Patent Application
20240173437
The present invention relates to a compound and a labeled biological substance using the compound.
In order to observe in vivo changes in response to various stimuli (diseases, environmental changes, and the like), fluorescently labeled biological substances obtained by labeling a biological molecule (an antibody or the like) having a binding property to a target substance to be detected, with a fluorescent compound (a fluorescent dye), are often used.
For example, also in Western blotting (hereinafter, also abbreviated as WB) that detects a specific protein from a protein mixture, a fluorescence method in which the presence or absence or the abundance of the specific protein is detected using a fluorescently labeled antibody having a binding property to this protein is used.
In addition, in bioimaging technology for analyzing the dynamics and functions of biological molecules, cells, tissues, and the like in a living body, in vivo fluorescence imaging in which a specific portion of a living body visualized by fluorescence labeling is observed is used as one of the techniques for the living body observation.
In the fluorescence labeling, an organic fluorescent dye molecule is generally used, and the brightness (fluorescence intensity) is increased generally by using a fluorescently labeled biological substance to which a plurality of fluorescent dye molecules are bonded. However, since most of the organic dyes exhibiting fluorescence, such as a cyanine dye and a rhodamine dye, have an aromatic chromophore having high planarity, an interaction between the dyes easily occurs, and as a result, a decrease in the fluorescence intensity after labeling due to an interaction such as self-association between the dyes easily occurs. In addition, as the number of molecules (degree of fluorescence labeling: DOL) of the fluorescent dye per one molecule of the biological molecule increases, the fluorescence intensity due to self-association or the like tends to further decrease.
On the other hand, in a field different from the fluorescence labeling, a dye compound that utilizes a fluorescence resonance energy transfer (FRET) phenomenon is known. As a dye compound that utilizes such a FRET phenomenon, for example, a compound in which a phosphor moiety I (an energy donor) that is excited with excitation light is linked, by a group containing proline, to another phosphor moiety II (an energy acceptor) that receives energy from the phosphor moiety I and emits light or is quenched is described as described in “Synthesis of Polyproline Spacers between NIR Dye Pairs for FRET to Enhance Photoacoustic Imaging”, WO2010/117420A, JP2003-508080A, JP2004-508838A, and WO99/002544A.
A dye that is used for fluorescence labeling is required to exhibit an excellent fluorescence intensity in various states of being in a solution, a membrane, a stained cell, and the like. However, in such dye compounds described above, which utilize the FRET phenomenon, as described in “Synthesis of Polyproline Spacers between NIR Dye Pairs for FRET to Enhance Photoacoustic Imaging”, WO2010/117420A, JP2003-508080A, JP2004-508838A, and WO99/002544A, the energy is transferred to the phosphor moiety II instead of emitting fluorescence from the phosphor moiety I, and thus the fluorescence intensity of the compound is decreased.
An object of the present invention is to provide a compound that makes it possible to obtain a labeled biological substance exhibiting an excellent fluorescence intensity in states of being in a solution and a membrane or a state of being in a stained cell. In addition, another object of the present invention is to provide a labeled biological substance obtained by bonding the compound to a biological substance.
That is, the above objects of the present invention have been achieved by the following means.
[1]
A compound comprising:
two or more phosphor moieties of which light absorption characteristics are equivalent to each other,
wherein the phosphor moieties adjacent to each other are each linked through a group including a structure represented by General Formula (I),
in the formula, X1 to X3 represent —O—, —S—, >NR1, or >CR2R3,
R1 to R3 represent a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an acyl group, —NR8R9, —OR10, or an anionic group,
R8 to R10 represent a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an acyl group, an aryl group, a heteroaryl group, or an anionic group,
n is an integer of 2 or more, and
* represents a bonding site.
[2]
The compound according to [1], in which the n is an integer of 3 or more.
[3]
The compound according to [1] or [2], in which the compound is represented by General Formula (II),
in the formula, R4 and R5 represent a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an acyl group, an amino group, a hydroxy group, an alkoxy group, a sulfanyl group, an aryl group, or a heteroaryl group,
R6 and R7 represent a hydrogen atom, a hydroxy group, a sulfanyl group, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, an alkoxy group, an amino group, an acyl group, a heteroaryl group, an anionic group, a cationic group, or Q, where Q represents a carboxy group, a substituent capable of being bonded to a biological substance, or a substituent capable of being bonded to a solid support,
L1 to L7 represents a single bond or a divalent linking group,
M represents a phosphor moiety, a physiologically active substance moiety, a prodrug moiety, or a radioactive isotope-containing moiety,
Y represents the structure represented by General Formula (I), and
m is an integer of 1 or more,
provided that at least two of M's represent the phosphor moieties of which the light absorption characteristics are equivalent to each other.
[4]
The compound according to [3], in which the compound is represented by General Formula (III),
in the formula, Y1 to Y3, Z1 to Z3, and W1 to W3 represent a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an acyl group, an amino group, a hydroxy group, an alkoxy group, a sulfanyl group, an aryl group, a heteroaryl group, or an anionic group,
LL3 and LL4 represent a divalent linking group,
s, t, and u are an integer of 0 or more, and
R4 to R7, L1, L2, L5, L6, X1 to X3, M, n, and m respectively have the same meanings as
R4 to R7, L1, L2, L5, L6, X1 to X3, M, n, and m described above.
[5]
The compound according to [4], in which the compound is represented by General Formula (IV),
in the formula, Y4 and Y5 represent a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an acyl group, an amino group, a hydroxy group, an alkoxy group, a sulfanyl group, an aryl group, a heteroaryl group, or an anionic group, and
R4 to R7, L2, L5, X1 to X3, Y1 to Y3, Z1 to Z3, W1 to W3, M, n, m, s, t, and u respectively have the same meanings as R4 to R7, L2, L5, X1 to X3, Y1 to Y3, Z1 to Z3, W1 to W3, M, n, m, s, t, and u described above.
[6]
The compound according to [5], in which the compound is represented by General Formula (V),
in the formula, R6A and R7A represent a hydrogen atom, a hydroxy group, a sulfanyl group, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, an alkoxy group, a heteroaryl group, an amino group, an acyl group, an anionic group, a cationic group, or Q, provided that at least one of R6A or R7A represents Q,
L8 and L9 represent a linking group,
provided that L9 in a case where R6A is Q is a linking group in which the number of shortest-distance atoms that connect >NY4 to R6A is 3 or more, and L8 in a case where R7A is Q is a linking group in which the number of shortest-distance atoms that connect >C═O to R7A is 3 or more, and
R4, R5, L2, L5, X1 to X3, Y1 to Y5, Z1 to Z3, W1 to W3, M, Q, n, m, s, t, and u respectively have the same meanings as R4, R5, L2, L5, X1 to X3, Y1 to Y5, Z1 to Z3, W1 to W3, M, Q, n, m, s, t, and u described above.
[7]
The compound according to any one of [4] to [6], in which at least one of the following condition (Z1), (Z2), or (Z3) is satisfied,
the condition (Z1): the s is an integer of 1 or more, and the Z1 is a group including an anionic group,
the condition (Z2): the t is an integer of 1 or more, and the Z2 is a group including an anionic group,
the condition (Z3): the u is an integer of 1 or more, and the Z3 is a group including an anionic group.
[8]
The compound according to any one of [1] to [7], in which the structure represented by General Formula (I) includes a structure in which X1 to X3 are >CR2R3.
[9]
The compound according to [8], in which in the structure in which X1 to X3 are >CR2R3, where the structure is included in the structure represented by General Formula (I), at least one R2 is —NR8R9, —OR10, or an anionic group.
[10]
The compound according to [9], in which in a structure in which X1 to X3 are >CR2R3 and at least one R2 is —NR8R9, —OR10, or an anionic group, where the structure is included in the structure represented by General Formula (I), at least one of R8, R9, or R10 is a group including an anionic group.
[11]
The compound according to [3], in which the compound is represented by General Formula (VI),
in the formula, X4 to X9 represent —O—, —S—, >NR101, or >CR102R113 provided that one of X4, X5, or X6 is >NR101 or >CR102R103, where R101 is -L10-M in a case where one of X4, X5, or X6 is >NR101 and R102 or R103 is -L10-M in a case where one of X4, X5, or X6 is >CR102R103,
one of X7, X8, or X9 is >NR101 or >CR102R103, where R11 is -L11-M in a case where one of X7, X8, or X9 is >NR101 and R102 or R103 is -L11-M in a case where one of X7, X8, or X9 is >CR102R103,
R101 to R103, which are neither -L10-M nor -L11-M, represent a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an acyl group, —NR8R9, —OR10, or an anionic group,
L10 and L11 represent a single bond or a divalent linking group,
n1 is an integer of 2 or more, and
R6 to R10, X1 to X3, M, and m respectively have the same meanings as R6 to R10, X1 to X3, M, and m described above.
[12]
The compound according to [11], in which the compound is represented by General Formula (VII),
in the formula, R6A and R7A represent a hydrogen atom, a hydroxy group, a sulfanyl group, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, an alkoxy group, a heteroaryl group, an amino group, an acyl group, an anionic group, a cationic group, or Q,
provided that at least one of R6A or R7A represents Q,
L12 and L13 represent a linking group,
na and nb are integers of 0 or more, and
L10, L11, X1 to X9, M, Q, n1, and m respectively have the same meanings as L10, L11, X1 to X9, M, Q, n1, and m described above.
[13]
The compound according to [12], in which (A) the na is an integer of 1 or more, and the R6A is the Q, and/or (B) the nb is an integer of 1 or more, and the R7A is the Q, provided that the number of shortest linking atoms of the L13 is 7 or less in a case of the (A), and the number of shortest linking atoms of the L12 is 7 or less in a case of the (B).
[14]
The compound according to any one of [11] to [13], in which the structure represented by General Formula (I) includes a structure in which X1 to X3 are >CR2R3.
[15]
The compound according to [14], in which in the structure in which X1 to X3 are >CR2R3, where the structure is included in the structure represented by General Formula (I), at least one R2 is —NR8R9, —OR10, or an anionic group.
[16]
The compound according to [15], in which in a structure in which X1 to X3 are >CR2R3 and at least one R2 is —NR8R9, —OR10, or an anionic group, where the structure is included in the structure represented by General Formula (I), at least one of R8, R9, or R10 is a group including an anionic group.
[17]
A labeled biological substance that is obtained by bonding the compound according to any one of [1] to [16] to a biological substance.
[18]
The labeled biological substance according to [17], in which the biological substance is any one of a protein, an amino acid, a nucleic acid, a nucleotide, a sugar chain, or a phospholipid.
The compound according to the aspect of the present invention makes it possible to obtain a labeled biological substance exhibiting an excellent fluorescence intensity in states of being in a solution and a membrane or a state of being in a stained cell. In addition, the labeled biological substance according to the aspect of the present invention exhibits an excellent fluorescence intensity.
In the present invention, in a case where there is a plurality of substituents, linking groups, structural units, or the like (hereinafter, referred to as substituents or the like), which are represented by a specific symbol or Formula, or in a case where a plurality of substituents or the like are regulated at the same time, the substituents or the like may be the same or different from each other, unless otherwise specified. The same applies to the regulation of the number of substituents or the like. In addition, in a case where a plurality of substituents or the like come close to each other (particularly in a case where they are adjacent to each other), they may be linked to each other to form a ring, unless otherwise specified. In addition, unless otherwise specified, rings such as an alicyclic ring, an aromatic ring, and a heterocyclic ring may be fused to form a fused ring.
For example, in the present invention, a structure represented by General Formula (I) described below means that n (n is an integer of 2 or more) pieces of structures represented by General Formula (i) are connected. In this case, the n pieces of structures represented by General Formulae (i) may be the same or different from each other. It is noted that X1 to X3 in General Formula (i) respectively have the same meanings as X1 to X3 in General Formula (I) described later. The same applies to a structure parenthesized in ( )s, a structure parenthesized in ( )t, a structure parenthesized in ( )u, a structure parenthesized in ( )m, a structure parenthesized in ( )n1, a structure parenthesized in ( )na, and a structure parenthesized in ( )nb, where s pieces of structures may be the same or different from each other, t pieces of structures may be the same or different from each other, u pieces of structures may be the same or different from each other, m pieces of structures may be the same or different from each other, n1 pieces of structures may be the same or different from each other, na pieces of structures may be the same or different from each other, and nb pieces of structures may be the same or different from each other.
In the present invention, in a case where an E type and a Z type of a double bond are present in a molecule, any one of the E type or the Z type, or a mixture thereof may be used unless otherwise specified. In addition, unless otherwise specified, in a case where a chiral carbon atom or a chiral center is present in a compound, the steric conformation may be any of R or S in the R—S notation, and a mixture thereof may be may be allowed.
In the present invention, the denotation of a compound or substituent is meant to include not only the compound itself but also a salt thereof, and an ion thereof. For example, the dissociable anionic group such as the carboxy group, the sulfo group, and the phosphono group (—P(═O)(OH)2) may have an ionic structure by a hydrogen ion being dissociated therefrom, or they may have a salt structure. That is, in the present invention, the “carboxy group” is meant to include a group of an ion or salt of a carboxylic acid, the “sulfo group” is meant to include a group of an ion or salt of a sulfonic acid, and the “phosphono group” is meant to include a group of an ion or salt of a phosphonic acid. The monovalent or polyvalent cation in forming the salt structure is not particularly limited. Examples thereof include an inorganic cation and an organic cation, and specific examples thereof include alkali metal cations such as Na+, Li+, and K+, alkaline earth metal cations such as Mg2+, Ca2+, and Ba2+, and organic ammonium cations such as a trialkylammonium cation and a tetraalkylammonium cation.
In a case of a salt structure, the kind of the salt may be one kind, two or more kinds thereof may be mixed, a salt-type group and a group having a free acid structure may be mixed in a compound, and a compound having a salt structure and a compound having a free acid structure compound may be mixed.
Any compound according to the embodiment of the present invention is a neutral compound. In the present invention, the fact that the compound is neutral means that the compound is electrically neutral. Specifically, the charge of the compound as a whole is adjusted to be 0 by a group having a charge or by a counterion in the compound. For example, in the cyanine dye represented by General Formula (α), which is exemplified as an example of the dye that constitutes the phosphor moiety, the formal charge of the nitrogen atom to which R42 is bonded is +1, and the dissociable group such as a sulfo group in another structure of the cyanine dye or the compound according to the embodiment of the present invention has an ionic structure such as a sulfonate ion to be paired with this formal charge, whereby the compound according to the embodiment of the present invention is to be a compound having a charge of 0 as a whole.
In each general formula pertaining to the cyanine dye, which is defined in the present invention, the positive charge possessed by the compound is specified and indicated, for convenience, as a structure of a specific nitrogen atom. However, since the cyanine dye defined in the present invention has a conjugated system, another atom other than the nitrogen atom actually may be capable of being positively charged, and thus any cyanine dye capable of adopting a structure represented by each general formula as one of the chemical structures is included in the cyanine dye represented by each general formula. This also applies to the negative charge.
In addition, it is meant to include those in which a part of the structure is changed within the scope that does not impair the effect of the present invention. Further, it is meant that a compound, which is not specified to be substituted or unsubstituted, may have any substituent within the scope that does not impair the effect of the present invention. The same applies to a substituent (for example, a group represented by “alkyl group”, “methyl group”, “methyl”) and a linking group (for example, a group represented by “alkylene group”, “methylene group”, “methylene”). Among such substituents, a preferred substituent in the present invention is a substituent selected from a substituent group T described later.
In the present invention, in a case where the number of carbon atoms of a certain group is specified, this number of carbon atoms means the number of carbon atoms of the entire group thereof unless otherwise specified in the present invention or the present specification. That is, in a case where this group has a form that further has a substituent, it means the total number of carbon atoms, to which the number of carbon atoms of this substituent is included.
In addition, in the present invention, the numerical range represented by using “to” means a range including the numerical values before and after “to” as the lower limit value and the upper limit value, respectively.
The compound according to the embodiment of the present invention is a compound containing two or more phosphor moieties of which light absorption characteristics are equivalent to each other, where it is a compound in which the phosphor moieties adjacent to each other are each linked through a group having a structure represented by General Formula (I). Although the details of the reason why the compound according to the embodiment of the present invention makes it possible to obtain a labeled biological substance exhibiting an excellent fluorescence intensity in both states of being in a solution and a membrane or in a state of being in a stained cell are not clear, they can be conceived as follows.
The compound according to the embodiment of the present invention is a compound containing two or more phosphor moieties of which light absorption characteristics are equivalent to each other, where the compound has a structure in which the phosphor moieties adjacent to each other are each linked through a group having a structure represented by General Formula (I). The structure represented by General Formula (I) has a repeating unit (the repetition number is 2 or more) including a nitrogen-containing saturated 5-membered ring such as a ring structure derived from proline and functions as a linker that is rigid with respect to the two phosphor moieties linked by a group including a structure represented by General Formula (I), and thus it is possible to effectively suppress the quenching due to the intramolecular or intermolecular association of the phosphor moieties. As a result, it is conceived that a labeled biological substance obtained by using the compound according to the embodiment of the present invention can exhibit an excellent fluorescence intensity.
Hereinafter, the compound according to the embodiment of the present invention will be described in detail.
<Compound According to Embodiment of Present Invention>
The compound according to the embodiment of the present invention is a compound containing two or more phosphor moieties of which light absorption characteristics are equivalent to each other, where it is a compound in which the phosphor moieties adjacent to each other are each linked through a group having a structure represented by General Formula (I).
The compound according to the embodiment of the present invention may be a compound classified into a polymer or an oligomer.
In the present invention, the phrase “phosphor moieties of which light absorption characteristics are equivalent to each other” means those that satisfy a relationship in which a difference in the maximum absorption wavelength between the respective phosphor moieties in the absorption spectra is within 15 nm.
In the present invention, the compound is preferably such that all the phosphor moieties contained in the compound satisfy a relationship in which a difference between the maximum absorption wavelength on the lowest wavelength side and the maximum absorption wavelength on the highest wavelength side among the maximum absorption wavelengths in the absorption spectra of the respective phosphor moieties is within 15 nm.
As a compound having two phosphor moieties in the compound, the compound that causes the FRET phenomenon is known as described above. In this compound, a difference in maximum absorption wavelength generally exceeds 15 nm between an absorption spectrum of a phosphor moiety I (an energy donor) that is excited with excitation light and an absorption spectrum of another phosphor moiety II (an energy acceptor) that receives energy from the phosphor moiety I and emits light or is quenched. In such a compound, the energy is transferred to the phosphor moiety II instead of emitting fluorescence from the phosphor moiety I, and thus the fluorescence intensity of the compound is decreased.
On the other hand, as described above, the compound according to the embodiment of the present invention has phosphor moieties of which light absorption characteristics are equivalent to each other, and thus the FRET phenomenon does not occur, and it is possible to exhibit a fluorescence intensity proportional to the number of phosphor moieties.
The chemical structures of the above-described phosphor moieties of which the light absorption characteristics are equivalent to each other are not particularly limited as long as the above-described difference in maximum absorption wavelength is satisfied, and they preferably have the same structure in terms of the main skeleton of the phosphor moiety. However, the steric conformation, chain length, and the like of the substituent may be different from each other, and in a case where an anionic group or a cationic group is contained, a counter ion thereof may be different from each other. The difference in the maximum absorption wavelength is preferably within 10 nm and more preferably within 5 nm.
It is noted that the absorption spectrum of the phosphor moiety is a spectrum that is obtained by measuring, by using a spectrophotometer, a simple body of a phosphor constituting the phosphor moiety, which is diluted with a PBS buffer solution (phosphate-buffered saline).
In addition, it is preferable that “the compound in which phosphor moieties adjacent to each other are each linked through a group having a structure represented by General Formula (I)” is a compound in which two structures having a phosphor moiety as a substituent are linked through a group including a structure represented by General Formula (I).
It is noted that the structure having the above-described phosphor moiety as a substituent is not particularly limited as long as the phosphor moieties adjacent to each other are each linked through a group having a structure represented by General Formula (I). For example, the structure may have, as a substituent, a group in which a 5-membered ring contains a phosphor moiety, where the 5-membered ring described in the structure represented by General Formula (I) is formed to have a carbon atom, a nitrogen atom, and X1 to X3 as ring-constituting atoms by a combination of L1 and L2, a combination of L1 and L3, a combination of L4 and L5, or a combination of L4 and L6, as described in detail in General Formula (II) described later. That is, the structure having a phosphor moiety as a substituent may be a structure in which the 5-membered ring described in the structure represented by General Formula (I), which has a carbon atom, a nitrogen atom, and X1 to X3 as ring-constituting atoms, has a group in which a phosphor moiety is contained as a substituent as long as the phosphor moieties adjacent to each other are each linked through a group having a structure represented by General Formula (I). Examples of such a compound include a compound represented by General Formula (VI) or (VII) described later.
In the compound according to the embodiment of the present invention, the number of the phosphor moieties is 2 or more. The upper limit value thereof is not particularly limited and can be set to, for example, 30 or less, and it is preferably 20 or less and more preferably 15 or less.
In addition to the above, the description, the specific examples, and the like of the phosphor moiety in General Formula (II) described later can be applied to the phosphor moiety contained in the compound according to the embodiment of the present invention.
(Structure Represented by General Formula (I))
In the formula, X1 to X3 represent —O—, —S—, >NR1, or >CR2R3.
R1 to R3 represent a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an acyl group, —NR8R9, —OR10, or an anionic group.
R8 to R10 represent a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an acyl group, an aryl group, a heteroaryl group, or an anionic group.
n is an integer of 2 or more.
* represents a bonding site.
In the present invention, the structure represented by General Formula (I) is preferably a structure represented by any one of General Formula (IA) or (IB) in consideration of stereoisomers. It is noted that X1 to X3 and n in the following general formula respectively have the same meanings as X1 to X3 and n in General Formula (I).
X1 to X3 represent —O—, —S—, >NR1, or >CR2R3, and R1 to R3 represent a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an acyl group, an aryl group, a heteroaryl group, —NR8R9, —OR10, or an anionic group.
The alkyl group, the alkenyl group, the alkynyl group, the acyl group, the aryl group, and the heteroaryl group, which can be adopted as R1 to R3, respectively have the same meanings as the alkyl group, the alkenyl group, the alkynyl group, the acyl group, the aryl group, and the heteroaryl group in the substituent group T which will be described later, and the same also applies to the preferred ranges thereof.
The alkyl group, the alkenyl group, the alkynyl group, the acyl group, the aryl group, and the heteroaryl group, which can be adopted as R1 to R3, may be unsubstituted or may have a substituent.
Examples of the substituent which may be contained in the alkyl group, the alkenyl group, the alkynyl group, the acyl group, the aryl group, and the heteroaryl group, as R1 to R3, include substituents in the substituent group T which will be described later. For example, a halogen atom or an anionic group is preferable.
In —NR8R9 and —OR10, which can be adopted as R1 to R3, R8 to R10, represent a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an acyl group, an aryl group, a heteroaryl group, or an anionic group.
The alkyl group, the alkenyl group, the alkynyl group, the acyl group, the aryl group, and the heteroaryl group, which can be adopted as R8 to R10, respectively have the same meanings as the alkyl group, the alkenyl group, the alkynyl group, the acyl group, the aryl group, and the heteroaryl group in the substituent group T which will be described later, and the same also applies to the preferred ranges thereof.
The alkyl group, the alkenyl group, the alkynyl group, the acyl group, the aryl group, the heteroaryl group, which can be adopted as R8 to R10, may be unsubstituted or may have a substituent.
Examples of the substituent which may be contained in the alkyl group, the alkenyl group, the alkynyl group, the acyl group, the aryl group, and the heteroaryl group, as R8 to R10, include substituents in the substituent group T which will be described later. For example, a halogen atom, a carbamoyl group, an acylamino group, an alkoxy group (preferably, an alkoxy group having an anionic group), or an anionic group is preferable, and a carbamoyl group, an acylamino group, an alkoxy group (preferably, an alkoxy group having an anionic group), or an anionic group is more preferable.
R1 is preferably a hydrogen atom, an alkyl group, —NR8R9, —OR10, or an anionic group.
R2 and R3 are preferably a hydrogen atom, —NR8R9, —OR10, or an anionic group, where it is more preferable that R2 is a hydrogen atom, —NR8R9, —OR10, or an anionic group, and R3 is a hydrogen atom.
R8 to R10 are preferably a hydrogen atom, an alkyl group, or an acyl group. The alkyl group may be an alkyl group having an anionic group, and the acyl group may be an acyl group having an anionic group (an acyl group having an anionic group or an acyl group substituted with an alkoxy group having an anionic group,). The above-described alkyl group and acyl group may have a substituent other than the anionic group, and examples thereof include substituents in the substituent group T which will be described later. For example, a carbamoyl group or an acylamino group is preferable.
Regarding X1 to X3, it is preferable that at least any one thereof is >CR2R3, and it is more preferable that at least two thereof are >CR2R3 and the remaining one is —O—, —S—, or >CR2R3.
In the present invention, from the viewpoint of making the structure represented by General Formula (I) a more rigid structure, it is preferable that the structure represented by General Formula (I) includes a structure in which X1 to X3 are >CR2R3. That is, “the structure represented by General Formula (I) includes a structure in which X1 to X3 are >CR2R3” means that all of X1 to X3 are >CR2R3 in at least one structure represented by General Formula (i) among n pieces of the consecutive structures represented by General Formula (i) described above.
The proportion of the number of the structures in which X1 to X3 are >CR2R3 among the structures represented by General Formula (I) is preferably 30% or more, more preferably 60% or more, and still more preferably 80%. The upper limit of the ratio is not particularly limited and may be 100% or less. It is noted that it is also preferable that all of the structures represented by General Formula (I) are structures in which X1 to X3 described above are >CR2R3.
From the viewpoint of suppressing the intermolecular interaction and furthermore, the intramolecular interaction between structures represented by General Formula (I) in a case where a plurality of structures are present, at least one R2 in the above-described structure in which X1 to X3 is >CR2R3 is preferably —NR8R9, —OR10, or an anionic group, and it is more preferably —NR8R9, —OR10, or an anionic group, where at least one of R8, . . . , or R10 is a group including an anionic group.
It is noted that the phrase “at least one R2 in structure in which X1 to X3 are >CR2R3 is —NR8R9, —OR10, or an anionic group, where at least one of R8, . . . , or R10 is a group including an anionic group” means that, in other words, at least one of R8 or R9 is a group including an anionic group in a case where R2 is —NR8R9, and R10 is a group including an anionic group in a case where R2 is —OR10.
In addition, the description that R10 is a group including an anionic group means that R10 is an anionic group or is a group having an anionic group as a substituent. The same applies to a case where at least one of R8 or R9 is a group including an anionic group.
n is an integer of 2 or more. Since n is an integer of 2 or more, the compound according to the embodiment of the present invention can impart the rigidity that is effective for suppressing the association of dyes (phosphor moieties), to a structure represented by General Formula (I), and a decrease in the fluorescence intensity can be suppressed.
In the present invention, in a case where the number of phosphor moieties is 2, the lower limit value of n is preferably an integer of 3 or more, more preferably an integer of 7 or more, still more preferably an integer of 9 or more, and particularly preferably an integer of 12 or more, from the viewpoint of further improving the fluorescence intensity. The upper limit value thereof is not particularly limited and can be set to, for example, an integer of 72 or less, and it is preferably an integer of 36 or less, more preferably an integer of 24 or less, and still more preferably an integer of 18 or less.
On the other hand, in a case where the number of phosphor moieties is 3 or more, a certain degree of association of dyes can be allowed since the number of phosphor moieties (the number of dyes) per one molecule is large as compared with a case where the number of phosphor moieties is 2, and in a case where the number of phosphor moieties is an integer of 3 or more, the fluorescence intensity can be further improved, which is preferable. Among the above, in a case where n is an integer of 6, there is a certain degree of effect of suppressing the association, which is insufficient. Therefore, n is more preferably an integer of 7 or more. In a case where the number of phosphor moieties is 3 or more, the upper limit value of n is, for example, preferably an integer of 72 or less, more preferably an integer of 36 or less, and still more preferably an integer of 24 or less.
<Compound Represented by General Formula (II)>
The compound according to the embodiment of the present invention is preferably represented by General Formula (II).
In the formula, R4 and R5 represent a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an acyl group, an amino group, a hydroxy group, an alkoxy group, a sulfanyl group, an aryl group, or a heteroaryl group.
R6 and R7 represent a hydrogen atom, a hydroxy group, a sulfanyl group, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, an alkoxy group, an amino group, an acyl group, a heteroaryl group, an anionic group, a cationic group, or Q.
Q represents a carboxy group, a substituent capable of being bonded to a biological substance, or a substituent capable of being bonded to a solid support.
L1 to L7 represents a single bond or a divalent linking group.
M represents a phosphor moiety, a physiologically active substance moiety, a prodrug moiety, or a radioactive isotope-containing moiety.
Y represents the structure represented by General Formula (I) described above.
m is an integer of 1 or more.
However, at least two of M's represent the above-described phosphor moieties of which the light absorption characteristics are equivalent to each other. In other words, this means that at least two of M's are phosphor moieties, where individual phosphor moieties adjacent to each other are the above-described phosphor moieties of which the light absorption characteristics are equivalent to each other.
R4 and R5 represent a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an acyl group, an amino group, a hydroxy group, an alkoxy group, a sulfanyl group, an aryl group, or a heteroaryl group.
The alkyl group, the alkenyl group, the alkynyl group, the acyl group, the amino group, the alkoxy group, the aryl group, and the heteroaryl group, which can be adopted as R4 or R5, respectively have the same meanings as the alkyl group, the alkenyl group, the alkynyl group, the acyl group, the amino group, the alkoxy group, the aryl group, and the heteroaryl group in the substituent group T which will be described later, and the same also applies to the preferred ranges thereof.
The alkyl group, the alkenyl group, the alkynyl group, the acyl group, the amino group, the alkoxy group, the aryl group, and the heteroaryl group, which can be adopted as R4 or R5, may be unsubstituted or may have a substituent, and examples of the substituent which may be contained therein include substituents in the substituent group T which will be described later. For example, a halogen atom is preferable.
From the viewpoint of ease of synthesis, R4 and R5 are preferably a hydrogen atom. R4 and R5 are often a hydrogen atom in a case where an amino acid is used as a raw material; however, the substituents in R4 and R5 do not greatly contribute to the exhibition of the excellent fluorescence intensity by the compound according to the embodiment of the present invention, and thus R4 and R5 may be another substituent (an alkyl group, an alkenyl group, an alkynyl group, an acyl group, an amino group, a hydroxy group, an alkoxy group, a sulfanyl group, an aryl group, or a heteroaryl group) other than the hydrogen atom.
R6 and R7 represent a hydrogen atom, a hydroxy group, a sulfanyl group, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, an alkoxy group, an amino group, an acyl group, a heteroaryl group, an anionic group, a cationic group, or Q.
The alkyl group, the alkenyl group, the alkynyl group, the aryl group, the alkoxy group, the amino group, the acyl group, the heteroaryl group, the anionic group, and the cationic group, which can be adopted as R6 or R7, respectively have the same meanings as the alkyl group, the alkenyl group, the alkynyl group, the aryl group, the alkoxy group, the amino group, the acyl group, the heteroaryl group, the anionic group, and the cationic group in the substituent group T which will be described later, and the same also applies to the preferred ranges thereof.
The alkyl group, the alkenyl group, the alkynyl group, the aryl group, the alkoxy group, the amino group, the acyl group, and the heteroaryl group, which can be adopted as R6 or R7, may be unsubstituted or may have a substituent.
Examples of the substituent which may be contained in the alkyl group, the alkenyl group, the alkynyl group, the aryl group, the alkoxy group, the amino group, the acyl group, and the heteroaryl group, as R6 or R7, include substituents in the substituent group T which will be described later, where an alkyl group, an acyl group, an alkoxy group, an amino group, an anionic group, a cationic group, -(L-O)gRE, or Q, or a substituent obtained by combining two or more thereof is preferable, and an alkyl group, an acyl group, an alkoxy group, an amino group, -(L-O)gRE, or Q, or a substituent obtained by combining two or more of these substituents is more preferable.
Q, which can be adopted as R6 or R7, represents a carboxy group, a substituent capable of being bonded to a biological substance, or a substituent capable of being bonded to a solid support.
As the substituent capable of being bonded to a biological substance, the description of the substituent capable of being bonded to a biological substance described later can be applied, and as the substituent capable of being bonded to a solid support, the description of the substituent capable of being bonded to a solid support described later can be applied.
For R6, the description of the substituent represented by -L9R6A or -L13R6A described later is preferably described, and For R7, the description of the substituent represented by -L8R7A or -L12R7A described later is preferably described.
In the present invention, from the viewpoint that a proper hydrophilicity and a proper excluded volume effect can be provided and an excellent fluorescence intensity can be obtained, it is preferable that any one of R6 or R7 preferably includes a structure represented by -(L-O)gRE, and it is more preferable that R7 includes a structure represented by -(L-O)gRE.
L2 to L5 and L7 represent a single bond or a divalent linking group and are preferably a single bond or a linking group obtained by combining one or two or more of an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, —O—, —S—, >C═O, >NRA, >S═O, >S(═O)2, and >P(═O)ORB. RA and RB respectively have the same meanings as RA and RB described later and represent a hydrogen atom or a substituent.
L1 and L6 represent a single bond or a divalent linking group and are preferably a single bond, an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, —O—, —S—, >C═O, >NRA, >S═O, >S(═O)2, or >P(═O)ORB. RA and RB respectively have the same meanings as RA and RB described later and represent a hydrogen atom or a substituent.
The alkylene group that can constitute L1 to L7 has the same meaning as the group in which one hydrogen atom is further removed from the alkyl group selected from the substituent group T, which is described later, and the same applies to the preferred one thereof.
The alkenylene group that can constitute L1 to L7 has the same meaning as the group in which one hydrogen atom is further removed from the alkenyl group selected from the substituent group T, which is described later, and the same applies to the preferred one thereof.
The alkenylene group that can constitute L1 to L7 has the same meaning as the group in which one hydrogen atom is further removed from the alkynyl group selected from the substituent group T, which is described later, and the same applies to the preferred one thereof.
The arylene group that can constitute L1 to L7 has the same meaning as the group in which one hydrogen atom is further removed from the aryl group selected from the substituent group T, which is described later, and the same applies to the preferred one thereof.
The heteroarylene group that can constitute L1 to L7 is synonymous with the group in which one hydrogen atom is further removed from the heteroaryl group selected from the substituent group T, which is described later, and the same applies to the preferred one thereof.
The alkylene group, the alkenylene group, the alkynylene group, the arylene group, and the heteroarylene group, which can constitute L1 to L7, may be an unsubstituted group or a group having a substituent.
The substituent which may be contained in the alkylene group, the alkenylene group, the alkynylene group, the arylene group, and the heteroarylene group, which can constitute L1 to L7, is not particularly limited, and it is preferably selected from a substituent group T described later. More preferred examples thereof include a halogen atom, an alkyl group, an acylamino group, and a carbamoyl group. In addition, the substituent which may be contained in the alkylene group, the alkenylene group, the alkynylene group, the arylene group, and the heteroarylene group, which can constitute L1 to L7, may be substituted with a substituent selected from the substituent group T which will be described later, and it is, for example, preferably an amino group.
In addition, the number of substituents which may be contained in the alkylene group, the alkenylene group, the alkynylene group, the arylene group, and the heteroarylene group, which can constitute L1 to L7, is not particularly limited as long as it can be adopted as a structure. The number thereof can be set to at least one or more, the upper limit thereof is not particularly limited, and for example, all hydrogen atoms in the alkylene group, the alkenylene group, the alkynylene group, the arylene group, and the heteroarylene group may be substituted with a substituent.
The substituent which can be adopted as RA in >NRA and as RB in >P(═O)ORB, which can constitute L1 to L7, is not particularly limited, and it is preferably selected from the substituent group T which will be described later. RA is preferably a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or an anionic group, more preferably a hydrogen atom or an alkyl group, and still more preferably a hydrogen atom. RB is preferably a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, or a heteroaryl group, more preferably a hydrogen atom or an alkyl group, and still more preferably a hydrogen atom.
It is noted that all the alkyl group, the alkenyl group, the alkynyl group, the aryl group, and the heteroaryl group, which can be adopted as RA and RB, may be an unsubstituted group or a group having a substituent.
In the linking group obtained by combining two or more of an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, —O—, —S—, >C═O, >NRA, >S═O, >S(═O)2, and >P(═O)ORB, which can constitute L2 to L5 and L7, the kind of the group to be combined is not particularly limited as long as the group to be combined has a reasonable chemical structure; however, it is preferably 2 to 6 kinds and more preferably 2 to 4 kinds. It is noted that in a case of counting, as one kind, each of the alkylene group, the alkenylene group, the alkynylene group, the arylene group, the heteroarylene group, —O—, —S—, >C═O, >NRA, >S═O, >S(═O)2, and >P(═O)ORB, the maximum number of kinds is 12.
It is noted that in the linking group obtained by combining two or more of an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, —O—, —S—, >C═O, >NRA, >S═O, >S(═O)2, and >P(═O)ORB, which can constitute L2 to L5 and L7, the number of groups to be combined is not particularly limited; however, examples thereof preferably include 2 to 10 groups, more preferably include 2 to 6 groups, and still more preferably 2 to 4 groups.
(i) L1 and L6
L1 is more preferably >C═O, >NRA, an arylene group, an alkylene group, —O—, or still more preferably >C═O, >NRA, an arylene group, or an alkylene group, and particularly preferably >C═O or >NRA.
L6 is more preferably >C═O, >NRA, or an arylene group and still more preferably >C═O or >NRA.
(ii) L3, L4, and L7
L3, L4, and L7 are more preferably a single bond, >C═O, >NRA, an alkylene group, an alkenylene group, an alkynylene group, an arylene group, or a heteroarylene group, or a combination of at least one of an alkylene group, an alkenylene group, an alkynylene group, an arylene group, or a heteroarylene group, and >C═O and >NRA, and still more preferably a single bond, >C═O, >NRA, an alkylene group, an alkenylene group, an alkynylene group, an arylene group, or a heteroarylene group, or a group that links —C(═O)NRA— to >C═O, or >NRAC(═O)— to >NRA by at least one of an alkylene group, an alkenylene group, an alkynylene group, an arylene group, or a heteroarylene group.
(iii) L2 and L5
L2 and L5 are more preferably a group obtained by combining one or two or more of an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, —O—, —S—, >C═O, and >NRA, and are still more preferably a group represented by *-Lx-Ly-**.
Lx is a single bond or a group obtained by combining one or two or more of an alkylene group, an alkenylene group, an alkynylene group, an arylene group, and a heteroarylene group, and LU is a single bond, —O, —, —S—, >C═O, or >NRA. * represents a bonding site to a carbon atom to which L1 and L3 are bonded or a carbon atom to which L4 and L6 are bonded, and ** represents a bonding site to M. However, in a case where LU is a single bond, U is a heteroarylene group or a group consisting of a group obtained by combining one or two or more of an alkylene group, an alkenylene group, an alkynylene group, and an arylene group, which are located on the * side, and a heteroarylene group which is located on the ** side.
Among the groups represented by * -Lx-Ly-**, L2 and L5 are preferably a group in which L is a single bond, —S—, >C═O, or >NRA, more preferably a group in which Ly is >C═O or >NRA, and still more preferably a group in which U is an alkylene group and Ly is >C═O or >NRA.
It is noted that in the compound represented by General Formula (II), it suffices that the number of shortest-distance atoms in the linking chain (each linking chain of the linking chain including L2 and the linking chain including L5) that connects M to a carbon atom to which L1 and L3 are bonded or a carbon atom to which L4 and L6 are bonded is, for example, 1 to 60, where 1 to 40 is preferable. In a case where M is a phosphor moiety, the above-described number of shortest-distance atoms means the number of atoms that constitute the shortest chain in the linking chain that connects a conjugated structural moiety for exhibiting fluorescence in the phosphor moiety M to a carbon atom to which L1 and L3 are bonded or a carbon atom to which L4 and L6 are bonded.
It is noted that in the compound represented by General Formula (II), it is also preferable that a structure represented by —(CH2—CH2—O)b— described later (b is also as described later) is provided at any portion of the linking chain represented by “-linking group ZZZ-L2-” in the structure represented by “the conjugated structural moiety of M—the linking group ZZZ-L2-described later” and any portion of the linking chain represented by “-linking group ZZZ-L5-” in the structure represented by “the conjugated structural moiety of M—the linking group ZZZ-L5-described later”.
In the compound represented by General Formula (II), adjacent groups may be bonded to each other to form a ring. Examples of the combination of adjacent groups which may be bonded to each other to form a ring include a combination of L1 and L2, a combination of L1 and L3, a combination of L2 and R4, a combination of L4 and L5, a combination of L4 and L6, or a combination of L5 and R5.
The above-described ring which may be formed by bonding adjacent groups to each other may be any one of an aromatic ring or an aliphatic ring or may be any one of a hydrocarbon ring or a hetero ring, and it is preferably a 5- or 6-membered ring.
The preferred examples of the aliphatic ring include a cyclopentane ring, a cyclohexane ring, or a 5-membered ring having a carbon atom, a nitrogen atom, and X1 to X3 as ring-constituting atoms, which is described in the structure represented by General Formula (I) described above, where a 5-membered ring having a carbon atom, a nitrogen atom, and X1 to X3 as ring-constituting atoms, which is described in the structure represented by General Formula (I) described above, is more preferable.
The aromatic ring is preferably a benzene ring or a nitrogen-containing aromatic heterocyclic ring, more preferably a benzene ring or a nitrogen-containing aromatic heterocyclic ring in which the ring-constituting atom is composed of a carbon atom and a nitrogen atom, and still more preferably a benzene ring or a pyridine ring.
These rings may have a substituent, and the substituent which may be contained is not particularly limited and is selected from the substituent group T.
The ring formed by the combination of L2 and R4 or the combination of L5 and R5 may be any one of the above-described aliphatic ring or aromatic ring, where the above-described aliphatic ring is preferable.
The ring formed by a combination of L1 and L2, a combination of L1 and L3, a combination of L4 and L5, or a combination of L4 and L6, may be any one of the above-described aliphatic ring or aromatic ring, where a 5-membered ring having a carbon atom, a nitrogen atom, and X1 to X3 as ring-constituting atoms, which is described in the structure represented by General Formula (I) described above, or a benzene ring is preferable.
For example, the following structures surrounded by broken lines in General Formula (II)
can be set to structures including the following structures. In the following structures, * indicates a connecting portion.
m is an integer of 1 or more. The upper limit value thereof is not particularly limited and can be set to, for example, an integer of 30 or less, and it is preferably an integer of 20 or less, more preferably an integer of 15 or less, and still more preferably an integer of 10 or less.
M represents a phosphor moiety, a physiologically active substance moiety, a prodrug moiety, or a radioactive isotope-containing moiety. However, at least two of M's represent phosphor moieties of which the light absorption characteristics are equivalent to each other.
The phosphor moiety which can be adopted as M (hereinafter, also referred to as the phosphor moiety M) can be used without particular limitation as long as it is a structural moiety consisting of an organic compound that exhibits fluorescence. In addition, the phosphor moiety M may be a structural moiety in which a structural moiety consisting of an organic compound exhibiting fluorescence further has a linking group. Such a linking group is not particularly limited; however, examples thereof include a linking group ZZZ described later. For example, in the compound represented by General Formula (II), preferred examples of the compound include a compound in which the phosphor moiety M is bonded to L2 or L5 by this linking group ZZZ.
Examples of the phosphor moiety M include a structural moiety consisting of at least one dye of a xanthene dye, a rhodamine dye, a coumarin dye, a cyanine dye, a pyrene dye, an oxazine dye, a squarylium dye, a pyridyloxazole dye, or a pyrromethene dye, which is preferable.
As the xanthene dye, the rhodamine dye, the coumarin dye, the cyanine dye, the pyrene dye, the oxazine dye, the squarylium dye, the pyridyloxazole dye, and the pyrromethene dye, dyes that are generally known as these dyes can be used without particular limitation.
As one aspect, the phosphor moiety M is preferably a structural moiety consisting of a pyrromethene dye. Examples of the pyrromethene dye include a dipyrromethene boron complex. As the dipyrromethene boron complex, it is possible to use a fluorescent compound (a dipyrromethene boron complex) represented by General Formula (1) or (4) described in WO2019/230963A, or a compound (a dipyrromethene boron complex) represented by General Formula (1) described in WO2021/100814A, and the description thereof can be incorporated into the present specification by reference.
It is noted that the incorporation is made such that the dye that constitutes the phosphor moiety M does not have a substituent capable of being bonded to a biological substance.
As another aspect, the phosphor moiety M is preferably a structural moiety consisting of a cyanine dye, and it is more preferably a structural moiety consisting of a cyanine dye represented by General Formula (α).
In the formula, R1 to R4 represent an alkyl group or —(CH2—CH2—O)b—R21. b is 1 to 50, and R21 represents an alkyl group.
R11 to R13 represent a hydrogen atom, an alkyl group, an alkoxy group, an aryloxy group, an alkylthio group, an arylthio group, an amino group, or a halogen atom, where adjacent groups may be bonded to each other to form a 5-membered or 6-membered ring.
R22 to R25 and R32 to R35 represent a hydrogen atom, an alkyl group, an alkoxy group, an aryl group, a sulfo group, a sulfamoyl group, a carboxy group, an alkoxycarbonyl group, an aryloxycarbonyl group, an acyloxy group, a carbamoyl group, an acylamino group, a nitro group, or a halogen atom.
R41 and R42 represent an alkyl group or —(CH2—CH2—O)b—R21. R21 and b respectively have the same meanings as R21 and b described above. R41 and R42 may be bonded to each other to form a ring.
a is an integer of 1 to 3.
In a case where one hydrogen atom is removed from any of R1 to R4, R11 to R13, R22 to
R25, R32 to R35, R41, or R42 described above, a monovalent structural moiety is provided.
However, the cyanine dye represented by Formula (α) is neutral.
Depending on the length of the methine chain having a repetition number of 2a+3, which is connected by a conjugated double bond, the cyanine dyes represented by General Formula (α) respectively have an excitation absorption wavelength in a wavelength range of 520 to 600 nm (in the vicinity of 585 nm) in a case of a=1, in a wavelength range of 620 to 700 nm (in the vicinity of 685 nm) in a case of a=2, and in a wavelength range of 740 to 830 nm (in the vicinity of 785 nm) in a case of a=3. As a result, each of the compounds according to the embodiment of the present invention which has, as the phosphor moiety M, a structural moiety consisting of a cyanine dye represented by General Formula (α) can be used as a compound exhibiting an excellent fluorescence intensity, in the fluorescence labeling in which a light source having any wavelength in a wavelength range of about 500 to 800 nm (for example, in the vicinity of 600 nm, in the vicinity of 700 nm, or in the vicinity of 800 nm) matching with the absorption excitation wavelength of the compound is used as an excitation light source.
In multicolor WB, a plurality of luminescence colors are detected in the range from the visible range to the near infrared range. As a result, it is necessary to select wavelengths so that the absorption and luminescence waveforms of a plurality of dyes have a suitable wavelength relationship so that crosstalk does not occur due to mutual interference in a case where the dyes are excited to emit light. Ideally, it should be adjusted so that only one dye emits light at one excitation light and the other dyes do not emit light. From this point of view, two kinds of excitation light sources having wavelengths separated to some extent, for example, in the vicinity of 700 nm and in the vicinity of 800 nm, are used for luminescence in the near infrared range of the multicolor WB.
As compared with the detection by visible light excitation, the fluorescence detection by near-infrared light excitation can suppress the autofluorescence of the membrane, that is, the background fluorescence, and thus it is easy to increase the signal to noise ratio (the S/N ratio) and it is possible to detect a target protein with high sensitivity. As a result, in recent years, there has been an increasing need for fluorescence detection WB using luminescence in the near infrared range in the analytical research on the trace amount of proteins.
However, in the near infrared range, the fluorescence quantum yield of the fluorescent dye is generally low, and thus it is difficult to obtain a high signal amount. Among the compounds according to the embodiment of the present invention which has, as the phosphor moiety M, a structural moiety consisting of a cyanine dye represented by General Formula (α), the compound in which a=2 or 3 can be used as a compound that exhibits an excellent fluorescence intensity even in the multicolor WB having the above-described two kinds of excitation light sources in the vicinity of 700 nm and in the vicinity of 800 nm, and in particular, it can exhibit an excellent fluorescence intensity even with respect to a request for observing and detecting proteins with higher sensitivity, as compared with the fluorescence labeling using cyanine dyes in the related art.
(i) R1 to R4
R1 to R4 represent an alkyl group or —(CH2—CH2—O)b—R21.
The alkyl group which can be adopted as R1 to R4 has the same meaning as the alkyl group in the substituent group T which will be described later.
The unsubstituted alkyl group preferably has 1 to 6 carbon atoms, more preferably has 1 to 4 carbon atoms, and still more preferably has 1 or 2 carbon atoms.
In a case where the alkyl group has a substituent, the alkyl group moiety of the alkyl group having a substituent preferably has 1 to 10 carbon atoms, more preferably has 1 to 8 carbon atoms, still more preferably has 2 to 6 carbon atoms, and particularly preferably has 2 to 5 carbon atoms. In addition, the number of atoms that constitute the longest chain of the alkyl group having a substituent is preferably 3 to 35, more preferably 3 to 25, still more preferably 3 to 15, and particularly preferably 3 to 11.
In the present invention, the “number of carbon atoms of the alkyl group moiety of the alkyl group having a substituent” means the number of carbon atoms excluding the substituent moiety contained in the alkyl group.
In the present invention, the “number of atoms that constitute the longest chain of the alkyl group having a substituent” means the number of atoms including the substituent moiety (that is, the number of atoms obtained by subtracting the number of atoms of the molecular chain that does not constitute the longest chain, from the number of total atoms). It is noted that in a case where a substituent having a dissociative hydrogen atom such as a sulfo group or a carboxy group constitutes the longest chain, the calculation is carried out including the hydrogen atom regardless of the presence or absence of dissociation. In addition, the number of atoms in the substituent moiety capable of being bonded to a biological substance described later is not included.
Examples of the substituent which may be contained in the alkyl group which can be adopted as R1 to R4 include an alkoxy group, a carboxy group, an alkoxycarbonyl group, an acyloxy group, a carbonyl group, an acylamino group, a sulfo group, a phosphono group, and —(CH2—CH2—O)b—R21, as well as a group consisting of a combination of these substituents.
The alkyl group having a substituent, which can be adopted as R1 to R4, is not particularly limited as long as it is the above-described alkyl group having a substituent.
The alkyl group which can be adopted as R1 to R4 is preferably an unsubstituted alkyl group.
(—(CH2—CH2—O)b—R21)
In —(CH2—CH2—O)b—R21 which can be adopted as R1 to R4, b is 1 to 50, and R21 represents an alkyl group.
b means an average repetition number (simply, also referred to as a repetition number), and it is preferably 1 to 24, more preferably 1 to 12, still more preferably 1 to 10, particularly preferably 4 to 10, and most preferably 4 to 8.
The average repetition number can be calculated from the average integrated value obtained by subjecting a compound to 1H-NMR measurement. The average repetition number defined in the present invention means a number that is obtained by rounding off the first decimal place of the average repetition number calculated according to the above method.
To the alkyl group as R21, the description of the alkyl group which can be adopted as R1 to R4 described above can be applied.
The —(CH2—CH2—O)b—R21 which can be adopted as R1 to R4 and —(CH2—CH2—O)—R21 which can be adopted as a substituent by an alkyl group as R1 to R4 are preferably an alkyl group of —(CH2—CH2—Ob-unsubstituted.
From the viewpoint of further improving the fluorescence intensity of the fluorescent dye itself, it is preferable that at least one of R1, . . . , or R4 includes a structure represented by —(CH2—CH2—O)b—, and it is more preferable at least one of R1 or R2 and at least one of R3 or R4 include a structure represented by —(CH2—CH2—O)b—.
It is still more preferable that the entire phosphor moiety M in the compound according to the embodiment of the present invention is a structural moiety consisting of a cyanine dye represented by General Formula (α), where at least one of R1 or R2 and at least one of R3 or R4 includes a structure represented by —(CH2—CH2—O)b—.
It is preferable that the structure represented by —(CH2—CH2—O)b— is introduced by employing —(CH2—CH2—O)b —R21 as R1 to R4.
The b in —(CH2—CH2—O)b— described above has the same meaning as the b in —(CH2—CH2—O)b—R21 described above.
Since the substituents of R1 to R4 protrude in a direction perpendicular to the cyanine dye skeleton (plane), it is presumed that in a case of including a structure represented by —(CH2—CH2—O)b— as this substituent, the fused ring portion is difficult to undergo the 71-71 interaction (the effect of suppressing the association is strengthened), and thus the decrease in the fluorescence intensity due to the association can be suppressed.
(ii) R11 to R13
R11 to R13 each independently represent a hydrogen atom, an alkyl group, an alkoxy group, an aryloxy group, an alkylthio group, an arylthio group, an amino group, or a halogen atom. Adjacent groups may be bonded to each other to form a 5- or 6-membered ring.
The alkyl group, the alkoxy group, the aryloxy group, the alkylthio group, the arylthio group, the amino group, and the halogen atom, which can be adopted as R11 to R13, respectively have the same meanings as the alkyl group, the alkoxy group, the aryloxy group, the alkylthio group, the arylthio group, the amino group, and the halogen atom in the substituent group T described later, and the same applies to the preferred ranges thereof.
Examples of the substituent which may be contained in the alkyl group, the alkoxy group, the aryloxy group, the alkylthio group, the arylthio group, and the amino group, as R11 to R13, include the substituents in the substituent group T described below.
Among R11 to R13, the 5- or 6-membered ring formed by bonding adjacent groups to each other may be either aromatic or aliphatic, and it is preferably aliphatic. In addition, it is preferable to form a 6-membered ring. The number of the above-described 5- or 6-membered rings in the compound is not particularly limited; however, it is preferably 1 or 2 and more preferably 1.
In a case of taking a case of a=3 as an example, preferred examples of the structure having a ring formed by bonding adjacent groups among R11 to R13 include the following structures. It is noted that in the following examples, R11 to R13 that do not form a ring structure are a hydrogen atom, and the ring structure is described as a structure that does not have a substituent, which are not limited thereto. It is noted that, hereinafter, the structure beyond the wavy line will be omitted.
R11 and R13 possessed by the carbon atom bonded to the indolenine ring are preferably a hydrogen atom.
R12, and R13 other than those described are preferably a hydrogen atom or an alkyl group.
Among R11 to R13, adjacent groups in R12 and R13 other than R11 and R13 possessed by the carbon atom bonded to the indolenine ring (that is, adjacent groups of R13 and R12 other than R13 possessed by the carbon atom bonded to the indolenine ring) are preferably bonded to each other to form a 5- or 6-membered ring and more preferably to form a 6-membered ring. In addition, it is preferable that the 5- or 6-membered ring is formed at the central portion of the bond that connects the indoline ring and the indolenine ring. The ring formed in the central portion of the bond connecting the indoline ring and the indolenine ring means a ring containing carbon atoms as ring-constituting atoms so that the numbers of bonded atoms from the indoline ring and the indolenine ring are the same.
In a case where one hydrogen atom is removed from any of R1 to R4, R11 to R13, R22 to R25, R32 to R35, R41, or R42 described above, the phosphor moiety M is a monovalent structural moiety.
Specifically, a monovalent structural moiety is provided in a case where one hydrogen atom is removed from a substituent which can be adopted as R1 to R4, R11 to R13, R22 to R25, R32 to R35, R41, or R42, or a hydrogen atom which can be adopted as R11 to R13, R22 to R25, or R32 to R35 is removed to provide a monovalent structural moiety which has a bonding site on the carbon atom to which R11 to R13, R22 to R25, or R32 to R35 has been bonded.
Among the above, it is preferable that the phosphor moiety M is to be a monovalent structural moiety by removing one hydrogen atom from the ring formed by the bonding of R41 and R42 described above or is to be a monovalent structural moiety by removing one hydrogen atom from a substituent which can be adopted as R12 (preferably R12 on the carbon atom at which the numbers of bonded atoms from the indoline ring and the indolenine ring are the same).
(iii) R22 to R25 and R32 to R35
R22 to R25 and R32 to R35 represent a hydrogen atom, an alkyl group, an alkoxy group, an aryl group, a sulfo group, a sulfamoyl group, a carboxy group, an alkoxycarbonyl group, an aryloxycarbonyl group, an acyloxy group, a carbamoyl group, an acylamino group, a nitro group, or a halogen atom. Regarding this R22 to R25 and R32 to R35, adjacent groups may be bonded to each other to form a fused ring.
The alkyl group, the alkoxy group, the aryl group, the sulfo group, the sulfamoyl group, the carboxy group, the alkoxycarbonyl group, the aryloxycarbonyl group, the acyloxy group, the carbamoyl group, the acylamino group, the nitro group, and the halogen atom, which can be adopted as R22 to R25 and R32 to R35, respectively have the same meanings as the alkyl group, the alkoxy group, the aryl group, the sulfo group, the sulfamoyl group, the carboxy group, the alkoxycarbonyl group, the aryloxycarbonyl group, the acyloxy group, the carbamoyl group, the acylamino group, the nitro group, and the halogen atom in the substituent group T which will be described later.
The fused ring formed by bonding adjacent groups among R22 to R25 and R32 to R35 to each other is not particularly limited. However, examples thereof include a naphthalene ring (a naphthalene ring that is formed together with a benzene ring to which R22 to R25 are bonded or a benzene ring to which R32 to R35 are bonded in a case where adjacent groups are bonded to each other to form the benzene ring). From the viewpoint of suppressing association, it is preferable that adjacent groups among R22 to R25 and R32 to R35 are not bonded to each other and do not form a fused ring.
From the viewpoint of improving water solubility and suppressing association, it is preferable that at least one of R22, . . . , or R25 and at least one of R32, . . . , or R35 have a hydrophilic group, and it is more preferable that at least one hydrophilic group is contained per the number of rings of one, to which R22 to R25 are bonded and rings to which R32 to R35 are bonded. For example, in a case where adjacent groups among R22 to R25 and R32 to R35 are bonded to each other to form a naphthalene ring as a fused ring, the number of rings to which R22 to R25 are bonded is two, and the number of rings to which R32 to R35 are bonded to each other is two, which means that it is more preferable that at least two of R22 to R25 and at least two of R32 to R35 have a hydrophilic group. The upper limit value thereof is not particularly limited as long as it is allowed in terms of structure, and it can be appropriately adjusted in accordance with the number of hydrophilic groups in the compound as a whole, which will be described later.
The hydrophilic group is not particularly limited; however, examples thereof include an alkoxy group having a substituent, a carboxy group, a sulfo group, and a phosphono group, where a sulfo group is preferable.
R22 to R25 and R32 to R35 are preferably a hydrogen atom, an alkyl group, a sulfo group, a nitro group, or a halogen atom, more preferably a hydrogen atom, an alkyl group, a sulfo group, or a halogen atom, and still more preferably a hydrogen atom, an alkyl group, or a sulfo group.
(iv) R41 and R42
R41 and R42 represent an alkyl group or —(CH2—CH2—O)b—R21. R21 and b respectively have the same meanings as R21 and b described above.
Examples of the substituent which may be contained in the alkyl groups as R41 and R42 include an alkoxy group, a carboxy group, an alkoxycarbonyl group, an acyloxy group, a carbamoyl group, an acylamino group, a sulfo group, and a phosphono group, as well as a group consisting of a combination of these substituents.
The alkyl group which can be adopted as R41 and R42 has the same meaning as the alkyl group in the substituent group T which will be described later.
The unsubstituted alkyl group preferably has 1 to 6 carbon atoms, more preferably has 1 to 4 carbon atoms, and still more preferably has 1 to 3 carbon atoms.
The alkyl group moiety of the alkyl group having a substituent preferably has 1 to 10 carbon atoms, more preferably has 1 to 8 carbon atoms, still more preferably has 1 to 7 carbon atoms, even still more preferably has 1 to 6 carbon atoms, and even further still more preferably has 1 to 5 carbon atoms. In addition, the number of atoms that constitute the longest chain of the alkyl group having a substituent is preferably 3 to 14, more preferably 3 to 12, and still more preferably 3 to 10.
The alkyl group having a substituent, which can be adopted as R41 and R42, is preferably an alkyl group having, as a substituent, at least one of an alkoxy group, a carboxy group, a sulfo group, or a phosphono group, and more preferably an alkyl group having, as a substituent, at least one of a carboxy group or a sulfo group, from the viewpoint of further improving water solubility. It is noted that it may be an alkyl group having a substituent consisting of a combination of the above-described preferred substituents (the alkoxy group, the carboxy group, the sulfo group, and the phosphono group) and a group other than these substituents.
In addition, the form of the alkyl group having a substituent, which can be adopted by R1 to R4, can be also preferably applied.
To —(CH2—CH2—O)b—R21 which can be adopted as R41 and R42, the description of —(CH2—CH2—O)b—R21 in R1 to R4 can be preferably applied.
R41 and R42 may be bonded to each other to form a ring.
Among the cyanine dyes represented by General Formula (α), preferred examples of the structure in which R41 and R42 are bonded to each other to form a ring include a cyanine dye represented by General Formula ((3).
In the formula, Lx to Ly represent an alkylene group or —(CH2—CH2—O)b-alkylene-*. * represents a bonding position to U.
The linking group U represents a divalent linking group having 1 to 100 atoms.
R1 to R4, R11 to R13, R22 to R21, R32 to R35, b, and a respectively have the same meanings as R1 to R4, R11 to R13, R22 to R25, R32 to R35, b, and a in General Formula (α), and the same also applies to the preferred ranges thereof unless otherwise specified.
At least one of R1, R2, R3, R4, U, or U includes a structure represented by —(CH2—CH2—O)b—. m has the same meaning as m described above.
However, the cyanine dye represented by Formula (β) is neutral.
The alkylene group which can be adopted as Lx and Ly corresponds to an alkylene group obtained by removing one hydrogen atom or one substituent from an alkyl group having a substituent which can be adopted as R41 and R42.
For the number of carbon atoms of the alkylene group moiety of the alkylene group which can be adopted as Lx to Ly, the description of the number of carbon atoms of the alkyl group moiety of the alkyl group having a substituent, as R41 and R42, can be preferably applied.
The —(CH2—CH2—O)b-alkylene-* which can be adopted as Lx and Ly corresponds to —(CH2—CH2—O)b-alkylene obtained by removing one hydrogen atom or one substituent from the alkyl group as R21, among the —(CH2—CH2—O)b—R21 which can be adopted as R41 and R42 (R21 represents an alkyl group having a substituent).
In the —(CH2—CH2—O)b-alkylene-* which can be adopted as Lx and Ly, b is preferably 1 to 10 and more preferably 1 to 8, and as the number of carbon atoms of the alkylene group moiety, the description of the number of carbon atoms of the alkyl group moiety of the alkyl group having a substituent in R41 to R42 can be preferably applied.
From the viewpoint of further improving the fluorescence intensity, it is preferable that both Lx and Ly include a structure represented by —(CH2—CH2—O)b—.
The total number of atoms constituting the linking group U is 1 to 100, and it is preferably 10 to 90, more preferably 20 to 90, and still more preferably 30 to 80.
The linking group U is preferably a divalent linking group formed by bonding three or more selected from an alkylene group, —O—, —NR50—, —COO—, —CONR50—, and —SO2NR50—. R50 represents a hydrogen atom or an alkyl group.
The number of carbon atoms in the alkylene moiety of the alkylene group which can be adopted as the linking group U is preferably 1 to 10, more preferably 1 to 8, still more preferably 1 to 7, particularly preferably 1 to 6, and most preferably 1 to 5.
In the present invention, “the number of carbon atoms in the alkylene moiety of the alkylene group” means the number of carbon atoms excluding the substituent moiety contained in the alkylene group.
As the alkyl group which can be adopted as R50, the description of the alkyl group as R1 to R4 can be preferably applied.
R50 is preferably a hydrogen atom.
The number of the above-described alkylene group, —O—, —NR50—, —COO—, —CONR50—, and —SO2NR50—, constituting the linking group U, is preferably 3 to 11, more preferably 3 to 7, still more preferably 3 to 5, and particularly preferably 3.
In the linking group U, the connecting portion to Lx to Ly is preferably —O—, —NR50—, —COO—, —CONR50—, or —SO2NR50—. That is, the linking group U is preferably bonded to the alkylene groups of Lx to Ly through —O—, —NR50—, —COO—, —CONR50—, or —SO2NR50—, which constitutes the linking group U. In the linking group U, it is more preferable that connecting portions to Lx to Ly are —O—, —NR50—, —COO—, —CONR50—, or —SO2NR50—, where the linking group U is a divalent linking group in which the connecting portions are connected to each other through an alkylene group.
It is preferable that the linking group U is to be a monovalent structural moiety, that is, a phosphor moiety M, by removing one hydrogen atom therefrom. In the linking group U, examples of the position where one hydrogen atom is removed include an alkylene group and an alkyl group as R50, where an alkylene group is preferable.
In a case where in the linking group U, one hydrogen atom is removed in the alkylene group or the alkyl group as R50, one hydrogen atom may be directly removed from the alkylene group or the alkyl group as R50, and the linking group ZZZ may be bonded to the alkylene group or the alkyl group as R50, thereby the linking group ZZZ serving as a bonding site.
Examples of the linking group ZZZ include an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, —O—, —S—, >C═O, >NR60, >S═O, >S(═O)2, >P(═O)OR70, —COO—, —CONR60—, and —(CH2—CH2—O)p—, as well as a group consisting of a combination of these substituents. The number of those to be combined is not particularly limited; however, it can be set to, for example, 2 to 20, and it is preferably 2 to 7 and more preferably 2 to 5.
R60 and R70 are a hydrogen atom or an alkyl group and are preferably a hydrogen atom. To the alkyl group which can be adopted as R60 or R70, the description of the alkyl group as R50 can be preferably applied.
p represents the repetition number, and it is preferably 1 to 10, more preferably 1 to 8, and still more preferably 1 to 4.
(v) a
a is an integer of 1 to 3, where it is preferably an integer of 2 or 3.
In the cyanine dye represented by General Formula (α), it is preferable that at least one of R1, R2, R3, R4, R41, or R42 includes a structure represented by —(CH2—CH2—O)b—. b has the same meaning as b described above. It is conceived that this makes it possible for the compound according to the embodiment of the present invention, which has a structural moiety consisting of a cyanine dye represented by General Formula (α), to have a proper hydrophilicity and a proper excluded volume effect, whereby a labeled biological substance to be obtained can exhibit an excellent fluorescence intensity.
In addition, from the viewpoint that sufficient hydrophilicity is imparted as the compound according to the embodiment of the present invention, the cyanine dye represented by General Formula (α) is such that the number of hydrophilic groups per one molecule of the cyanine dye represented by General Formula (α) is preferably 2 or more, more preferably 2 to 8, still more preferably 2 to 6, and particularly preferably 3 to 6.
To the hydrophilic group, the description of the hydrophilic group which can be adopted by R22 to R25 and R32 to R35 described above can be applied.
The position of the hydrophilic group is not particularly limited unless specified otherwise, and examples of the group having the hydrophilic group preferably include R11 to R13, R22 to R25, R32 to R35, R41, or R42.
It is noted that in the structure represented by General Formula (α) or (β), one hydrogen atom may be removed from any substituent to provide a monovalent structural moiety (the phosphor moiety M); however, it is preferable that, for example, one hydrogen atom is removed from the linking group U to provide a monovalent structural moiety, or one hydrogen atom is removed from a substituent, which can be adopted as R12 (preferably R12 on the carbon atom at which the numbers of bonded atoms from the indoline ring and the indolenine ring are the same), to provide a monovalent structural moiety.
The physiologically active substance moiety which can be adopted as M can be used without particular limitation as long as it is a structural moiety consisting of a physiologically active substance. Examples of the physiologically active substance include a vitamin, a coenzyme, a hormone, an antibiotic, a neurotransmitter, and a cytokine. More specific examples thereof are calicheamicin, doxorubicin, daunorubicin, mitomycin C, bleomycin, cyclocytidine, vincristine, vinblastine, methotrexate, cisplatin or a derivative thereof, auristatin or a derivative thereof, maytansine or a derivative thereof, taxol or a derivative thereof, and camptothecin or a derivative thereof, which are described in paragraph [0095] of JP2021-020956A, and the description of paragraphs [0095] to [0099] of JP2021-020956A can be applied thereto.
The prodrug moiety which can be adopted as M can be used without particular limitation as long as it is a structural moiety consisting of a compound that is metabolized in vivo to be changed to a physiologically active substance. To the prodrug, for example, the description (a prodrug form of 2-pyrrolinodoxorubicin) in paragraph [0003] of JP2020-105187A can be applied.
The radioactive isotope-containing moiety which can be adopted as M can be used without particular limitation as long as it is a structural moiety containing a radioactive isotope that is capable of being used in the medical field. Examples of the radioactive isotope include iodine 131, indium 111, yttrium 90, lutetium 177, and copper 64, which are not limited thereto. The description of paragraph [0225] of JP2021-11483A can be applied thereto. Examples of the structural moiety containing a radioactive isotope include a structural moiety in which the radioactive isotope is bonded or coordinated to a nitrogen atom of an amino group or a tertiary amine, a sulfanyl group, an aryl group, a heteroaryl group, or the like. Examples of the structural moiety in which a nitrogen atom of a tertiary amine is coordinated to the radioactive isotope) include a structural moiety in which 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or the like is coordinated to the radioactive isotope to form a complex, and examples of the structural moiety in which a sulfanyl group is coordinated to the radioactive isotope include a structural moiety consisting of a complex such as copper (II) diacetylbis(N(4)-methylthiosemicarbazone).
<Compound Represented by General Formula (III)>
The compound represented by General Formula (II) is preferably represented by General Formula (III).
In the formula, Y1 to Y3, Z1 to Z3, and W1 to W3 represent a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an acyl group, an amino group, a hydroxy group, an alkoxy group, a sulfanyl group, an aryl group, a heteroaryl group, or an anionic group.
LL3 and LL4 represent a divalent linking group.
s, t, and u are an integer of 0 or more.
R4 to R7, L1, L2, L5, L6, X1 to X3, M, n, and m respectively have the same meanings as R4 to R7, L1, L2, L5, L6, X1 to X3, M, n, and m in General Formula (II) described above.
However, the structure parenthesized by s, t, or u is not allowed to be the structure represented by General Formula (I) described above. That is, Y1 is not allowed to be bonded to W1 or Z1, Y2 is not allowed to be bonded to W2 or Z2, or Y3 is not allowed to be bonded to W3 or Z3 to form a structure represented by General Formula (I).
Y1 to Y3, Z1 to Z3, and W1 to W3 represent a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an acyl group, an amino group, a hydroxy group, an alkoxy group, a sulfanyl group, an aryl group, a heteroaryl group, or an anionic group.
The alkyl group, the alkenyl group, the alkynyl group, the acyl group, the amino group, the alkoxy group, the aryl group, the heteroaryl group, and the anionic group, which can be adopted as Y1 to Y3, Z1 to Z3, or W1 to W3, respectively have the same meanings as the alkyl group, the alkenyl group, the alkynyl group, the acyl group, the amino group, the alkoxy group, the aryl group, the heteroaryl group, and the anionic group in the substituent group T which will be described later, and the same also applies to the preferred ranges thereof.
The alkyl group, the alkenyl group, the alkynyl group, the acyl group, the amino group, the alkoxy group, the aryl group, and the heteroaryl group, which can be adopted as Y1 to Y3, Z1 to Z3, or W1 to W3, may be unsubstituted or may have a substituent, and examples of the substituent which may be contained therein include substituents in the substituent group T which will be described later. For example, an aryl group, a halogen atom, or an anionic group is preferable.
In addition, the substituent which may be contained in the above-described alkyl group, alkenyl group, alkynyl group, acyl group, amino group, alkoxy group, aryl group, and heteroaryl group, which can constitute Y1 to Y3, Z1 to Z3, or W1 to W3, may be substituted with a substituent selected from the substituent group T which will be described later, and for example, an anionic group is preferable.
Y1 to Y3 are preferably a hydrogen atom, an alkyl group, an aryl group, or a heteroaryl group, and more preferably a hydrogen atom or an alkyl group.
W1 to W3 are preferably a hydrogen atom, an alkyl group, an aryl group, or a heteroaryl group, and more preferably a hydrogen atom.
Z1 to Z3 are preferably an alkyl group, an aryl group, or a heteroaryl group, and more preferably an alkyl group.
In order to suppress the interaction between the structures represented by General Formula (I), the compound represented by General Formula (III) preferably has a group having a charge repulsive action, and from this viewpoint, at least one of Z1, Z2, or Z3 is preferably a group including an anionic group, and more preferably an alkyl group including an anionic group.
Here, the “at least one of Z1, Z2, or Z3 is a group including an anionic group” means that at least one of the following conditions (Z1) to (Z3) is satisfied.
In addition, the phrase “at least one of Z1, Z2, or Z3 is an alkyl group including an anionic group” means that in a case where “a group including an anionic group” in the conditions (Z1) to (Z3) is read as “an alkyl group including an anionic group”, at least one of the following condition (Z1), (Z2), or (Z3) is satisfied.
LL3 and LL4 represent a divalent linking group and are more preferably a divalent linking group obtained by combining one or two or more of an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, —O—, >C═O, and >NRA.
RA represents a hydrogen atom or a substituent.
To the alkylene group, the alkenylene group, the alkynylene group, the arylene group, the heteroarylene group, and >NRA, which can constitute LL3 and LL4, the description of the alkylene group, the alkenylene group, the alkynylene group, the arylene group, the heteroarylene group, and >NRA, which can constitute L1 to L7 described above, can be preferably applied.
LL3 is preferably >C═O, >NRA, an alkylene group, an arylene group, or a heteroarylene group, and more preferably >C═O or NRA. It is noted that RA is preferably a hydrogen atom.
LL4 is preferably >C═O, >NRA, an alkylene group, an arylene group, or a heteroarylene group, and more preferably >C═O or >NRA. It is noted that RA is preferably a hydrogen atom.
s, t, and u are an integer of 0 or more.
The upper limit value of each of s, t, and u is not particularly limited and can be set to, for example, 20 or less, and it is preferably 10 or less and more preferably 5 or less. Among the above, s, t, and u are preferably an integer of 0 or 1.
It is noted that in a case where a structure can be classified into both a structure parenthesized by t and a structure parenthesized by u, it is preferentially classified into a structure parenthesized by u.
<Compound represented by General Formula (IV)>
The compound represented by General Formula (III) is preferably represented by General Formula (IV).
In the formula, Y4 and Y5 represent a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an acyl group, an amino group, a hydroxy group, an alkoxy group, a sulfanyl group, an aryl group, a heteroaryl group, or an anionic group.
R4 to R7, L2, L5, X1 to X3, Y1 to Y3, Z1 to Z3, W1 to W3, M, n, m, s, t, and u respectively have the same meanings as R4 to R7, L2, L5, X1 to X3, Y1 to Y3, Z1 to Z3, W1 to W3, M, n, m, s, t, and u in General Formula (III) described above.
Y4 and Y5 represent a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an acyl group, an amino group, a hydroxy group, an alkoxy group, a sulfanyl group, an aryl group, a heteroaryl group, or an anionic group.
The alkyl group, the alkenyl group, the alkynyl group, the acyl group, the amino group, the alkoxy group, the aryl group, the heteroaryl group, and the anionic group, which can be adopted as Y4 or Y5, respectively have the same meanings as the alkyl group, the alkenyl group, the alkynyl group, the acyl group, the amino group, the alkoxy group, the aryl group, the heteroaryl group, and the anionic group in the substituent group T which will be described later, and the same also applies to the preferred ranges thereof.
It is noted that all the alkyl group, the alkenyl group, the alkynyl group, the acyl group, the amino group, the alkoxy group, the aryl group, and the heteroaryl group, which can be adopted as Y4 or Y5, may be an unsubstituted group or a group having a substituent.
Y4 and Y5 are preferably a hydrogen atom or an alkyl group and more preferably a hydrogen atom.
<Compound Represented by General Formula (V)>
The compound represented by General Formula (IV) is preferably represented by General Formula (V).
In the formula, R6A and R7A represent a hydrogen atom, a hydroxy group, a sulfanyl group, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, an alkoxy group, a heteroaryl group, an amino group, an acyl group, an anionic group, a cationic group, or Q. However, at least one of R6A or R7A represents Q.
L8 and L9 represent a linking group. However, L9 in a case where R6A is Qis a linking group in which the number of shortest-distance atoms that connect >NY4 to R6A is 3 or more, and L8 in a case where R7A is Q is a linking group in which the number of shortest-distance atoms that connect >C═O to R7A is 3 or more.
R4, R5, L2, L5, X1 to X3, Y1 to Y5, Z1 to Z3, W1 to W3, M, Q, n, m, s, t, and u respectively have the same meanings as R4, R5, L2, L5, X1 to X3, Y1 to Y5, Z1 to Z3, W1 to W3, M, Q, n, m, s, t, and u in General Formula (IV) described above.
R6A or R7A and L9 or L8 are each determined such that R6A or R7A is an unsubstituted group and L9 or L8 is the longest group. However, in a case where the group represented by -L9R6A or -L1R7A has an anionic group, a cationic group, or Q, it is determined such that the anionic group, the cationic group, or Q, which is located on the most terminal side (the R6A side in -L9R6A or the R7A side in -L8R7A), is R6A or R7A.
R6A and R7A represent a hydrogen atom, a hydroxy group, a sulfanyl group, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, an alkoxy group, a heteroaryl group, an amino group, an acyl group, an anionic group, a cationic group, or Q. However, at least one of R6A or R7A represents Q.
The alkyl group, the alkenyl group, the alkynyl group, the aryl group, the alkoxy group, the heteroaryl group, the amino group, the acyl group, the anionic group, and the cationic group, which can be adopted as R6A and R7A, respectively have the same meanings as the alkyl group, the alkenyl group, the alkynyl group, the aryl group, the alkoxy group, the heteroaryl group, the amino group, the acyl group, the anionic group, and the cationic group in the substituent group T which will be described later, and the same also applies to the preferred ranges thereof. However, all the groups are unsubstituted groups.
Q which can be adopted as R6A and R7A has the same meaning as Q described above, and the same applies to the preferred range thereof.
R6A is preferably an alkyl group, a sulfanyl group, an aryl group, a heteroaryl group, or Q and more preferably an alkyl group or Q.
R7A is preferably an alkyl group, a sulfanyl group, an aryl group, a heteroaryl group, or Q, and more preferably an alkyl group or Q.
L8 and L9 represent a linking group.
The linking group which can be adopted as L8 and L9 is, for example, more preferably a divalent linking group obtained by combining one or two or more of an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, —O—, >C═O, and >NRA. RA represents a hydrogen atom or a substituent.
For the alkylene group, the alkenylene group, the alkynylene group, the arylene group, the heteroarylene group, and >NRA, which can constitute L8 or L9, the description of the alkylene group, the alkenylene group, the alkynylene group, the arylene group, the heteroarylene group, and >NRA, which can constitute L1 to L7 described above, can be preferably applied.
The substituent which may be contained in the alkylene group, the alkenylene group, the alkynylene group, the arylene group, and the heteroarylene group, which can constitute L8 or L9, is not particularly limited, and it is preferably selected from a substituent group T which will be described later. More preferred examples thereof include a halogen atom, an aryl group, and an alkyl group. In addition, the substituent which may be contained in the alkylene group, the alkenylene group, the alkynylene group, the arylene group, and the heteroarylene group, which can constitute L8 or L9, may be substituted with a substituent selected from the substituent group T which will be described later, and it is, for example, preferably an anionic group.
In addition, the number of substituents which may be contained in the alkylene group, the alkenylene group, the alkynylene group, the arylene group, and the heteroarylene group, which can constitute L8 or L9, is not particularly limited as long as it can be adopted as a structure. The number thereof can be set to at least one or more, the upper limit thereof is not particularly limited, and for example, all hydrogen atoms in the alkylene group, the alkenylene group, the alkynylene group, the arylene group, and the heteroarylene group may be substituted with a substituent.
However, L9 in a case where R6A is Q is a linking group in which the number of shortest-distance atoms that connect >NY4 to R6A is 3 or more, and L8 in a case where R7A is Q is a linking group in which the number of shortest-distance atoms that connect >C═O to R7A is 3 or more.
Regarding the divalent linking group L9, “the number of shortest-distance atoms that connect >NY4 to R6A” means the number of atoms that constitute the shortest chain connecting >NY4 to R6A, and regarding the above-described divalent linking group L8, “the number of shortest-distance atoms that connect >C═O to R7A” means the number of atoms that constitute the shortest chain connecting >C═O to R7A. It is noted that >NY4 means >NY4 that is directly bonded to L9, and >C═O means >C═O that is directly bonded to L8. For example, in a compound (2) that is used in Examples described later, since L8 is —NH(C2H4O)4C2H4— and R7A is —COOH, the number of atoms that constitute the shortest chain connecting >C═O to R7A is 15.
In the compound represented by General Formula (V), at least one of R6A or R7A is Q. In a case where Q is linked to >C═O or >NY4 by a linking group having the number of shortest-distance atoms of 3 or more, the distance from a bonding point between the main chain connecting R6A to R7A and M (that is, the carbon atom to which L5 and R5 are bonded or the carbon atom to which L2 and R4 are bonded) is increased, and the steric hindrance around Q is reduced, whereby the reactivity of the dye multimer in which the number of phosphor moieties M in the compound is 2 or more is improved with respect to the antibody, and the degree of fluorescence labeling (DOL) can be improved.
At least one of R6A or R7A described above is Q, where the number of shortest-distance atoms of the linking group that links this Q to >C═O or >NY4 (the number of shortest-distance atoms that connect >NY4 to R6A in a case where R6A is Q) and the number of shortest-distance atoms that connect >C═O to R7A in a case where R7A is Q) is preferably 3 to 60, more preferably 12 to 40, and still more preferably 15 to 40.
In the present invention, from the viewpoint of ease of synthesis, it is preferable that any one of L8 or L9 is a group including -(L-O)g—, and it is more preferable that L8 is a group including -(L-O)g—.
L1 is more preferably a linking group obtained by combining one or two or more of an alkylene group, —O—, >C═O, and >NRA, and it is still more preferably an alkylene group, —O—, >C═O, >NRA, a group in which —NRA-alkylene group is bonded to a right side of a repeating unit (preferably having the repetition number of 1 to 20) represented by —[NRA-alkylene-C(═O)]—, —NRA-(L-O)g-alkylene, or —C(═O)-(L-O)g alkylene.
L9 is more preferably a linking group obtained by combining one or two or more of an alkylene group, —O—, >C═O, and >NRA, and it is still more preferably an alkylene group, —O—, >C═O, >NRA, a group in which —NRA-alkylene group is bonded to a right side of a repeating unit (preferably having the repetition number of 1 to 20) represented by —[NRA-alkylene-C(═O)]—, —NRA-(L-O)g-alkylene, or —C(═O)-(L-O)g alkylene.
<Compound Represented by General Formula (VI)>
The compound represented by General Formula (II) is also preferably represented by General Formula (VI). The compound represented by General Formula (VI) corresponds to a compound in which L1 and L4 are >NH and L3 and L6 are >C═O, L7 is a single bond, R4 and R5 are a hydrogen atom, and L1 and L2, and L4 and L5 are respectively bonded to each other to form a specific 5-membered ring in the compound represented by General Formula (II).
In the formula, X4 to X9 represent —O—, —S—, >NR101, or >CR102R103.
However, one of X4, X5, or X6 is >NR101 or >CR102R103, where R101 is -L10-M in a case where one of X4, X5, or X6 is >NR101 and R102 or R103 is -L10-M in a case where one of X4, X5, or X6 is >CR102R103. One of X7, X8, or X9 is >NR101 or >CR102R103, where R101 is -L11-M in a case where one of X7, X8, or X9 is >NR101 and R102 or R103 is -L11-M in a case where one of X7, X8, or X9 is >CR102R103.
R101 to R103, which are neither -L10-M nor -L11-M, represent a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an acyl group, —NR8R9, —OR10, or an anionic group.
L10 and L11 represent a single bond or a divalent linking group.
n1 is an integer of 2 or more.
R6 to R10, X1 to X3, M, and m respectively have the same meanings as R6 to R10, X1 to X3, M, and m in General Formula (II) described above.
To X4 to X6, the description of X1 to X3 in General Formula (I) described above can be applied unless otherwise specified, except that one of X4, X5, or X6 is >NR101 or >CR102R103 where R101 is -L10-M in a case where one of X4, X5, or X6 is >NR101 and R102 or R103 is -L10-M in a case where one of X4, X5, or X6 is >CR102R103.
In addition, the description of X1 to X3 in General Formula (I) described above can be applied to X7 to X9 unless otherwise specified, except that one of X7, X8, or X9 is >NR101 or >CR102R103, where R101 is -L11-M in a case where one of X7, X8, or X9 is >NR101 or R102 or R103 is -L10-M in a case where one of X7, X8, or X9 is >CR102R103.
That is, the description of X1 described above is applied to X4 and X7, the description of X2 described above is applied to X5 and X8, and the description of X3 described above is applied to X6 and X9, and the descriptions of R1, R2, and R3 in General Formula (I) described above can be respectively applied to R101, R102, and R103 which are neither -L10-M nor -L11-M.
In >NR101 and >CR102R103 among X4 to X9, which have neither -L1O-M nor -L11-M, R101 is preferably an alkyl group, and R102 and R103 are preferably a hydrogen atom.
Regarding the two groups among X4 to X6, which do not have -L10-M described above, it is preferable that at least one thereof is >CR102R103, it is more preferable that at least one thereof is >CR102R103 and the remaining one is —O—, —S—, or >CR102R103, and it is still more preferable that both the two are >CR102R103
Regarding the two groups among X7 to X9, which do not have -L11-M described above, it is preferable that at least one thereof is >CR102R103, it is more preferable that at least one thereof is >CR102R103 and the remaining one is —O—, —S—, or >CR102R103, and it is still more preferable that both the two are >CR102R103.
Among X4 to X6, >NR101 or >CR102R103 which has -L10-M as any one of R101 to R103 is preferably a group represented by >CR102R103 where R103 is -L10-M, and it is more preferably a group represented by >CR102R103 where R102 is a hydrogen atom and R103 is -L10-M.
Among X7 to X9, >NR101 or >CR102R103 which has -L11-M as any one of R101 to R103 is preferably a group represented by >CR102R103 where R103 is -L11-M, and it is more preferably a group represented by >CR102R103 where R102 is a hydrogen atom and R103 is -L11-M.
Among X4 to X6, the group having -L10-M is not particularly limited; however, it is preferably X5.
Among X7 to X9, the group having -L11-M is not particularly limited; however, it is preferably X8.
L10 and L11 are preferably a single bond, or a group obtained by combining one or two or more of an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, —O—, —S—, >C═O, and >NRA, more preferably a group obtained by combining one or two or more of an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, —O—, —S—, >C═O, and >NRA, and still more preferably a group represented by * -Lx1-Ly1-**.
To the alkylene group, the alkenylene group, the alkynylene group, the arylene group, the heteroarylene group, and >NRA, which can constitute L10 and L11, the description of the alkylene group, the alkenylene group, the alkynylene group, the arylene group, the heteroarylene group, and >NRA, which can constitute L2 and L5 described above, can be applied unless otherwise specified.
Lx1 is a single bond or a group obtained by combining one or two or more of an alkylene group, an alkenylene group, an alkynylene group, an arylene group, and a heteroarylene group, and Ly1 is a single bond, —O, —, —S—, >C═O, or >NRA. It is noted that in *-Lx1-Ly1-**, * represents a bonding site to X4 to X9, and ** represents a bonding site to M.
Among the groups represented by * -Lx1-Ly1-**, L10 and L11 are preferably a group in which Ly1 is a single bond, —S—, >C═O, or >NRA, more preferably a group in which Ly1 is >C═O or >NRA, and still more preferably a group in which Lx1 is a single bond and Ly1 is >C═O or >NRA.
It is noted that in the compound represented by General Formula (VI), it suffices that the number of shortest-distance atoms in the linking chain (each linking chain of the linking chain including L10 and the linking chain including L11) that connects M to any one of X4 to X9 is, for example, 1 to 60, where 1 to 40 is preferable. In a case where M is a phosphor moiety, the above-described number of shortest-distance atoms means the number of atoms that constitute the shortest chain in the linking chain that connects a conjugated structural moiety for exhibiting fluorescence to any one of X4 to X9 in the phosphor moiety M.
It is noted that in the compound represented by General Formula (VI), it is also preferable that the structure represented by —(CH2—CH2—O)b— described above (b is also as described above) is provided at any portion of the linking chain represented by “-linking group ZZZ-L10-” in the structure represented by “the conjugated structural moiety of M—the linking group ZZZ-L10- described above” and any portion of the linking chain represented by “-linking group ZZZ-L11-” in the structure represented by “the conjugated structural moiety of M—the linking group ZZZ-L11- described above”.
n1 is an integer of 2 or more.
In the compound represented by General Formula (VI), the linker main chain connecting the two phosphor moieties is rigid and the dye association can be further suppressed as compared with the compound represented by any one of General Formulae (III) to (V) described above. Therefore, the lower limit value of n1 is preferably an integer of 3 or more, and it is more preferably an integer of 5 or more from the viewpoint that a sufficient effect of improving the fluorescence intensity is obtained. The same applies even in a case where the number of phosphor moieties is two or more and a case where the number thereof is 2 or more.
The upper limit value of n1 is, for example, preferably an integer of 36 or less, more preferably an integer of 24 or less, and still more preferably an integer of 18 or less.
<Compound Represented by General Formula (VII)>
The compound represented by General Formula (VI) is preferably represented by General Formula (VII).
In the formula, R6A and R7A represent a hydrogen atom, a hydroxy group, a sulfanyl group, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, an alkoxy group, a heteroaryl group, an amino group, an acyl group, an anionic group, a cationic group, or Q. However, at least one of R6A or R7A represents Q.
L12 and L13 represent a linking group.
na and nb are an integer of 0 or more.
L10, L11, X1 to X9, M, Q, n1, and m respectively have the same meanings as L10, L11, X1 to X9, M, Q, n1, and m in General Formula (VI) described above.
Unless otherwise specified, R6A and R7A have the same meanings as R6A and R7A in General Formula (V) described above. That is, the description of R6A and R7A in General Formula (V) can be applied to R6A and R7A.
R6A or R7A and L12 or L13 are each determined such that R6A or R7A is an unsubstituted group and L12 or L13 is the longest group. However, in a case where the group represented by -L13R6A or -L12R7A has an anionic group, a cationic group, or Q, it is determined such that the anionic group, the cationic group, or Q, which is located on the most terminal side (the R6A side in -L13R6A or the R7A side in -L12R7A), is R6A or R7A.
L12 and L13 represent a linking group.
The linking group which can be adopted as L12 and L13 is, for example, more preferably a divalent linking group obtained by combining one or two or more of an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, —O—, >C═O, and >NRA. RA represents a hydrogen atom or a substituent.
To the alkylene group, the alkenylene group, the alkynylene group, the arylene group, the heteroarylene group, and >NRA, which can constitute L12 and L13, the description of the alkylene group, the alkenylene group, the alkynylene group, the arylene group, the heteroarylene group, and >NRA, which can constitute L8 or L9 in General Formula (V) described above, can be applied.
In the compound represented by General Formula (VII), it is preferable that (A) na is an integer of 1 or more and R6A is Q, and/or (B) nb is an integer of 1 or more and R7A is Q.
However, the number of shortest linking atoms of L13 is 7 or less in a case of the (A), and the number of shortest linking atoms of L12 is 7 or less in a case of the (B).
Regarding the divalent linking group L13, “the number of shortest linking atoms in L13” means the number of atoms that constitute the shortest chain connecting N directly bonded to L13 to R6A, in a case where na is an integer of 1 or more, where the N is one of those indicated in the structure parenthesized in ( )na, and it means the number of atoms that constitute the shortest chain connecting N directly bonded to L13 to R6A, in a case where na is 0, where the N is one of those indicated in the structure parenthesized in ( )m.
In addition, Regarding the divalent linking group L12, “the number of shortest linking atoms in L12” means the number of atoms that constitute the shortest chain connecting >C═O directly bonded to L12 to R7A, in a case where nb is an integer of 1 or more, where the >C═O is one of those indicated in the structure parenthesized in ( )nb, and it means the number of atoms that constitute the shortest chain connecting >C═O to R7A, which is indicated on the left side of the structure parenthesized in ( )nb in General Formula (VII), in a case where nb represents 0.
For example, in a compound (6) that is used in Examples described later, since L12 is —NHC2H4— and R7A is —COOH, the number of shortest linking atoms of L12 is 3.
In the compound represented by General Formula (VII), at least one of R6A or R7A is Q. In particular, it is conceived that in a case of satisfying (A) and/or (B) described above, the mobility of the linker main chain is reduced, and thus the dye association can be further suppressed.
The number of shortest linking atoms of L12 and the number of shortest linking atoms of L13, which are described, are preferably 1 to 5 and more preferably 1 to 4.
In the present invention, from the viewpoint of ease of synthesis, it is also preferable that any one of L12 or L13 is a group including -(L-O)g—, and it is also more preferable that L12 is a group including -(L-O)g—.
L12 is more preferably a linking group obtained by combining one or two or more of an alkylene group, —O—, >C═O, and >NRA, more preferably an —NRA-alkylene group or an —NRA-(L-O)g-alkylene group, and still more preferably an —NRA-alkylene group.
L13 is more preferably a linking group obtained by combining one or two or more of an alkylene group, —O—, >C═O, and >NRA, and still more preferably >NRA or >C═O.
na and nb are an integer of 0 or more.
In a case where R6A is Q, na is preferably 0 to 20, more preferably 2 to 20, and still more preferably 4 to 18.
In a case where R7A is Q, nb is preferably 0 to 20, more preferably 2 to 20, and still more preferably 4 to 18. It is noted that the lower limit value of nb may be 0, and in this case, nb is preferably 0 to 20 and more preferably 0 to 18.
In a case where R6A is not Q, na is preferably 0 to 20, more preferably 0 to 10, and still more preferably 0 to 5.
In a case where R7A is not Q, nb is preferably 0 to 20, more preferably 0 to 10, and still more preferably 0 to 5.
In a case where a compound represented by any one of General Formulae (II) to (VII) among the compounds according to the embodiment of the present invention is obtained by using a peptide synthesis method, the structure is, in general, such that the C-terminal structure is on the right side of the paper surface, and the N-terminal structure is on the left side of the paper surface.
The compound according to the embodiment of the present invention preferably contains at least one substituent represented by Q, that is, at least one of a carboxy group, a substituent capable of being bonded to a biological substance, or a substituent capable of being bonded to a solid support.
The compound according to the embodiment of the present invention can be bonded to a biological substance by the carboxy group or a substituent capable of being bonded to a biological substance described later, whereby a targeted labeled biological substance can be obtained. It is noted that a substituent capable of being bonded to a biological substance can be easily derived from a carboxy group by a conventional method.
In addition, the compound according to the embodiment of the present invention can be bonded to a solid support such as microparticles by the carboxy group or a substituent capable of being bonded to a solid support described later, whereby a targeted labeled microparticles can be obtained. The microparticle is not particularly limited; however, examples thereof include small particles that are useful for bonding to the compound according to the embodiment of the present invention, including a glass bead, a non-polymer bead such as a magnetic bead, and a polymer bead. In a certain embodiment, the microparticle includes a polystyrene bead. The small particle is not particularly limited as long as it has a size that is commonly used in the fluorescence labeling; however, the average particle diameter thereof is generally 10 nm to 10 m. It is noted that a substituent capable of being bonded to a solid support can be easily derived from a carboxy group by a conventional method.
In the present invention, for convenience, the substituent capable of being bonded to a biological substance and the substituent capable of being bonded to a solid support do not include a carboxy group, where “the substituent capable of being bonded to a biological substance” includes a substituent capable of being bonded to a biological substance, which is derived from a carboxy group, and “the substituent capable of being bonded to a solid support” includes a substituent capable of being bonded to a solid support, which is derived from a carboxy group. However, as described above, it is also possible to carry out bonding to a biological substance or a solid support by a carboxy group.
In the compound according to the embodiment of the present invention, a position where the substituent represented by Q is provided is not particularly limited; however, the substituent represented by Q is preferably provided in a structure other than the structure represented by General Formula (I) and the phosphor moiety and more preferably provided in at least one of R6 or R7 in the compound represented by General Formula (II).
It suffices that the number of substituents represented by Q in the compound according to the embodiment of the present invention is at least 1 or more in total, and it is preferably 1 to 3, more preferably 1 or 2, and still more preferably 1, from the viewpoint of the quantification of the target substance to be detected.
In addition, from the viewpoint of imparting sufficient hydrophilicity as a compound, the compound according to the embodiment of the present invention also preferably has an anionic group be described later at a position other than the phosphor moiety, and for example, it preferably has one or more anionic groups, more preferably 1 to 8 anionic groups, and still more preferably 1 to 6 anionic groups.
The position of the anionic group is not particularly limited unless specified otherwise, and examples of the group having the anionic group preferably include Z1 to Z3 or X1 to X3 in the compound represented by General Formula (III).
Specific examples of the compound according to the embodiment of the present invention will be shown below; however, the present invention is not limited to these compounds. In the following specific examples, the sulfo group may adopt a salt structure in which a hydrogen ion is dissociated. In the following specific examples, Dye indicates a phosphor moiety.
Examples of the preferred form according to the present invention include a compound satisfying the following form I among the compounds represented by General Formula (III) and a compound satisfying the following form II among the compounds represented by General Formula (VI).
(Form I)
Y1 to Y3: A hydrogen atom, an alkyl group, an aryl group, or a heteroaryl group
Z1 to Z3: An alkyl group, an aryl group, or a heteroaryl group
W1 to W3: A hydrogen atom, an alkyl group, an aryl group, or a heteroaryl group
LL3 and LL4: >C═O, >NRA, an alkylene group, an arylene group, or a heteroarylene group
s, t, and u: An integer of 0 or 1
R4 and R5: A hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an acyl group, an amino group, a hydroxy group, an alkoxy group, a sulfanyl group, an aryl group, or a heteroaryl group
R6: A substituent represented by -L9R6A (L9 is a linking group obtained by combining one or two or more of an alkylene group, —O—, >C═O, and >NRA, and R6A is an alkyl group, a sulfanyl group, an aryl group, a heteroaryl group, or Q.)
R7: A substituent represented by -L8R7A (L8 is a linking group obtained by combining one or two or more of an alkylene group, —O—, >C═O, and >NRA, and R7A is an alkyl group, a sulfanyl group, an aryl group, a heteroaryl group, or Q.)
L1: >C═O, >NRA, an arylene group, an alkylene group, —O—, or
L2 and L5: A single bond, or a group obtained by combining one or two or more of an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, —O—, —S—, >C═O, and >NRA
L6: >C═O, >NRA, or an arylene group
X1 to X3: At least two thereof are >CR2R3, and the remaining one is —O—, —S—, or >CR2R3. (R2 and R3 are preferably a hydrogen atom, —NR8R9, —OR10, or an anionic group.)
Phosphor moiety in M: A structural moiety consisting of at least one dye of a xanthene dye, a rhodamine dye, a coumarin dye, a cyanine dye, a pyrene dye, an oxazine dye, a squarylium dye, a pyridyloxazole dye, or a pyrromethene dye
n: An integer of 2 or more
m: An integer of 1 to 30
(Form II)
X4 to X6: A group in which a group having -L10-M is a group represented by >CR102R103 (R102 is a hydrogen atom, and R103 is -L10-M), where at least one of two groups which do not have L10-M is >CR102R103 and the remaining one is —O—, —S—, or >CR102R103 (R102 and R103 are preferably a hydrogen atom, —NR8R9, —OR10, or an anionic group.).
X7 to X9: A group in which a group having -L11-M is a group represented by >CR102R103 (R102 is a hydrogen atom, and R103 is -L10-M), where at least one of two groups which do not have L11-M is >CR102R103 and the remaining one is —O—, —S—, or >CR102R103 (R102 and R103 are preferably a hydrogen atom, —NR8R9, —OR10, or an anionic group.).
L10 and L11: A single bond, or a group obtained by combining one or two or more of an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, —O—, —S—, >C═O, and >NRA
n1: It is an integer of 2 or more.
R6: A substituent represented by -L13R6A (L13 is a linking group obtained by combining one or two or more of an alkylene group, —O—, >C═O, and >NRA, and R6A is an alkyl group, a sulfanyl group, an aryl group, a heteroaryl group, or Q.)
R7: A substituent represented by -L12R7A (L12 is a linking group obtained by combining one or two or more of an alkylene group, —O—, >C═O, and >NRA, and R7A is an alkyl group, a sulfanyl group, an aryl group, a heteroaryl group, or Q.)
X1 to X3: At least two thereof are >CR2R3, and the remaining one is —O—, —S—, or >CR2R3. (R2 and R3 are preferably a hydrogen atom, —NR8R9, —OR10, or an anionic group.)
Phosphor moiety in M: A structural moiety consisting of at least one dye of a xanthene dye, a rhodamine dye, a coumarin dye, a cyanine dye, a pyrene dye, an oxazine dye, a squarylium dye, a pyridyloxazole dye, or a pyrromethene dye
m: An integer of 1 to 30
In these forms, the above-described preferred descriptions pertaining to Y1 to Y3, Z1 to Z3, W1 to W3, LL3, LL4, s, t, u, R4 to R7, L1, L2, L5, L6, X1 to X3, M, n, and m can be applied to Y1 to Y3, Z1 to Z3, W1 to W3, LL3, LL4, s, t, u, R4 to R7, L1, L2, L5, L6, X1 to X3, M, n, and m in General Formula (III). In addition, to X4 to X9, L10, L11, n1, R6, R7, X1 to X3, M, and m in General Formula (VI), the above-described preferred descriptions pertaining to X4 to X9, L10, L11, n1, R6, R7, X1 to X3, M, and m can be applied.
The compound according to the embodiment of the present invention can be bonded to a biological substance such as a protein (including a peptide), an amino acid, a nucleic acid, a nucleotide, a sugar chain, or a lipid, by at least one substituent capable of being bonded to a biological substance, where the substituent is contained in the compound, and the compound can be used as a labeled biological substance.
The substituent capable of being bonded to a biological substance can be used without particular limitation as long as it is a group for acting (including adhering) or bonding to a biological substance, and examples thereof include the substituents described in WO2002/026891A.
Specific examples of “the substituent capable of being bonded to a biological substance” include the following structures.
X means a halogen atom such as an iodine atom or a bromine atom. * represents a bonding site.
In addition to the above, it is possible to use a peptide structure (a polyamino acid structure), a long-chain alkyl group, or the like can be used as the “substituent capable of being bonded to a biological substance”.
Among them, preferred examples thereof include an N-hydroxysuccinimide ester structure (an NHS ester structure), a succinimide structure, a maleimide structure, an azide group, an acetylene group, a peptide structure (a polyamino acid structure), a long-chain alkyl group (preferably having 12 to 30 carbon atoms), and a quaternary ammonium group.
Among the compounds according to the embodiment of the present invention, specific examples of the compound having at least one substituent capable of being bonded to a biological substance also include, as a specific example, a form in which the carboxy group in the above-described exemplary compound according to the present invention is appropriately replaced with the substituent capable of being bonded to a biological substance described above. It is noted that the present invention is not limited to these compounds. For example, in the specific examples thereof, a group having a dissociative hydrogen atom such as a carboxy group or a sulfo group may adopt a salt structure by a hydrogen atom being dissociated therefrom.
The compound according to the embodiment of the present invention can be bonded to a solid support such as the above-described microparticles by a substituent capable of being bonded to at least one solid support, the substituent being contained in the compound, and it can be used as a solid support reagent.
The substituent that can be bonded to a solid support can be used without particular limitation as long as it is a group for acting on (including adhering to) or bonding to a solid support, and examples thereof preferably include the substituents described as the above-described substituent capable of being bonded to a biological substance. Among them, preferred examples thereof include an N-hydroxysuccinimide ester (NHS ester) structure, a succinimide structure, and a maleimide structure.
Among the compounds according to the embodiment of the present invention, specific examples of the compound having at least one substituent capable of being bonded to a solid support also include, as a specific example, a form in which the carboxy group in the above-described exemplary compound of the compound according to the embodiment of the present invention is appropriately replaced with the substituent capable of being bonded to a solid support described above. It is noted that the present invention is not limited to these compounds. For example, in the specific examples thereof, a group having a dissociative hydrogen atom such as a carboxy group or a sulfo group may adopt a salt structure by a hydrogen atom being dissociated therefrom.
The compound according to the embodiment of the present invention can be synthesized according to a conventional method. For example, it can be synthesized based on the peptide synthesis such as solid phase peptide synthesis, and a method that uses an automatic peptide synthesizer described in WO2018/174078A can also be preferably applied. The phosphor moiety, the physiologically active substance moiety, the prodrug moiety, and the radioactive isotope-containing moiety can also be synthesized based on a conventional method and introduced into the compound according to the embodiment of the present invention.
A compound having a substituent capable of being bonded to a biological substance can also be synthesized according to a conventional method. For example, Bioconjugate Techniques (Third Edition, written by Greg T. Hermanson) can be referred to.
<<Labeled Biological Substance>>
The labeled biological substance according to the embodiment of the present invention is a substance in which the compound according to the embodiment of the present invention, is bonded to a biological substance. Since the compound according to the embodiment of the present invention has fluorescence due to the phosphor moiety and exhibits an excellent fluorescence intensity, it can be preferably used for a labeled biological substance. The bond between the compound according to the embodiment of the present invention and a biological substance may have a form in which the compound according to the embodiment of the present invention and the biological substance are directly bonded or a form of being linked via a linking group.
Preferred examples of the biological substance include a protein (including a peptide), an amino acid, a nucleic acid, a nucleotide, a sugar chain, and a lipid. Preferred examples of the protein include an antibody, and preferred examples of the lipid include a phospholipid, a fatty acid, and sterol, where a phospholipid is more preferable.
Among the above biological substances, the clinically useful substance is not particularly limited; however, examples thereof include immunoglobulins such as immunoglobulin (Ig) G, IgM, IgE, IgA, and IgD; blood plasma proteins such as complement, C-reactive protein (CRP), ferritin, α1 microglobulin, β2 microglobulin, and antibodies thereof; tumor markers such as α-fetoprotein, carcinoembryonic antigen (CEA), prostate acid phosphatase (PAP), carbohydrate antigen (CA) 19-9, and CA-125, and antibodies thereof; hormones such as luteinizing hormone (LH), follicle-stimulating hormone (FSH), human ciliated gonadotropin (hCG), estrogen, and insulin, and antibodies thereof; and viral infection-related substances of viruses such HIV and ATL, hepatitis B virus (HBV)-related antigens (HBs, HBe, and HBc), human immunodeficiency virus (HIV), adult T-cell leukemia (ATL), and antibodies thereof.
The examples thereof further include bacteria such as Corynebacterium diphtheriae, Clostridium botulinum, mycoplasma, and Treponema pallidum, and antibodies thereof; protozoa such as Toxoplasma, Trichomonas, Leishmania, Trypanosoma, and malaria parasites, and antibodies thereof; embryonic stem (ES) cells such as ELM3, HM1, KH2, v6.5, v17.2, v26.2 (derived from mice, 129, 129/SV, C57BL/6, and BALB/c), and antibodies thereof; antiepileptic drugs such as phenytoin and phenobarbital; cardiovascular drugs such as quinidine and digoxin; anti-asthma drugs such as theophylline; drugs such as antibiotics such as chloramphenicol and gentamicin, and antibodies thereof; and enzymes, extracellular toxins (for example, styrelidine O), and the like, and antibodies thereof. In addition, antibody fragments such as Fab′2, Fab, and Fv can also be used.
The specific form in which the compound according to the embodiment of the present invention and a biological substance interact with each other to be bonded to each other includes, for example, the forms described below.
However, in the form of the i) described above, the peptide in the compound of the present invention is not particularly limited as long as it is a peptide that is capable of forming a non-covalent bond or a covalent bond with a peptide in the biological substance, and preferred examples of the position where such a peptide is provided include R6 or R7 in General Formula (II).
In addition to the forms i) to v) described above, the bonding can be formed, for example, in the form 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. In addition, the method described in the same document can be appropriately referred to for the preparation of the labeled biological substance according to the embodiment of the present invention.
Among the compounds according to the embodiment of the present invention, the labeled biological substance according to the embodiment of the present invention, which is obtained from a compound having a substituent capable of being bonded to a biological substance and a biological substance that is bonded to the compound by an interaction includes the compound in which a moiety other than the substituent capable of being bonded to a biological substance is replaced with the compound according to the embodiment of the present invention, and a product thereof, in the description of the compound example and the product in paragraph 0038 of JP2019-172826A. However, the present invention is not limited to these labeled biological substances and the like.
<Reagent Containing Labeled Biological Substance>
In the reagent containing the labeled biological substance according to the embodiment of the present invention, the form of the labeled biological substance according to the embodiment of the present invention, for example, a solution form dissolved in an aqueous medium such as saline or a phosphate buffer solution, and a solid form such as a fine particle powder or a freeze-dried powder, is not particularly limited and can be appropriately selected depending on the purpose of use.
For example, in a case where the labeled biological substance according to the embodiment of the present invention is used as a fluorescence labeling reagent, it can be used as a reagent containing the labeled biological substance having any one of the forms described above.
<Use Application of Labeled Biological Substance>
The labeled biological substance according to the embodiment of the present invention, which is obtained from the compound according to the embodiment of the present invention, can exhibit an excellent fluorescence intensity and stably detect fluorescence emitted from the labeled biological substance excited by light irradiation. As a result, the labeled biological substance according to the embodiment of the present invention can be applied to various techniques using the fluorescence labeling, and it can be suitably used, for example, as a fluorescence labeling reagent in a multicolor WB or dot blotting or as a reagent for in vivo fluorescence imaging.
The fluorescence detection carried out using the labeled biological substance according to the embodiment of the present invention usually includes the following processes (i) to (iii) or (iv) to (vii). The fluorescence detection including the processes (i) to (iii) corresponds to the direct method using a primary antibody fluorescently labeled with the compound according to the embodiment of the present invention, and the fluorescence detection including the processes (iv) to (vii) corresponds to the indirect method using a secondary antibody fluorescently labeled with the compound according to the embodiment of the present invention.
Examples of the biological substance (the primary biological substance) capable of binding to the target biological substance and the biological substance (the secondary biological substance) capable of binding to the primary biological substance include the biological substances in the labeled biological substance according to the embodiment of the present invention. The above biological substance can be appropriately selected in accordance with the target biological substance (a biological substance in the test object) or the primary biological substance, and a biological substance capable of specifically binding to the biological substance in the test object or to the primary biological substance can be selected.
Examples of the protein among the target biological substances include a protein, which is a so-called disease marker. The disease marker is not particularly limited; however, examples thereof include α-fetoprotein (AFP), protein induced by vitamin K absence or antagonist II (PIVKA-II), breast carcinoma-associated antigen (BCA) 225, basic fetoprotein (BFP), carbohydrate antigen (CA) 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-seminoprotein (γ-Sm), prostate specific antigen (PSA), prostatic acid phosphatase (PAP), nerve 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), cytokeratin (CYFRA) pepsinogen (PG), C-reactive protein (CRP), serum amyloid A protein (SAA), myoglobin, creatine kinase (CK), troponin T, and ventricular muscle myosin light chain I.
The target biological substance may be a bacterium. Examples of the bacterium include a bacterium to be subjected to a cellular and microbiological test, which is not particularly limited. Specific examples thereof include Escherichia coli, Salmonella, Legionella, and bacteria causing problems in public health.
The target biological substance may be a virus. Although the virus is not particularly limited, examples of the virus antigen include hepatitis virus antigens such as hepatitis C and B virus antigens, p24 protein antigen of HIV virus, and pp65 protein antigen of cytomegalovirus (CMV), and E6 and E7 proteins of human papillomavirus (HPV).
In the above (i) or (iv), the sample containing the target biological substance is not particularly limited and can be prepared according to a conventional method.
In addition, the labeled biological substance according to the embodiment of the present invention is not particularly limited and can be prepared by bonding a biological substance capable of binding to a target biological substance to the compound according to the embodiment of the present invention, according to a conventional method. The form of the bond and the reaction to form the bond are as described above in the labeled biological substance according to the embodiment of the present invention.
In the above (v), the target biological substance may be directly bonded to the primary biological substance or may be bonded through another biological substance which is different from the target biological substance and the primary biological substance. In addition, in the above (vi), the primary biological substance in the conjugate b may be directly bonded to the secondary biological substance in the labeled biological substance B according to the embodiment of the present invention or may be bonded through another biological substance which is different from the primary biological substance and the secondary biological substance.
The labeled biological substance according to the embodiment of the present invention can be used as a fluorescently labeled antibody in both the direct method and the indirect method but is preferably used as a fluorescently labeled antibody in the indirect method.
In the above (ii) or (v) and the above (vi), the binding of the labeled biological substance or the like according to the embodiment of the present invention to the target biological substance is not particularly limited and can be carried out according to a conventional method.
In the above (iii) or (vii), the wavelength for exciting the labeled biological substance according to the embodiment of the present invention is not particularly limited as long as the wavelength is a luminescence wavelength (excitation wavelength) capable of exciting the labeled biological substance according to the embodiment of the present invention. Generally, the wavelength for excitation is preferably 300 to 1,000 nm and more preferably 400 to 800 nm.
The fluorescence excitation light source used in the present invention is not particularly limited as long as it emits light having a luminescence wavelength (excitation wavelength) capable of exciting the labeled biological substance according to the embodiment of the present invention, and for example, various laser light sources can be used. In addition, various optical filters can be used to obtain a preferred excitation wavelength or detect only fluorescence.
Other matters in the above (i) to (vii) are not particularly limited, and conditions of a method, a reagent, a device, and the like, which are generally used in the fluorescence detection using fluorescence labeling, can be appropriately selected.
In addition, also regarding the processes other than the above (i) to (vii), conditions of a method, a reagent, a device, and the like, which are generally used, can be appropriately selected in accordance with various methods using fluorescence labeling.
For example, in the multicolor WB using the labeled biological substance according to the embodiment of the present invention, it is possible to detect a target biological substance with excellent fluorescence intensity by preparing a blotted membrane according to a method generally used for a target biological substance (protein separation by electrophoresis, blotting to a membrane, and blocking of a membrane) and using the labeled biological substance according to the embodiment of the present invention as a labeled antibody (preferably, as a secondary antibody). In the dot blotting using the labeled biological substance according to the embodiment of the present invention, as in the case of the multicolor WB, it is possible to detect a target biological substance with excellent fluorescence intensity by preparing a blotted nitrocellulose membrane, a blotted PVDF (polyvinylidene fluoride) membrane, or the like according to a method generally used for a target biological substance and using the labeled biological substance according to the embodiment of the present invention as a labeled antibody (preferably, as a secondary antibody).
—Substituent Group T—
In the present invention, the preferred substituents include those selected from the following substituent group T.
In addition, in the present invention, in a case of being simply described as a substituent, the substituent refers to this substituent group T, and in a case where an individual group, for example, an alkyl group is only described, a corresponding group in the substituent group T is preferably applied.
Further, in the present invention, in a case where an alkyl group is described separately from a cyclic (cyclo)alkyl group, the alkyl group is meant to include a linear alkyl group and a branched alkyl group. On the other hand, in a case where an alkyl group is not described separately from a cyclic alkyl group, and unless otherwise specified, the alkyl group is meant to include a linear alkyl group, a branched alkyl group, and a cycloalkyl group. The same applies to groups (alkoxy group, alkylthio group, alkenyloxy group, and the like) containing a group capable of having a cyclic structure (alkyl group, alkenyl group, alkynyl group, and the like) and compounds containing a group capable of having a cyclic structure. In a case where a group is capable of forming a cyclic skeleton, the lower limit of the number of atoms of the group forming the cyclic skeleton is 3 or more and preferably 5 or more, regardless of the lower limit of the number of atoms specifically described below for the group that can adopt this structure.
In the following description of the substituent group T, a group having a linear or branched structure and a group having a cyclic structure, such as an alkyl group and a cycloalkyl group, are sometimes described separately for clarity.
The groups included in the substituent group T include the following groups.
An alkyl group (preferably having 1 to 30 carbon atoms, more preferably having 1 to 20 carbon atoms, still more preferably having 1 to 12 carbon atoms, still more preferably having 1 to 8 carbon atoms, still more preferably having 1 to 6 carbon atoms, and particularly preferably having 1 to 3 carbon atoms), an alkenyl group (preferably having 2 to 30 carbon atoms, more preferably having 2 to 20 carbon atoms, still more preferably having 2 to 12 carbon atoms, still more preferably having 2 to 6 carbon atoms, and even still more preferably having 2 to 4 carbon atoms), an alkynyl group (preferably having 2 to 30 carbon atoms, still more preferably having 2 to 20 carbon atoms, still more preferably having 2 to 12 carbon atoms, still more preferably having 2 to 6 carbon atoms, and even still more preferably having 2 to 4 carbon atoms), a cycloalkyl group (preferably having 3 to 20 carbon atoms), a cycloalkenyl group (preferably having 5 to 20 carbon atoms), an aryl group (it may be a monocyclic group or may be a fused ring group (preferably a fused ring group of 2 to 6 rings); in a case of a fused ring group, the fused ring group consists of a 5- to 7-membered ring; and the aryl group preferably has 6 to 40 carbon atoms, more preferably has 6 to 30 carbon atoms, still more preferably has 6 to 26 carbon atoms, and particularly preferably has 6 to 10 carbon atoms), a heterocyclic group (it has, as a ring-constituting atom, at least one of a nitrogen atom, oxygen atom, a sulfur atom, a phosphorus atom, a silicon atom, or selenium atom, may be a monocyclic ring, or may be a fused ring group (preferably a fused ring group of 2 to 6 rings); in a case of a monocyclic group, the monocyclic ring is preferably a 5- to 7-membered ring and more preferably a 5-membered or 6-membered ring; the heterocyclic group preferably has 2 to 40 carbon atoms and more preferably having 2 to 20 carbon atoms; and the heterocyclic group includes an aromatic heterocyclic group (a heteroaryl group) and an aliphatic heterocyclic group), an alkoxy group (preferably having 1 to 20 carbon atoms, and more preferably having 1 to 12 carbon atoms), an alkenyloxy group (preferably having 2 to 20 carbon atoms, and more preferably having 2 to 12 carbon atoms), and an alkynyloxy group (preferably having 2 to 20 carbon atoms, and more preferably having 2 to 12 carbon atoms), a cycloalkyloxy group (preferably having 3 to 20 carbon atoms), an aryloxy group (preferably having 6 to 40 carbon atoms, more preferably having 6 to 26 carbon atoms, and still more preferably having 6 to 14 carbon atoms), a heterocyclic oxy group (preferably having 2 to 20 carbon atoms), a polyalkyleneoxy group,
an alkoxycarbonyl group (preferably having 2 to 20 carbon atoms), a cycloalkoxycarbonyl group (preferably having 4 to 20 carbon atoms), an aryloxycarbonyl group (preferably having 6 to 20 carbon atoms), an amino group (preferably having 0 to 20 carbon atoms; the amino group includes an unsubstituted amino group (—NH2), a (mono- or di-) alkylamino group, a (mono- or di-) alkenylamino group, a (mono- or di-) alkynylamino group, a (mono- or di-) cycloalkylamino group, a (mono- or di-) cycloalkenylamino group, a (mono- or di-) arylamino group, or a (mono- or di-) heterocyclic amino group, where each of the above groups substituting an unsubstituted amino group has the same definition as the corresponding group in the substituent group T), a sulfamoyl group (preferably having 0 to 20 carbon atoms; it is preferably an alkyl, cycloalkyl, or aryl sulfamoyl group), an acyl group (preferably having 1 to 20 carbon atoms, and more preferably having 2 to 15 carbon atoms; it includes —C(═O)H, an alkylcarbonyl group, a cycloalkylcarbonyl group, an arylcarbonyl group, and a heterocyclic carbonyl group), an acyloxy group (preferably having 1 to 20 carbon atoms), a carbamoyl group (preferably having 1 to 20 carbon atoms; it is preferably an alkyl, cycloalkyl, or aryl carbamoyl group),
an acylamino group (preferably having 1 to 20 carbon atoms), a sulfonamide group (preferably having 0 to 20 carbon atoms and preferably an alkyl, cycloalkyl, or aryl sulfonamide group), an alkylthio group (preferably having 1 to 20 carbon atoms and more preferably having 1 to 12 carbon atoms), a cycloalkylthio group (preferably having 3 to 20 carbon atoms), an arylthio group (preferably having 6 to 40 carbon atoms, more preferably having 6 to 26 carbon atoms, and still more preferably having 6 to 14 carbon atoms), a heterocyclic thio group (preferably having 2 to 20 carbon atoms), an alkyl, cycloalkyl, or aryl sulfonyl group (preferably having 1 to 20 carbon atoms),
a silyl group (preferably having 1 to 30 carbon atoms and more preferably having 1 to 20 carbon atoms; it is preferably a silyl group at which an alkyl, aryl, alkoxy, or aryloxy has been substituted), a silyloxy group (preferably having 1 to 20 carbon atoms: it is preferably a silyloxy group at which an alkyl, aryl, alkoxy, or aryloxy has been substituted), a hydroxy group, a cyano group, a nitro group, a halogen atom (for example, a fluorine atom, a chlorine atom, a bromine atom or an iodine atom), an oxygen atom (specifically, >CH2 which constitutes a ring is replaced with >C═O), a carboxy group (—CO2H), a phosphono group [—PO(OH)2], a phosphonooxy group [—O—PO(OH)2], a sulfo group (—SO3H), a borate group [—B(OH)2], an onio group (also referred to as a cationic group; it includes an ammonio group including a cyclic ammonio group, a sulfonio group, and a phosphonio group, and it preferably has 0 to 30 carbon atoms and more preferably has 1 to 20 carbon atoms), a sulfanyl group (—SH), a guanidino group (—NHC(═NH)NH2), an amino acid residue, and a polyamino acid residue.
(Anionic group)
In the present invention, the anionic group may be any group having an anion. Examples of such an anionic group include a carboxy group, a phosphono group (a phosphonate group, —PO(OH)2), a phosphonooxy group (a phosphate group, —OPO(OH)2), and a sulfo group, where a phosphono group, a phosphonooxy group, or a sulfo group is preferable, and a phosphonooxy group or a sulfo group is more preferable.
The anionic group may dissociate a hydrogen ion to have an ionic structure, or it may have a salt structure. To the monovalent or polyvalent cation in a case where the anionic group has a salt structure, the description of the monovalent or polyvalent cation in the description of the salt structure described above can be preferably applied.
(Cationic Group)
In the present invention, the cationic group may be any group having a cation. Examples of such a cationic group include a group having a quaternary ammonium ion and a group having a quaternary phosphonium ion, where a group having a quaternary ammonium ion is preferable. It is noted that preferred examples of the substituent possessed by N+ in the group having a quaternary ammonium ion and the substituent possessed by P+ in the group having a quaternary phosphonium ion include an alkyl group and an aryl group, where groups in which all the substituents possessed by N+ and P+ are alkyl groups are preferable.
The cationic group may have a salt structure in addition to the ionic structure. Examples of the monovalent or polyvalent anion in a case where the cationic group has a salt structure include halide ions such as F− and Cl−, and monovalent or polyvalent organic anions such as BF4−, PF6−, and a bis(trifluoromethylsulfonyl)imide ion.
(Polyalkyleneoxy Group)
In the present invention, the polyalkyleneoxy group may be any group represented by -(L-O)gRE.
The L represents an alkylene group obtained by removing one hydrogen atom from an alkyl group in the substituent group T described above, where it preferably has 2 to 4 carbon atoms, more preferably has 2 or 3 carbon atoms, and still more preferably has 2 carbon atoms. The number of carbon atoms contained in the shortest chain that links two carbon atoms which are bonding sites of the group is preferably 0 to 2, more preferably 0 or 1, and still more preferably 0. That is, L is most preferably an ethylene group.
the g means an average repetition number (simply, also referred to as a repetition number), which is preferably 1 to 24, more preferably 1 to 12, and still more preferably 4 to 12. Even in a case where the repetition number is small, for example, even in a case where g is 1, a proper hydrophilicity and a proper excluded volume effect can be exhibited.
The RE represents a hydrogen atom or an alkyl group. For the alkyl group which can be adopted as RE, the description of the alkyl group in the above-described substituent group T can be preferably applied, and among the above, an alkyl group having 1 to 3 carbon atoms is preferable. The alkyl group which can be adopted as RE may have a substituent.
In addition, examples of the group obtained by combining a plurality of substituents selected from the substituent group T include the above-described alkyl group, alkenyl group, alkynyl group, cycloalkyl group, cycloalkenyl group, aryl group, heterocyclic group, alkoxy group, alkenyloxy group, alkynyloxy group, cycloalkyloxy group, aryloxy group, heterocyclic oxy group, alkoxycarbonyl group, cycloalkoxycarbonyl group, aryloxycarbonyl group, amino group, sulfamoyl group, acyl group, acyloxy group, carbamoyl group, acylamino group, sulfonamide group, alkylthio group, cycloalkylthio group, arylthio group, heterocyclic thio group, and an alkyl, cycloalkyl, or aryl sulfonyl group, which have, as a substituent, an anionic group (a carboxy group, a phosphono group, or a sulfo group), a cationic group (an onio group), an amino acid residue, a polyamino acid residue, or a —(CH2—CH2—O)b-alkyl group (b has the same meaning as b in R1 to R4 in General Formula (α) described above).
The substituent selected from the substituent group T is more preferably an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, a heterocyclic group, an alkoxy group, a cycloalkoxy group, an aryloxy group, an acyl group, an alkoxycarbonyl group, a cycloalkoxycarbonyl group, an amino group, an acylamino group, a carbamoyl group, a cyano group, a halogen atom, an anionic group, or a cationic group, and particularly preferably an alkyl group, an alkenyl group, an aryl group, a heterocyclic group, an alkoxy group, an acyl group, an alkoxycarbonyl group, an amino group, an acylamino group, a carbamoyl group, a cyano group, an anionic group, or a cationic group.
In addition to the group obtained by combining a plurality of substituents selected from the substituent group T described above, the substituent selected from the substituent group T also includes a group obtained by combining a plurality of the above groups, unless otherwise specified. For example, in a case where a compound, a substituent, or the like contains an alkyl group, an alkenyl group, or the like, the alkyl group, the alkenyl group, or the like may be substituted or unsubstituted. In addition, in a case where a compound, a substituent, or the like contains an aryl group, a heterocyclic group, or the like, the aryl group, the heterocyclic group, or the like may be a monocyclic ring or a fused ring moiety, and may be substituted or unsubstituted.
Hereinafter, the present invention will be described in more detail based on Examples, but the present invention is not limited thereto. It is noted that room temperature means 25° C.
Compounds (1) to (4) and (6) to (9) according to the embodiment of the present invention and a comparative compound (1) are shown below.
It is noted that M1 to M4 in the compounds indicate the phosphor moieties, which consist of structures represented by the following structural formulae, respectively, and * represents a bonding site. In addition, in each compound, the sulfo group or the phosphonooxy group may include a salt structure (for example, a potassium salt, a sodium salt, a triethylammonium (TEA) salt, or an N,N-diisopropylethylammonium (DIPEA) salt), even unless otherwise specified.
The compound (2) and the comparative compound (1) are compounds in which the distance between two M1's is about the same.
In addition, the fluorescence intensity of the phosphor moieties M1 and M2 alone is about the same, and it is conceived that the difference in the phosphor moiety does not significantly contribute to the evaluation results of various fluorescence intensities pertaining to the labeled antibody described later.
The method of synthesizing each compound and a labeled antibody will be described in detail below; however, the starting materials, the dye intermediates, and the synthetic routes are not limited thereto.
It is noted that the abbreviations used in the synthesis of the respective compounds described below are as follows.
In addition, % v/v means a percentage in terms of volume.
Unless otherwise specified, SNAP Ultra C18 (product name, manufactured by Biotage, LLC) or Sfar C18 (product name, manufactured by Biotage, LLC) was used as the carrier in the reverse phase column chromatography, and Hi-Flash Column (product name, manufactured by Yamazen Corporation) was used as a carrier in the normal phase column chromatography.
The mixing ratio in the eluent used in the reverse phase column chromatography or the normal phase column chromatography is in terms of the volume ratio. For example, “acetonitrile:water=from 0:100 to 20:80” means that the eluent of “acetonitrile:water=0:100” was changed to an eluent of “acetonitrile:water=20:80”.
For the preparative high performance liquid chromatography (HPLC), 2767 (product name, manufactured by Waters Corporation) was used.
The MS spectrum was measured by ACQUITY SQD LC/MS System [product name, manufactured by Waters Corporation, ionization method: electrospray Ionization (ESI)] or LCMS-2010EV [product name, manufactured by Shimadzu Corporation, ionization method: an ionization method simultaneously carrying out ESI and atmospheric pressure chemical ionization (APCI)].
It is noted that in the synthesis of each compound, the synthesis of the peptide chain was carried out according to the general method of the peptide solid phase method described in WO2018/174078A.
[General Method of Solid Phase Peptide Synthesis Method Using Automatic Peptide Synthesizer]
Solid phase peptide synthesis was carried out using an automatic peptide synthesizer (product name: SyroI, manufactured by biotage, LLC). The synthesizer was set with Rink Amide-ChemMatrix (registered trade name, manufactured by Biotage, LLC), an N-methyl-2-pyrrolidone (NMP) solution of Fmoc amino acid (0.5 mol/L), an NMP solution of cyano-hydroxyimino-acetic acid ethyl ester (1.0 mol/L) and diisopropylethylamine (0.1 mol/L), an NMP solution of diisopropylcarbodiimide (1.0 mol/L), an NMP solution of piperidine (20% v/v), and an NMP solution of acetic anhydride (20% v/v), and synthesis was carried out according to the manual. A procedure of Fmoc deprotection (20 minutes), washing with NMP, condensation of Fmoc amino acids (1 hour), and washing with NMP was set as one cycle, and this cycle was repeated to elongate the peptide chain.
[Synthesis of Compound (M1-1)]
A compound (M1-1) was synthesized based on the following scheme. It is noted that the compound (1-C) is the same as the compound (1-C) in the synthesis of a compound (M2-1) described later. The results of the MS measurement of the compound (5) were as follows.
MS (ESI m/z): (M+H+)+=1,723, (M−H+)−=1,721
<Synthesis of Compound (1)>
A compound (1) was synthesized based on the following scheme.
1) Synthesis of Compound (1-1)
Solid phase peptide synthesis was carried out using H-Gly-Trt(2-Cl)-Resin (manufactured by Watanabe Chemical Industries, Ltd., 0.93 mmol/g, 53.8 mg) as a starting raw material. The elongation that was carried out by using N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycine (Fmoc-Gly-OH) was repeated for 4 cycles. Nε-(tert-butoxycarbonyl)-Nα-[(9H-fluoren-9-ylmethoxy)carbonyl)-L-lysine (Fmoc-Lys(Boc)-OH) was subjected to elongation, and the elongation that was carried out by using N-(9-fluorenylmethoxycarbonyl)-L-proline (Fmoc-Pro-OH) was repeated for 12 cycles. Nε-(tert-butoxycarbonyl)-Nα-[(9H-fluoren-9-ylmethoxy)carbonyl)-L-lysine (Fmoc-Lys(Boc)-OH) was subjected to elongation, and then, an NMP solution of piperidine (20% v/v) was added thereto to carry out a reaction for 20 minutes, thereby carrying out the deprotection of the Fmoc group, and then, an NMP solution of acetic anhydride (20% v/v) was added thereto to carry out a reaction for 10 minutes, thereby subjecting the N-terminal amino group to acetylation. After completion of the elongation, the resin was washed with dichloromethane, and then the solvent was distilled off under reduced pressure. 2.0 mL of a mixed solution of TFA:triisopropylsilane:water=95:2.5:2.5 was added thereto, and the peptide was cut out and deprotected. After 2 hours, the resin was filtered out, and methyl-t-butyl ether (12 mL) was added to the filtrate to generate a solid. The solid was precipitated by centrifugation, and then the supernatant was removed. The solid was washed with methyl-t-butyl ether, and then the solvent was distilled off under reduced pressure to obtain 65.2 mg of a white solid compound (1-1).
2) Synthesis of Compound (1)
1.5 mg of the compound (1-1), 150 μL of N,N-dimethylformamide (DMF), 1 μL of triethylamine (Et3N), and 3.75 mg of the compound (M1-1) were placed in an eggplant flask having a capacity of 10 mL, and stirring was carried out at room temperature for 1 hour. Thereafter, the reaction solution was concentrated and purified by preparative HPLC, and freeze drying was followed to obtain 2.2 mg of a compound (1). The results of the MS measurement of the compound (1) were as follows.
MS (ESI m/z): (M+H+)+=5,174, (M−H+)−=5,172
3) Synthesis of Compound (1-NHS)
220 μL of N,N-dimethylformamide (DMF), 1 mg of N,N,N′,N′-tetramethyl-O—(N-succinimidyl)uronium hexafluorophosphate, and 1.3 μL of triethylamine (Et3N) were added to 2.2 mg of the compound (1), and stirring was carried out for 1 hour. Then, the solvent was distilled off under reduced pressure, ethyl acetate was added, the supernatant was removed, and vacuum drying was carried out to obtain a compound (1-NHS).
<Synthesis of Compound (2)>
1) Synthesis of Compound (2-7)
A compound (2-7) was synthesized according to the following scheme.
(i) Synthesis of Compound (2-2)
300 mg of the compound (4-1) described in WO2020/175473A as the compound (2-1), 3 mL of tetrahydrofuran (THF), 229.8 mg of Nε-(tert-butoxycarbonyl)-Nα-[(9H-fluoren-9-ylmethoxy)carbonyl)-L-lysine (Fmoc-Lys(Boc)-OH), 76.8 μL of diisopropylcarbodiimide, and 8.0 mg of 4-dimethylaminopyridine were placed in an eggplant flask having a capacity of 50 mL, and stirring was carried out at room temperature for 2 hours. A solid precipitated by adding acetonitrile (30 mL) was subjected to filtration and drying under reduced pressure to obtain 350 mg of a compound (2-2).
(ii) Synthesis of Compound (2-3)
Solid phase peptide synthesis was carried out using H-Pro-Trt(2-Cl)-Resin (manufactured by Watanabe Chemical Industries, Ltd., 0.94 mmol/g, 53.2 mg) as a starting raw material. The elongation that was carried out by using N-(9-fluorenylmethoxycarbonyl)-L-proline (Fmoc-Pro-OH) was repeated for 11 cycles to elongate Nε-(tert-butoxycarbonyl)-Nα-[(9H-fluoren-9-ylmethoxy)carbonyl)-L-lysine (Fmoc-Lys(Boc)-OH). Subsequently, an NMP solution of piperidine (20% v/v) was added thereto to carry out a reaction for 20 minutes, thereby carrying out the deprotection of the Fmoc group, and then, an NMP solution of acetic anhydride (20% v/v) was added thereto to carry out a reaction for 10 minutes, thereby subjecting the N-terminal amino group to acetylation. After completion of the elongation, the resin was washed with dichloromethane, and then the solvent was distilled off under reduced pressure. 2.0 mL of a mixed solution of hexafluoro-2-propanol (HFIP):dichloromethane (CH2Cl2)=1:4 was added thereto, and the peptide was cut out. After 30 minutes, the resin was filtered out to concentrate the filtrate, which was subsequently purified by reverse phase column chromatography to obtain 59.3 mg of a white solid compound (2-3).
(iii) Synthesis of Compound (2-4) 20 mg of the compound (2-2), 400 μL of chloroform (CHCl3), and 4.4 μL of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) were placed in an eggplant flask having a capacity of 10 mL, and stirring was carried out at 35° C. for 1 hour. 1.9 μL of methanesulfonic acid (MsOH), 15.3 μL of N,N-diisopropylethylamine (DIPEA), 25.5 mg of the compound (2-3), and 23.1 mg of (7-azabenzotriazole-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP) were placed therein, and stirring was carried out at 35° C. for 1 hour. A solid precipitated by adding acetonitrile (4 mL) was subjected to filtration and drying under reduced pressure to obtain 34.0 mg of a compound (2-4).
(iv) Synthesis of Compound (2-5)
30.0 mg of the compound (2-4), 600 μL of chloroform (CHCl3), 60 μL of 2,2,2-trifluoroethanol (TFE), and 6 μL of trifluoroacetic acid (TFA) were placed in an eggplant flask having a capacity of 10 mL, and stirring was carried out at room temperature for 4 hours. Thereafter, the reaction solution was filtered, and 6 mL of methanol (MeOH) was added to the filtrate to generate a precipitate, which was subsequently centrifuged. The recovered compound was purified by preparative HPLC and subjected to freeze drying to obtain 16.5 mg of a compound (2-5).
(v) Synthesis of Compound (2-6)
10 mg of the compound (2-5), 200 μL of water, 200 μL of N,N-dimethylformamide (DMF), 3.9 mg of tert-butyl 1-amino-3,6,9,12-tetraoxadecane-15-ate, and 6.7 mg of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) were placed in an eggplant flask having a capacity of 10 mL, and stirring was carried out at room temperature for 2 hours. Thereafter, the reaction solution was concentrated and purified by preparative HPLC, and freeze drying was followed to obtain 8.9 mg of a compound (2-6).
(vi) Synthesis of Compound (2-7)
7.0 mg of the compound (2-6) and 200 μL of trifluoroacetic acid (TFA) were placed in an eggplant flask having a capacity of 10 mL, and stirring was carried out at room temperature for 1 hour. Thereafter, the reaction solution was concentrated and purified by preparative HPLC, and freeze drying was followed to obtain 5.9 mg of a compound (2-7).
2) Synthesis of Compound (2)
3.2 mg of the above-described compound (2) was synthesized from 2.0 mg of the compound (2-7) in the same manner, except that in the synthesis of the compound (1) described above, the compound (2-7) was used instead of the compound (1-1). The results of the MS measurement of the compound (2) were as follows.
MS (ESI m/z): (M+H+)+=5,136, (M−H+)−=5,134
3) Synthesis of Compound (2-NHS)
The following compound (2-NHS) was synthesized in the same manner, except that in the synthesis of the compound (1-NHS) described above, the compound (2) was used instead of the compound (1).
<Synthesis of Compound (3)>
1) Synthesis of Compound (3-6)
A compound (3-6) was synthesized according to the following scheme.
(i) Synthesis of Compound (3-1)
Solid phase peptide synthesis was carried out using H-Pro-Trt(2-Cl)-Resin (0.94 mmol/g, 638.4 mg) as a starting raw material. The elongation that was carried out by using N-(9-fluorenylmethoxycarbonyl)-L-proline (Fmoc-Pro-OH) was repeated for 5 cycles to elongate N-(ter-butoxycarbonyl)-Nα-[(9H-fluoren-9-ylmethoxy)carbonyl)-L-lysine (Fmoc-Lys(Boc)-OH). After completion of the elongation, the resin was washed with dichloromethane, and then the solvent was distilled off under reduced pressure. A 2.0 mL of a mixed solution of hexafluoro-2-propanol (HFIP):dichloromethane (CH2Cl2) 1:4 was added thereto, and the peptide was cut out. After 30 minutes, the resin was filtered out to concentrate the filtrate, which was subsequently purified by reverse phase column chromatography to obtain 479.3 mg of a white solid compound (3-1).
(ii) Synthesis of Compound (3-2)
98 mg of the compound (2-2), 1 mL of chloroform (CHCl3), and 21.5 μL of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) were placed in an eggplant flask having a capacity of 50 mL, and stirring was carried out at 35° C. for 1 hour. 9.5 μL of methanesulfonic acid (MsOH), 50.2 μL of N,N-diisopropylethylamine (DIPA), 50.0 mg of the compound (3-1), and 49.6 mg of (7-azabenzotriazole-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP) were placed therein, and stirring was carried out at 35° C. for 3 hours. A solid precipitated by adding acetonitrile (10 mL) was subjected to filtration and drying under reduced pressure.
The same operation was repeated twice to carry out the elongation of the compound (3-1), thereby obtaining 230 mg of a compound (3-2).
(iii) Synthesis of Compound (3-3)
230 mg of the compound (3-2), 5 mL of chloroform (CHCl3), and 18.1 μL of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) were placed in an eggplant flask having a capacity of 100 mL, and stirring was carried out at 35° C. for 1 hour. 7.9 μL of methanesulfonic acid (MsOH), 84.4 μL of N,N-diisopropylethylamine (DIPEA), and 34.3 μL of acetic anhydride (Ac2O) were placed therein, and stirring was carried out at 35° C. for 1 hour. A solid precipitated by adding acetonitrile (50 mL) was subjected to filtration and drying under reduced pressure to obtain 209 mg of a compound (3-3).
(iv) Synthesis of Compounds (3-4) to (3-6)
100 mg of a compound (3-4) was obtained from 209 mg of the compound (3-3) in the same manner as in the synthesis of the compound (2-5).
84.2 mg of a compound (3-5) was obtained from 100 mg of the compound (3-4) in the same manner as in the synthesis of the compound (2-6).
75.9 mg of a compound (3-6) was obtained from 84.2 mg of the compound (3-5) in the same manner as in the synthesis of the compound (2-7).
2) Synthesis of Compound (3)
3.6 mg of the above-described compound (3) was synthesized from 2.0 mg of the compound (3-6) in the same manner, except that in the synthesis of the compound (1) described above, the compound (3-6) was used instead of the compound (1-1). The results of the MS measurement of the compound (3) were as follows.
MS (ESI m/z): (M+H+)+=9,382, (M−H+)−=9,380
3) Synthesis of Compound (3-NHS)
The following compound (3-NHS) was synthesized in the same manner, except that in the synthesis of the compound (1-NHS) described above, the compound (3) was used instead of the compound (1).
<Synthesis of Compound (4)>
Solid phase peptide synthesis was carried out using H-Gly-Trt(2-Cl)-Resin (manufactured by Watanabe Chemical Industries, Ltd., 0.93 mmol/g, 53.8 mg) as a starting raw material. The elongation that was carried out by using N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycine (Fmoc-Gly-OH) was repeated for 4 cycles. Nε-(tert-butoxycarbonyl)-Nα-[(9H-fluoren-9-ylmethoxy)carbonyl)-L-lysine (Fmoc-Lys(Boc)-OH) was subjected to elongation, and the elongation that was carried out by using N-(9-fluorenylmethoxycarbonyl)-L-proline (Fmoc-Pro-OH) was repeated for 12 cycles. After subjecting N-(9-fluorenylmethoxycarbonyl)-O-phospho-L-tyrosine (Fmoc-Tyr(PO3H2)—OH) and Nε-(tert-butoxycarbonyl)-Nα-[(9H-fluoren-9-ylmethoxy)carbonyl)-L-lysine (Fmoc-Lys(Boc)-OH) to elongation, an NMP solution of piperidine (20% v/v) was added thereto to carry out a reaction for 20 minutes, thereby carrying out the deprotection of the Fmoc group, and then, an NMP solution of acetic anhydride (20% v/v) was added thereto to carry out a reaction for 10 minutes, thereby subjecting the N-terminal amino group to acetylation. After completion of the elongation, the resin was washed with dichloromethane, and then the solvent was distilled off under reduced pressure. 2.0 mL of a mixed solution of TFA:triisopropylsilane:water=95:2.5:2.5 was added thereto, and the peptide was cut out and deprotected. After 2 hours, the resin was filtered out, and methyl-t-butyl ether (12 mL) was added to the filtrate to generate a solid. The solid was precipitated by centrifugation, and then the supernatant was removed. The solid was washed with methyl-t-butyl ether, and then the solvent was distilled off under reduced pressure to obtain 25.2 mg of the following white solid compound (4-1).
2) Synthesis of Compound (4)
3.5 mg of the above-described compound (4) was synthesized from 2.0 mg of the compound (4-1) in the same manner, except that in the synthesis of the compound (1) described above, the compound (4-1) was used instead of the compound (1-1). The results of the MS measurement of the compound (4) were as follows.
MS (ESI m/z): (M+H+)+=5,417, (M−H+)−=5,415
3) Synthesis of Compound (4-NHS)
The following compound (4-NHS) was synthesized in the same manner, except that in the synthesis of the compound (1-NHS) described above, the compound (4) was used instead of the compound (1).
<Synthesis of Comparative Example Compound (1)>
1) Synthesis of Compound (5-7)
A compound (5-7) was synthesized according to the following scheme.
(i) Synthesis of Compound (5-3)
750 mg of NF-(1-(4,4-dimethyl-2,6-dioxocyclohexa-1-ylidene)-3-methylbutyl)-Nα-[(9H-fluoren-9-ylmethoxy)carbonyl)-L-lysine (Fmoc-Lys(ivDde)-OH) as the compound (5-1), 15 mL of tetrahydrofuran (THF), 461 mg of tert-butyl 1-amino-3,6,9,12-tetraoxadecane-15-ate, 595 mg of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), and 273 μL of N,N-diisopropylethylamine (DIPEA) were placed in an eggplant flask having a capacity of 50 mL, and stirring was carried out at room temperature for 4 hours. Thereafter, liquid separation was carried out with ethyl acetate and saturated saline, and then the organic layer was distilled off under reduced pressure to obtain a compound (5-2) as a crude product.
The compound (5-2), 7.5 mL of tetrahydrofuran (THF), and 258 μL of piperidine were placed in an eggplant flask having a capacity of 50 mL, and stirring was carried out at room temperature for 12 hours. Thereafter, liquid separation was carried out with ethyl acetate and saturated saline, the organic layer was distilled off under reduced pressure, and the purification was carried out by normal phase column chromatography to obtain 855 mg of a compound (5-3).
(ii) Synthesis of Compound (5-4)
300 mg of the compound (5-3), 7.7 mL of dichloromethane (CH2Cl2), 83.2 μL of acetic anhydride (Ac2O), and 139 μL of triethylamine (TEA) were placed in an eggplant flask having a capacity of 50 mL, and stirring was carried out at room temperature for 1 hour. Thereafter, liquid separation was carried out with chloroform (CHCl3) and saturated saline, and then the organic layer was distilled off under reduced pressure to obtain a crude product.
The obtained crude product and 3.5 mL of trifluoroacetic acid (TFA) were placed in an eggplant flask having a capacity of 50 mL, and stirring was carried out at room temperature for 30 minutes. Thereafter, the reaction solution was concentrated and purified by reverse phase column chromatography, and freeze drying was followed to obtain 287 mg of a compound (5-4).
(iii) Synthesis of Compound (5-5)
287 mg of the compound (5-4), 5.7 mL of tetrahydrofuran (THF), 172 mg of tert-butyl 1-amino-3,6,9,12-tetraoxadecane-15-ate, 255 mg of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), and 117 μL of N,N-diisopropylethylamine (DIPEA) were placed in an eggplant flask having a capacity of 50 mL, and stirring was carried out at room temperature for 4 hours. Thereafter, liquid separation was carried out with ethyl acetate and saturated saline, and then the organic layer was distilled off under reduced pressure to obtain a compound as a crude product.
The obtained crude product and 2.5 mL of trifluoroacetic acid (TFA) were placed in an eggplant flask having a capacity of 50 mL, and stirring was carried out at room temperature for 30 minutes. Thereafter, the reaction solution was concentrated and purified by reverse phase column chromatography, and freeze drying was followed to obtain 281 mg of a compound (5-5).
(iv) Synthesis of Compound (5-6)
140 mg of the compound (5-5), 2.8 mL of tetrahydrofuran (THF), 103 mg of the compound (5-3), 71.9 mg of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), and 33.0 μL of N,N-diisopropylethylamine (DIPEA), were placed in an eggplant flask having a capacity of 50 mL, and stirring was carried out at room temperature for 4 hours. Thereafter, liquid separation was carried out with ethyl acetate and saturated saline, and then the organic layer was distilled off under reduced pressure to obtain a compound as a crude product.
The obtained crude product, 2.8 mL of tetrahydrofuran (THF), and 30.6 μL of hydrazine monohydrate were placed in an eggplant flask having a capacity of 50 mL, and stirring was carried out at room temperature for 12 hours. Thereafter, the reaction solution was concentrated and purified by reverse phase column chromatography, and freeze drying was followed to obtain 152 mg of a compound (5-6).
(v) Synthesis of Compound (5-7)
108 mg of the compound (5-6) and 3 mL of trifluoroacetic acid (TFA) were placed in an eggplant flask having a capacity of 50 mL, and stirring was carried out at room temperature for 1.5 hours. Thereafter, the reaction solution was concentrated and purified by reverse phase column chromatography, and freeze drying was followed to obtain 100 mg of a compound (5-7).
2) Synthesis of Comparative Compound (1)
3.9 mg of the above-described comparative compound (1) was synthesized from 2.2 mg of the compound (5-7) in the same manner, except that in the synthesis of the compound (1) described above, the compound (5-7) was used instead of the compound (1-1). The results of the MS measurement of the comparative compound (1) were as follows.
MS (ESI m/z): (M+H+)+=4,466, (M−H+)−=4,464
3) Synthesis of Comparative Compound (1-NHS)
The following comparative compound (1-NHS) was synthesized in the same manner, except that in the synthesis of the compound (1-NHS) described above, the comparative compound (1) was used instead of the compound (1).
[Synthesis of Compound (M2-1)]
A compound (M2-1) was synthesized based on the following scheme. The results of the MS measurement of the compound (M2-13) were as follows.
MS (ESI m/z): (M+H+)+=1,384, (M−H+)−=1,382
<Synthesis of Compound (6)>
1) Synthesis of Compound (6-8)
A compound (6-8) was synthesized according to the following scheme.
(i) Synthesis of Compound (6-1)
Solid phase peptide synthesis was carried out using H-Pro-Trt(2-Cl)-Resin (manufactured by Watanabe Chemical Industries, Ltd., 0.93 mmol/g, 53.8 mg) as a starting raw material. N-(9-fluorenylmethoxycarbonyl)-L-proline (Fmoc-Pro-OH) and (2S,4S)-(tert-butoxycarbonyl)-4-amino-1-(9H-fluoren-9-ylmethoxy)carbonyl)-pyrrolidine-2-carboxylic acid (Fmoc-L-Pro(4-NHBoc)-OH(2S,4S)) were subjected to elongation, and the elongation that was carried out by using N-(9-fluorenylmethoxycarbonyl)-L-proline (Fmoc-Pro-OH) was repeated for 3 cycles. (2S,4S)-(tert-butoxycarbonyl)-4-amino-1-(9H-fluoren-9-ylmethoxy)carbonyl)-pyrrolidine-2-carboxylic acid (Fmoc-L-Pro(4-NHBoc)-OH (2S,4S)) was subjected to elongation, and the elongation using N-(9-fluorenylmethoxycarbonyl)-L-proline (Fmoc-Pro-OH) was repeated for 3 cycles. (2S,4S)-(tert-butoxycarbonyl)-4-amino-1-(9H-fluoren-9-ylmethoxy)carbonyl)-pyrrolidine-2-carboxylic acid (Fmoc-L-Pro(4-NHBoc)-OH(2S,4S)) and N-(9-fluorenylmethoxycarbonyl)-L-proline (Fmoc-Pro-OH) were subjected to elongation. After completion of the elongation, the resin was washed with dichloromethane, and then the solvent was distilled off under reduced pressure. 2.0 mL of a mixed solution of TFA:triisopropylsilane:water=95:2.5:2.5 was added thereto, and the peptide was cut out and deprotected. After 2 hours, the resin was filtered out, and methyl-t-butyl ether (12 mL) was added to the filtrate to generate a solid. The solid was precipitated by centrifugation, and then the supernatant was removed. The solid was washed with methyl-t-butyl ether, and then the solvent was distilled off under reduced pressure to obtain 51.2 mg of a white solid compound (6-1).
(ii) Synthesis of Compound (6-2)
352 mg of the compound (6-1), 3.5 mL of chloroform (CHCl3), 423 μL of N-diisopropylethylamine (DIPEA), and 115 μL of acetic anhydride (Ac2O) were placed in an eggplant flask having a capacity of 50 mL, and stirring was carried out at room temperature for 1 hour. Thereafter, the reaction solution was concentrated and purified by preparative HPLC, and freeze drying was followed to obtain 314 mg of a compound (6-2).
(iii) Synthesis of Compound (6-3)
593 mg of the above-described compound (6-3) was synthesized from 500 mg of the compound (2-1) in the same manner, except that in the synthesis of the compound (2-2) described above, N-[(9H-fluoren-9-ylmethoxy)carbonyl]-β-alanine (Fmoc-βAla-OH) was used instead of Nε-(tert-butoxycarbonyl)-Nα-[(9H-fluoren-9-ylmethoxy)carbonyl)-L-lysine (Fmoc-Lys(Boc)-OH).
108 mg of the compound (6-3), 2.2 mL of chloroform (CHCl3), and 26.8 μL of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) were placed in an eggplant flask having a capacity of 50 mL, and stirring was carried out at 35° C. for 1 hour. 11.7 μL of methanesulfonic acid (MsOH), 93.3 μL of N,N-diisopropylethylamine (DIPEA), 169 mg of the compound (6-2), and 141 mg of (7-azabenzotriazole-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP) were placed therein, and stirring was carried out at 35° C. for 3 hours. A solid precipitated by adding acetonitrile (10 mL) was subjected to filtration and drying under reduced pressure to obtain 219 mg of a compound (6-4).
(v) Synthesis of Compound (6-5)
233 mg of the above-described compound (6-5) was synthesized from 219 mg of the compound (6-4) in the same manner, except that in the synthesis of the compound (6-4) described above, (2S,4S)-(tert-butoxycarbonyl)-4-amino-1-(9H-fluoren-9-ylmethoxy)carbonyl)-pyrrolidine-2-carboxylic acid (Fmoc-L-Pro(4-NHBoc)-OH(2S,4S)) was used instead of the compound (6-2) and the compound (6-4) was used instead of the compound (6-3).
(vi) Synthesis of Compound (6-6)
The same operation as the above-described synthesis of the compounds (6-4) and (6-5) was repeated twice to synthesize 190 mg of a compound (6-6) from 100 mg of the compound (6-5).
(vii) Synthesis of Compound (6-7)
33.7 mg of the above-described compound (6-7) was synthesized from 40.2 mg of the compound (6-6) in the same manner, except that in the synthesis of the compound (3-3) described above, the compound (6-6) was used instead of the compound (3-2).
(viii) Synthesis of Compound (6-8)
30.1 mg of the compound (6-7) and 1.0 mL of a mixed solution of TFA:triisopropylsilane:water=95:2.5:2.5 were added to an eggplant flask having a capacity of 10 mL, and stirring was carried out at room temperature for 1 hour. 10 mL of methanol (MeOH) was added to the reaction solution to generate a solid to be removed, and then the solid was removed by filtration. The filtrate was concentrated and purified by preparative HPLC, and freeze drying was followed to obtain 10.6 mg of a compound (6-8).
2) Synthesis of Compound (6)
0.85 mg of the above-described compound (6) was synthesized from 2.0 mg of the compound (6-8) in the same manner, except that in the synthesis of the compound (1) described above, the compound (6-8) was used instead of the compound (1-1) and the compound (M2-1) was used instead of the compound (M1-1). The results of the MS measurement of the compound (6) were as follows.
MS (ESI m/z): (M+H+)+=8,571, (M−H+)−=8,569
3) Synthesis of Compound (6-NHS)
The following compound (6-NHS) was synthesized in the same manner, except that in the synthesis of the compound (1-NHS) described above, the compound (6) was used instead of the compound (1).
<Synthesis of Compound (7)
A compound (7) was synthesized according to the following scheme:
(i) Synthesis of Compound (7-1)
25.9 mg of the above-described compound (7-1) was synthesized from 30.1 mg of the compound (2-5) in the same manner, except that in the synthesis scheme of the compound (2-7) described above, tert-butyl amino-PEGS-ate was used instead of tert-butyl 1-amino-3,6,9,12-tetraoxadecane-15-ate.
(ii) Synthesis of Compound (7)
0.96 mg of the above-described compound (7) was synthesized from 2.0 mg of the compound (7-1) in the same manner, except that in the synthesis of the compound (1) described above, the compound (7-1) was used instead of the compound (1-1) and the compound (M2-1) was used instead of the compound (M1-1).
The results of the MS measurement of the compound (7) were as follows.
MS (ESI m/z): (M+H+)+=4,636, (M−H+)−=4,634
(iii) Synthesis of Compound (7-NHS)
The above-described compound (7-NHS) was synthesized in the same manner, except that in the synthesis of the compound (1-NHS) described above, the compound (7) was used instead of the compound (1).
[Synthesis of Compound (M3-1)]
A compound (M3-1) was synthesized based on the following scheme. The results of the MS measurement of the compound (3-10) were as follows.
MS (ESI m/z): (M+H+)+=1,579, (M−H+)−=1,577
<Synthesis of Compound (8)>
(i) Synthesis of Compound (8)
11.0 mg of the above-described compound (8) was synthesized from 8.9 mg of the compound (6-8) in the same manner, except that in the synthesis of the compound (6) described above, the compound (M3-1) was used instead of the compound (M2-1). The results of the MS measurement of the compound (8) were as follows.
MS (ESI m/z): (M+H+)+=9,160, (M−H+)−=9,158
(ii) Synthesis of Compound (8-NHS)
The following compound (8-NHS) was synthesized in the same manner, except that in the synthesis of the compound (1-NHS) described above, the compound (8) was used instead of the compound (1).
<Synthesis of Compound (9)>
1) Synthesis of Compound (9-3)
A compound (9-3) was synthesized according to the following scheme.
1H-NMR (CDCl3, 300 MHz) δ=0.88 (911, t), 1.18-1.53 (93H, m), 1.67-1.87 (7H, m), 3.89-4.05 (6H, m), 4.75-4.86 (1H, m), 6.56 (2H, s).
2) Synthesis of Compound (9-18)
A compound (9-18) was synthesized according to the following scheme.
1) Synthesis of Compound (9-4)
110 mg of the above-described compound (9-4) was synthesized from 248 mg of the compound (6-1) in the same manner, except that in the synthesis scheme of the compound (6-2) described above, benzyloxycarbonyl chloride was used instead of acetic anhydride (Ac2O).
2) Synthesis of Compound (9-8)
1.0 g of 3,5-dihydroxybenzoic acid (a compound (9-5)), 4.0 mL of N,N-dimethylformamide (DMF), 3.3 mL of 1,3-propane sultone, and 2.6 g of potassium carbonate (K2CO3) dissolved in 2 mL of water were placed in an eggplant flask having a capacity of 50 mL, and stirring was carried out at 80° C. for 2 hours. Thereafter, liquid separation was carried out with ethyl acetate, 0.75 g of sodium hydroxide (NaOH) was subsequently added to the aqueous layer from which the organic layer had been removed, and stirring was carried out at 80° C. for 2 hours. Thereafter, cooling was carried out to 0° C., and neutralization was carried out with hydrochloric acid. The reaction solution was purified by reverse phase column chromatography, and freeze drying was followed to obtain 126 mg of a compound (9-7).
A compound (9-8) was synthesized in the same manner, except that in the synthesis of the compound (1-NHS) described above, the compound (9-7) was used instead of the compound (1).
3) Synthesis of Compound (9-18)
(i) Synthesis of Compound (9-9)
3.2 g of a compound (9-9) was synthesized from 2.5 g of the compound (9-3) in the same manner, except that in the synthesis of the compound (2-2) described above, the compound (9-3) was used instead of the compound (2-1) and N-[(9H-fluoren-9-ylmethoxy)carbonyl]-β-alanine (Fmoc-βAla-OH) was used instead of Nε-(tert-butoxycarbonyl)-Nα-[(9H-fluoren-9-ylmethoxy)carbonyl)-L-lysine (Fmoc-Lys(Boc)-OH).
(ii) Synthesis of Compound (9-10)
3.2 g of the compound (9-9), 32 mL of tetrahydrofuran (THF), and 795 μL of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) were placed in an eggplant flask having a capacity of 100 mL, and stirring was carried out at room temperature for 10 minutes. The reaction solution was cooled to 5° C. or lower, and 2.8 mL of N,N-diisopropylethylamine (DIPEA), 347 L of methanesulfonic acid (MsOH) diluted in 7.0 mL of tetrahydrofuran (THF), 1.5 g of (2S,4S)-(tert-butoxycarbonyl)-4-amino-1-(9H-fluoren-9-ylmethoxy)carbonyl)-pyrrolidine-2-carboxylic acid (Fmoc-L-Pro(4-NHBoc)-OH(2S,4S)), and 4.2 g of (7-azabenzotriazole-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP) were placed therein, and stirring was carried out at room temperature for 2 hours. A solid precipitated by adding acetonitrile (320 mL) was subjected to filtration and drying under reduced pressure to obtain 3.9 g of a compound (9-10).
(iii) Synthesis of Compound (9-11)
299 mg of a compound (9-11) was synthesized from 250 mg of the compound (9-10) in the same manner, except that in the synthesis of the compound (9-10) described above, the compound (9-10) was used instead of the compound (9-9) and the compound (6-2) was used instead of (2S,4S)-(tert-butoxycarbonyl)-4-amino-1-(9H-fluoren-9-ylmethoxy)carbonyl)-pyrrolidine-2-carboxylic acid (Fmoc-L-Pro(4-NHBoc)-OH(2S,4S)).
(iv) Synthesis of Compound (9-12)
253 mg of a compound (9-12) was synthesized from 299 mg of the compound (9-11) in the same manner, except that in the synthesis of the compound (9-10) described above, the compound (9-11) was used instead of the compound (9-9).
(v) Synthesis of Compound (9-13)
287 mg of a compound (9-13) was synthesized from 253 mg of the compound (9-12) in the same manner, except that in the synthesis of the compound (9-10) described above, the compound (9-12) was used instead of the compound (9-9) and the compound (9-4) was used instead of (2S,4S)-(tert-butoxycarbonyl)-4-amino-1-(9H-fluoren-9-ylmethoxy)carbonyl)-pyrrolidine-2-carboxylic acid (Fmoc-L-Pro(4-NHBoc)-OH(2S,4S)).
(vi) Synthesis of Compound (9-14)
268 mg of a compound (9-14) was synthesized from 287 mg of the compound (9-13) in the same manner, except that in the synthesis of the compound (9-10) described above, the compound (9-13) was used instead of the compound (9-9).
(vii) Synthesis of Compound (9-15)
139 mg of a compound (9-15) was synthesized from 150 mg of the compound (9-14) in the same manner, except that in the synthesis of the compound (3-3) described above, the compound (9-14) was used instead of the compound (3-2).
(viii) Synthesis of Compound (9-16)
69.0 mg of a compound (9-16) was synthesized from 150 mg of the compound (9-15) in the same manner, except that in the synthesis of the compound (2-5) described above, the compound (9-15) was used instead of the compound (2-4).
(ix) Synthesis of Compound (9-17)
69.0 mg of the compound (9-16), 3.0 mL of methanol (MeOH), and 3.0 mg of palladium/carbon (Pd: 10%) (about 55% wet product) were placed in an autoclave container, and after hydrogen substitution, stirring was carried out under 0.9 MPa at room temperature for 7 hours. The reaction solution was filtered, and then the solvent was distilled off under reduced pressure. The purification was carried out by preparative HPLC, and freeze drying was followed to obtain 15.4 mg of a compound (9-17).
(x) Synthesis of Compound (9-18)
were placed 15.4 mg of the compound (9-17), 465 μL of dimethyl sulfoxide, 6.48 μL of triethylamine (Et3N), and 7.60 mg of the compound (9-8) in an eggplant flask having a capacity of 10 mL, stirring was carried out at 50° C. for 3 hours. Thereafter, 465 μL of trifluoroacetic acid (TFA) was placed therein, and stirring was carried out at room temperature for 1 hour. A solid precipitated by adding ethyl acetate was subjected to filtration and drying under reduced pressure. The purification was carried out by preparative HPLC, and freeze drying was followed to obtain 3.0 mg of a compound (9-18).
<Synthesis of Compound (9-NHS)>
A compound (9-NHS) was synthesized according to the following scheme.
1) Synthesis of Compound (9)
0.43 mg of the above-described compound (9) was synthesized from 3.0 mg of the compound (9-18) in the same manner, except that in the synthesis of the compound (6) described above, the compound (M4-1) described in WO2021/100814A was used instead of the compound (M2-1). The results of the MS measurement of the compound (9) were as follows.
MS (ESI m/z): (M+H+)+=5,922, (M−H+)−=5,920
2) Synthesis of Compound (9-NHS)
The following compound (9-NHS) was synthesized in the same manner, except that in the synthesis of the compound (1-NHS) described above, the compound (9) was used instead of the compound (1).
For each of the above-described compounds, the degree of fluorescence labeling, the solution fluorescence intensity of the labeled antibody, the dot blot fluorescence intensity, the Western blotting fluorescence intensity, and the fluorescence intensity in stained cells were evaluated.
[0] Preparation of Fluorescently Labeled Antibody
104 μL of an anti-rabbit IgG antibody (2.3 mg/ml) and 10.4 μL of a carbonate buffer were added to a microtube, the resultant mixture was shaken and stirred. Then, a dimethyl sulfoxide solution of the compound (1-NHS) was added thereto so that the molar equivalent ratio of the compound (1-NHS) with respect to 1 equivalent of the antibody was as shown in Table A, and further, the resultant mixture was shaken and stirred. After being allowed to stand at 4° C. for 24 hours, the reaction solution was subjected to purification, by using a centrifugal ultrafiltration filter (product name: Amicon Ultra UFC 510096, manufactured by Merck KGaA) and a PBS solution (phosphate-buffered saline), to obtain an IgG antibody labeled with the compound (1). Regarding each of the compounds (2) to (4), (6), and (7), and the comparative compound (1), a labeled antibody was obtained in the same manner as in the case of the compound (1). A labeled antibody was obtained in the same manner, except that regarding each of the compounds (8) and (9), an anti-mouse IgG antibody was used instead of the anti-rabbit IgG antibody and the reaction condition was set to room temperature for 1 hour. The degree of fluorescence labeling (DOL) of the obtained labeled antibody was calculated according to the method described below. The results are summarized in Table A.
As the method for calculating the degree of fluorescence labeling, such a general method as described below was used. The description in [ ] indicates a unit, and [-] means that there is no unit. In the present test, protein means an anti-rabbit IgG antibody regarding the compounds (1) to (4), (6), and (7), and the comparative compound (1), and means an anti-mouse IgG antibody regarding the compounds (8) and (9).
Degree of fluorescence labeling=fluorescent dye concentration/protein concentration
The fluorescent dye concentration means the total molar concentration [M] of the labeled fluorescent dye, and the protein concentration means the molar concentration [M] of the fluorescently labeled protein. They are respectively calculated according to the following expressions.
Fluorescent dye concentration=Dyemax/εdye
Protein concentration=(IgG280−(Dyemax×CF))/εprotein
Each symbol in the above expressions is as follows.
From the results in Table A above, the following facts can be seen.
Even in a case where the compounds (1) to (4), which are the compounds according to the embodiment of the present invention, are added at any molar equivalent ratio of 10 equivalents, 15 equivalents, or 20 equivalents, with respect to 1 equivalent of the antibody, they exhibit the degree of fluorescence labeling equal to or higher than that in a case where the comparative compound (1) which does not have a structure represented by General Formula (I) is used, and the binding property to the antibody is at a sufficient level without any problem in practical use. This fact can be read from the comparison between No. c01 and Nos. 001 to 004. In addition, even in a case where the compound (6), which is the compound according to the embodiment of the present invention, is added at any molar equivalent ratio of 15 equivalents or 30 equivalents, with respect to 1 equivalent of the antibody, the degree of fluorescence labeling is 3.5 or more, and the binding property to the antibody is at a sufficient level without any problem in practical use. In addition, even in a case where the compound (7), which is the compound according to the embodiment of the present invention, is added at any molar equivalent ratio of 15 equivalents or 20 equivalents, with respect to 1 equivalent of the antibody, the degree of fluorescence labeling is 4.5 or more, and the binding property to the antibody is at a sufficient level without any problem in practical use. In addition, even in a case where the compound (8), which is the compound according to the embodiment of the present invention, is added at any molar equivalent ratio of 40 equivalents or 80 equivalents, with respect to 1 equivalent of the antibody, the degree of fluorescence labeling is 3.5 or more, and even in a case where the compound (9), which is the compound according to the embodiment of the present invention, is added at any molar equivalent ratio of 20 equivalents or 40 equivalents, with respect to 1 equivalent of the antibody, the degree of fluorescence labeling is 3.9 or more, and the binding property to the antibody is at a sufficient level without any problem in practical use.
It is noted that in the following evaluation of the fluorescence intensity, it is possible to compare the fluorescence intensities in a state where the numbers of phosphor moieties bonded to proteins are arranged to be about the same by comparing examples in which the product of the number of phosphor moieties in the compound and the degree of fluorescence labeling (DOL) is about the same.
[1] Evaluation of Solution Fluorescence Intensity
A solution of the labeled antibody prepared as described above was prepared to have a protein concentration of 0.005 mg/mL, and the integrated value of the fluorescence intensity in a fluorescence wavelength range of 810 to 840 nm was calculated by using a spectroscopic fluorescence intensity meter (product name: RF-5300, manufactured by Shimadzu Corporation) with excitation light of 785 nm and unified the exposure conditions. Using the integrated value of the fluorescence intensity of the comparative compound (1)—IgG having DOL of 3.0 (addition of 10 equivalents of dye) in a fluorescence wavelength range of 810 to 840 nm as the reference value, the ratio to this reference value (the integrated value of the fluorescence intensity of the labeled antibody in a fluorescence wavelength range of 810 to 840 nm/the reference value) was calculated, and evaluation was carried out based on the following evaluation standards. The results are summarized in Table 1.
—Evaluation Standards for Fluorescence Intensity (Integrated Value)—
[2] Evaluation of Dot Blot Fluorescence Intensity
Human-derived transferrin (20 mg/mL) was adjusted to 50 ng/mL with a TBS-T buffer solution, and 2 μL thereof was carefully spotted on a nitrocellulose membrane. The membrane was dried and then blocked in TBS-T with a Fish Gelatin blocking buffer solution. Subsequently, 6 μL of a polyclonal rabbit anti-human transferrin antibody was added to 30 mL of a PBS-T buffer solution, the membrane was immersed therein, and shaking was carried out for 1 hour. Then, the membrane was taken out and washed with a TBS-T buffer solution four times. Then, 15 μL of the labeled antibody (anti-rabbit IgG) was added to 30 mL of a TBS-T buffer solution, the membrane was immersed therein, and incubation was carried out at room temperature for 1 hour with stirring. The membrane was washed 3 times with a TBS-T buffer solution for 10 minutes and finally washed with a TBS buffer solution for 10 minutes. The obtained membrane was dried on a hot plate at 40° C. for 1 hour and imaged using an Amersham Typhoon scanner (manufactured by Cytiva) with excitation light of 785 nm under the uniform exposure conditions, thereby calculating the fluorescence intensity in a fluorescence wavelength range of 810 to 840 nm. Using the integrated value of the fluorescence intensity of the comparative compound (1)—IgG having DOL of 3.0 (addition of 10 equivalents of dye) in a fluorescence wavelength range of 810 to 840 nm as the reference value, the ratio to this reference value (the integrated value of the fluorescence intensity of the labeled antibody in a fluorescence wavelength range of 810 to 840 nm/the reference value) was calculated, and evaluation was carried out based on the following evaluation standards. The results are summarized in Table 2.
—Evaluation Standards for Fluorescence Intensity (Integrated Value)—
[3] Evaluation of Fluorescence Intensity in Western Blotting
Transferrin (manufactured by Merck KGaA) was diluted with a Fluorescent Compatible Sample Buffer (4×, non-reducing) (manufactured by Thermo Fisher Scientific, Inc.) to 1 ng/uL, 0.3 ng/uL, or 0.1 ng/μL, followed by heating treatment at 95° C. for 5 minutes. The above-described transferrin sample and a PageRuler Prestained NIR Protein Ladder (manufactured by Thermo Fisher Scientific, Inc.) were loaded on Novex 4-20% Tris-Glycine Mini Gels (manufactured by Thermo Fisher Scientific, Inc.), and then electrophoresis was carried out at a constant voltage of 225 V for 45 minutes. The gel after electrophoresis and a nitrocellulose membrane (manufactured by Cytiva) were superimposed, protein transfer was carried out at a constant voltage of 12 V for 1 hour, and then the membrane was immersed in a Western Blot Blocking Buffer (Fish Gelatin) (manufactured by TAKARA Bio Inc.) and allowed to stand at 4° C. for 12 hours. After washing with a TBS-T buffer, the membrane was immersed in a solution containing a primary antibody for transferrin (manufactured by Dako Corporation) (diluted 5,000 times), shaking was carried out for 1 hour, and the membrane was washed with the TBS-T buffer. A solution of the comparative compound (1)—IgG (diluted 25,000 times) was prepared, and the membrane was immersed and shaken for 1 hour in a light-shielded state, followed by washing with TBS-T. The membrane was shielded from light for 1 hour in a constant-temperature tank at 40° C. and then dried. Imaging was carried out using an Amersham Typhoon scanner (manufactured by Cytiva), and with excitation light of 785 nm under the uniform measurement conditions, the fluorescence intensity of a fraction of 3.24 mm2 in a fluorescence wavelength range of 810 to 840 nm was measured and used as a signal fluorescence intensity. The fluorescence intensity of the antibodies labeled with other compounds was measured in the same manner as described above.
Using the integrated value of the signal fluorescence intensity of the comparative compound (1)—IgG having DOL of 3.0 (addition of 10 equivalents of dye) in a fluorescence wavelength range of 810 to 840 nm as the reference value, the ratio to this reference value (the integrated value of the signal fluorescence intensity of the labeled antibody in a fluorescence wavelength range of 810 to 840 nm/the reference value) was calculated, and evaluation was carried out based on the following evaluation standards. The results are summarized in Table 3.
—Evaluation Standards for Fluorescence Intensity (Integrated Value)—
From the results in Tables 1 to 3 above, the following facts can be seen.
The comparative compound (1) is not the compound defined in the present invention in that two phosphor moieties of which light absorption characteristics are equivalent to each other are not linked through a group having a structure represented by General Formula (I). In the labeled antibody obtained by using this comparative compound (1), all of the fluorescence intensity in the solution, the fluorescence intensity in the dot blot, and the fluorescence intensity in the Western blotting are low (Nos. c11, c21, and c31).
On the other hand, in at least any form of the solution, the dot blot, or the Western blotting, all of the antibodies labeled with the compounds (1) to (4), (6) and (7), which are the compounds according to the embodiment of the present invention, have a fluorescence intensity of 1.1 times or more with respect to the fluorescence intensity of the antibody labeled with the comparative compound (1) and exhibit an excellent fluorescence intensity. Moreover, the decrease in the fluorescence intensity associated with the increase in DOL is also suppressed (Nos. 101 to 106 with respect to No. c11, Nos. 201 to 204 with respect to No. c21, and Nos. 301 to 305 with respect to No. c31).
[4] Evaluation of Fluorescence Intensity of Labeled Antibody in Stained Cells
Cell staining was carried out as follows by using the labeled antibody synthesized as described above. The following characteristics were evaluated for the prepared stained cells. The results are summarized in Table 4.
[Preparation of Stained Cell Sample]
HeLa cells [manufactured by European Collection of Authenticated Cell Cultures, Inc.]were seeded on a 96-well plate [manufactured by Thermo Fisher Scientific, Inc.] and cultured in an incubator for 20 hours in a D-MEM culture medium containing 10% fetal calf serum, 1% Penicilline/streptomycin, and 1% MEM non-essential amino acids [all of which are manufactured by FUJIFILM Wako Pure Chemical Corporation].
Subsequently, the culture medium was removed, and methanol was used to carry out treatment at −20° C. for 5 minutes, thereby carrying out fixation, washing was subsequently carried out with PBS [manufactured by Thermo Fisher Scientific, Inc.], and a PBS solution containing, as a blocking agent and a membrane permeating agent, Triton X-100 (polyethylene glycol mono-p-isooctylphenyl ether) [manufactured by Sigma-Aldrich Co., LLC] having a concentration of 0.2% and bovine serum albumin (BSA) [manufactured by Biological Industries, Inc.] having a concentration of 2%, was added to the washed cells, which were subsequently allowed to stand for 1 hour. The blocking and the membrane permeating agent was removed, a diluted solution of an anti-α-Tubulin antibody [mouse monoclonal, manufactured by FUJIFILM Wako Pure Chemical Corporation] was added to the cells as a primary antibody, and the cells were allowed to stand at room temperature for 1 hour at a final antibody concentration of 1 μg/mL. After washing with PBS-T, an aqueous solution of 1 μg/mL of the compound (8)—IgG was added to the cells as a secondary antibody, the cells were allowed to stand at room temperature for 1 hour while being shielded from light and then washed again with PBS-T. Further, after washing with PBS, one drop of Prolong Gold [manufactured by Thermo Fisher Scientific, Inc.] was added as an antifading agent to each well with a dropper to obtain each stained cell sample. Immediately after the preparation of the stained cell sample, the following evaluation of the fluorescence intensity was carried out.
[Evaluation of Fluorescence Intensity in Stained Cells]
Using a plate reader EnSight manufactured by PerkinElmer, Inc., the fluorescence intensity of the obtained stained cells was measured under the following conditions.
Well scan mode, excitation wavelength: 633 nm, fluorescence wavelength: 670 to 720 nm (data interval: 5 nm), band width: 8 nm, 100 times of integration, 3×3 points, measurement height: 3 mm, point distance: 1 mm) (Measurement condition)
The sum (integrated value) of the fluorescence intensities in a fluorescence wavelength range of 670 to 720 nm was defined as the fluorescence intensity of the sample. Using an integrated value of the fluorescence intensity in a fluorescence wavelength range of 670 to 720 nm as the reference value, the integrated value being a value in a case where a goat anti-mouse secondary antibody labeled with Alexa Fluor Plus 647 [manufactured by Thermo Fisher Scientific, Inc.] was used instead of the aqueous solution of the compound (8)—IgG, the ratio to this reference value (the integrated value of the fluorescence intensity in a fluorescence wavelength range of 670 to 720 nm/the reference value) was calculated, and evaluation was carried out based on the following evaluation standards.
—Evaluation Standards for Fluorescence Intensity (Integrated Value)—
From the results in Table 4, it has been found that the antibody labeled with the compound (8) according to the embodiment of the present invention, which has a fluorescence wavelength at 670 to 720 nm exhibits an excellent fluorescence intensity as compared with a labeled antibody labeled with Alexa Fluor Plus 647 [manufactured by Thermo Fisher Scientific, Inc.], which is a commercially available fluorescent compound having an excitation maximum wavelength at about the same wavelength.
[5] Evaluation of Fluorescence Intensity of Labeled Antibody
Using the labeled antibody synthesized as described above, the fluorescence intensity was measured as follows. The results are summarized in Table 5.
[Preparation of Labeled Antibody Sample]
The labeled antibody was diluted with PBS [manufactured by Thermo Fisher Scientific, Inc.] to 0.15, 0.31, 0.63, 1.25, 2.5, 5 μg/mL and transferred to a 96-well plate [manufactured by Thermo Fisher Scientific, Inc.].
[Evaluation of Fluorescence Intensity of Labeled Antibody Sample]
Using a plate reader EnSight manufactured by PerkinElmer, Inc., the fluorescence intensity of the obtained labeled antibody sample was measured under the following conditions.
Well scan mode, excitation wavelength: 488 nm, fluorescence wavelength: 505 to 550 nm (data interval: 1 nm), band width: 8 nm, 100 times of integration, 1 point, measurement height: 9.5 mm) (Measurement condition)
The sum (integrated value) of the fluorescence intensities in a fluorescence wavelength range of 505 to 550 nm was defined as the fluorescence intensity of each sample, and the slope of the regression line was determined. Using an integrated value in a fluorescence wavelength range of 505 to 550 nm as the fluorescence intensity of each sample, the integrated value being a value in a case where a goat anti-mouse secondary antibody labeled with Alexa Fluor Plus 488 [manufactured by Thermo Fisher Scientific, Inc.] was used instead of the compound (9)—IgG, and using the slope of the regression line as the reference value, the ratio to this reference value (the slope of the regression line calculated from the fluorescence intensities of the respective concentrations of the labeled antibody/the reference value) was calculated, and evaluation was carried out based on the following evaluation standards.
—Evaluation Standards for Fluorescence Intensity (Slope of Regression Line)—
From the results in Table 5, it has been found that the antibody labeled with the compound (9) according to the embodiment of the present invention, which has a fluorescence wavelength at 505 to 550 nm exhibits an excellent fluorescence intensity as compared with a labeled antibody labeled with Alexa Fluor Plus 488 [manufactured by Thermo Fisher Scientific, Inc.], which is a commercially available fluorescent compound having an excitation maximum wavelength at about the same wavelength.
As described above, the compound according to the embodiment of the present invention is a compound which contains two or more phosphor moieties of which light absorption characteristics are equivalent to each other and is a compound in which the phosphor moieties adjacent to each other are each linked through a specific group having a structure represented by General Formula (I), and thus it can impart an excellent fluorescence intensity to a labeled biological substance to be obtained, in at least any form of the solution, the dot blot, the Western blotting, or the stained cell. In particular, this fact can be clearly understood from the comparison between the compound (2) and the comparative compound (1), where the compound (2) is such that the distance in the compound between two phosphor moieties having light absorption characteristics equivalent to each other is about the same.
The present invention has been described together with the embodiments of the present invention. However, the inventors of the present invention do not intend to limit the present invention in any part of the details of the description unless otherwise specified, and it is conceived that the present invention should be broadly construed without departing from the spirit and scope of the invention shown in the attached “WHAT IS CLAIMED IS”.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2021-141998 | Aug 2021 | JP | national |
| 2022-100572 | Jun 2022 | JP | national |
This application is a Continuation of PCT International Application No. PCT/JP2022/032640 filed on Aug. 30, 2022, which claims priority under 35 U.S.C. § 119 (a) to Japanese Patent Application No. 2021-141998 filed in Japan on Aug. 31, 2021, and Japanese Patent Application No. 2022-100572 filed in Japan on Jun. 22, 2022. Each of the above applications is hereby expressly incorporated by reference, in its entirety, into the present application.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/JP2022/032640 | Aug 2022 | WO |
| Child | 18411057 | US |
