Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/13—Labelling of peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof

Definitions

• the present invention relates to compounds and labeled biological materials using the same.
• fluorescently labeled biological materials are often used, in which biological molecules (e.g. antibodies) that are capable of binding to the target substance to be detected are labeled with a fluorescent compound (fluorescent dye).
• a fluorescent compound fluorescent dye
• WB Western blotting
• WB Western blotting
• a fluorescent method for detecting the presence or absence or amount of the specific protein using a fluorescently labeled antibody that binds to the protein.
• bioimaging technology that analyzes the dynamics and functions of biomolecules, cells, tissues, etc. in a living body
• biofluorescence imaging which observes specific parts of a living body that have been visualized by fluorescent labeling, is used as one of the techniques for observing living bodies.
• organic fluorescent dye molecules are generally used, and usually, by using fluorescently labeled biological material with multiple fluorescent dye molecules bound thereto, the brightness (fluorescence intensity) is increased.
• organic dyes that exhibit fluorescence such as cyanine dyes, rhodamine dyes, and dipyrromethene dyes, have aromatic chromophores with high planarity, and therefore are prone to interaction between dyes, and as a result, the fluorescence intensity is likely to decrease due to interactions such as self-association between dyes after labeling.
• fluorescent labeling rate DOL
• the fluorescence intensity tends to decrease due to self-association, etc.
• dye multimers having multiple fluorescent dyes in one molecule are also known as dyes used for fluorescent labeling.
• the fluorescent dyes are also likely to associate with each other within a molecule and between molecules, which tends to reduce the fluorescence intensity.
• the fluorescent dyes are more likely to associate with each other, which tends to reduce the fluorescence intensity.
• Patent Document 1 describes a polymer in which first and second fluorescent or colored moieties are linked by a linking group having two or more charged moieties on the polymer backbone, and it is described that in the polymer described in Patent Document 1, repulsion of the two or more charged moieties present between the first and second colored or fluorescent moieties causes the polymer to assume a more linear or "extended” conformation, thereby maintaining sufficient spatial distance between the fluorescent and/or colored moieties and preventing or reducing intramolecular quenching (association quenching within a molecule).
• dye compounds utilizing the FRET phenomenon are known.
• FRET Fluorescence Resonance Energy Transfer
• dye compounds utilizing the FRET phenomenon for example, as described in Non-Patent Document 1 and Patent Documents 2 to 4, compounds in which a phosphor part I (energy donor) excited by excitation light and another phosphor part II (energy acceptor) that receives energy from the phosphor part I and emits or quenches light are linked by a group containing a peptide such as polyproline are described.
• Patent Document 5 describes a glycosaminoglycan-mimetic molecule that includes a polyproline backbone, an anionic group on the proline side chain, and one fluorescent moiety. It is described that the glycosaminoglycan-mimetic molecule described in Patent Document 5 has improved cellular internalization efficiency due to the anionic group on the proline side chain.
• the present invention aims to provide a compound that, in a solution state, is excellent in suppressing a decrease in fluorescence quantum yield due to dye multimerization and exhibits excellent fluorescence brightness, and also aims to provide a labeled biological material obtained by binding this compound to a biological material.
• X 1 to X 3 represent —O—, —S—, >NR 1 or >CR 2 R 3 .
• R 1 to R 3 and R 11 each represent a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an acyl group, -NR 8 R 9 , -OR 10 or a group selected from the following hydrophilic group Pi.
• R 8 to R 10 each represent a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an acyl group, an aryl group, a heteroaryl group, or a group selected from the following hydrophilic group Pi.
• the structure enclosed by ( ) n does not contain the above phosphor moiety and contains at least one group selected from the following hydrophilic group Pi.
• n is an integer of 2 or more, and * indicates a bond.
• L represents an alkylene group
• R E has the same meaning as R 8 to R 10 above, and g is 2 to 50. However, R E is not -(OL) g R E or -(LO) g R E among the groups selected from the hydrophilic group Pi.
• n is an integer of 3 or more.
• R 4 and R 5 each 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 each 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, or Q.
• Q represents a group selected from the above hydrophilic group Pi, a substituent capable of binding to a biological substance, or a substituent capable of binding to a solid support.
• L 1 to L 7 each represent a single bond or a divalent linking group.
• M represents a fluorescent moiety, a biologically active substance moiety, a prodrug moiety, or a radioisotope-containing moiety.
• Y represents a structure represented by the above general formula (I).
• m is an integer of 1 or more.
• at least two of M represent phosphor moieties.
• X 4 to X 9 represent —O—, —S—, >NR 101 or >CR 102 R 103 .
• at least one of X 4 to X 6 is >N-L 10 -M or >C(R 102 )-L 10 -M
• at least one of X 7 to X 9 is >N-L 11 -M or >C(R 102 )-L 11 -M.
• R 101 to R 103 which are neither -L 10 -M nor -L 11 -M each represent a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an acyl group, -NR 8 R 9 , -OR 10 or a group selected from the above hydrophilic group Pi.
• L 10 and L 11 each represent a single bond or a divalent linking group.
• the structure enclosed by ( ) n1 does not contain the above phosphor moiety and contains at least one type of group selected from the above hydrophilic group Pi.
• n1 is an integer of 2 or more
• q is an integer of 0 or 1.
• R 4 to R 11 , X 1 to X 3 , M and m have the same meanings as R 4 to R 11 , X 1 to X 3 , M and m above.
• L and R E are defined as L and R E above, and g is 25 to 50. However, at least one of R E is a group containing at least one group selected from a polyol residue, a sugar residue, and a polyamino acid residue.
• a labeled biological substance comprising the compound according to any one of [1] to [14] and a biological substance bound thereto.
• the labeled biological substance according to [15], wherein the biological substance is any one of a protein, an amino acid, a nucleic acid, a nucleotide, a sugar chain, and a phospholipid.
• the compound of the present invention is excellent in suppressing the decrease in fluorescence quantum yield caused by forming a dye multimer in a solution state, and exhibits excellent fluorescence brightness. Furthermore, the labeled biological material of the present invention is obtained from a compound that exhibits excellent fluorescence brightness in a solution state.
• substituents when there are a plurality of substituents, linking groups, or structural units, etc., represented by a specific code or formula (hereinafter referred to as substituents, etc.), or when a plurality of substituents, etc., are specified at the same time, unless otherwise specified, the respective substituents, etc. may be the same or different from each other. This also applies to the definition of the number of substituents, etc.
• substituents, etc. when a plurality of substituents, etc., are adjacent to each other (particularly, when adjacent), they may be linked to each other to form a ring, unless otherwise specified.
• rings such as alicyclic rings, aromatic rings, and heterocyclic rings, may be further condensed to form a condensed ring.
• the structure represented by the general formula (I) described below means that n structures represented by the general formula (i) below (n is an integer of 2 or more) are connected. In this case, the n structures represented by the general formula (i) may be the same or different from each other.
• X 1 to X 3 and R 11 in the general formula (i) below have the same meaning as X 1 to X 3 and R 11 in the general formula (I) described below.
• the structure grouped by ( ) m the structure grouped by ( ) n1 , the structure grouped by ( ) na , and the structure grouped by ( ) nb , and the m structures may be the same or different from each other, the n1 structures may be the same or different from each other, the na structures may be the same or different from each other, and the nb structures may be the same or different from each other.
• the indication of the compound and the substituent is used to include not only the compound itself and the substituent itself, but also their salts and ions.
• the term “carboxy group” is used to include a carboxylate ion or a salt group thereof
• the term “sulfo group” is used to include a sulfonate ion or a salt group thereof
• the term “phosphono group” is used to include a phosphonate ion or a salt group thereof.
• the monovalent or polyvalent cation in constituting the above salt structure is not particularly limited, and examples thereof include inorganic cations, organic cations, etc., and specific examples thereof include cations of alkali metals such as Na + , Li + , and K + , cations of alkaline earth metals such as Mg 2+ , Ca 2+ , and Ba 2+ , organic ammonium cations such as trialkylammonium cations and tetraalkylammonium cations, and organic phosphonium cations such as quaternary phosphonium ions.
• alkali metals such as Na + , Li + , and K +
• alkaline earth metals such as Mg 2+ , Ca 2+ , and Ba 2+
• organic ammonium cations such as trialkylammonium cations and tetraalkylammonium cations
• organic phosphonium cations such as quaternary phosphonium ions
• the type of salt may be one type or two or more types may be mixed, the salt type and the free acid structure group may be mixed in the compound, and further, a compound of a salt structure and a compound of a free acid structure may be mixed.
• All of the compounds of the present invention are neutral compounds.
• a compound being neutral means that it is electrically neutral. Specifically, the charge of the compound as a whole is adjusted to be 0 by a group or counter ion having a charge in the compound.
• a cyanine dye which is an example of a dye constituting a phosphor portion, has a formal charge of +1 on a nitrogen atom as shown in the exemplified compound described below, and a dissociative group such as a sulfo group in the cyanine dye or in other structures in the compound of the present invention has an ionic structure such as a sulfonate ion in order to be paired with this formal charge, so that the compound of the present invention is a compound with a charge of 0 as a whole.
• it includes those in which a part of the structure has been changed, as long as the effect of the present invention is not impaired.
• substituents for example, groups expressed as “alkyl group”, “methyl group”, “methyl”, etc.
• linking groups for example, groups expressed as “alkylene group”, “methylene group”, “methylene”, etc.
• the preferred substituents in the present invention are those selected from the substituent group T described below.
• this carbon number means the total number of carbon atoms of the group unless otherwise specified in the present invention or this specification.
• this group further has a substituent, it means the total number of carbon atoms including the substituent.
• a numerical range expressed using " ⁇ ” means a range that includes the numerical values written before and after " ⁇ " as the lower and upper limits.
• the compound of the present invention is a compound having at least two phosphor moieties and a structure represented by the following general formula (I).
• the reason why the compound of the present invention is excellent in suppressing the decrease in fluorescence quantum yield caused by forming a dye multimer in a solution state and shows excellent fluorescence brightness is not clear in detail, but it is thought to be as follows. It is known that a compound having at least two fluorescent moieties in a molecule is generally more susceptible to quenching due to intermolecular or intramolecular association of the fluorescent moieties, i.e., a decrease in fluorescence intensity, than a compound having one fluorescent moiety in a molecule.
• the compound of the present invention is a compound having at least two fluorescent moieties, and contains a relatively rigid structure consisting of a repeating unit (the number of repeats n is an integer of 2 or more) containing a nitrogen-containing saturated five-membered ring such as a ring structure derived from proline, as represented by the general formula (I) described below, and therefore can suppress quenching due to association of the fluorescent moieties.
• the structure represented by the general formula (I) described below contains at least one specific hydrophilic group selected from the hydrophilic group group Pi described below in addition to the repeating unit containing the nitrogen-containing saturated five-membered ring, the specific hydrophilic group present in the vicinity of the relatively rigid structure can effectively exhibit the excluded volume effect.
• the compound of the present invention can effectively suppress association of the fluorescent moieties, is excellent in suppressing a decrease in the fluorescence quantum yield in a solution state (a solution state in which the compound is dissolved in an aqueous medium), and can exhibit excellent fluorescence brightness, and it is considered that the labeled biological material obtained using the compound of the present invention exhibits excellent fluorescence intensity.
• the first and second fluorescent or colored moieties are linked by a linking group having two or more positively charged moieties on a chain-like polymer backbone.
• the linking group specifically described in Patent Document 1 is inferior in suppressing the decrease in fluorescence quantum yield by forming dye multimers in a solution state, and is inferior in suppressing the decrease in fluorescence brightness, because the positively charged moieties present on the chain-like polymer backbone cause dye association due to polymer aggregation caused by electrostatic interaction with the positively charged sites.
• the compound of the present invention is a compound having at least two phosphor moieties and a structure represented by general formula (I) described below.
• the compounds of the present invention may be classified as polymers or oligomers.
• the phosphor moiety may be contained in the compound of the present invention as a structure separate from the structure represented by the following general formula (I), in a state of being bound directly or via a linking group to the bond * in the following general formula (I).
• Examples of the form in which the phosphor moiety is bound directly or via a linking group to the bond * in general formula (I) include compounds represented by the below-described general formula (II).
• it is preferable that adjacent phosphor moieties are linked via a structure represented by the following general formula (I), that is, that is, the compound is one in which two structures having phosphor moieties as substituents are linked via a group including a structure represented by the following general formula (I).
• the structure having a phosphor moiety bonded to the bond * in the general formula (I) as a substituent is not particularly limited.
• a combination of L 1 and L 2 , a combination of L 1 and L 3 , a combination of L 4 and L 5 , or a combination of L 4 and L 6 may form a 5-membered ring having carbon atoms, nitrogen atoms, and X 1 to X 3 as ring constituent atoms, as described in the structure represented by the general formula (I) below, and this 5-membered ring may have a group containing a phosphor moiety as a substituent.
• the structure having a phosphor moiety as a substituent may be a 5-membered ring having carbon atoms, nitrogen atoms, and X 1 to X 3 as ring constituent atoms, as described in the structure represented by the general formula (I) below, having a group containing a phosphor moiety as a substituent.
• Examples of such compounds include compounds represented by the general formulas (III) and (IV) described below.
• the description and specific examples of the phosphor moiety in general formula (II) described below can be applied.
• X 1 to X 3 represent —O—, —S—, >NR 1 or >CR 2 R 3 .
• R 1 to R 3 and R 11 each represent a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an acyl group, -NR 8 R 9 , -OR 10 or a group selected from the following hydrophilic group Pi.
• R 8 to R 10 each represent a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an acyl group, an aryl group, a heteroaryl group, or a group selected from the following hydrophilic group Pi.
• n is an integer of 2 or more, and * indicates a bond.
• L represents an alkylene group, R E has the same meaning as R 8 to R 10 above, and g is 2 to 50. However, R E is not -(OL) g R E or -(LO) g R E among the groups selected from the hydrophilic group Pi.
• the structure represented by the above general formula (I) is preferably a structure represented by either of the following general formulas (IA) or (IB), taking into consideration stereoisomers, where X 1 to X 3 , R 11 and n in the following general formulas are synonymous with X 1 to X 3 , R 11 and n in the above general formula (I).
• X 1 to X 3 each represent -O-, -S-, >NR 1 or >CR 2 R 3
• R 1 to R 3 and R 11 each represent a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an acyl group, an aryl group, a heteroaryl group, -NR 8 R 9 , -OR 10 or a group selected from the hydrophilic group Pi.
• the alkyl group, alkenyl group, alkynyl group, acyl group, aryl group and heteroaryl group which can be taken as R 1 to R 3 and R 11 , as well as the carboxy group, sulfo group, phosphono group, betaine residue, polyol residue, sugar residue, polyamino acid residue, -(O-L) g R E and -(L-O) g R E in the above-mentioned hydrophilic group group Pi, have the same meaning as the alkyl group, alkenyl group, alkynyl group, acyl group, aryl group and heteroaryl group, as well as the carboxy group, sulfo group, phosphono group, betaine residue, polyol residue, sugar residue, polyamino acid residue, -(O-L) g R E and -(L-O) g R E in the anionic group in the substituent group T described later, and the preferred ranges are also the same.
• alkyl group, alkenyl group, alkynyl group, acyl group, aryl group and heteroaryl group which can be taken as R 1 to R 3 and R 11 , as well as the betaine residue, polyol residue, sugar residue, polyamino acid residue, -(OL) g R E and -(L-O) g R E in the hydrophilic group group Pi may be unsubstituted or may have a substituent.
• Substituents that may be possessed by the alkyl group, alkenyl group, alkynyl group, acyl group, aryl group and heteroaryl group in R 1 to R 3 and R 11 , and the betaine residue, polyol residue, sugar residue, polyamino acid residue, -(O-L) g R E and -(L-O) g R E in the above hydrophilic group group Pi include the substituents in the below-mentioned substituent group T, and for example, a halogen atom or a group selected from the above hydrophilic group group Pi is preferable.
• R 8 to R 10 each represent a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an acyl group, an aryl group, a heteroaryl group or a group selected from the above hydrophilic group Pi.
• alkyl group, alkenyl group, alkynyl group, acyl group, aryl group and heteroaryl group which can be taken as R 8 to R 10 , as well as the betaine residue, polyol residue, sugar residue, polyamino acid residue, -(OL) g R E and -(L-O) g R E in the above hydrophilic group group Pi, may be unsubstituted or may have a substituent.
• Substituents that may be possessed by the alkyl group, alkenyl group, alkynyl group, acyl group, aryl group and heteroaryl group in R 8 to R 10 , and the betaine residue, polyol residue, sugar residue, polyamino acid residue, -(O-L) g R E and -(L-O) g R E in the above hydrophilic group group Pi, include the substituents in the substituent group T described below.
• a halogen atom, a carbamoyl group, an acylamino group, an alkoxy group (preferably an alkoxy group having a group selected from the above hydrophilic group group Pi), a heteroaryl group or a group selected from the above hydrophilic group group Pi is preferred, and a carbamoyl group, an acylamino group, an alkoxy group (preferably an alkoxy group having a group selected from the above hydrophilic group group Pi), a heteroaryl group or a group selected from the above hydrophilic group group Pi is more preferred.
• R 1 is preferably a hydrogen atom, an alkyl group, --NR 8 R 9 , --OR 10 or a group selected from the above hydrophilic group Pi.
• R2 and R3 are preferably a hydrogen atom, -NR 8 R 9 , -OR 10 or a group selected from the above hydrophilic group Pi, and it is more preferable that R2 is a hydrogen atom, -NR 8 R 9 , -OR 10 or a group selected from the above hydrophilic group Pi, and R3 is a hydrogen atom.
• R 11 is preferably a hydrogen atom, an alkyl group, —NR 8 R 9 , —OR 10 or a group selected from the above hydrophilic group Pi, and more preferably a hydrogen atom.
• R 8 to R 10 are preferably a hydrogen atom, an alkyl group, or an acyl group.
• the alkyl group may be an alkyl group having at least one type of group selected from the hydrophilic group group Pi
• the acyl group may be an acyl group containing at least one type of group selected from the hydrophilic group group Pi.
• the acyl group containing at least one of the groups selected from the hydrophilic group group Pi may preferably be an acyl group having at least one of the groups selected from the hydrophilic group group Pi, or an acyl group substituted with an alkoxy group or a heteroaryl group having at least one of the groups selected from the hydrophilic group group Pi.
• the alkyl group and acyl group may have a substituent other than the group selected from the hydrophilic group group Pi, and examples of the substituents include those in the substituent group T described below, and for example, a carbamoyl group or an acylamino group is preferable.
• -(OL) g R E and -(L-O) g R E are included as divalent groups obtained by removing one hydrogen atom from a hydrogen atom or a substituent that can be taken as R E , and may further have a carboxy group, a sulfo group, a phosphono group, a betaine residue, a polyol residue, a sugar residue or a polyamino acid residue bonded thereto.
• Examples of a divalent group obtained by removing one hydrogen atom from a hydrogen atom or a substituent that can be taken as R E from -(O-L) g R E or -(L-O) g R E include -(O-L) g -, -(O-L) g -NHCO-, -(O-L) g -CONH-, -(O-L) g -CONHalkylene-, -(O-L) g -NHCOalkylene-, -(L-O) g -, -(L-O) g -NHCO-, -(L-O) g -CONH-, -(L-O) g -CONHalkylene- and -(L-O) g -NHCOalkylene-.
• at least one is preferably >CR 2 R 3 , and more preferably at least two are >CR 2 R 3 and the remaining
• the structure represented by the above general formula (I) contains a structure in which X 1 to X 3 are >CR 2 R 3.
• the structure represented by general formula (I) contains a structure in which X 1 to X 3 are >CR 2 R 3 " means that, among the n structures represented by the above general formula (i) in which X 1 to X 3 are linked, all of X 1 to X 3 in at least one structure represented by general formula (i) are >CR 2 R 3 .
• the proportion of the number of structures in which X 1 to X 3 are >CR 2 R 3 is preferably 30% or more, more preferably 60% or more, and even more preferably 80% or more. There is no particular limit to the upper limit, and it can be 100% or less. It is also preferable that all of the structures represented by general formula (I) are structures in which X 1 to X 3 are >CR 2 R 3 .
• At least one R 2 is preferably -NR 8 R 9 , -OR 10 or a group selected from the above hydrophilic group Pi, and more preferably -NR 8 R 9 , -OR 10 or a group selected from the above hydrophilic group Pi, and at least one of R 8 to R 10 is a group containing at least one of groups selected from the above hydrophilic group Pi.
• At least one R 2 in the structure in which X 1 to X 3 are >CR 2 R 3 is -NR 8 R 9 , -OR 10 or a group selected from the above hydrophilic group Pi, and at least one of R 8 to R 10 is a group containing at least one of the groups selected from the above hydrophilic group Pi means that when R 2 is -NR 8 R 9 , at least one of R 8 and R 9 is a group containing at least one of the groups selected from the above hydrophilic group Pi, and when R 2 is -OR 10 , R 10 is a group containing at least one of the groups selected from the above hydrophilic group Pi.
• R 10 is a group containing at least one of the groups selected from the hydrophilic group group Pi
• R 10 is a group selected from the hydrophilic group group Pi or a group having at least one of the groups selected from the hydrophilic group group Pi as a substituent.
• R 8 and R 9 is a group containing at least one of the groups selected from the hydrophilic group group Pi.
• the structure represented by the above general formula (I) includes a structure in which X 1 to X 3 are >CR 2 R 3 , and at least one R 2 in the structure in which X 1 to X 3 are >CR 2 R 3 is -NR 8 R 9 , -OR 10 or a group selected from the above hydrophilic group Pi
• the structure represented by the above general formula (I) includes a group selected from the above hydrophilic group Pi, and more preferably, the group selected from the hydrophilic group Pi is specifically one of the following groups:
• at least one of the groups selected from the hydrophilic group group Pi contained in the structure represented by the above general formula (I) is preferably a carboxy group, a sulfo group, or a phosphono group, and it is more preferable that the structure represented by the above general formula (I) contains two or more groups selected from a carboxy group, a sulfo group, and a phosphono group.
• At least one of the groups selected from the hydrophilic group Pi in the structure represented by the general formula (I) is preferably a betaine residue, a polyol residue, a sugar residue, a polyamino acid residue, -(OL) g R E , or -(L-O) g R E.
• L, R E , and g are synonymous with the above L, R E , and g.
• at least one of the groups selected from the hydrophilic group Pi in the structure represented by general formula (I) is preferably a polyol residue, a sugar residue, a polyamino acid residue, -(OL) g R E , or -(L-O) g R E.
• L, R E , and g are synonymous with the above L, R E , and g.
• g is preferably 25 to 50, and more preferably g is 25 to 50 and at least one of R E is a group containing at least one group selected from a polyol residue, a sugar residue, and a polyamino acid residue.
• at least one group selected from the hydrophilic group group Pi possessed by the structure represented by the above general formula (I) is a betaine residue.
• n is an integer of 2 or more.
• n is an integer of 2 or more, and thus it is possible to impart rigidity to the structure represented by general formula (I) that is effective in suppressing the aggregation of the dye (phosphor moiety), and thus it is possible to suppress a decrease in the fluorescence intensity.
• the lower limit of n is preferably an integer of 3 or more, more preferably an integer of 6 or more, even more preferably an integer of 9 or more, and particularly preferably an integer of 12 or more, from the viewpoint of further suppressing dye association.
• n is preferably an integer of 3 to 72, more preferably an integer of 6 to 36, even more preferably an integer of 9 to 24, and particularly preferably an integer of 12 to 18.
• the number of the phosphor parts is 3 or more, the number of phosphor parts (number of dyes) per molecule is larger than when the number of the phosphor parts is 2, so a certain amount of dye association can be tolerated, and an integer of 3 or more is preferable because the fluorescence intensity can be further improved.
• n is an integer of 6, a certain amount of association suppression effect is obtained but is insufficient, so n is more preferably an integer of 7 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 even more preferably an integer of 24 or less. That is, when the number of the phosphor parts is 3 or more, n is preferably an integer of 3 to 72, more preferably an integer of 7 to 36, and even more preferably an integer of 7 to 24.
• R 4 and R 5 each 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 each 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, or Q.
• Q represents a group selected from the above hydrophilic group Pi, a substituent capable of binding to a biological substance, or a substituent capable of binding to a solid support.
• L 1 to L 7 each represent a single bond or a divalent linking group.
• M represents a fluorescent moiety, a biologically active substance moiety, a prodrug moiety, or a radioisotope-containing moiety.
• Y represents a structure represented by the above general formula (I). m is an integer of 1 or more. However, at least two of M represent the above phosphor moieties.
• R4 and R5 each 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, alkenyl group, alkynyl group, acyl group, amino group, alkoxy group, aryl group and heteroaryl group which can be taken as R4 or R5 have the same meaning as the alkyl group, alkenyl group, alkynyl group, acyl group, amino group, alkoxy group, aryl group and heteroaryl group in the substituent group T described later, and the preferred ranges are also the same.
• the alkyl group, alkenyl group, alkynyl group, acyl group, amino group, alkoxy group, aryl group and heteroaryl group which can be taken as R4 or R5 may be unsubstituted or may have a substituent.
• R 4 and R 5 are often hydrogen atoms, but since the substituents in R 4 and R 5 do not significantly contribute to the excellent association inhibition effect of the compound of the present invention in a solution state, R 4 and R 5 may be other substituents (alkyl group, alkenyl group, alkynyl group, acyl group, amino group, hydroxy group, alkoxy group, sulfanyl group, aryl group, or heteroaryl group) other than hydrogen atoms.
• R6 and R7 each 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, or Q.
• the alkyl group, alkenyl group, alkynyl group, aryl group, alkoxy group, amino group, acyl group and heteroaryl group which can be taken as R6 or R7 have the same meaning as the alkyl group, alkenyl group, alkynyl group, aryl group, alkoxy group, amino group, acyl group and heteroaryl group in the substituent group T described later, and the preferred ranges are also the same.
• the alkyl group, alkenyl group, alkynyl group, aryl group, alkoxy group, amino group, acyl group and heteroaryl group which can be taken as R6 or R7 may be unsubstituted or may have a substituent.
• Substituents which the alkyl group, alkenyl group, alkynyl group, aryl group, alkoxy group, amino group, acyl group and heteroaryl group in R6 or R7 may have include the substituents in the substituent group T described later.
• an alkyl group, an acyl group, an alkoxy group, an amino group or Q, or a substituent which is a combination of two or more of these is preferred, and an alkyl group, an acyl group, an alkoxy group, an amino group, -(O-L) g R E , -(L-O) g R E , a carboxy group, a substituent capable of binding to a biological substance or a substituent capable of binding to a solid support, or a substituent which is a combination of two or more of these substituents is more preferred.
• Q which can be taken as R6 or R7 represents a group selected from the above hydrophilic group Pi, a substituent capable of binding to a biological substance, or a substituent capable of binding to a solid support.
• substituent capable of binding to a biological substance the description of the substituent capable of binding to a biological substance described below can be applied, and as the substituent capable of binding to a solid support, the description of the substituent capable of binding to a solid support described below can be applied.
• Q is preferably a carboxy group, a substituent capable of binding to a biological substance, or a substituent capable of binding to a solid support, and more preferably a substituent capable of binding to a biological substance or a substituent capable of binding to a solid support.
• R 6 include the substituent represented by -L 13 R 6A described below
• preferred examples of R 7 include the substituent represented by -L 12 R 7A described below.
• the description of the substituent represented by -L 13 R 6A refers to a substituent obtained by combining a group that can be taken as L 13 and a group that can be taken as R 6A .
• L 2 to L 5 and L 7 each represent a single bond or a divalent linking group, and are preferably a single bond or a linking group consisting of one or a combination of two or more of an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, -O-, -S-, >C ⁇ O, >NR A , >S ⁇ O, >S( ⁇ O) 2 and >P( ⁇ O)OR B.
• R A and R B are synonymous with R A and R B described below and represent a hydrogen atom or a substituent.
• R A and R B are synonymous with R A and R B described below, and represent a hydrogen atom or a substituent.
• the alkylene group which can constitute L 1 to L 7 has the same meaning as the group in which one hydrogen atom has been removed from an alkyl group selected from the substituent group T described below, and the preferred examples are also the same.
• the alkenylene group which can constitute L 1 to L 7 has the same meaning as the group in which one hydrogen atom has been removed from an alkenyl group selected from the substituent group T described later, and the preferred examples are also the same.
• the alkynylene group which can constitute L 1 to L 7 has the same meaning as the group in which one hydrogen atom has been removed from an alkynyl group selected from the below-mentioned substituent group T, and the preferred examples are also the same.
• the arylene group which can constitute L 1 to L 7 has the same meaning as the group in which one hydrogen atom has been removed from an aryl group selected from the substituent group T described later, and the preferred examples are also the same.
• the heteroarylene group which can constitute L 1 to L 7 has the same meaning as the group in which one hydrogen atom has been removed from a heteroaryl group selected from the substituent group T described later, and the preferred examples are also the same.
• the alkylene group, alkenylene group, alkynylene group, arylene group and heteroarylene group which can constitute L 1 to L 7 may be an unsubstituted group or a group having a substituent.
• the substituents which may be possessed by the alkylene groups, alkenylene groups, alkynylene groups, arylene groups and heteroarylene groups which may constitute L 1 to L 7 are not particularly limited and are preferably selected from the substituent group T described below, more preferably a halogen atom, an alkyl group, an acylamino group or a carbamoyl group.
• the substituents which may be possessed by the alkylene groups, alkenylene groups, alkynylene groups, arylene groups and heteroarylene groups which may constitute L 1 to L 7 may be further substituted with a substituent selected from the substituent group T described below, for example, an amino group is preferred.
• the number of substituents that may be possessed by the alkylene group, alkenylene group, alkynylene group, arylene group, and heteroarylene group that may constitute L 1 to L 7 is not particularly limited as long as it can be adopted as a structure, and may be at least 1 or more.
• the upper limit is not particularly limited, and for example, all hydrogen atoms in the alkylene group, alkenylene group, alkynylene group, arylene group, and heteroarylene group may be substituted with substituents.
• R A 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 even more preferably a hydrogen atom.
• R B 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 even more preferably a hydrogen atom.
• the alkyl group, alkenyl group, alkynyl group, aryl group and heteroaryl group which can be taken as R 1 A and R 1 B may be either an unsubstituted group or a group having a substituent.
• the types of groups to be combined are not particularly limited as long as a suitable chemical structure is obtained, but for example, 2 to 6 types are preferable, and 2 to 4 types are more preferable.
• the number of groups to be combined is not particularly limited, and is preferably, for example, 2 to 10, more preferably 2 to 6, and even more preferably 2 to 4.
• L 3 , L 4 , L 7 L 3 , L 4 and L 7 are each preferably a single bond, >C ⁇ O, >NR A , 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 and a heteroarylene group with >C ⁇ O and >NR A , and more preferably a single bond, >C ⁇ O, >NR A , an alkylene group, an alkenylene group, an alkynylene group, an arylene group or a heteroarylene group, or a group connecting -C( ⁇ O)NR A - and >C ⁇ O, or -NR A C( ⁇ O)- and >NR A via at least one of an alkylene group, an alkenylene group, an alkynylene group, an arylene group and a heteroarylene group,
• L x is a single bond, or a group consisting of one or more of an alkylene group, an alkenylene group, an alkynylene group, an arylene group, and a heteroarylene group
• L x is a heteroarylene group, or a group consisting of a group consisting of one or more of an alkylene group, an alkenylene group, an alkynylene group, and an arylene group located on the * side, and a heteroarylene group located on the ** side.
• the shortest number of atoms in the linking chain (each of the linking chains including L2 and L5 ) linking M to the carbon atom to which L1 and L3 are bonded, or the carbon atom to which L4 and L6 are bonded, may be, for example, 1 to 60, and is preferably 1 to 40.
• M is a phosphor moiety
• the shortest number of atoms means the number of atoms constituting the shortest chain of the linking chains linking the conjugated structure portion for exhibiting fluorescence in the phosphor moiety M to the carbon atom to which L1 and L3 are bonded, or the carbon atom to which L4 and L6 are bonded.
• any part of the connecting chain represented by "-linking group ZZZ-L 2 -" in the structure represented by the conjugated structure portion of M-linking group ZZZ-L 2 -, and any part of the connecting chain represented by "-linking group ZZZ-L 5 -" in the structure represented by the conjugated structure portion of M-linking group ZZZ-L 5 - have a structure represented by -(CH 2 -CH 2 -O) b -.
• the above b is 1 to 50, 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 repeat number can be calculated from the average integral value by performing 1H -NMR measurement on the compound.
• the average repeat number specified in the present invention means a number obtained by rounding off the average repeat number calculated by the above method to the first decimal place.
• R 60 and R 70 are a hydrogen atom or an alkyl group, preferably a hydrogen atom.
• the alkyl group which can be taken as R 60 or R 70 has the same meaning as the alkyl group in the substituent group T described later.
• p represents the number of repetitions, preferably 1 to 10, more preferably 1 to 8, and even more preferably 1 to 4.
• adjacent groups may be bonded to each other to form a ring.
• Examples of the combination of adjacent groups that may be bonded to each other to form a ring include the combination of L1 and L2 , the combination of L1 and L3 , the combination of L2 and R4 , the combination of L4 and L5 , the combination of L4 and L6 , or the combination of L5 and R5 .
• the ring which may be formed by bonding adjacent groups to each other may be either an aromatic ring or an aliphatic ring, or either a hydrocarbon ring or a hetero ring, and is preferably a 5- or 6-membered ring.
• the aliphatic ring is preferably a cyclopentane ring, a cyclohexane ring, or a 5-membered ring having a carbon atom, a nitrogen atom, and X 1 to X 3 as ring-constituting atoms as described in the structure represented by the above general formula (I), and more preferably a 5-membered ring having a carbon atom, a nitrogen atom, and X 1 to X 3 as ring-constituting atoms as described in the structure represented by the above general formula (I).
• the aromatic ring is preferably a benzene ring or a nitrogen-containing aromatic heterocycle, more preferably a benzene ring or a nitrogen-containing aromatic heterocycle whose ring constituent atoms are carbon atoms and nitrogen atoms, and further preferably a benzene ring or a pyridine ring.
• These rings may have a substituent, and the substituent that may be had is selected from the substituent group T without any particular limitation.
• the ring formed by the combination of L2 and R4 or the combination of L5 and R5 may be either the above-mentioned aliphatic ring or aromatic ring, and is preferably the above-mentioned aliphatic ring.
• the ring formed by the combination of L1 and L2 , the combination of L1 and L3 , the combination of L4 and L5 , or the combination of L4 and L6 may be any of the above-mentioned aliphatic rings and aromatic rings, and is preferably a 5-membered ring having a carbon atom, a nitrogen atom, and X1 to X3 as ring-constituting atoms, or a benzene ring, as described in the structure represented by the above-mentioned general formula (I).
• the following structure enclosed by the dashed line in general formula (II) is
• the structure may include the following structure.
• * indicates a linking portion.
• n is an integer of 1 or more. There is no particular upper limit, and for example, it can be an integer of 30 or less, preferably an integer of 20 or less, more preferably an integer of 15 or less, even more preferably an integer of 10 or less, and particularly preferably an integer of 3 or less. In other words, m can be an integer from 1 to 30, preferably an integer of 1 to 20, more preferably an integer of 1 to 15, and even more preferably an integer of 1 to 10.
• M represents a fluorescent moiety, a biologically active substance moiety, a prodrug moiety, or a radioisotope-containing moiety, provided that at least two of M represent fluorescent moieties.
• the phosphor part (hereinafter also referred to as phosphor part M) that can be taken as M can be used without any particular limitation as long as it is a structural part made of an organic compound that exhibits fluorescence.
• the phosphor part M may also be a structural part in which the structural part made of an organic compound that exhibits fluorescence further has a linking group.
• a linking group is not particularly limited, but may be, for example, the above-mentioned linking group ZZZ.
• a compound in which the phosphor part M is bonded to L2 or L5 by this linking group ZZZ is preferably exemplified.
• the fluorescent portion M include structural portions formed of at least one dye selected from the group consisting of xanthene dyes, rhodamine dyes, coumarin dyes, cyanine dyes, pyrene dyes, oxazine dyes, squarylium dyes, pyridyloxazole dyes, and pyrromethene dyes, and are preferred.
• xanthene dyes As the above-mentioned xanthene dyes, rhodamine dyes, coumarin dyes, cyanine dyes, pyrene dyes, oxazine dyes, squarylium dyes, pyridyloxazole dyes and pyrromethene dyes, dyes that are commonly known as these dyes can be used without any particular limitation.
• the phosphor portion M is preferably a structural portion made of a pyrromethene dye.
• pyrromethene dyes include dipyrromethene boron complexes.
• dipyrromethene boron complex a fluorescent compound represented by the general formula (1) or (4) described in WO 2019/230963 (dipyrromethene boron complex) and a compound represented by the general formula (1) described in WO 2021/100814 (dipyrromethene boron complex) can be used, and these descriptions can be incorporated by citation into this specification.
• the dye constituting the phosphor portion M is incorporated so as not to have any substituent group capable of binding to a biological substance.
• a structural portion made of a commercially available fluorescent dye can be used without any particular restrictions.
• commercially available fluorescent dyes include Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, all of which are product names manufactured by Thermo Fisher Scientific.
• the relationship between the light absorption properties of two or more phosphor moieties M present in a compound is not particularly limited, and it is preferable that the compound is one in which at least two of the phosphor moieties are phosphor moieties whose light absorption properties are equivalent to each other.
• phosphor moieties having equivalent light absorption characteristics means that the difference in maximum absorption wavelength in the absorption spectrum of each phosphor moiety satisfies the relationship of being within 15 nm.
• the phosphor portions are phosphor portions having equivalent light absorption properties
• compounds that cause the FRET phenomenon are known as compounds having two fluorescent moieties in a compound.
• the difference in maximum absorption wavelength in the absorption spectrum between the fluorescent moiety I (energy donor) excited by excitation light and the other fluorescent moiety II (energy acceptor) that receives energy from the fluorescent moiety I and emits or quenches light usually exceeds 15 nm.
• the fluorescent intensity of the compound is low because energy is transferred to the fluorescent moiety II instead of the fluorescent moiety I emitting fluorescence. Therefore, when all the phosphor moieties contained in the compound satisfy the relationship that the 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 spectrum of each phosphor moiety is within 15 nm, the FRET phenomenon does not occur, and the compound can exhibit a fluorescence intensity proportional to the number of phosphor moieties.
• the chemical structures of the phosphor parts having the above-mentioned equivalent light absorption characteristics are not particularly limited as long as the difference in the maximum absorption wavelength is satisfied, and preferably, the structures of the main skeletons of the phosphor parts are the same.
• the steric configuration and chain length of the substituents may be different, and when an anionic group or a cationic group is present, the counter ions may be different.
• the difference in the above-mentioned maximum absorption wavelength is preferably within 10 nm, more preferably within 5 nm.
• it is also preferable that at least two of the phosphor moieties are compounds having phosphor moieties which have unequal light absorption properties.
• phosphor moieties having mutually unequal light absorption characteristics means that the difference in maximum absorption wavelengths in the absorption spectra of the respective phosphor moieties exceeds 15 nm.
• the phosphor portions are phosphor portions having non-equivalent light absorption properties, it is preferable that all of the phosphor portions contained in the compound satisfy the relationship that the 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 spectrum of each phosphor portion is more than 15 nm.
• the chemical structures of the phosphor moieties having the above-mentioned non-equivalent light absorption characteristics are not particularly limited as long as they satisfy the above-mentioned difference in maximum absorption wavelength, and preferably have different main skeleton structures, that is, different types of phosphor moieties.
• the difference in maximum absorption wavelength is preferably more than 20 nm, and more preferably more than 30 nm.
• the absorption spectrum of the phosphor part is a spectrum measured by using a spectrophotometer to measure the phosphor alone constituting the phosphor part diluted with PBS (phosphate-buffered saline).
• the number of the phosphor moieties M is 2 or more. There is no particular upper limit, and it can be, for example, 30 or less, preferably 20 or less, more preferably 15 or less, even more preferably 10 or less, and particularly preferably 4 or less. In other words, the number of the phosphor moieties is preferably 2 to 30, more preferably 2 to 20, even more preferably 2 to 15, particularly preferably 2 to 10, and of these, 2 to 4 is preferred.
• the physiologically active substance portion that can be used as M can be any structural portion consisting of a physiologically active substance, without any particular limitation.
• physiologically active substances include vitamins, coenzymes, hormones, antibiotics, neurotransmitters, cytokines, and the like.
• the physiologically active substances described in paragraph [0095] of JP-A-2021-020956 include 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, camptothecin or a derivative thereof, and the descriptions in paragraphs [0095] to [0099] of JP-A-2021-020956 can be applied.
• the prodrug moiety that can be used as M can be any structural part consisting of a compound that is metabolized in vivo and converted into a physiologically active substance.
• the description in paragraph [0003] of JP 2020-105187 A prodrug form of 2-pyrrolinodoxorubicin
• the radioisotope-containing moiety that can be used as M can be any structural part containing a radioisotope that can be used in the medical field, without particular limitation. Examples of radioisotopes include, but are not limited to, iodine-131, indium-111, yttrium-90, lutetium-177, and copper-64.
• JP 2021-11483 A can be applied.
• structural parts containing radioisotopes include structural parts in which the radioisotope is bound or coordinated to an amino group or a nitrogen atom of a tertiary amine, a sulfanyl group, an aryl group, or a heteroaryl group.
• An example of a structural portion in which a nitrogen atom of a tertiary amine is coordinated to the radioisotope is a structural portion in which DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) or the like is coordinated to the radioisotope to form a complex
• an example of a structural portion in which a sulfanyl group is coordinated to the radioisotope is a structural portion made of a complex such as diacetylbis(N(4)-methylthiosemicarbazonane)copper(II).
• the compound represented by the above general formula (II) is preferably represented by the following general formula (III):
• X 4 to X 9 represent —O—, —S—, >NR 101 or >CR 102 R 103 .
• at least one of X 4 to X 6 is >N-L 10 -M or >C(R 102 )-L 10 -M
• at least one of X 7 to X 9 is >N-L 11 -M or >C(R 102 )-L 11 -M.
• R 101 to R 103 which are neither -L 10 -M nor -L 11 -M each represent a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an acyl group, -NR 8 R 9 , -OR 10 or a group selected from the above-mentioned hydrophilic group Pi.
• L 10 and L 11 each represent a single bond or a divalent linking group.
• the structure enclosed by ( ) n1 does not contain a phosphor moiety and contains at least one type of group selected from the above-mentioned hydrophilic group group Pi.
• n1 is an integer of 2 or more, and q is an integer of 0 or 1.
• R 4 to R 11 , X 1 to X 3 , M and m have the same meanings as R 4 to R 11 , X 1 to X 3 , M and m in the above-mentioned formula (II
• X 1 to X 3 in the above general formula (I) can be applied to X 4 to X 6 , except that at least one of X 4 to X 6 is >N-L 10 -M or >C(R 102 )-L 10 -M.
• X 7 to X 9 the descriptions of X 1 to X 3 in the above general formula (I) can be applied, unless otherwise specified, except that at least one of X 7 to X 9 is >N-L 11 -M or >C(R 102 )-L 11 -M.
• R 101 is preferably an alkyl group
• R 102 and R 103 are preferably hydrogen atoms.
• at least one is >CR 102 R 103
• more preferably at least one is >CR 102 R 103 and the remaining one is -O-, -S- or >CR 102 R 103
• further preferably both are >CR 102 R 103 .
• At least one is >CR 102 R 103 , more preferably at least one is >CR 102 R 103 and the remaining one is -O-, -S- or >CR 102 R 103 , and further preferably both are >CR 102 R 103 .
• X 4 to X 6 as >N-L 10 -M or >C(R 102 )-L 10 -M, >C(R 102 )-L 10 -M is preferable, and >CH-L 10 -M is more preferable.
• X 7 to X 9 as >N-L 11 -M or >C(R 102 )-L 11 -M, >C(R 102 )-L 11 -M is preferable, and >CH-L 11 -M is more preferable.
• the group having -L 10 -M is not particularly limited, but is preferably X 5 .
• X 7 to X 9 the group having -L 11 -M is not particularly limited, but is preferably X 8 .
• the alkylene group, alkenylene group, alkynylene group, arylene group, heteroarylene group, and >NR A which can constitute L10 and L11 can be the same as the alkylene group, alkenylene group, alkynylene group, arylene group, heteroarylene group, and >NR A which can constitute L2 and L5 described above.
• L x1 is a single bond or a group consisting of one or a combination of two or more of an alkylene group, an alkenylene group, an alkynylene group, an arylene group and a heteroarylene group
• the shortest number of atoms in the linking chain (each of the linking chains including L10 and L11 ) linking M to any of X4 to X9 may be, for example, 1 to 60, and preferably 1 to 40.
• the shortest number of atoms means the number of atoms constituting the shortest chain among the linking chains linking the conjugated structure portion for exhibiting fluorescence in the phosphor moiety M to any of X4 to X9 .
• the conjugated structure portion of M--any part of the linking chain represented by "-linking group ZZZ-L 10 -" in the structure represented by the above linking group ZZZ-L 10 -, and the conjugated structure portion of M--any part of the linking chain represented by "-linking group ZZZ-L 11 -" in the structure represented by the above linking group ZZZ-L 11 -, have the structure represented by the above-mentioned -(CH 2 -CH 2 -O) b - (b is also as defined above).
• n1 is an integer of 2 or more.
• the linker main chain connecting the two phosphor moieties is rigid and dye association can be more suppressed than in the compound in which L1 and L2 , and L4 and L5 in the above general formula (II) are not bonded to each other to form a specific 5-membered ring, so that the lower limit of n1 is preferably an integer of 3 or more, more preferably an integer of 6 or more, even more preferably an integer of 9 or more, and particularly preferably an integer of 12 or more. This is true whether the number of the phosphor moieties is 2 or 2 or more.
• n1 is, for example, preferably an integer of 36 or less, more preferably an integer of 24 or less, and even more preferably an integer of 18 or less. That is, n1 is preferably an integer of 2 to 36, more preferably an integer of 3 to 36, still more preferably an integer of 6 to 24, particularly preferably an integer of 9 to 24, and among these, an integer of 12 to 18 is preferable.
• q is an integer of 0 or 1, with 0 being preferred.
• R 6A and R 7A 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, or Q, provided that at least one of R 6A and R 7A represents a carboxy group, a substituent capable of binding to a biological material, or a substituent capable of binding to a solid support.
• L 12 and L 13 each represent a single bond or a linking group.
• Each of na and nb is an integer of 0 or more
• each of r and v is an integer of 0 or 1.
• R 4 , R 5 , R 11 , L 10 , L 11 , X 1 to X 9 , M, Q, q, n1 and m are synonymous with R 4 , R 5 , R 11 , L 10 , L 11 , X 1 to X 9 , M, Q, q, n1 and m in the above general formula (III).
• the structure enclosed by ( ) n1 does not contain a phosphor moiety and contains at least one type of group selected from the above-mentioned hydrophilic group group Pi.
• R 6A or R 7A and L 12 or L 13 are determined so that R 6A or R 7A is an unsubstituted group and L 12 or L 13 is the longest group, respectively, provided that when the group represented by -L 13 R 6A or -L 12 R 7A has Q, the Q located at the most terminal side (the R 6A side in -L 13 R 6A , the R 7A side in -L 12 R 7A ) is determined so that R 6A or R 7A .
• R 6A and R 7A each represent a hydrogen atom, a hydroxyl 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, or Q, provided that at least one of R 6A and R 7A represents a carboxyl group, a substituent capable of binding to a biological material, or a substituent capable of binding to a solid support.
• the alkyl group, alkenyl group, alkynyl group, aryl group, alkoxy group, heteroaryl group, amino group and acyl group which can be taken as R 6A and R 7A have the same meaning as the alkyl group, alkenyl group, alkynyl group, aryl group, alkoxy group, heteroaryl group, amino group and acyl group in the substituent group T described later, and the preferred ranges are also the same, however, all are unsubstituted groups.
• Q which can be adopted as R 6A and R 7A has the same meaning as Q described above, and the preferred range is also the same.
• R 6A is preferably an alkyl group, a sulfanyl group, an aryl group, a heteroaryl group or Q, more preferably an alkyl group or Q, and even more preferably an alkyl group, a carboxy group, a substituent capable of binding to a biological substance or a substituent capable of binding to a solid support.
• R 7A is preferably an alkyl group, a sulfanyl group, an aryl group, a heteroaryl group or Q, more preferably an alkyl group or Q, and even more preferably an alkyl group, a carboxy group, a substituent capable of binding to a biological substance or a substituent capable of binding to a solid support.
• L 12 and L 13 each represent a single bond or a linking group.
• R A represents a hydrogen atom or a substituent.
• alkylene group alkenylene group, alkynylene group, arylene group, heteroarylene group, and >NR A which can constitute L 12 and L 13
• the descriptions of the alkylene group, alkenylene group, alkynylene group, arylene group, heteroarylene group, and >NR A which can constitute L 1 to L 7 described above can be preferably applied.
• the substituents which may be possessed by the alkylene group, alkenylene group, alkynylene group, arylene group and heteroarylene group which may constitute L 12 and L 13 are not particularly limited and are preferably selected from the substituent group T described below, more preferably a halogen atom, an aryl group or an alkyl group.
• the substituents which may be possessed by the alkylene group, alkenylene group, alkynylene group, arylene group and heteroarylene group which may constitute L 12 and L 13 may be further substituted with a substituent selected from the substituent group T described below, and for example, a carboxy group, a sulfo group, a phosphono group, a cationic group, a betaine residue, a polyol residue, a sugar residue, a polyamino acid residue, -(O-L) g R E or -(L-O) g R E is preferable.
• the number of substituents that may be possessed by the alkylene group, alkenylene group, alkynylene group, arylene group, and heteroarylene group that may constitute L12 and L13 is not particularly limited as long as it can be adopted as a structure, and may be at least 1 or more.
• the upper limit is not particularly limited, and for example, all hydrogen atoms in the alkylene group, alkenylene group, alkynylene group, arylene group, and heteroarylene group may be substituted with substituents.
• n a is an integer of 0 or more (preferably an integer of 1 or more) and R 6A is a carboxy group, a substituent capable of binding to a biological substance, or a substituent capable of binding to a solid support, and/or (B) the sum of n b and v is an integer of 0 or more (preferably an integer of 1 or more) and R 7A is a carboxy group, a substituent capable of binding to a biological substance, or a substituent capable of binding to a solid support.
• the number of atoms linked by the shortest distance in L 13 is 7 or less
• the number of atoms linked by the shortest distance in L 12 is 7 or less.
• the "shortest number of linked atoms of L13" means, when na is an integer of 1 or more, the number of atoms constituting the shortest chain connecting N directly bonded to L13 and R6A among Ns shown in the structure grouped by ( ) na , and when na is 0, means the number of atoms constituting the shortest chain connecting N directly bonded to L13 and R6A among Ns shown in the structure grouped by ( ) m .
• the "shortest number of linked atoms of L12” means the number of atoms constituting the shortest chain connecting R7A and the atom to which L12 is bonded that is located to the left of L12 on the paper, similar to the "shortest number of linked atoms of L13 " above.
• L 12 is --NHC 2 H 4 -- and R 7A is --COOH, so that the shortest number of connecting atoms in L 12 is 3.
• R 6A and R 7A is a carboxy group, a substituent capable of binding to a biological substance, or a substituent capable of binding to a solid support.
• the shortest number of linked atoms in L 12 and the shortest number of linked atoms in L 13 are preferably 1-5, and more preferably 1-4.
• L 12 or L 13 is a group containing -(L-O) g -, and it is more preferable that L 12 is a group containing -(L-O) g -.
• na and nb are an integer of 0 or more.
• r and v is an integer of 0 or 1.
• R 6A is a carboxy group, a substituent capable of binding to a biological substance, or a substituent capable of binding to a solid support
• na is preferably an integer of 0 to 40, more preferably an integer of 0 to 20, and even more preferably an integer of 0 to 15.
• R 7A is a carboxy group, a substituent capable of binding to a biological substance, or a substituent capable of binding to a solid support
• the sum of nb and v is preferably an integer of 0 to 40, more preferably an integer of 0 to 20, and even more preferably an integer of 0 to 15.
• na is preferably an integer of 0 to 20, more preferably an integer of 0 to 10, and even more preferably an integer of 0 to 5.
• R 7A is not a carboxy group, a substituent capable of binding to a biological substance, or a substituent capable of binding to a solid support, the sum of nb and v is preferably an integer of 0 to 20, more preferably an integer of 0 to 10, and even more preferably an integer of 0 to 5.
• r is preferably 0 and v is preferably 0.
• the right side of the page is usually the C-terminal structure
• the left side of the page is usually the N-terminal structure.
• the compound of the present invention preferably contains at least one of the substituents represented by Q, which is a carboxy group, a substituent capable of binding to a biological substance, or a substituent capable of binding to a solid support.
• Q which is a carboxy group, a substituent capable of binding to a biological substance, or a substituent capable of binding to a solid support.
• the compound of the present invention can be bound to a biological substance via the carboxy group or a substituent capable of binding to a biological substance described below, to obtain a target labeled biological substance.
• the carboxy group can be easily induced to a substituent capable of binding to a biological substance by a conventional method.
• the compound of the present invention can be bound to a solid support such as a microparticle by a carboxy group or a substituent capable of binding to a solid support described below, to obtain the desired labeled microparticles.
• the microparticles are not particularly limited, but include small particles useful for binding to the compound of the present invention, including non-polymer beads such as glass beads and magnetic beads, and polymer beads.
• the microparticles include polystyrene beads.
• the small particles are not particularly limited as long as they are of a size commonly used in fluorescent labeling, but typically have an average particle size of 10 nm to 10 ⁇ m.
• the carboxy group can be easily induced to a substituent capable of binding to a solid support by a conventional method.
• the substituents capable of binding to a biological material and the substituents capable of binding to a solid support do not include carboxy groups
• the "substituents capable of binding to a biological material” include substituents derived from carboxy groups capable of binding to a biological material
• the "substituents capable of binding to a solid support” include substituents derived from carboxy groups capable of binding to a solid support.
• the carboxy group is located in a structure other than the structure represented by general formula (I) and the fluorescent body portion, and in the compound represented by general formula (II), it is preferable that the carboxy group is located in at least one of R6 and R7 .
• the number of the above-mentioned carboxy groups, substituents capable of binding to a biological substance, or substituents capable of binding to a solid support in the compound of the present invention may be at least one in total, and from the viewpoint of quantification of the substance to be detected, the number is preferably 1 to 3, more preferably 1 or 2, and even more preferably 1.
• the compound of the present invention has a group selected from the above-mentioned hydrophilic group group Pi at a position other than the phosphor portion.
• the compound has one or more groups, more preferably 1 to 8 groups, and even more preferably 1 to 6 groups.
• the position of the group selected from the hydrophilic group Pi is not particularly limited unless otherwise specified, and preferred examples of the group selected from the hydrophilic group Pi include X 1 to X 3 or R 11 in the compound represented by general formula (III).
• X + in -SO 3 - X + represents a monovalent cation such as a hydrogen ion or a polyvalent cation.
• examples of the monovalent cation and polyvalent cation that can be taken as X + include the above-mentioned alkali metal cations, alkaline earth metal cations, organic ammonium cations, organic phosphonium cations, etc.
• the compounds of the present invention can be bound to biological substances such as proteins (including peptides), amino acids, nucleic acids, nucleotides, sugar chains, and lipids via at least one substituent capable of binding to a biological substance possessed by the compounds, and can be used as labeled biological substances.
• the substituent capable of binding to a biological material can be used without any particular limitation as long as it is a group that acts (including attachment) or binds to a biological material, and examples thereof include the substituents described in WO 2002/026891, etc.
• electrophilic groups and nucleophilic groups described in Table 2 of WO 2002/026891, and the reactive groups Rx described in WO 2002/026891, page 18, line 16 to page 19, line 13 can be applied to the present invention.
• reactive groups Rx described in WO 2002/026891, page 18, line 16 to page 19, line 13 can be applied to the present invention.
• Specific examples of the "substituent capable of binding to a biological substance” include the following structures.
• X represents a halogen atom such as an iodine atom or a bromine atom.
• * represents a bond.
• peptide structures polyamino acid structures
• long-chain alkyl groups, etc. can also be used as the "substituent capable of binding to a biological substance".
• preferred examples include an NHS ester structure (N-hydroxysuccinimide 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.
• the present invention is not limited to these compounds.
• groups that have a dissociable hydrogen atom such as a carboxy group and a sulfo group
• the hydrogen atom may dissociate to form a salt structure.
• the compound of the present invention can be bound to a solid support such as the above-mentioned microparticles via at least one substituent of the compound capable of binding to a solid support, and can be used as a solid support reagent.
• the substituent capable of binding to a solid support can be used without any particular limitation as long as it is a group that acts (including attachment) or binds to a solid support, and preferred examples thereof include the substituents exemplified above as the substituent capable of binding to a biological substance, etc.
• preferred examples include an NHS ester structure (N-hydroxysuccinimide ester), a succinimide structure, and a maleimide structure.
• the present invention is not limited to these compounds.
• groups that have a dissociable hydrogen atom such as a carboxy group and a sulfo group
• the hydrogen atom may dissociate to form a salt structure.
• the compound of the present invention can be synthesized by a conventional method. For example, it can be synthesized based on peptide synthesis such as solid-phase peptide synthesis, and a method using an automatic peptide synthesizer described in International Publication No. 2018/174078 can also be preferably applied.
• the fluorescent body part, the physiologically active substance part, the prodrug part, and the radioisotope-containing part can also be synthesized based on a conventional method and introduced into the compound of the present invention.
• Compounds having a substituent capable of binding to a biological substance can also be synthesized by a conventional method, for example, Bioconjugate Techniques (Third Edition, by Greg T. Hermanson).
• the labeled biological material of the present invention is a material in which the compound of the present invention is bound to a biological material.
• the compound of the present invention has fluorescence due to the fluorescent part and shows an excellent aggregation suppression effect in a solution state, so it can be preferably used as a labeled biological material.
• the bond between the compound of the present invention and the biological material may be in the form of a direct bond between the compound of the present invention and the biological material, or may be in the form of a bond between the compound of the present invention and the biological material via a linking group.
• Preferred examples of the biological material include proteins (including peptides), amino acids, nucleic acids, nucleotides, sugar chains, and lipids.
• Preferred examples of the protein include antibodies, and preferred examples of the lipid include phospholipids, fatty acids, and sterols, with phospholipids being more preferred.
• the clinically pathologically useful materials are not particularly limited, but include, for example, immunoglobulins such as Ig (Immunoglobulin) G, IgM, IgE, IgA, and IgD, complement, C-reactive protein (CRP), ferritin, plasma proteins such as ⁇ 1 microglobulin and ⁇ 2 microglobulin and antibodies thereto, ⁇ -fetoprotein, carcinoembryonic antigen (CEA), prostatic acid phosphatase (PAP), CA (carbohydrate tumor markers such as CA-19-9, CA-125, and antibodies thereof; hormones such as luteinizing hormone (LH), follicle-stimulating hormone (FSH), human chorionic gonadotropin (hCG), estrogen, insulin, and antibodies thereof; substances related to viral infections such as hepatitis B virus (HBV)-related antigens (HBs, HBe, HBc), human immunodeficiency virus (HIV), adult T-cell leukemia
• bacteria such as Corynebacterium diphtheriae, Clostridium botulinum, Mycoplasma, Treponema pallidum, and antibodies thereof; protozoa such as Toxoplasma, Trichomonas, Leishmania, Trypanosoma, Plasmodium, and antibodies thereof; ES cells (embryonic stem cells) such as ELM3, HM1, KH2, v6.5, v17.2, v26.2 (derived from mice 129, 129/SV, C57BL/6, BALB/c) and antibodies thereof; antiepileptic drugs such as phenytoin, phenobarbital, and the like; cardiovascular drugs such as quinidine, digoxin, and the like; antiasthmatic drugs such as theophylline, and antibiotics such as chloramphenicol, gentamicin, and the like; other enzymes; and exotoxins (such as styrelidin O) and antibodies thereof.
• antibody fragments such as Fab'2, Fab
• binding can be performed in the form described in, for example, 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, pp. 62-83.
• the method described in the same document can be appropriately referred to.
• the labeled biological substance of the present invention obtained from a compound having a substituent capable of binding to a biological substance and a biological substance that binds to the compound through interaction includes compounds and products in which the parts other than the substituent capable of binding to a biological substance are replaced with the compounds of the present invention in the description of the compound examples and products in paragraph 0038 of JP2019-172826A.
• the present invention is not limited to these labeled biological substances, etc.
• the labeled biological material of the present invention may be in the form of a solution dissolved in an aqueous medium such as physiological saline or phosphate buffer, or in the form of a solid such as a fine particle powder or a freeze-dried powder, without any particular limitation, and the form may be appropriately selected depending on the purpose of use, etc.
• an aqueous medium such as physiological saline or phosphate buffer
• a solid such as a fine particle powder or a freeze-dried powder
• the labeled biological material of the present invention obtained from the compound of the present invention can exhibit excellent fluorescence intensity due to the excellent aggregation suppression effect of the compound of the present invention in a solution state, and it is considered that the fluorescence emitted from the labeled biological material excited by light irradiation can be stably detected. Therefore, the labeled biological material of the present invention can be applied to various techniques using fluorescent labels, and can be suitably used, for example, as a fluorescent labeling reagent in multicolor WB or dot blotting or a biological fluorescent imaging reagent.
• Fluorescence detection using the labeled biological material of the present invention generally comprises the following steps (i) to (iii) or (iv) to (vii): Fluorescence detection comprising steps (i) to (iii) corresponds to a direct method using a primary antibody fluorescently labeled with the compound of the present invention, and fluorescence detection comprising steps (iv) to (vii) corresponds to an indirect method using a secondary antibody fluorescently labeled with the compound of the present invention.
• a sample containing a target biological material hereinafter also referred to as a "target biological material”
• a labeled biological material of the present invention in which a biological material capable of binding to the target biological material in (a) (hereinafter also referred to as a "primary biological material”) is bound to a compound of the present invention (hereinafter also referred to as a "labeled biological material A of the present invention”).
• conjugate b preparing a conjugate in which the target biological material in (c) is bound to the primary biological material in (d) above;
• conjugate B2 preparing a conjugate in which the primary biological material in the conjugate b is bound to the secondary biological material in the labeled biological material B of the present invention; and
• the above-mentioned biological substances capable of binding to the target biological substance (primary biological substances) and biological substances capable of binding to the primary biological substances (secondary biological substances) include the biological substances in the labeled biological substances of the present invention. They can be appropriately selected according to the target biological substance (biological substance in a specimen) or the primary biological substance, and a biological substance capable of specifically binding to the biological substance in a specimen or the primary biological substance can be selected.
• proteins include so-called disease markers.
• Disease markers are not particularly limited, but include, for example, ⁇ -fetoprotein (AFP), PIVKA-II (protein induced by vitamin K absorption or antagonist II), BCA (breast carcinoma-associated antigen) 225, basic fetoprotein (BFP), CA (carbohydrate antigen) 15-3, CA19-9, CA72-4, CA125, CA130, CA602, CA54/61 (CA546), carcinoembryonic antigen (CEA), DUPAN-2, elastase 1, immunosuppressive acidic protein (IAP), NCC-ST-439, gamma-seminoprotein (gamma-Sm), prostate-specific antigen (PSA), prostatic acid phosphatase (PAP), neuron-specific enolase (NSE), Iba1, amyloid beta, tau, flotillin, squamous cell carcinoma-associated antigen (SCC antigen), sialyl LeX-
• AFP
• the target biological material may be bacteria, including, but not limited to, bacteria that are the subject of cellular microbiological testing, such as E. coli, Salmonella, Legionella, and bacteria that cause public health problems.
• the target biological material may be a viral antigen
• viral antigens include, but are not limited to, hepatitis virus antigens such as hepatitis C and B virus antigens, p24 protein antigens of HIV virus, pp65 protein antigens of CMV (cytomegalovirus), and E6 and E7 protein antigens of HPV (human papillomavirus).
• the sample containing the target biological material is not particularly limited and can be prepared according to a conventional method.
• the labeled biological substance of the present invention is also not particularly limited and can be prepared by binding a biological substance capable of binding to a target biological substance to the compound of the present invention in a conventional manner. The form of binding and the reaction for forming the bond are as explained for the labeled biological substance of the present invention.
• the target biological material and the primary biological material may be directly bonded to each other, or may be bonded to each other via another biological material different from the target biological material and the primary biological material.
• the primary biological material in the conjugate b and the secondary biological material in the labeled biological material B of the present invention may be directly bonded to each other, or may be bonded to each other via another biological material different from the primary biological material and the secondary biological material.
• the labeled biological material of the present invention can be used as a fluorescently labeled antibody in either the direct method or the indirect method, but is preferably used as a fluorescently labeled antibody in the indirect method.
• the binding between the labeled biological substance of the present invention and the target biological substance is not particularly limited and can be carried out according to a conventional method.
• the wavelength for exciting the labeled biological material of the present invention is not particularly limited as long as it is light of a wavelength capable of exciting the labeled biological material of the present invention. In general, 300 to 1000 nm is preferred, and 400 to 800 nm is more preferred.
• the fluorescence excitation light source used in the present invention is not particularly limited as long as it emits light of a wavelength capable of exciting the labeled biological material of the present invention, and for example, various laser light sources can be used.
• various optical filters can be used to obtain a preferred excitation wavelength or to detect only the fluorescence.
• a blot membrane is prepared by a method normally used for the target biological material (protein separation by electrophoresis, blotting to a membrane, blocking of the membrane), and the labeled biological material of the present invention is used as a labeled antibody (preferably a secondary antibody), thereby enabling detection of the target biological material with excellent fluorescence intensity.
• a blot nitrocellulose membrane or a blot PVDF (polyvinylidene fluoride) membrane is prepared by a method normally used for the target biological material, and the labeled biological material of the present invention is used as a labeled antibody (preferably a secondary antibody), thereby enabling detection of the target biological material with excellent fluorescence intensity.
• a labeled antibody preferably a secondary antibody
• substituent group T-- preferred examples of the substituent include those selected from the following substituent group T.
• substituent group T when a substituent is merely described, the description of the corresponding substituent in the substituent group T can be referred to and applied.
• substituent group T when only an "alkyl group” is described, the description of the "alkyl group” in the substituent group T can be referred to and applied. This also applies to other substituents other than the "alkyl group”.
• substituents selected from the following substituent group T examples of the substituent that a certain substituent such as an "alkyl group” may have include substituents selected from the following substituent group T.
• the alkyl group is described in distinction from the cyclic (cyclo) alkyl group, the alkyl group is used to include linear alkyl groups and branched alkyl groups.
• the alkyl group is used to include linear alkyl groups, branched alkyl groups, and cycloalkyl groups.
• groups alkoxy groups, alkylthio groups, alkenyloxy groups, etc.
• groups that can have a cyclic structure alkyl groups, alkenyl groups, alkynyl groups, etc.
• compounds that contain groups that can have a cyclic structure.
• the lower limit of the number of atoms of the group that forms 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 groups that can have this structure.
• substituent group T a group having a linear or branched structure and a group having a cyclic structure are sometimes described separately, for example, an alkyl group and a cycloalkyl group, in order to clarify the difference.
• the groups contained 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, even more preferably having 1 to 12 carbon atoms, even more preferably having 1 to 8 carbon atoms, even 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, even more preferably having 2 to 12 carbon atoms, even more preferably having 2 to 6 carbon atoms, and even more preferably having 2 to 4 carbon atoms
• an alkynyl group preferably having 2 to 30 carbon atoms, more preferably having 2 to 20 carbon atoms, and even more preferably having 1 to 3 carbon atoms.
• the aryl group has 6 to 40 carbon atoms, more preferably 6 to 30 carbon atoms, even more preferably 6 to 26 carbon atoms, and particularly preferably 6 to 10 carbon atoms), a cycloalkyl group (preferably 3 to 20 carbon atoms), a cycloalkenyl group (preferably 5 to 20 carbon atoms), an aryl group (which may be a monocyclic group or a condensed ring group (preferably a condensed ring group of 2 to 6 rings). If the condensed ring group, it is composed of a 5- to 7-membered ring, etc.
• the aryl group preferably has 6 to 40 carbon atoms, more preferably 6 to 30 carbon atoms, even more preferably 6 to 26 carbon atoms, and particularly preferably 6 to 10 carbon atoms), a heterocyclic group (a It has at least one nitrogen atom, oxygen atom, sulfur atom, phosphorus atom, silicon atom or selenium atom as a constituent atom, and may be a monocyclic group or a condensed ring group (preferably a condensed ring group of 2 to 6 rings). In the case of a monocyclic group, the number of ring members is preferably 5 to 7, more preferably 5 or 6. The number of carbon atoms in the heterocyclic group is preferably 2 to 40, more preferably 2 to 20.
• Heterocyclic groups include aromatic heterocyclic groups (heteroaryl groups) and aliphatic heterocyclic groups (aliphatic heterocyclic groups). ), alkoxy group (preferably having 1 to 20 carbon atoms, more preferably having 1 to 12 carbon atoms), alkenyloxy group (preferably having 2 to 20 carbon atoms, more preferably having 2 to 12 carbon atoms), alkynyloxy group (preferably having 2 to 20 carbon atoms, more preferably having 2 to 12 carbon atoms), cycloalkyloxy group (preferably having 3 to 20 carbon atoms), aryloxy group (preferably having 6 to 40 carbon atoms, more preferably having 6 to 26 carbon atoms, even more preferably having 6 to 14 carbon atoms), heterocyclic oxy group (preferably having 2 to 20 carbon atoms), polyalkyleneoxy group,
• Alkoxycarbonyl group (preferably having 2 to 20 carbon atoms), cycloalkoxycarbonyl group (preferably having 4 to 20 carbon atoms), aryloxycarbonyl group (preferably having 6 to 20 carbon atoms), amino group (preferably having 0 to 20 carbon atoms, unsubstituted amino group (-NH 2 ), (mono- or di-) alkylamino group, (mono- or di-) alkenylamino group, (mono- or di-) alkynylamino group, (mono- or di-) cycloalkylamino group, (mono- or di-) cycloalkenylamino group, (mono- or di-) arylamino group, (mono- or di-) heterocyclic amino group.
• Acylamino group (preferably 1 to 20 carbon atoms), sulfonamido group (preferably 0 to 20 carbon atoms, preferably an alkyl, cycloalkyl or aryl sulfonamido group), alkylthio group (preferably 1 to 20 carbon atoms, more preferably 1 to 12 carbon atoms), cycloalkylthio group (preferably 3 to 20 carbon atoms), arylthio group (preferably 6 to 40 carbon atoms, more preferably 6 to 26 carbon atoms, even more preferably 6 to 14 carbon atoms), heterocyclic thio group (preferably 2 to 20 carbon atoms), alkyl, cycloalkyl or arylsulfonyl group (preferably 1 to 20 carbon atoms),
• Silyl group (preferably a silyl group having 1 to 30 carbon atoms, more preferably a silyl group having 1 to 20 carbon atoms and substituted with an alkyl, aryl, alkoxy or aryloxy), silyloxy group (preferably a silyloxy group having 1 to 20 carbon atoms and substituted with an alkyl, aryl, alkoxy or aryloxy), hydroxy group, cyano group, nitro group, halogen atom (e.g., fluorine atom, chlorine atom, bromine atom or iodine atom), oxygen atom (specifically, >CH 2 constituting the ring is replaced with >C ⁇ O), carboxy group (—CO 2 H), phosphono group [—PO(OH) 2 ], phosphonooxy group [—O—PO(OH) 2 ], sulfo group (—SO 3 H), boronic acid group [—B(OH) 2 ], aniono group (also called a cationic group,
• the anionic group may be any group having an anion.
• examples of such anionic group include a carboxy group, a phosphono group (phosphonic acid group, -PO(OH) 2 ), a phosphonooxy group (phosphate group, -OPO(OH) 2 ), and a sulfo group, with the phosphono group, phosphonooxy group, and sulfo group being preferred, and the phosphonooxy group and sulfo group being more preferred.
• the anionic group may have an ionic structure in which a hydrogen ion is dissociated, or may have a salt structure.
• the monovalent or polyvalent cation may be the same as that described above for the salt structure.
• the cationic group may be a group having a cation. Examples of such cationic groups include a group having a quaternary ammonium ion (ammonio group) and a group having a quaternary phosphonium ion (phosphonio group), and the like, and the group having a quaternary ammonium ion is preferred.
• the substituent of N + in the group having a quaternary ammonium ion and the substituent of P + in the group having a quaternary phosphonium ion are preferably alkyl groups and aryl groups, and it is more preferred that all the substituents of N + and P + are alkyl groups.
• the cationic group may have a salt structure in addition to an ionic structure.
• examples of the monovalent or polyvalent anion include halide ions such as F ⁇ and Cl ⁇ , and monovalent or polyvalent organic anions such as BF 4 ⁇ , PF 6 ⁇ , and bis(trifluoromethylsulfonyl)imide ion.
• the betaine residue means a group obtained by removing one hydrogen atom from a compound having a betaine structure.
• the compound having a betaine structure may be any compound containing an anionic group and a cationic group in the same molecule, and is preferably a compound containing at least one anionic group selected from a carboxy group, a phosphono group, a phosphonooxy group, and a sulfo group, and at least one cationic group selected from an ammonio group and a phosphonio group in the same molecule.
• a group obtained by removing one hydrogen atom from a compound that does not contain an anionic group and a cationic group in the same molecule does not correspond to a betaine residue, and is classified as the above-mentioned anionic group having a salt structure or cationic group having a salt structure.
• the polyol residue means a group obtained by removing one hydrogen atom from a polyol compound having two or more hydroxy groups in the molecule, which does not correspond to either a sugar residue or a polyalkyleneoxy group described below.
• the polyol residue may be a chain-like group or a group having a cyclic structure, and an example of the polyol residue is a cyclodextrin residue.
• the sugar residue means a group obtained by removing one hydrogen atom from a sugar compound.
• the sugar compound may be any of monosaccharides, polysaccharides in which two or more sugars are bound, sugar alcohols, and chemically modified sugars in which epichlorohydrin or the like is copolymerized with a sugar.
• polyol residues that correspond to sugar residues are classified as sugar residues.
• sugar residues include glucose, sucrose, maltose, lactose, trehalose, ribitol, sorbitol, mannitol, maltitol, lactitol, xylitol, fructose, 1-kestose, nystose trihydrate, fucose, dulcitol, galactooligosaccharide, 4'-galactosyllactose, isomaltooligosaccharide, lactulose, palatinite, palatinose monohydrate, raffinose pentahydrate, arabinose, dihydroxyacetone dimer, galactose, glyceraldehyde dimer, mannose, ribose, xylose, lactose,
• sugar compounds include those obtained by removing one hydrogen atom from a sugar compound such as rufructoside, erythritol, dulcoside A, isosteviol, rebaudioside
• the polyamino acid residue means a group obtained by removing one hydrogen atom from an amino acid compound or a polyamino acid compound formed by binding two or more amino acids.
• the polyamino acid residue is a group that is adjusted and used so as to be neutral (the sum of the charges is 0) when the compound of the present invention and the labeled biological substance are used.
• the polyalkyleneoxy group may be a group represented by -(OL) g R E or -(LO) g R E.
• those which correspond to a polyalkyleneoxy group are classified as a polyalkyleneoxy group.
• the above L represents an alkylene group obtained by removing one hydrogen atom from an alkyl group in the above substituent group T, and the number of carbon atoms is preferably 2 to 4, more preferably 2 or 3, and still more preferably 2, and the number of carbon atoms contained in the shortest chain connecting two carbon atoms which are bonds of the alkylene 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 above g means the average number of repetitions (also simply referred to as the number of repetitions), and is preferably from 2 to 50, more preferably from 12 to 50, and even more preferably from 24 to 50.
• R E has the same meaning as R 8 to R 10 described above, and represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an acyl group, an aryl group, a heteroaryl group, an anionic group, a betaine residue, a polyol residue, a sugar residue, or a polyamino acid residue, and is preferably a hydrogen atom or an alkyl group.
• the alkyl group that can be taken as R E can be preferably the same as described for the alkyl group in the above substituent group T, and among them, an alkyl group having 1 to 3 carbon atoms is preferable.
• the alkyl group that can be taken as R E may have a substituent.
• hydrophilic group group Pi “carboxy group, sulfo group, phosphono group, betaine residue, polyol residue, sugar residue, polyamino acid residue, -(O-L) gR E , and -(L-O) gR E " is described as all or a part of options of a specific substituent, unless otherwise specified, the hydrophilic group group Pi: "carboxy group, sulfo group, phosphono group, betaine residue, polyol residue, sugar residue, polyamino acid residue, -(O-L) gR E , and -(L-O) gR E " in the options of the specific substituent is the hydrophilic group group Pi1: "carboxy group, sulfo group, phosphono group, polyol residue, sugar residue, polyamino acid residue, -(O-L) gR E , and -(L-O) gR E " is preferably replaced with
• hydrophilic group group Pi "a carboxy group, a sulfo group, a phosphono group, a betaine residue, a polyol residue, a sugar residue, a polyamino acid residue, -(O-L) g R E , and -(L-O) g R E " is described as all or part of the preferred range of the substituent.
• Examples of groups formed by combining a plurality of substituents selected from the substituent group T include the above-mentioned 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, alkyl, cycloalkyl or arylsulfonyl group, each of which has as a
• 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, a carboxy group, a sulfo group, a phosphono group, a betaine residue, a polyol residue, a sugar residue, a polyamino acid residue, -(O-L) g R E or -(L-O) g R E , and particularly preferably an alkyl group, an alkenyl group, an aryl group, a heterocyclic group, an alkoxy group, an acyl group,
• the substituents selected from the substituent group T include, in addition to groups formed by combining multiple substituents selected from the above-mentioned substituent group T, groups formed by combining multiple of the above groups, unless otherwise specified.
• groups formed by combining multiple of the above groups unless otherwise specified.
• the compound or the substituent contains an alkyl group, an alkenyl group, etc., these may be substituted or unsubstituted.
• an aryl group, a heterocyclic group, etc. are contained, they may be monocyclic or condensed, and may be substituted or unsubstituted.
• room temperature means 25°C.
• X + in the sulfo group represents a monovalent cation such as a hydrogen ion or a polyvalent cation.
• the sulfo group may contain a salt structure, for example, a metal salt such as a potassium salt or a sodium salt, an organic ammonium salt such as a TEA (triethylammonium) salt, a DIPEA (N,N-diisopropylethylammonium) salt, or a phosphonium salt, or an organic phosphonium salt. This also applies to the description of the synthesis examples described below.
• Comparative compound (1) is Alexa Fluor 488 (trade name, manufactured by Thermo Fisher Scientific). Comparative compound (2) corresponds to compound (1) described in WO 2021/100814, synthesized based on Synthesis Example 1 described in the Examples of WO 2021/100814.
• %v/v means percent by volume
• %w/v means percent by mass
• the carrier used in reverse phase column chromatography was SNAP Ultra C18 (trade name, manufactured by Biotage) or Sfar C18 (trade name, manufactured by Biotage), and the carrier used in normal phase column chromatography was Hi-Flash Column (trade name, manufactured by Yamazen).
• the mixing ratio of the eluent used in reverse phase column chromatography or normal phase column chromatography is a volume ratio.
• MS spectra were measured using an ACQUITY SQD LC/MS System (trade name, manufactured by Waters, ionization method: ESI (ElectroSpray Ionization)) or an LCMS-2010EV (trade name, manufactured by Shimadzu Corporation, ionization method: simultaneous ESI and APCI (Atmospheric Pressure Chemical Ionization)).
• ACQUITY SQD LC/MS System trade name, manufactured by Waters, ionization method: ESI (ElectroSpray Ionization)
• LCMS-2010EV trade name, manufactured by Shimadzu Corporation, ionization method: simultaneous ESI and APCI (Atmospheric Pressure Chemical Ionization)
• the synthesis apparatus was set with Rink Amide-ChemMatrix (registered trademark, manufactured by Biotage), 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 performed according to the manual.
• One cycle consisted of Fmoc deprotection (20 minutes), washing with NMP, condensation of Fmoc amino acid (1 hour), and washing with NMP.
• the peptide chain was extended by repeating this cycle.
• the white solid was then transferred to a 100 mL recovery flask, and 15 mL of chloroform, 8 mL of N,N-diisopropylethylamine (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and 3.55 g of azidoacetic acid-NHS ester (manufactured by BroadPharm Co., Ltd.) were added, and the mixture was stirred at room temperature for 15 minutes. After the reaction was completed, an aqueous hydrochloric acid solution was added under ice cooling, the mixture was extracted with chloroform, and the organic layer was washed three times with an aqueous hydrochloric acid solution. The organic layer was then added to 300 mL of MTBE, and filtered under reduced pressure to obtain 4.5 g of compound (P-1) as a white solid.
• compound (P-1) was elongated, and the elongation using N-(9-fluorenylmethoxycarbonyl)-L-proline (Fmoc-Pro-OH) was repeated for three cycles.
• compounds (1) to (8) are compounds having three or four phosphor moieties consisting of a pyrromethene dye (comparative compound (2)) and a structure represented by general formula (I), and are compounds defined in the present invention.
• the fluorescence quantum yield of the solutions of these compounds (1) to (8) was maintained high, at 0.73 or more for the compounds having three phosphor moieties and 0.25 or more for the compounds having four phosphor moieties, and the fluorescent brightness (converted brightness) of the compounds was superior to that of comparative compound (2), which is a dye monomer.
• the compound of the present invention is a compound having two or more fluorescent moieties and a structure represented by general formula (I), and is therefore believed to be able to effectively suppress self-association and association quenching of the compound of the present invention in a solution state, maintain a high fluorescence quantum yield, and achieve excellent fluorescence brightness.

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• 2024
  • 2024-02-27 WO PCT/JP2024/007133 patent/WO2024181456A1/ja not_active Ceased
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