [Chemical Formula 3]

[0050]

[0051] In the above formula, R 61 to R 67 represent a hydrogen atom or a substituent. R 68 and R 69 represent a hydrogen atom, a hydroxyl group, a halogen atom, an alkoxy group, an alkyl group, an aryl group, -CºCR b or a cyano group. R b represents a hydrogen atom, an alkyl group or an aryl group.

[0052] The dye represented by the above formula becomes an n-valent FL by removing n hydrogen atoms from any one of R 61 to R 69 .

[0053] 〔9〕

[0054] The fluorescent compound according to the item [8], wherein,

[0055] The R 63 and R 66 are an alkyl group or an aryl group.

[0056] 〔10〕

[0057] The fluorescent compound according to the item [8], wherein,

[0058] The above-mentioned dipyrromethene boron complex dye is represented by the following general formula (7).

[0059] [Chemical Formula 4]

[0060]

[0061] In the above formula, R 61 , R 62 , R 64 , R 65 , and R 67 to R 69 have the same meanings as the above-mentioned R 61 , R 62 , R 64 , R 65 , and R 67 to R 69 . R 70 to R 79 represent a hydrogen atom or a substituent.

[0062] The dye represented by the above formula becomes an n-valent FL by removing n hydrogen atoms from any one of R 61 , R 62 , R 64 , R 65 , R 67 to R 69 , and R 70 to R 79 .

[0063] 〔11〕

[0064] A fluorescently labeled biological substance, which is obtained by bonding the fluorescent compound according to any one of <1> to <10> to a biological substance.

[0065] 〔12〕

[0066] The fluorescently labeled biological substance according to <11>, wherein

[0067] The above-mentioned biological substance is any one of a protein, an amino acid, a nucleic acid, a sugar chain, and a lipid.

[0068] 〔13〕

[0069] The fluorescently labeled biological substance according to <11> or <12>, wherein

[0070] The bonding of the above-mentioned fluorescent compound to the above-mentioned biological substance is any one of the following i) to v) based bonding:

[0071] i) a non-covalent bond or a covalent bond between peptides,

[0072] ii) Van der Waals interaction of long-chain alkyl group in the compound with lipid bilayer or lipid in the biological substance,

[0073] iii) Amide bond formed by reaction of N-hydroxysuccinimide ester in the compound with amino group in the biological substance,

[0074] iv) Thioether bond formed by reaction of maleimide group in the compound with sulfanyl group in the biological substance,

[0075] v) Bond formed by click reaction of azido group in the compound with ethynyl group in the biological substance or of ethynyl group in the compound with azido group in the biological substance, accompanied by formation of triazole ring.

[0076] Effects of the Invention

[0077] The fluorescent compound of the present application has high water solubility required for fluorescent labeling, and is high in molar absorption coefficient and fluorescence quantum yield, and is excellent in brightness and light resistance, and a fluorescently labeled biological substance excellent in light resistance can be obtained. Also, the fluorescently labeled biological substance of the present application exhibits excellent light resistance. DETAILED DESCRIPTION

[0078] In the present application, when a plurality of substituents or linking groups represented by a specific symbol or formula or the like (hereinafter, referred to as substituents or the like) are present, or when a plurality of substituents or the like are simultaneously specified, each of the substituents or the like can be the same or different from each other, unless otherwise specified. The same is true for the number of substituents or the like. Also, when a plurality of substituents or the like are close to each other (particularly, when adjacent), they can be connected to each other to form a ring, unless otherwise specified. Also, when a ring is formed, an unsaturated ring or an aromatic ring can be formed by removing a hydrogen atom, unless otherwise specified. Also, a ring, such as an alicyclic ring, an aromatic ring, and a heterocyclic ring, can be further fused to form a fused ring, unless otherwise specified.

[0079] In the present application, unless otherwise specified, with respect to a double bond, in the case where E type and Z type are present within a molecule, either of them can be present, and a mixture thereof can also be present. Also, unless otherwise specified, in the case where diastereomers and enantiomers are present as a compound, either of them can be present, and a mixture thereof can also be present.

[0080] In the present application, regarding the expression of the compound and the substituent, in addition to the compound itself and the substituent itself, the meaning including the salt thereof, the ion thereof is also used. For example, carboxyl group, sulfo group, and phosphono group (-P(=0)(OH)2) and the like can adopt an ionic structure by hydrogen ion dissociation, or can adopt a salt structure. That is, in the present application, "carboxyl group" is used in the meaning including carboxylate ion or a salt thereof, "sulfo group" is used in the meaning including sulfonate ion or a salt thereof, and "phosphono group" is used in the meaning including phosphonate ion or a salt thereof. As the monovalent or polyvalent cation constituting the above-mentioned salt structure, there is no particular limitation, and inorganic cations, organic cations and the like can be mentioned, and specifically, alkali metal cations such as Na + , Li + , and K + , alkaline earth metal cations such as Mg 2+ , Ca 2 + , and Ba 2+ , and organic ammonium cations such as trialkylammonium cation, tetraalkylammonium cation can be mentioned.

[0081] In the case of the salt structure, the kind of the salt can be one kind, or two or more kinds can be mixed, or a group of salt type and free acid structure can be mixed in the compound, and a compound of salt structure and a compound of free acid structure can also be mixed.

[0082] The compound of the present application is all an electrically neutral compound. Specifically, the electric charge of the entire compound is adjusted to 0 by a group having an electric charge within the compound or a counter ion. For example, in the case where the FL has a structural part including a cyanine dye, the formal charge of the nitrogen atom is +1, and in order to pair with this formal charge, the dissociable group such as sulfo group in the cyanine dye has an ionic structure of sulfonate ion and the like, whereby the compound of the present application including the structural part of the cyanine dye becomes a compound having an electric charge of 0 as the entire compound.

[0083] Further, the compound of the present application includes a compound in which a part of the structure is changed within a range not impairing the effects of the present application. Furthermore, regarding a compound which is not explicitly described as substituted or unsubstituted, it is indicated that it can have an arbitrary substituent within a range not impairing the effects of the present application. This is the same regarding the substituent (for example, a group expressed as "alkyl group", "methyl group", "methyl", and the like) and the linking group (for example, a group expressed as "alkylene group", "methylene group", "methylene", and the like). Among such arbitrary substituents, in the present application, it is preferable that the substituent is a substituent selected from the substituent group T described later.

[0084] In the present application, in the case where the number of carbon atoms of a certain group is specified, the number of carbon atoms means the number of carbon atoms of the group as a whole unless otherwise specified in the present application or the specification. That is, when the group further has a substituent, it means the total number of carbon atoms including the substituent.

[0085] Also, in the present application, the numerical range indicated using "~" means a range including the numerical values recited before and after "~" as lower and upper limits.

[0086] The fluorescent compound of the present application is represented by the following general formula (1). The fluorescent compound of the present application has high water solubility required for fluorescent labeling, high molar absorption coefficient and high fluorescence quantum yield, excellent fluorescence intensity (brightness), and the reason why it can exhibit excellent light resistance as a compound or a fluorescently labeled biological substance is not yet clear, but it is believed as follows.

[0087] As shown in general formula (1), the fluorescent compound of the present application is a compound having a specific phenyl group substituted with substituents R 11 ~R 15 in which two groups adjacent to each other among the substituents R 11 ~R 15 are a sulfo group (-SO3 - X + ) and a substituent. Thereby, it is believed that a steric repulsion effect of the substituent protruding at least one of the meta position and the para position of the phenyl group with respect to the planar skeleton of the fluorophore portion FL and an electronic repulsion effect by the sulfo group are obtained, and thus the interaction of at least the fluorophore portions FL of the molecules with each other is inhibited to be able to inhibit self-association or aggregation of the fluorescent compound of the present application, and the light emission efficiency (molar absorption coefficient x fluorescence quantum yield) is improved to be able to improve the fluorescence intensity (brightness), and water solubility based on the sulfo group can be obtained. Furthermore, it is believed that by one of the two groups adjacent to each other among the substituents R 11 ~R 15 being a sulfo group, sufficient hydrophilicity can be ensured, and the oxidation potential of the fluorophore portion is deepened, and thus it is difficult to be oxidized, and excellent light resistance can be exhibited. That is, regarding the fluorescent compound of the present application, in a wavelength region according to the width of the pigment skeleton possessed by the fluorophore portion FL, the absorption emission wavelength is adjusted to a desired range, and it has high water solubility required for fluorescent labeling, high molar absorption coefficient and high fluorescence quantum yield, excellent fluorescence intensity (brightness), and as a compound or a fluorescently labeled biological substance, it can exhibit excellent light resistance.

[0088] Hereinafter, the fluorescent compound of the present application represented by general formula (1) will be described in detail.

[0089]

[0090] The fluorescent compound represented by General Formula (1) of the present application (hereinafter, also referred to as "the compound of the present application") is as described below.

[0091] [Chemical Formula 5]

[0092]

[0093] In the above formula, FL represents an n-valent fluorescent body portion, and n is an integer of 1 or more.

[0094] R 11 ~R 15 represents a hydrogen atom or a substituent. Among them, at least one of the combination of R 11 and R 12 and the combination of R 12 and R 13 is a combination in which one is -SO3 - X + and the other is a substituent. X + represents a hydrogen ion or a metal ion.

[0095] (R 11 ~R 15 )

[0096] R 11 ~R 15 represents a hydrogen atom or a substituent.

[0097] The substituent that can be adopted as R 11 ~R 15 is not particularly limited, and the substituents in Substituent Group T described later can be cited, and among them, a hydroxyl group, an alkoxy group, an aryloxy group, a heteroaryloxy group, a sulfo group, an alkyl group, an aryl group, a heteroaryl group, or an amino group can also be preferably cited.

[0098] The substituent that the above-mentioned alkoxy group, aryloxy group, heteroaryloxy group, alkyl group, aryl group, heteroaryl group, and amino group can have can be cited from the substituents in Substituent Group T described later. Also, an alkylene glycol structure can also be preferably cited.

[0099] The above-mentioned alkylene glycol structure is specifically a structure represented by -(L-O) m - and is preferably a structure represented by -O(L-O) m -R 21 . For example, in the case where the alkoxy group has an alkylene glycol structure, the alkyl moiety in the alkoxy group can have an alkylene glycol structure, and is preferably a structure represented by -O(L-O) m -R 21 . ​

[0100] The L mentioned above refers to an alkylene obtained by removing one hydrogen atom from the alkyl group T described later. The number of carbon atoms is preferably 2 to 4, more preferably 2 or 3, and even more preferably 2. The number of carbon atoms contained in the shortest chain of the alkylene bond that connects two carbon atoms is preferably 0 to 2, more preferably 0 or 1, and even more preferably 0.

[0101] The above m represents the average number of repetitions (also simply called the number of repetitions), preferably 1 to 24, more preferably 1 to 12, even more preferably 1 to 10, particularly preferably 1 to 8, and most preferably 2 to 4.

[0102] The above R 21 The alkyl group represented has a carbon number of 1 to 10, more preferably 1 to 6, even more preferably 1 to 4, and particularly preferably 1 to 2.

[0103] The above sulfonyl group is composed of -SO3 - X + The group represented, X + Represents hydrogen ions or metal ions. As X + The metal ions that can be used include the cations of the aforementioned alkali metals or alkaline earth metals.

[0104] As R 11 ~R 15 Preferably, it is a hydrogen atom, hydroxyl group, alkoxy group, aryloxy group, heteroaryloxy group or sulfonyl group, more preferably a hydrogen atom, alkoxy group or sulfonyl group, and even more preferably an alkoxy group or hydrogen atom or sulfonyl group having an alkylene glycol structure.

[0105] Among them, in R 11 ~R 15 In the middle, R 11 With R 12 Combinations and R 12 With R 13 At least one of the combinations is -SO3 - X + And the other is a combination of substituents (hereinafter also referred to as "ortho-substituents"). X + With the above X + The meanings are the same.

[0106] As the ortho-substituent in the above combination, hydroxyl, alkoxy, aryloxy or heteroaryloxy are preferred, more preferably alkoxy, aryloxy or heteroaryloxy, and even more preferably alkoxy. From the viewpoint of excellent effect in inhibiting the association of pigments with each other, excellent inhibition of non-specific bonding, imparting high water solubility to the compound, and excellent labeling bonding to biological substances, alkoxy with an alkylene glycol structure is particularly preferred.

[0107] -SO3 - X + R 12 -SO3 - X + R 11 R 13 R 12 R 13 -SO3 - X + R 12 -SO3 - X + R 11 R 13 R 12 -SO3 - X + R 11 R

[0108] In addition, in R 11 to R 15 , with respect to the group which does not belong to the above -SO3 - X + combination with the ortho substituent group, there is no particular limitation and can be selected from the hydrogen atom or the substituent which R 11 to R 15 can take as described above.

[0109] For example, in R 12 -SO3 - X + and R 11 is the combination of the ortho substituent group, it is preferable that R 13 to R 15 are all hydrogen atoms, or R 13 and R 14 are hydrogen atoms and R 15 is a substituent. In R 12 -SO3 - X + and R 13 is the combination of the ortho substituent group, it is preferable that R 11 , R 14 and R 15 are all hydrogen atoms, or R 11 is a hydrogen atom and one of R 14 and R 15 is a hydrogen atom and the other is a substituent. In R 12 is the ortho substituent group and R 13 -SO3 - X+ one of R 11 is a hydrogen atom and the other is a substituent. 14 one of R 15 is a hydrogen atom and the other is a substituent.

[0110] The compound of the present application is preferably represented by the following general formula (2).

[0111] [Chemical Formula 6]

[0112]

[0113] In the above formula, R 1 and R 2 represent a hydrogen atom, an alkyl group, an aryl group or a heteroaryl group. FL represents an n-valent fluorophore moiety, and n is an integer of 1 or more.

[0114] (R 1 and R 2 )

[0115] The alkyl group that can be used as R 1 and R 2 corresponds to the alkyl group in the alkoxyl group described above as R 11 to R 15 , and the description of the alkyl group can be preferably applied.

[0116] The aryl group that can be used as R 1 and R 2 corresponds to the aryl group in the aryloxy group described above as R 11 to R 15 , and the description of the aryl group can be preferably applied.

[0117] The heteroaryl group that can be used as R 1 and R 2 corresponds to the heteroaryl group in the heteroaryloxy group described above as R 11 to R 15 , and the description of the heteroaryl group can be preferably applied.

[0118] R 1 and R 2 are preferably an alkyl group, an aryl group or a heteroaryl group, and more preferably an alkyl group. From the viewpoints of excellent effects of inhibiting association of pigments with each other, excellent effects of inhibiting non-specific bonding, excellent effects of imparting high water solubility to the compound, excellent effects of bonding to biological substances, and excellent effects of exhibiting a more excellent fluorescence quantum yield, R 1 and R 2 are further preferably an alkyl group having a structure of an alkylene glycol.

[0119] Unless otherwise specified, R 13 , R14 , X + and n have the same meanings as the aforementioned R 13 , R 14 , X + and n. Among them, as R 13 and R 14 , a hydrogen atom is preferred.

[0120] (FL, n)

[0121] In the aforementioned general formulae (1) and (2), FL represents an n-valent fluorescent body portion, and n is an integer of 1 or more.

[0122] As the fluorescent body portion that can be adopted as FL, there is no particular limitation as long as it is a structural portion including an organic compound that exhibits fluorescence, and a generally known pigment can be used as such.

[0123] As FL, for example, a structural portion including a dipyrromethene pigment, a rhodamine pigment, a xanthene pigment, a pyrrolopyrylo pigment, a pyrene pigment, a coumarin pigment or a cyanine pigment can be cited, and a structural portion including a dipyrromethene pigment, a pyrrolopyrylo pigment, a pyrene pigment, a coumarin pigment or a cyanine pigment is preferred.

[0124] As the aforementioned dipyrromethene pigment, rhodamine pigment, xanthene pigment, pyrrolopyrylo pigment, pyrene pigment, coumarin pigment or cyanine pigment, there is no particular limitation and a generally known pigment can be used as such.

[0125] n is preferably an integer of 1 or more, preferably an integer of 1 to 8, more preferably an integer of 1 to 6, further preferably an integer of 2 to 4, and particularly preferably an integer of 2.

[0126] From the viewpoint of general use in bioimaging, the compound of the present application preferably has at least one carboxyl group or a substituent capable of bonding to a biological substance described later, and more preferably has at least one carboxyl group or a substituent capable of bonding to a biological substance described later in FL.

[0127] The compound of the present application is bonded to a biological substance through the aforementioned carboxyl group or substituent capable of bonding to a biological substance, and a target labeled biological substance can be obtained. In addition, the carboxyl group can be easily derivatized into a substituent capable of bonding to a biological substance by a conventional method.

[0128] In the present application, the "substituent capable of bonding to a biological substance" includes a substituent capable of bonding to a biological substance derived from a carboxyl group.

[0129] The number of carboxyl groups or substituents capable of bonding to a biological substance possessed by the compound of the present application is preferably at least one or more in total, more preferably 1 to 3, further preferably 1 or 2, and particularly preferably 1, from the viewpoint of quantification of the detection target substance.

[0130] The FL is preferably a structural unit including a dipyrromethene chromophore. In addition, the dipyrromethene chromophore can form a complex.

[0131] The FL is more preferably a structural unit including a dipyrromethene complex chromophore formed by complexing with any metal atom or semimetal atom, further preferably a structural unit including a dipyrromethene complex chromophore in which a dipyrromethene ligand is coordinated to a boron atom, a zinc atom, an aluminum atom, or a phosphorus atom, and particularly preferably a structural unit including a dipyrromethene boron complex chromophore, and most preferably a structural unit including a dipyrromethene boron complex chromophore represented by the following general formula (6).

[0132] [Chemical Formula 7]

[0133]

[0134] In the above formula, R 61 ~R 67 represent a hydrogen atom or a substituent. R 68 and R 69 represent a hydrogen atom, a hydroxyl group, a halogen atom, an alkoxy group, an alkyl group, an aryl group, -C≡CR b or a cyano group, and R b represents a hydrogen atom, an alkyl group, or an aryl group.

[0135] The chromophore represented by the above formula becomes an n-valent FL by removing n hydrogen atoms from any one of R 61 ~R 69 .

[0136] (i) R 61 ~R 67

[0137] R 61 ~R 67 represent a hydrogen atom or a substituent.

[0138] As the substituent of R 61 ~R 67 , the substituents in the following substituent group T can be mentioned.

[0139] As R 61 and R 64 , in the above, an alkyl group is also preferable.

[0140] As R 62 and R 65 , in the above, a hydrogen atom or an aryl group can also be preferable.

[0141] As R 62 or R 65 , in the above, a hydrogen atom or an aryl group can also be preferable.In the case of hydrogen atoms, it is preferable to form FL bonds by removing the hydrogen atom.

[0142] Among them, R is more preferred. 62 and R 65 The bonds are hydrogen atoms and are FL bonds formed by removing at least one of these hydrogen atoms. From the viewpoint of further improving the molar absorptivity by expanding or increasing the overlap integral of HOMO (Highest Occupied Molecular Orbital) and LUMO (Lowest Unoccupied Molecular Orbital), R is further preferred. 62 and R 65 The bonds are composed of hydrogen atoms and are formed by removing these two hydrogen atoms to create FL bonds.

[0143] As R 63 and R 66 Among the above, hydrogen atoms, alkyl groups, aryl groups, and -CR groups are also preferred examples. a1 =CR a2 R a3 or -C≡CR b .

[0144] Substituents that the aforementioned alkyl and aryl groups may have include those in the substituent group T described below, for example, a carboxyl group.

[0145] The above R a1 ~R a3 Representing a hydrogen atom, alkyl, or aryl group, examples of alkyl and aryl groups in the substituent group T described below are examples of alkyl and aryl groups. Examples of substituents that these alkyl and aryl groups can have are substituents in the substituent group T described below, such as alkyl, alkoxy, and amino groups. R a1 and R a2 Preferably, hydrogen atoms, R a3 Preferably, it is a hydrogen atom or an aryl group, more preferably an aryl group.

[0146] The above R b Can preferentially apply R 68 and R 69 R in b The records.

[0147] In R 63 Or R 66 In the case of hydrogen atoms, it is preferable to form FL bonds by removing the hydrogen atom. Furthermore, in R... a3 In the case of hydrogen atoms, it is preferable to form FL bonds by removing the hydrogen atom.

[0148] wherein R 63 and R 66 is more preferably an alkyl group, an aryl group or -CH=CR a3 H (wherein R a3 is an aryl group).

[0149] As R 67 , in the above, a hydrogen atom, an alkyl group, an aryl group or a heteroaryl group is also preferable, and an aryl group is more preferable.

[0150] As the substituent group which the above-mentioned alkyl group, aryl group and heteroaryl group can have, the substituent group in the substituent group set T described later can be mentioned, and for example, a halogen atom (preferably a fluorine atom), an alkyl group, an alkoxy group, a carbamoyl group, a carboxyl group or a substituent group capable of bonding to a biological substance can be mentioned. Also, it is preferable to mention the above-mentioned group represented by -O(L-O) m -R 21 represents a group.

[0151] In the case where R 67 is a hydrogen atom, it is also preferable that the bonding bond becomes FL by removing the hydrogen atom.

[0152] wherein, from the viewpoint of being able to inhibit intermolecular association aggregation by a steric hindrance effect, further improving the fluorescence quantum yield, and further improving the light resistance, R 67 A phenyl group having a substituent at the ortho position is further preferable. In the phenyl group having a substituent at the ortho position, a substituent can or can not be present at the meta position and para position, but it is preferable that a carboxyl group or a substituent group capable of bonding to a biological substance is directly present or present via a linking group at the para position.

[0153] The adjacent 2 groups among R 61 to R 67 may also be bonded to form a 5- or 6-membered ring. At this time, the formed ring can also have an unsaturated bond.

[0154] For example, R 61 and R 62 may be bonded to form a 5- or 6-membered ring, preferably a benzene ring, R 64 and R 65 may be bonded to form a 5- or 6-membered ring, preferably a benzene ring.

[0155] Also, the 5- or 6-membered ring formed by the bonding of the adjacent 2 groups among R 61 to R 67 may have a substituent group, and as the substituent group which can be present, the substituent group in the substituent group set T described later can be mentioned, and for example, an alkoxy group and a carboxyl group can be mentioned.

[0156] Also, it is preferable that the bonding bond becomes FL by removing R 61~R 67 A hydrogen atom on a 5- or 6-membered ring formed by bonding of adjacent 2 groups becomes a bonding site of FL.

[0157] (ii)R 68 and R 69

[0158] R 68 and R 69 represents a hydrogen atom, a hydroxyl group, a halogen atom, an alkoxy group, an alkyl group, an aryl group, -C≡CR b or a cyano group, R b represents a hydrogen atom, an alkyl group or an aryl group.

[0159] As R 68 or R 69 The halogen atom, the alkoxy group, the alkyl group and the aryl group which can be employed can be exemplified by the halogen atom, the alkoxy group, the alkyl group and the aryl group in the substituent group T described later.

[0160] As R b The alkyl group and the aryl group which can be employed can be exemplified by the alkyl group and the aryl group in the substituent group T described later.

[0161] As R 68 or R 69 The alkoxy group, the alkyl group and the aryl group which can be employed and the alkyl group and the aryl group which can be possessed by R b may be exemplified by the substituents in the substituent group T described later, for example, a halogen atom (preferably a fluorine atom) and an alkyl group.

[0162] In the case where R 68 or R 69 is a hydrogen atom, it is preferable that a bonding site of FL be formed by removing the hydrogen atom. Also, in the case where R b is a hydrogen atom, it is preferable that a bonding site of FL be formed by removing the hydrogen atom.

[0163] As R 68 and R 69 , it is preferable that one be a halogen atom and the other be a halogen atom, an alkyl group, a halogenated alkyl group, an aryl group or a halogenated aryl group, more preferable that one be a halogen atom and the other be a halogen atom or a halogenated alkyl group, and further preferable that one be a halogen atom and the other be a halogenated alkyl group from the viewpoint of more excellent light resistance.

[0164] The structural portion including the dipyrromethene boron complex dye represented by the above general formula (6) is preferably provided with a carboxyl group or a substituent capable of bonding to a biological substance at at least any one of R 61 ~R 69 , more preferably at at least any one of R 61 ~R 67At least one of them has a carboxyl group or a substituent capable of bonding with biological material, and more preferably has a carboxyl group or a substituent in R. 67 It has a carboxyl group or a substituent that can bond with biological substances.

[0165] Furthermore, regarding the compounds of the present invention having a structural portion comprising a dipyrrole methyl borate complex pigment represented by the above general formula (6) as FL, from the viewpoint that the compounds of the present invention exhibit sufficient hydrophilicity, the number of hydrophilic groups in each molecule of the dipyrrole methyl borate complex pigment represented by general formula (6) is preferably 2 or more, more preferably 2 to 10, further preferably 3 to 9, and especially preferably 4 to 7.

[0166] There are no particular limitations on the hydrophilic group; examples include alkoxy, carboxyl, sulfonyl, and phosphonyl groups with substituents, with alkoxy or sulfonyl groups having substituents being preferred. Among alkoxy groups with substituents, alkoxy groups having an alkylene glycol structure are preferred.

[0167] As a hydrophilic group, as long as it is an R group defined by the general formula (1) above, 11 ~R 15 -SO3 on the phenyl group as a substituent - X + It can have either a sulfonate group or other hydrophilic groups. Preferably, each pigment molecule has two or more sulfonate groups.

[0168] The dipyrrole-methylboron complex pigment represented by the above general formula (6) is obtained from R 61 ~R 69 Any one of the atoms in R can be reduced to an n-valent FL by removing n hydrogen atoms. For example, by removing n hydrogen atoms from R. 62 Remove one hydrogen atom and from R 65 Removing one hydrogen atom results in a divalent FL. Furthermore, by removing one hydrogen atom from R... 63 Remove 2 hydrogen atoms and from R 66 It becomes tetravalent FL by removing two hydrogen atoms.

[0169] In this invention, it is preferable to use R 62 ~R 69 Any one of them can be made into an n-valent FL by removing n hydrogen atoms, more preferably by removing n hydrogen atoms from R. 62 and R 65 At least one of them is reduced by one hydrogen atom to become an n-valent FL, and further preferred by removing one hydrogen atom from R. 62 and R 65 Each FL is reduced by removing one hydrogen atom to become at least divalent.

[0170] As a compound of the present invention, the above-mentioned R 63 and R66 R is preferably an alkyl group or an aryl group.

[0171] R 63 and R 66 are an alkyl group, the compound of the present application having a structural portion including a dipyrromethene boron complex dye represented by general formula (6) as FL can exhibit green-yellow-orange fluorescent light. On the other hand, in the case where R 63 and R 66 are an aryl group, the compound of the present application having a structural portion including a dipyrromethene boron complex dye represented by general formula (6) as FL can exhibit orange-red fluorescent light.

[0172] Further, as the compound of the present application, it is also preferable that R 63 and R 66 be -CH=CR a3 H (wherein R a3 is an aryl group), that is, the above-described dipyrromethene boron complex dye be represented by the following general formula (7). The compound of the present application having a structural portion including a dipyrromethene boron complex dye represented by general formula (7) as FL can exhibit red-deep red fluorescent light. Furthermore, by introducing an electron-donating group such as an amino group, it is possible to further lengthen the wavelength of the absorption wavelength, and it is possible to exhibit fluorescent light in the near-infrared wavelength region.

[0173] [Chemical Formula 8]

[0174]

[0175] In the above formula, R 61 , R 62 , R 64 , R 65 , and R 67 to R 69 have the same meanings as R 61 , R 62 , R 64 , R 65 , and R 67 to R 69 in the above general formula (6). R 70 to R 79 represent a hydrogen atom or a substituent.

[0176] The dye represented by the above formula becomes an n-valent FL by removing n hydrogen atoms from any one of R 61 , R 62 , R 64 , R 65 , R 67 to R 69 , and R 70 to R 79 .

[0177] R 70 ~R 79 represents a hydrogen atom or a substituent.

[0178] R 70 ~R 79 As the substituent which can be employed, the substituents in the substituent group T to be described later can be mentioned, and a hydrogen atom, an alkyl group, an alkoxy group or an amino group can be preferably mentioned. Also, the above-mentioned group represented by -O(L-O) m -R 21 represents a group represented by -O(L-O)

[0179] R 70 ~R 79 As the substituent which can be employed for the alkyl group, the alkoxy group and the amino group, the substituents in the substituent group T to be described later can be mentioned, and for example, a halogen atom, an alkyl group and an aryl group can be mentioned.

[0180] R 70 ~R 79 In the case where R is a hydrogen atom, a bond by removal of the hydrogen atom to become FL is preferred. Also, in the case where the amino group is -NH2, a bond by removal of the hydrogen atom on the nitrogen atom to become FL is preferred.

[0181] R 70 ~R 79 is preferably a hydrogen atom.

[0182] Hereinafter, specific examples of the compounds of the present application will be shown, but the present application is not limited to these compounds. In the specific examples described below, -SO3 - X + and -COO - X + X in the above-mentioned X + has the same meaning as the above-mentioned X + represents a hydrogen ion or a metal ion. Also, Me represents a methyl group, and Ph represents a phenyl group.

[0183] [Chemical Formula 9]

[0184]

[0185] [Chemical Formula 10]

[0186]

[0187] [Chemical Formula 11]

[0188]

[0189] [Chemical Formula 12]

[0190]

[0191] [Chemical Formula 13]

[0192]

[0193] [Chemical Formula 14]

[0194]

[0195] [Chemical Formula 15]

[0196]

[0197] As a preferred embodiment of the present application, a compound represented by the above general formula (2) can be given, in which n is 2, FL is represented by the above general formula (6), and as a compound including a structural portion of a dipyrromethene boron complex dye satisfying the following Mode I or II.

[0198] (Mode I)

[0199] a mode in which R 63 and R 66 are an alkyl group or an aryl group, R 61 and R 64 are an alkyl group, R 62 and R 65 are a hydrogen atom, the bonding bond by removing these two hydrogen atoms becomes FL, R 67 is a phenyl group having a substituent (preferably a halogen atom or an alkoxy group) at an ortho position and a carboxyl group or a substituent capable of bonding to a biological substance at a para position directly or via a linking group, R 68 and R 69 are one halogen atom and the other is a halogen atom or a halogenated alkyl group.

[0200] (Mode II)

[0201] a mode in which R 63 and R 66 are -CH=CR a3 H (in which R a3 is an aryl group.), R 61 and R 64 are an alkyl group, R 62 and R 65 are a hydrogen atom, the bonding bond by removing these two hydrogen atoms becomes FL, R 67 is a phenyl group having a substituent (preferably a halogen atom or an alkoxy group) at an ortho position and a carboxyl group or a substituent capable of bonding to a biological substance at a para position directly or via a linking group, R 68 and R 69 are one halogen atom and the other is a halogen atom or a halogenated alkyl group.

[0202] In these modes, the description of R 61 ~R 69 above can be applied to the corresponding description of R 61 ~R 69 above. Also, regarding R 1 , R 2 , R 13 , R 14 , and X + in General Formula (2), the preferred description regarding R 1 , R 2 , R 13 , R 14 , and X + above can be applied, wherein it is preferred that R 1 and R 2 are alkyl groups and R 13 and R 14 are hydrogen atoms.

[0203] In the case where the compound of the present application has a substituent capable of bonding to a biological substance, the compound can be used as a marker for a biological substance by virtue of the at least one substituent capable of bonding to a biological substance possessed by the compound, which can bond to a protein (including a peptide), an amino acid, a nucleic acid, a sugar chain, a lipid, and the like.

[0204] As the substituent capable of bonding to a biological substance, any group can be used without particular limitation as long as it is a group for interacting (including attaching) or bonding with a biological substance, and examples thereof include the substituents described in International Publication No. 2002 / 026891 and the like. Among them, the NHS ester structure (N-hydroxysuccinimide ester structure), the succinimide structure, the maleimide structure, the azido group, the ethynyl group, the peptide structure (polyamino acid structure), the long-chain alkyl group (preferably, carbon number: 12 to 30), and the quaternary ammonium group can be preferably exemplified.

[0205] In the compound of the present application, as a specific example of the compound having at least one substituent capable of bonding to a biological substance, for example, a case where a carboxyl group in the exemplified compound of the present application is appropriately substituted with a substituent capable of bonding to a biological substance described later can be exemplified. In addition, the present application is not limited to these compounds. For example, in these specific examples, regarding a group having a dissociable hydrogen atom such as a carboxyl group and a sulfo group, a salt structure can be adopted by dissociation of the hydrogen atom.

[0206] Regarding the compound of the present application, in addition to setting the structure of the compound to the structure defined by General Formula (1), synthesis can generally be performed by referring to a known method. For example, regarding the R 11 ~R 15As a method for synthesizing the benzene ring structure as a substituent, there can be mentioned a method described in Nippon Kagaku Kaishi (Chemistry and Industry Chemistry), 1985, No. 1, p. 70-74, Acta Chemica Scandinavica, Series B: Organic Chemistry and Biochemistry, 1979, Vol. B33, p. 261-264, Journal of the Chemical Society, Perkin Transactions 2: Physical Organic Chemistry (1972-1999), 1985, No. 5, p. 659-667, and the like. As a method for synthesizing the dipyrromethene boron complex dye represented by the above general formula (6), there can be mentioned a method described in Patent Literature 1 or 2, and the like. Further, the synthesis can be performed in accordance with the synthesis method of each compound described in the Examples described later. In particular, the compound represented by the above general formula (2) can also be synthesized simply in accordance with the synthesis example described in the Examples of the present application, based on appropriate technical knowledge.

[0207] As for the compound having a substituent capable of bonding to a biological substance, the compound structure is set to the structure defined in the general formula (1), and other than this, it can be synthesized by a publicly known method. For example, reference can be made to Bioconjugate Techniques (Third Edition, Greg T. Hermanson).

[0208] <<Labeling a biological substance>>

[0209] The labeled biological substance of the present application is a substance in which the compound of the present application is bonded to a biological substance. The compound of the present application has fluorescence, exhibits high hydrophilicity and excellent fluorescence intensity, and exhibits excellent light resistance, and thus can be preferably used for labeling a biological substance. The bonding of the compound represented by the general formula (1) to a biological substance can be a direct bonding of the compound represented by the general formula (1) to a biological substance, or can be a bonding via a linking group.

[0210] As the above biological substance, there can be preferably mentioned a protein (including a peptide), an amino acid, a nucleic acid, a sugar chain, and a lipid. As the protein, there can be preferably mentioned an antibody, and as the lipid, there can be preferably mentioned a phospholipid, a fatty acid, and a sterol, more preferably a phospholipid.

[0211] Among the aforementioned biological substances, there are no particular limitations on substances that are useful in clinical pathology. Examples include immunoglobulins such as Ig (Immunoglobulin) G, IgM, IgE, IgA, and IgD; complement, C-reactive protein (CRP), ferritin, α1-microglobulin, β2-microglobulin, and their antibodies; alpha-fetoprotein, carcinoembryonic antigen (CEA), prostatic acid phosphatase (PAP), CA (carbohydrate antigen) 19-9, CA-125, and their antibodies; hormones such as luteinizing hormone (LH), follicle-stimulating hormone (FSH), human chorionic gonadotropin (hCG), estrogen, and insulin, and their antibodies; viral infection-related substances such as hepatitis B virus (HBV) related antigens (HBs, HBe, HBc), human immunodeficiency virus (HIV), and adult T-cell leukemia (ATL), and their antibodies.

[0212] In addition, examples include bacteria such as diphtheria bacteria, botulinum toxin, mycoplasma, and spirochetes and their antibodies; protozoa such as Toxoplasma gondii, Trichomonas vaginalis, Leishmaniasis, Trypanosoma cruzi, and Plasmodium and their antibodies; ES cells (embryonic stem cells) such as ELM3, HM1, KH2, v6.5, v17.2, and v26.2 (derived from mouse 129, 129 / SV, C57BL / 6, and BALB / c) and their antibodies; antiepileptic drugs such as phenytoin and phenobarbital; cardiovascular drugs such as quinidine and digoxin; anti-asthmatic drugs such as theophylline; antibiotics such as chloramphenicol and gentamicin and their antibodies; other enzymes; exotoxins (such as styrelidine O) and their antibodies; and antibody fragments such as Fab'2, Fab, and Fv.

[0213] As a specific way in which the compounds of the present invention interact with and bond with biological substances, the following methods can be cited as examples.

[0214] Examples include:

[0215] i) The peptides in the compounds of the present invention are bonded to peptides in biological substances by non-covalent bonds (e.g., hydrogen bonds, ionic bonds including chelate bonds) or covalent bonds.

[0216] ii) The van der Waals forces between the long-chain alkyl groups in the compounds of the present invention and lipid double membranes and lipids in biological substances,

[0217] iii) An amide bond is formed by the reaction of the NHS ester (N-hydroxysuccinimide ester) in the compound of the present invention with an amino group in biological material.

[0218] iv) a thioether bond resulting from the reaction of a maleimide group in the compound of the present application with a sulfanyl group (-SH) in the biological substance,

[0219] v) a triazole ring formed by the click reaction of an azido group in the compound of the present application with an ethynyl group in the biological substance or the click reaction of an ethynyl group in the compound of the present application with an azido group in the biological substance.

[0220] In addition to the above i) to v), for example, bonding can be performed by the method described in Lucas C. D. de Rezende and Flavio da Silva Emery,. A Review of the Synthetic Strategies for the Development of BODIPY Dyes for Conjugation with Proteins, Orbital: The Electronic Journal of Chemistry, 2013, Vol 5, No. 1, p. 62-83. Also, in the production of the labeled biological substance of the present application, the method described in this document and the like can be appropriately referred to.

[0221] Among the compounds of the present application, as to the labeled biological substance of the present application obtained from a compound having a substituent capable of bonding with a biological substance and a biological substance bonded by interaction therewith, the compound in which the portion other than the substituent capable of bonding with a biological substance in the compound example and the product described in paragraph 0038 of Japanese Patent Application Publication No. 2019-172826 is substituted with the pigment portion in the compound of the present application and the product thereof can be mentioned. Among them, the present application is not limited to these compounds and the like.

[0222] <Reagent containing a labeled biological substance>

[0223] In the reagent containing the labeled biological substance of the present application, the labeled biological substance of the present application is not particularly limited, and its form can be appropriately selected depending on the purpose of use and the like, such as a solution form in which it is dissolved in an aqueous medium such as physiological saline and a phosphate buffer, and a solid form such as a microparticulate powder and a freeze-dried powder.

[0224] For example, in the case where the labeled biological substance of the present application is used as a fluorescent labeling reagent, it can also be used as a reagent containing the labeled biological substance in any of the above forms.

[0225] <Use of a labeled biological substance>

[0226] The labeled biological substance of the present application obtained from the compound of the present application can exhibit excellent fluorescence intensity, and can stably detect fluorescence emitted from the labeled biological substance excited by light irradiation. Therefore, the labeled biological substance of the present application can be applied to various techniques using fluorescent labeling, for example, can be suitably used as a fluorescent labeling reagent in multicolor WB or dot blotting, or a live fluorescent imaging reagent.

[0227] The fluorescence detection using the labeled biological substance of the present application generally includes the procedures of (i) to (iii) or (iv) to (vii). The fluorescence detection including the procedures of (i) to (iii) corresponds to a direct method using a primary antibody fluorescently labeled with the compound of the present application, and the fluorescence detection including the procedures of (iv) to (vii) corresponds to an indirect method using a secondary antibody fluorescently labeled with the compound of the present application.

[0228] (i) the procedure of preparing the following (a) and (b) respectively

[0229] (a) a sample containing a biological substance as a target (hereinafter, also referred to as "target biological substance")

[0230] (b) a labeled biological substance of the present application (hereinafter, also referred to as "labeled biological substance A of the present application") in which a biological substance capable of binding to the target biological substance in the above (a) (hereinafter, also referred to as "primary biological substance") is bound to the compound of the present application

[0231] (ii) the procedure of preparing a bound body (hereinafter, also referred to as "fluorescently labeled bound body A") in which the target biological substance in the above (a) is bound to the primary biological substance in the above (b) of the labeled biological substance A of the present application

[0232] (iii) the procedure of irradiating the above fluorescently labeled bound body A with light in a wavelength region absorbed by the labeled biological substance A of the present application, and detecting fluorescence emitted from the labeled biological substance A of the present application

[0233] (iv) the procedure of preparing the following (c) to (e) respectively

[0234] (c) a sample containing a target biological substance

[0235] (d) a biological substance capable of binding to the target biological substance in the above (c) (hereinafter, also referred to as "primary biological substance")

[0236] (e) a labeled biological substance of the present application (hereinafter, also referred to as "labeled biological substance B of the present application") in which a biological substance capable of binding to the primary biological substance in the above (d) (hereinafter, also referred to as "secondary biological substance") is bound to the compound of the present application

[0237] (v) a step of preparing a bonded body in which the target biological substance in (c) above is bonded to the primary biological substance in (d) above (hereinafter, also referred to as "bonded body b")

[0238] (vi) a step of preparing a bonded body in which the primary biological substance in the bonded body b above is bonded to the secondary biological substance in the marker biological substance B of the present application (hereinafter, also referred to as "fluorescently labeled bonded body B2")

[0239] (vii) a step of irradiating the fluorescently labeled bonded body B2 with light in a wavelength region absorbed by the marker biological substance B of the present application, and detecting fluorescence emitted from the marker biological substance B of the present application

[0240] As the biological substance (primary biological substance) capable of bonding to the target biological substance and the biological substance (secondary biological substance) capable of bonding to the primary biological substance, the biological substance in the marker biological substance of the present application described above can be given. The biological substance capable of specifically bonding to the biological substance in the subject or the primary biological substance can be selected as appropriate in accordance with the target biological substance (biological substance in the subject) or the primary biological substance.

[0241] As the protein in the above-mentioned target biological substance, a so-called disease marker can be mentioned. As the disease marker, there is no particular limitation, and for example, α-fetoprotein (AFP), PIVKA-II (protein induced by vitamin K absence or antagonist II), BCA (breast carcinoma-associated antigen) 225, basic fetoprotein (BFP), CA (carbohydrate antigen) 15-3, CA19-9, CA72-4, CA125, CA130, CA602, CA54 / 61 (CA546), carcinoembryonic antigen (CEA), DUPAN-2, elastase 1, immunosuppressive acidic protein (IAP), NCC-ST-439, γ-precipitin (γ-Sm), prostate specific antigen (PSA), prostatic acid phosphatase (PAP), neuron specific enolase (NSE), Iba1, amyloid β, Tau, flotillin, squamous cell carcinoma-associated antigen (SCC antigen), sialyl LeX-i antigen (SLX), SPan-1, tissue polypeptide antigen (TPA), sialyl Tn antigen (STN), CYFRA (cytokeratin) pepsinogen (PG), C-reactive protein (CRP), serum amyloid A protein (SAA), myoglobin, creatine kinase (CK), troponin T, ventricular muscle myosin light chain I, and the like can be mentioned.

[0242] The above-mentioned target biological substance can be a bacterium, and as the bacterium, a bacterium as an object of cellular microbiology examination can be mentioned, and although there is no particular limitation, for example, Escherichia coli, Salmonella, respiratory tract bacteria, bacteria that cause problems in public health, and the like can be mentioned.

[0243] The above-mentioned target biological substance can be a virus, and as the antigen of the virus, there is no particular limitation, and for example, an antigen of a hepatitis virus such as a hepatitis C virus, a hepatitis B virus, a p24 protein antigen of an HIV virus, a pp65 protein antigen of a CMV (cytomegalovirus), and E6 and E7 proteins of an HPV (human papillomavirus) can be mentioned.

[0244] In the above-mentioned (i) or (iv), the sample containing the target biological substance is not particularly limited, and can be prepared according to a conventional method.

[0245] Also, the labeling biological substance of the present application is not particularly limited, and can be prepared by bonding a biological substance capable of bonding to the target biological substance to the compound of the present application according to a conventional method. The form of the bond and the reaction for forming the bond are as described above in the labeling biological substance of the present application.

[0246] In the above (v), the target biological substance can be directly bonded to the primary biological substance, or can be bonded via another biological substance different from the target biological substance and the primary biological substance. Also, in the above (vi), the primary biological substance in the bonding body b can be directly bonded to the secondary biological substance in the labeling biological substance B of the present application, or can be bonded via another biological substance different from the primary biological substance and the secondary biological substance.

[0247] The labeling biological substance of the present application can also be used as a fluorescently labeled antibody in either of the direct method and the indirect method, and is preferably used as a fluorescently labeled antibody in the indirect method.

[0248] In the above (ii) or (v) and (vi), the bonding of the labeling biological substance of the present application or the like to the target biological substance is not particularly limited, and can be performed according to a conventional method.

[0249] In the above (iii) or (vii), regarding the wavelength for exciting the labeling biological substance of the present application, it is not particularly limited as long as it is a wavelength capable of exciting the light emission wavelength (excitation wavelength) of the labeling biological substance of the present application. In general, it is preferably 300 to 1000 nm, and more preferably 400 to 800 nm.

[0250] As the fluorescent excitation light source used in the present application, it is not particularly limited as long as it is a light source emitting a wavelength capable of exciting the light emission wavelength (excitation wavelength) of the labeling biological substance of the present application, and various laser light sources can be used, for example. Also, various filters can be used to obtain a preferred excitation wavelength or to detect only fluorescence.

[0251] Regarding other matters in the above (i) to (vii), there is no particular limitation, and conditions such as methods, reagents, devices, and the like generally used in fluorescence detection using fluorescent labeling can be appropriately selected.

[0252] Also, regarding procedures other than the above (i) to (vii), conditions such as methods, reagents, devices, and the like generally used in various methods using fluorescent labeling can be appropriately selected.

[0253] For example, using the multicolor WB of the labeled biological substance of the present application, by making a blotting membrane using the method generally used as a target biological substance (separation of proteins using electrophoresis, blotting to a membrane, blocking of a membrane), by using the labeled biological substance of the present application as a labeled antibody (preferably a secondary antibody), it is possible to stably detect fluorescence emitted from a target biological substance with excellent fluorescence intensity. For the dot blotting using the labeled biological substance of the present application, as well as the multicolor WB, by making a blotting nitrocellulose membrane or a blotting PVDF (polyvinylidene fluoride) membrane or the like using the method generally used as a target biological substance, by using the labeled biological substance of the present application as a labeled antibody (preferably a secondary antibody), it is possible to stably detect fluorescence emitted from a target biological substance with excellent fluorescence intensity.

[0254] -Substituent Group T-

[0255] In the present application, as a preferable substituent, a substituent selected from the following Substituent Group T can be mentioned.

[0256] Also, in the present application, in the case of only a substituent being mentioned, reference is made to the Substituent Group T, and in the case of only a group such as an alkyl group being mentioned, the corresponding group of the Substituent Group T is preferably used.

[0257] Further, in the present specification, in the case of a straight chain alkyl group and a branched chain alkyl group being distinguished, an alkyl group is used in the meaning including a straight chain alkyl group and a branched chain alkyl group. On the other hand, in the case of an alkyl group not being distinguished from a cyclic alkyl group and in the case of no specific mention, an alkyl group is used in the meaning including a straight chain alkyl group, a branched chain alkyl group and a cyclic alkyl group. This is the same for a group (alkoxy group, alkylthio group, alkenyloxy group, etc.) containing a group (alkyl group, alkenyl group, alkynyl group, etc.) capable of adopting a cyclic structure, and a compound containing a group capable of adopting a cyclic structure. In the case of a group being capable of forming a cyclic skeleton, the lower limit of the number of atoms of the group forming the cyclic skeleton is not related to the lower limit of the number of atoms specifically mentioned in the following with respect to a group capable of adopting the structure, and is 3 or more, preferably 5 or more.

[0258] In the explanation of the following Substituent Group T, for example, as with an alkyl group and a cyclic alkyl group, in order to make clear a group of a straight chain or branched chain structure and a group of a cyclic structure, they are sometimes written separately.

[0259] As a group included in the Substituent Group T, the following groups can be mentioned.

[0260] alkyl (the number of carbon atoms is preferably from 1 to 20, more preferably from 1 to 12, further preferably from 1 to 8, particularly preferably from 1 to 6, and most preferably from 1 to 3. For example, methyl, ethyl, isopropyl, t-butyl, amyl, heptyl, 1-ethylpentyl, benzyl, 2-ethoxyethyl, 1-carboxymethyl, trifluoromethyl, etc.), alkenyl (the number of carbon atoms is preferably from 2 to 20, more preferably from 2 to 12, further preferably from 2 to 10, and particularly preferably from 2 to 8. For example, vinyl, allyl, oleyl, etc.), alkynyl (the number of carbon atoms is preferably from 2 to 20, more preferably from 2 to 12, further preferably from 2 to 10, and particularly preferably from 2 to 8. For example, ethynyl, diynyl, phenylethynyl, etc.), cycloalkyl (the number of carbon atoms is preferably from 3 to 20. For example, cyclopropyl, cyclopentyl, cyclohexyl, 4-methylcyclohexyl, etc.), cycloalkenyl (the number of carbon atoms is preferably from 5 to 20. For example, cyclopentenyl, cyclohexenyl, etc.), aryl (it can be a monocyclic group or a condensed ring group (preferably a 2- to 6-ringed condensed ring group). The number of carbon atoms is preferably from 6 to 26, more preferably from 6 to 18, and further preferably from 6 to 12. For example, phenyl, 1-naphthyl, 4-methoxyphenyl, 2-chlorophenyl, 3-methylphenyl, etc.), heterocyclic group (the number of carbon atoms is preferably from 2 to 20. At least one of the ring-constituting atoms is an oxygen atom, a sulfur atom or a nitrogen atom. It can be a monocyclic group or a condensed ring group (preferably a 2- to 6-ringed condensed ring group). In the case of a monocyclic group, the number of ring members is preferably from 5 to 7, and more preferably 5 or 6. The heterocyclic group includes aromatic heterocyclic groups (heteroaryl groups) and aliphatic heterocyclic groups (aliphatic heterocyclic groups). For example, 2-pyridyl, 4-pyridyl, 2-imidazolyl, 2-benzimidazolyl, 2-thiazolyl, 2-oxazolyl, etc.), alkoxy (the number of carbon atoms is preferably from 1 to 20, and more preferably from 1 to 12. For example, methoxy, ethoxy, isopropoxy, benzyloxy, etc.), alkenyloxy (the number of carbon atoms is preferably from 2 to 20, and more preferably from 1 to 12. For example, vinyloxy, allyloxy, etc.), alkynyloxy (the number of carbon atoms is preferably from 2 to 20, and more preferably from 1 to 12. For example, 2-propynyloxy, 4-butynyloxy, etc.), cycloalkoxy (the number of carbon atoms is preferably from 3 to 20. For example, cyclopropoxy, cyclopentoxy, cyclohexoxy, 4-methylcyclohexoxy, etc.), aryloxy (the number of carbon atoms is preferably from 6 to 26, and more preferably from 6 to 14. For example, phenoxy, 1-naphthoxy, 3-methylphenoxy, 4-methoxyphenoxy, etc.), heterocyclic-oxy (the heterocyclic group portion of the heterocyclic-oxy group can preferably be as described above. For example, imidazolyloxy, benzimidazolyloxy, thiazolyloxy, benzothiazolyloxy, triazinyloxy, purinyloxy), polyalkyleneoxy (the number of carbon atoms is preferably from 2 to 40, and more preferably from 2 to 20.),

[0261] Alkoxycarbonyl (preferably with 2 to 20 carbon atoms, e.g., ethoxycarbonyl, 2-ethylhexyloxycarbonyl, etc.), cycloalkoxycarbonyl (preferably with 4 to 20 carbon atoms, e.g., cyclopropoxycarbonyl, cyclopentoxycarbonyl, cyclohexyloxycarbonyl, etc.), aryloxycarbonyl (preferably with 6 to 20 carbon atoms, e.g., phenoxycarbonyl, naphthoxycarbonyl, etc.), amino (preferably with 0 to 20 carbon atoms, including alkylamino, alkenylamino, acetylenylamino, cycloalkylamino, cycloalkenylamino, arylamino, and heterocyclic amino). The above groups substituted for unsubstituted amino groups have the same meaning as the corresponding groups in substituent group T. For example, amino, N,N-dimethylamino, N,N-diethylamino, N-ethylamino, N-allylamino, N-(2-propynyl)amino, N-cyclohexylamino, N-cyclohexenylamino, aniline, pyridineamino, imidazoleamino, benzimidazoleamino, thiazoleamino, benzothiazoleamino, triazineamino, etc.), aminosulfonyl (preferably with 0 to 20 carbon atoms, preferably alkyl, cycloalkyl, or aryl aminosulfonyl, for example, N,N-dimethylaminosulfonyl, N-cyclohexylaminosulfonyl, N-phenylaminosulfonyl, etc.), acyl (preferably with 1 to 20 carbon atoms, for example, acetyl, cyclohexylcarbonyl, benzoyl, etc.), acyloxy (preferably with 1 to 20 carbon atoms, for example, acetoxy, cyclohexylcarbonyloxy, benzoyloxy, etc.), carbamoyl (preferably with 1 to 20 carbon atoms, preferably alkyl, cycloalkyl, or aryl carbamoyl, for example, N,N-dimethylcarbamoyl, N-cyclohexylcarbamoyl, N-phenylcarbamoyl, etc.),

[0262] Acylamino (preferably an acylamino group with 1 to 20 carbon atoms, such as acetamido, cyclohexylcarbonylamino, benzoylamino, etc.), amide group (aminocarbonyl), sulfonamide group (preferably a sulfonamide group with 0 to 20 carbon atoms, preferably an alkyl, cycloalkyl, or aryl group, such as methanesulfonamide, benzenesulfonamide, N-methylmethanesulfonamide, N-cyclohexylsulfonamide, N-ethylbenzenesulfonamide, etc.), alkylthio group (preferably with 1 to 20 carbon atoms, such as methylthio, ethylthio, etc.). The following groups are used: isopropylthio, benzylthio, etc.; cycloalkylthio (preferably with 3 to 20 carbon atoms, such as cyclopropylthio, cyclopentylthio, cyclohexylthio, 4-methylcyclohexylthio, etc.); arylthio (preferably with 6 to 26 carbon atoms, such as phenylthio, 1-naphthio, 3-methylphenylthio, 4-methoxyphenylthio, etc.); alkyl, cycloalkyl, or arylsulfonyl (preferably with 1 to 20 carbon atoms, such as methylsulfonyl, ethylsulfonyl, cyclohexylsulfonyl, benzylsulfonyl, etc.).

[0263] silyl group (preferably having 1 to 20 carbon atoms, preferably an alkyl-, aryl-, alkoxy-, and aryloxy-substituted silyl group, for example, triethylsilyl, triphenylsilyl, diethylbenzylsilyl, dimethylphenylsilyl, and the like), siloxy group (preferably having 1 to 20 carbon atoms, preferably an alkyl-, aryl-, alkoxy-, and aryloxy-substituted siloxy group, for example, triethylsiloxy, triphenylsiloxy, diethylbenzylsiloxy, dimethylphenylsiloxy, and the like), hydroxyl group, cyano group, nitro group, halogen atom (for example, fluorine atom, chlorine atom, bromine atom, iodine atom, preferably fluorine atom), carboxyl group, sulfo group, phosphono group, phosphoryl group, boronic acid group, sulfanyl group (-SH), amino acid residue, or polyamino acid residue. Also, the substituents selected from the substituent group T are more preferably alkyl group, alkenyl group, cycloalkyl group, aryl group, heterocyclic group, alkoxy group, cycloalkoxy group, aryloxy group, alkoxycarbonyl group, cycloalkoxycarbonyl group, the above-mentioned amino group, acylamino group, cyano group, or halogen atom, and particularly preferably alkyl group, alkenyl group, heterocyclic group, alkoxy group, alkoxycarbonyl group, amino group, acylamino group, or cyano group.

[0264] As for the substituents selected from the substituent group T, unless otherwise specified, a group formed by combining a plurality of the above-mentioned groups is also included. For example, when a compound or a substituent, etc. contains an alkyl group, alkenyl group, etc., these can be linear or branched, and these can be substituted or unsubstituted. Also, when containing an aryl group, heterocyclic group, etc., these can be monocyclic or fused ring, and these can be substituted or unsubstituted.

[0265] Examples

[0266] Hereinafter, the present application will be further explained in detail according to Examples, but the present application is not limited thereto. Also, room temperature means 25°C.

[0267] The compounds (1-1) to (1-5), (2-1) to (2-2), and comparative compounds (1) and (2) used in the Examples are shown below. Me represents methyl group.

[0268] Also, in the compound of the Examples, even in the case where not particularly described, the sulfo group can contain a salt structure (for example, potassium salt, sodium salt, and the like metal salt, TEA (triethylamine) salt or DIPEA (N,N-diisopropylethylamine) salt as a part of the structure).

[0269] [Chemical Formula 16]

[0270]

[0271] The comparative compound (1) is compound (4) described in the examples of Patent Literature 1, and was synthesized according to the method described in Patent Literature 1. The comparative compound (2) is a compound described in [Chemical Formula 72] from the second from the left, and was synthesized according to the method described in Patent Literature 2 and the following synthesis example of the compound. Also, regarding the labeling antibody of the comparative compound (2), in the following synthesis method of the labeling antibody of compound (1-1), compound (1-F) was changed to the comparative compound (2), and was synthesized in the same manner except for this.

[0272] Hereinafter, the synthesis method of each compound will be described in detail, but the starting material, the color intermediate, and the synthesis route are not limited to these.

[0273] In the case where not particularly described, the carrier in the reverse phase column chromatography used SNAP Ultra C18 (product name, manufactured by Biotage Japan Ltd.) or Sfar C18 (product name, manufactured by Biotage Japan Ltd.), and the carrier in the normal phase column chromatography used Hi-Flash Column (product name, manufactured by YAMAZEN CORPORATION).

[0274] The mixing ratio in the eluent used in the reverse phase column chromatography or the normal phase column chromatography is the volume ratio. For example, "acetonitrile: water = 0: 100 → 20: 80" means changing the eluent of "acetonitrile: water = 0: 100" to the eluent of "acetonitrile: water = 20: 80".

[0275] The separation and purification HPLC (High Performance Liquid Chromatography) used 2767 (product name, manufactured by Waters Corporation).

[0276] The MS spectrum was measured using ACQUITY SQD LC / MS System (product name, manufactured by Waters Corporation, ionization method: ESI (ElectroSpray Ionization)) or LCMS-2010EV (product name, manufactured by Shimadzu Corporation, ionization method: ESI and APCI (Atmospheric Pressure Chemical Ionization) ionization method) simultaneously.

[0277] (Synthesis Example 1: Synthesis of compound (1-1) and labeling antibody of compound (1-1))

[0278] [Chemical Formula 17]

[0279]

[0280] Into a 100-ml three-necked flask, compound (1-1A) i.e., tert-butyl 3,5-difluoro-4-formylbenzoate (manufactured by Combi-Blocks Inc.) 220 mg, dichloromethane 10 ml, 2,4-dimethylpyrrole 175 mg were placed, and stirring was performed under a stream of nitrogen. The reaction solution was ice-cooled in an ice bath, and 1 μl of trifluoroacetic acid (TFA) was added dropwise, and stirring was performed for 2 hours. Thereafter, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone 209 mg was added, and stirring was performed for 30 minutes. After 1 μl of triethylamine was added, the dichloromethane was concentrated under reduced pressure, and purification was performed by silica gel column chromatography with ethyl acetate / hexane as the eluent, thereby obtaining compound (1-1B).

[0281] Into a 100-ml three-necked flask, compound (1-1B) 170 mg, dichloromethane (super dehydrated) 3 ml, N-ethyldiisopropylamine (DIPEA) 0.86 ml were placed, and stirring was performed for 10 minutes or more. To this, a mixed solution of CF3BF3K 290 mg / TMSOTf 732 mg / acetonitrile (super dehydrated) 3.3 ml, which was prepared separately, was added dropwise, and stirring was performed under nitrogen for 1 hour. Thereafter, distilled water was added, liquid-liquid extraction was performed to extract the organic layer, and after concentration under reduced pressure, purification was performed by silica gel column chromatography with ethyl acetate / hexane as the eluent, thereby obtaining compound (1-1C).

[0282] Into a 100-ml three-necked flask, compound (1-1C) 170 mg, dichloromethane 1 ml were placed, and mixing and stirring were performed, and after cooling at 0 to 5°C, N-bromosuccinimide (NBS) 41 mg was added, and stirring was performed for 15 minutes. Purification was performed by silica gel column chromatography with dichloromethane / hexane as the eluent, thereby obtaining compound (1-1D).

[0283] Into a 100-ml three-necked flask, was placed compound (1-1D) 35 mg, 2,6-dimethoxyphenylboronic acid 66 mg, cesium fluoride 68 mg, distilled water 0.1 ml, and cyclopentyl methyl ether (CPME) 6 ml, and degassing and nitrogen substitution were performed while stirring. Thereafter, SPhos Pd G3 (product name, manufactured by Sigma-Aldrich Inc., Pd catalyst for cross coupling) 7 mg was added, and stirring was performed at 100°C. During the reaction, SPhos Pd G3 and distilled water were appropriately added, and heating and stirring were performed. After returning to room temperature, distilled water was added, liquid-liquid extraction was performed to extract the organic layer, and after concentration under reduced pressure, purification was performed by silica gel column chromatography using ethyl acetate / hexane as the eluent to obtain compound (1-1E).

[0284] Into a 50-ml eggplant-shaped flask, was placed compound (1-1E) 9 mg, dichloromethane (super dehydrated) 0.9 ml, and acetonitrile (super dehydrated) 0.3 ml, and stirring was performed under nitrogen. Chlorosulfonic acid 11 μl was added under ice cooling, and stirring was performed for 30 minutes. After adding potassium carbonate 72 mg, distilled water 2 ml was added dropwise, and stirring was performed. Purification was performed on the reaction solution using Sfar C18 (product name, manufactured by Biotage Japan Ltd.) with acetonitrile / water as the eluent to obtain compound (1-1F) 4.5 mg. The obtained compound was identified by ESI-MS. [M-2K+3H] + = 887, [M-2K] 2- / 2 = 442

[0285] Into a 10-ml test tube, was placed compound (1-1F) 4 mg, N,N-dimethylformamide (DMF) 0.35 ml, and N-hydroxysuccinimide 3 mg, and while stirring, N,N-dicyclohexylcarbodiimide 10 mg was added, and stirring was performed at room temperature for 1.5 hours. Purification was performed on the reaction solution using Sfar C18 (product name, manufactured by Biotage Japan Ltd.) with acetonitrile / water as the eluent, and the solvent was removed by freeze drying distillation to obtain compound (1-1) 1 mg.

[0286] To 400 μL of Anti-rabbit IgG antibody (Anti-IgG, Host: goat, 2.4 mg / mL, Catalog No. 111-005-003, manufactured by Jackson ImmunoResearch Laboratories, Inc.) was added 40 μL of carbonate pH standard solution (pH = 10.01) (manufactured by FUJIFILM Wako Pure Chemical Corporation) and 3.2 μL of a dimethyl sulfoxide (DMSO) solution of compound (1-1) at a concentration of 20 mM, and after stirring, the mixture was allowed to stand at room temperature for 1 hour. Subsequently, the reaction solution was directly applied to a Sephadex G-25 column (Catalog No. 17085101, manufactured by GE Healthcare), and purified using PBS (phosphate buffered saline, pH = 7.4, manufactured by FUJIFILM Wako Pure Chemical Corporation), thereby obtaining a labeled antibody of compound (1-1).

[0287] [Synthesis Example 2: Synthesis of compound (1-2) and a labeled antibody of compound (1-2)]

[0288] In the synthesis of compound (1-1), compound (1-1A) was replaced with 3,5-dimethoxy-4-formyl benzoic acid tert-butyl ester (synthesized according to the synthesis method of compound (7-C) described in International Publication No. 2019 / 230963), and otherwise, compound (1-2) was synthesized in the same manner. + = 911, [M-Su (succinimide)-2K+H] 2- / 2 = 454

[0289] In the synthesis of a labeled antibody of compound (1-1), compound (1-1) was changed to compound (1-2), and otherwise, a labeled antibody of compound (1-2) was synthesized in the same manner.

[0290] [Synthesis Example 3: Synthesis of compound (1-3)]

[0291] [Chemical Formula 18]

[0292]

[0293] To a 100-ml three-necked flask, compound (1-3A) (3,5-dimethoxy-4-formylbenzoic acid tert-butyl ester, synthesized according to the synthesis of compound (7-C) described in International Publication No. 2019 / 230963) 1 g, dichloromethane 100 ml, 2,4-dimethylpyrrole 686 mg were placed, and stirring was performed under a stream of nitrogen. The reaction liquid was ice-cooled in an ice bath, and trifluoroacetic acid (TFA) 19 μl was added dropwise, and stirring was performed for 2 hours. Thereafter, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone 844 mg was added, and stirring was performed for 30 minutes. After the addition of 3.46 ml of N-ethyldiisopropylamine (DIPEA), boron trifluoride diethyl ether complex 4.1 ml was added dropwise, and stirring was performed for 1 hour. The reaction liquid was gradually added while stirring was performed in 200 ml of a liquid prepared by adding ice to saturated aqueous sodium bicarbonate solution 100 ml, and stirring was performed for 10 minutes. Dichloromethane was concentrated under reduced pressure, and purification was performed by silica gel column chromatography with ethyl acetate / hexane as the eluent, thereby obtaining compound (1-3B).

[0294] In the synthesis of compound (1-1), compound (1-3B) was used instead of compound (1-1C), and otherwise, compound (1-3) was obtained in the same manner as in the synthesis of compound (1-1). + = 861, [M-Su(succinimide)-2K+H] 2- / 2 = 429

[0295] [Synthesis Example 4: Synthesis of compound (1-4)]

[0296] [Chemical Formula 19]

[0297]

[0298] To a 100-ml three-necked flask, 2-bromoresorcinol 2 g, acetonitrile 20 ml, triethylene glycol-2-bromoethyl ether 6.51 g, potassium carbonate 2.87 g were placed, and stirring was performed under a stream of nitrogen at 70°C for 11 hours, and a reaction was performed. Thereafter, liquid-liquid separation was performed using ethyl acetate and distilled water, the organic layer was extracted by liquid-liquid separation, and after concentration under reduced pressure, purification was performed by silica gel column chromatography with ethyl acetate as the eluent, thereby obtaining compound (1-4A) 2.0 g.

[0299] Into a 200-ml three-necked flask, there were put 1.8 g of compound (1-4A), 0.45 g of tetrahydroxydiborane (BBA), 40 ml of tetrahydrofuran (THF), 20 ml of methanol, 1.7 ml of N-ethyldiisopropylamine (DIPEA), and the mixture was subjected to degassing under reduced pressure with stirring, and was replaced with nitrogen. Next, 23 mg of dichlorobis [di-tert-butyl-(4-dimethylamino phenyl) phosphine] palladium (II) (Pd(Amphos)2Cl2) was added, and the mixture was heated and stirred at 58°C for 3 hours. After the completion of the reaction, the solvent was distilled off under reduced pressure with an evaporator after the mixture was left to stand at room temperature overnight. Purification by column chromatography on silica gel with ethyl acetate / methanol as an eluent gave 0.8 g of compound (1-4B).

[0300] In the synthesis of compound (1-1), compound (1-4B) was used instead of 2,6-dimethoxyphenylboronic acid, and otherwise, compound (1-4) was synthesized in the same manner as in the synthesis of compound (1-1). + = 1591.6, [M-Su(succinimide)-2K+2H] - = 1589.6

[0301] [Synthesis Example 5: Synthesis of compound (1-5)]

[0302] [Chemical Formula 20]

[0303]

[0304] Into a 50-ml three-necked flask, there were put 10 mg of compound (1-4), 10 mg of l-amino-3,6,9,12-tetraoxypentadecan-15-oic acid, 0.3 ml of DMF, and 6 μl of triethylamine, and the mixture was stirred at room temperature for 10 minutes. Saturated brine was added, and the reaction solution was purified using Sfar C18 (product name, manufactured by Biotage Japan Ltd.) with acetonitrile / water as an eluent, and the solvent was removed by freeze drying distillation to give 8 mg of compound (1-5A).

[0305] In the synthesis of compound (1-4), compound (1-5A) was used instead of compound (1-4D), and otherwise, compound (1-5) was synthesized in the same manner as in the synthesis of compound (1-4). + = 1838.7, [M-Su(succinimide)-2Na+H] 2- / 2 = 917.8

[0306] In the synthesis of the labeled antibody of compound (1-1), compound (1-1) was changed to compound (1-5), and otherwise, the labeled antibody of compound (1-5) was synthesized in the same manner.

[0307] [Synthesis Example 6: Synthesis of compound (2-1) and labeled antibody of compound (2-1)]

[0308] [Chemical Formula 21]

[0309]

[0310] Into a 100-ml three-necked flask, compound (1-1D) 25 mg, toluene (super dehydrated) 5 ml, benzaldehyde 30 μl, piperidine 22 μl, acetic acid (AcOH) 30 μl were put, and heating stirring was performed under nitrogen at 90-100°C. Distilled water was added, and the organic layer was extracted by liquid-liquid extraction, and after concentration under reduced pressure, compound (2-1E) was obtained by purification with a silica gel column chromatography using ethyl acetate / hexane as an eluent.

[0311] In the synthesis of compound (1-1), compound (2-1E) was used instead of compound (1-1D), and otherwise, compound (2-1) was synthesized in the same manner as the synthesis of compound (1-1).[M-Su(succinimide)-2K+4H] + = 1063

[0312] In the synthesis of the labeled antibody of compound (1-1), compound (1-1) was changed to compound (2-1), and otherwise, the labeled antibody of compound (2-1) was synthesized in the same manner.

[0313] [Synthesis Example 7: Synthesis of compound (2-2) and labeled antibody of compound (2-2)]

[0314] In the synthesis of compound (2-1), compound (1-1D) was replaced with a compound in which compound (1-1A) in the synthesis of compound (1-1D) was replaced with 3,5-dimethoxy-4-formylbenzoic acid tert-butyl ester (synthesized according to the synthesis of compound (7-C) described in International Publication No. 2019 / 230963), and otherwise, compound (2-2) was synthesized in the same manner.[M-Su(succinimide)-2K+4H] + = 1087

[0315] In the synthesis of the labeled antibody of compound (1-1), compound (1-1) was changed to compound (2-2), and otherwise, the labeled antibody of compound (2-2) was synthesized in the same manner.

[0316] Evaluation of molar absorption coefficient and fluorescence quantum yield of compound or labeled antibody

[0317] For each of the above-mentioned compounds, the following properties were evaluated, and the results are shown in Table 1.

[0318] In addition, each of the compounds (1-1) to (1-5) and (2-1) to (2-2) was dissolved in an aqueous solution at a sample concentration of 1 mg / ml, and showed high water solubility required from the viewpoint of practicality as a fluorescent compound.

[0319] [1] Evaluation of molar absorption coefficient ε of compound

[0320] Using a spectrophotometer UV-3600 (product name) manufactured by Shimadzu Corporation, the molar absorption coefficient ε (L / mol-cm) at the wavelength of maximum absorption of a PBS (phosphate buffered saline, pH 7.4) solution of the above-mentioned compound was measured, and was evaluated according to the following evaluation criteria.

[0321] With respect to the molar absorption coefficient, the higher the evaluation grade, the more excellent the molar absorption coefficient, and therefore it is very preferable, and in the present test, it is preferable that the molar absorption coefficient satisfies the evaluation grade of "B" or more (S to B) from the viewpoint of practicality as a fluorescent compound.

[0322] Evaluation criteria for molar absorption coefficient ε

[0323] S: 80000 or more

[0324] A: 70000 or more and less than 80000

[0325] B: 60000 or more and less than 70000

[0326] C: 40000 or more and less than 60000

[0327] D: less than 40000

[0328] In the above evaluation grade, the unit of ε is Lmol -1 cm -1 .

[0329] [2] Evaluation of fluorescence quantum yield

[0330] The PBS (pH 7.4) solution of the above-mentioned compound or labeled antibody was evaluated by the method described in the following reference.

[0331] "A Guide to Recording Fluorescence Quantum Yields" (HORIBA Scientific's material, available through https: / / www.horiba.com / fileadmin / uploads / Scientific / Documents / Fluorescence / quantumyieldstrad.pdf)

[0332] With respect to the fluorescence quantum yield, the fluorescence quantum yield is excellent if the evaluation grade is high, and thus it is very preferable, and in the present test, the fluorescence quantum yield satisfies the evaluation grade "C" or more is preferable from the viewpoint of the practicability of the fluorescent compound, the labeled antibody.

[0333] -Evaluation criteria for fluorescence quantum yield-

[0334] S: 0.7 or more

[0335] A: 0.6 or more and less than 0.7

[0336] B: 0.4 or more and less than 0.6

[0337] C: 0.3 or more and less than 0.4

[0338] D: less than 0.3

[0339] [Table 1]

[0340]

[0341] (Note)

[0342] The labeled antibodies of the compounds (1-1) to (1-5), (2-1) to (2-2) and the comparative compounds (1), (2), the compound (1-5) are each as shown above.

[0343] "-" in the column of molar absorption coefficient of the labeled antibody of the compound (1-5) means that it is not measured.

[0344] In the column of the bonding position to the biological substance, the case where the bonding position to the biological substance is written as "Yes", the case where it is not written as "No", and the case of the labeled antibody is written as "-".

[0345] <Example 2> Evaluation of light resistance of compounds and labeled antibodies

[0346] The light resistance of each of the compounds or the labeled antibodies shown above was evaluated in the following manner.

[0347] [1] Evaluation of light resistance in an aqueous solution

[0348] The above compound or labeled antibody (hereinafter, referred to as a sample) was dissolved in PBS at pH 7.4 in such a manner that the absorbance at the wavelength peak became 0.095 to 0.105. The absorbance at the wavelength peak of the sample was measured over time by a spectrometer (manufactured by Agilent Technologies, Inc., product name: Agilent 8453) in a state where the obtained solution was exposed to light using a carousel light irradiator.

[0349] In addition, a xenon lamp UXL-500D-O (product name) manufactured by Ushio Inc. was used, and a HA-50 filter and a Y44 filter (both product names) manufactured by HOYA Corporation were used as sharp cut filters, and exposure using a carousel light irradiator was performed at an exposure intensity of 22 mW / cm 2 (500 nm converted).

[0350] The absorbance at the wavelength peak before exposure was set to 100%, and the exposure time until the absorbance at the wavelength peak decreased by 50% (the absorbance at the wavelength peak reached 50%) was calculated. The ratio to the reference value (the exposure time of the compound or labeled antibody / the reference value) was calculated based on the results of the exposure time of the labeled antibody of Comparative Compound (1), (2), or Comparative Compound (2) as the reference value, and evaluation was performed according to the following evaluation criteria. Since the higher the evaluation grade is, the longer the stable time is, it is very preferable.

[0351] Evaluation Criteria for Light Resistance

[0352] S: 10 times or more relative to the reference value

[0353] A: 5 times or more and less than 10 times relative to the reference value

[0354] B: 3 times or more and less than 5 times relative to the reference value

[0355] C: 1.2 times or more and less than 3 times relative to the reference value

[0356] D: 1.0 times or more and less than 1.2 times relative to the reference value

[0357] E: Less than 1.0 times relative to the reference value

[0358] [Table 2]

[0359] N0. Compound or labeled antibody Light resistance 311 Compound (1-1) S 312 Compound (1-2) S 313 Compound (1-3) C 314 Compound (1-4) S 315 Compound (1-5) S 411 Compound (2-2) C 412 Labeled antibody of compound (2-2) C c31 Comparative compound (1) Reference value in No. 311 to 315 c41 Comparative compound (2) Reference value in No. 411 c42 Labeled antibody of comparative compound (2) Reference value in No. 412

[0360] The following was found from the results of Table 1 and Table 2 described above.

[0361] The FL is represented by General Formula (6), and R 63and R 66 Among the compounds having a structural portion including a dipyrromethene boron complex dye having an alkyl group or an aryl group, the compound of the present application having a substituent R 11 ~R 15 of the comparative compound (1) not having a specific phenyl group is poor in light resistance. In contrast, the compounds (1-1) to (1-5) having a specific phenyl group of the present application having a substituent R 11 ~R 15 exhibit excellent light resistance of 1.2 times or more relative to the comparative compound (1), and have high water solubility required for fluorescent labeling, exhibit the same degree of molar absorption coefficient and high fluorescence quantum yield, and are excellent in fluorescence intensity (brightness).

[0362] As FL, represented by General Formula (7), among the compounds having a structural portion including a dipyrromethene boron complex dye, the compound of the present application having a substituent R 11 ~R 15 of the comparative compound (2) not having a specific phenyl group is poor in molar absorption coefficient. In contrast, the compounds (2-1) to (2-2) having a specific phenyl group of the present application having a substituent R 11 ~R 15 exhibit excellent molar absorption coefficient and fluorescence quantum yield relative to the comparative compound (2). Also, with respect to the compound (2-2), the compound and the labeled biological substance obtained are excellent in light resistance.

[0363] The present application has been described together with embodiments thereof, but the present inventors believe that, unless specifically designated, the present application is not limited in any detail of the description, and should be interpreted broadly without departing from the spirit and scope of the present application shown in the appended technical solution.

[0364] This application claims priority based on Japanese Patent Application No. 2021-133510 filed on August 18, 2021 in Japan, which is hereby incorporated by reference, and the contents thereof are incorporated into the present specification as a part of the present specification.

Claims

1. A fluorescent compound represented by the following general formula (2), In the above formula, R 1 and R 2 It indicates a methoxy group or is composed of -O (LO). m -R 21 The structure of the alkylene glycol is represented by L, where L represents an alkylene group with 2 carbon atoms, m is 2 to 4, and R... 21 Indicates alkyl groups having 1 to 2 carbon atoms. n is 2, R 13 R 14 Represents a hydrogen atom. X + It represents hydrogen ions or metal ions. The FL is a structural part comprising a dipyrrole-methylboron complex pigment represented by the following general formula (6) or (7). In general formula (6), R 62 and R 65 The bond consists of hydrogen atoms and is formed by removing these two hydrogen atoms to create an FL bond. R 63 and R 66 Indicates methyl or -CH=CR a3 H, where R a3 It is a phenyl group. R 61 and R 64 Indicates methyl, R 67 This indicates a phenyl group having a fluorine atom or methoxy group as a substituent at the ortho position and a carboxyl group or a substituent capable of bonding with biological substances directly or indirectly through a linking group at the para position. The substituents capable of binding with biological substances are selected from NHS ester structures, succinimide structures, maleimide structures, azide groups, ethynyl groups, peptide structures, long-chain alkyl groups with 12 to 30 carbon atoms, and quaternary ammonium groups. R 68 and R 69 One of them is a fluorine atom and the other is a fluorine atom or a fluorinated methyl group. The pigment represented by general formula (6) is obtained from R 62 and R 65 It becomes FL with two hydrogen atoms removed; In general formula (7), R 61 R 62 R 64 R 65 and R 67 ~R 69 R in general formula (6) 61 R 62 R 64 R 65 and R 67 ~R 69 The meanings are the same, R 70 ~R 79 Represents a hydrogen atom. The pigment represented by general formula (7) is obtained from R 62 and R 65 Removing two hydrogen atoms results in a divalent FL.

2. A fluorescently labeled biological substance, which is formed by bonding the fluorescent compound of claim 1 to the biological substance.

3. The fluorescently labeled biological material according to claim 2, wherein, The biological substance is any one of protein, amino acid, nucleic acid, glycan and lipid.

4. The fluorescently labeled biological material according to claim 3, wherein, The binding of the fluorescent compound to the biological substance is based on any one of the following i) to v): i) Non-covalent or covalent bonds between peptides ii) The long-chain alkyl groups in the compound interact with the lipid double membrane or the van der Waals interaction of lipids in biological substances. iii) An amide bond formed by the reaction of N-hydroxysuccinimide ester in the compound with an amino group in biological material. iv) A thioether bond is formed by reacting the maleimide group in the compound with a thioalkyl group in the biological material. v) A bond formed by a click reaction between an azide group in a compound and an acetylene group in a biological substance, accompanied by the formation of a triazole ring.

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