US20090118471A1

US20090118471A1 - Novel coumarin derivatives

Info

Publication number: US20090118471A1
Application number: US12/337,327
Authority: US
Inventors: Tetsuo Nagano; Yasuteru Urano; Takuji Shoda
Original assignee: University of Tokyo NUC
Current assignee: University of Tokyo NUC
Priority date: 2006-02-06
Filing date: 2008-12-17
Publication date: 2009-05-07
Also published as: US7482473B2; US20070197778A1; JP2007204452A; JP4929452B2

Abstract

A compound represented by the following general formula (I) or a salt thereof [R¹represents hydrogen atom, or a C_1-12alkyl group; R²represents a C_1-12alkyl group having one or more maleimide groups, an aryl group having two or more maleimide groups, a group represented by the formula (A): —(CH₂)_n—CO—N(R¹¹)(R¹²) (R¹¹and R¹²represent hydrogen atom, a C_1-12alkyl group, a C_1-12alkyl group having one or more maleimide groups, or an aryl group having one or more maleimide groups, and n represents an integer of 1 to 6), or a group represented by the formula (B): —(CH₂)_m—NH—CO—R¹³(R¹³represents a C_1-12alkyl group having one or more maleimide groups, or an aryl group having one or more maleimide groups, and m represents an integer of 1 to 6); R³and R⁵represent hydrogen atom, or a halogen atom; R⁴represents hydroxyl group, a C_1-12acyloxy group, a C_1-12acyloxymethyloxy group, or an amino group], which is useful as a novel fluorescent labeling agent having a property of emitting intense fluorescence only after reacting with a thiol group.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of application Ser. No. 11/470,820, filed Sep. 7, 2006, the disclosure of which is expressly incorporated by reference herein in its entirety.
The present application also claims priority under 35 U.S.C. §119 of Japanese Patent Application No. 2006-027815, filed Feb. 6, 2006.

TECHNICAL FIELD

The present invention relates to a novel coumarin derivative. More specifically, the present invention relates to a coumarin derivative having a feature of being substantially non-fluorescent, per se, and emitting intense fluorescence after reaction with a thiol group.

BACKGROUND ART

Direct fluorescent visualization of intracellular localization and dynamic behavior of proteins in cells and tissues in a living state is extremely important for elucidation of physiological functions of the proteins, so techniques utilizing fusion proteins comprising green fluorescent protein (GFP) have been widely used in recent years. However, a possibility of not being able to monitor target protein's behavior precisely is pointed out due to extremely large molecular size of GFP per se, a time lag between intracellular expression of GFP and formation of fluorophore, and the like.
As a method for introducing a fluorescent tag into a protein, it is known to use a thiol-reactive fluorescent labeling agent such as CPM, MDCC, fluorescein-5-maleimide and TMR-5-maleimide utilizing thiol groups in proteins (for example, thiol group of cysteine).
Although the molecular size of these thiol-reactive fluorescent labeling agents is small, they have a problem that the difference of fluorescent intensities between before and after the reaction with a thiol group is small; specifically they may emit intense fluorescence before the reaction with thiol group, or they may have very weak fluorescence after being introduced into proteins by the reaction. Moreover, since the fluorescence tag is introduced by an addition reaction with arbitrary proteins containing cysteine residues with a thiol group, they have a problem that they cannot fluorescently label the target protein with fluorescence for fluorescent-based observation.
Under these circumstances, it has been desired to provide (1) a fluorescent labeling agent having small molecular size achieving significant difference of fluorescence intensities between before and after the reaction with thiol group, and (2) a means for introducing a fluorescent tag selectively only into target protein by fluorescent labeling agent whose molecular size is small to visualize the target protein fluorescently at high sensitivity.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a novel fluorescent labeling agent having a property of emitting intense fluorescence only after reaction with a thiol group. Another object of the present invention is to provide a means for specifically introducing a fluorescent tag whose molecular size is small only into a target protein to visualize the protein fluorescently at high sensitivity.
The inventors of the present invention conducted various researches to achieve the aforementioned objects. As a result, they found that, in a coumarin derivative into which a substituent having maleimide group is introduced, the maleimide group acted as a PeT (Photoinduced Electron Transfer) acceptor to quench fluorescence of the coumarin as fluorophore, and after the nucleophilic addition of a nucleophilic agent such as a thiol group (for example, thiol group of cysteine) to maleimide group, the maleimide group no longer acted as a PeT acceptor, and thus the fluorescence of the coumarin was recovered. On the basis of this finding, they found that a thiol-reactive fluorescent labeling agent was successfully provided which was capable of switching fluorescence on and off by a reaction with a thiol group.
The inventors of the present invention also found that a coumarin derivative into which a substituent having two or more maleimide groups were introduced was substantially non-fluorescent. This is because all of the maleimide groups functioned as PeT acceptors, thus the coumarin's fluorescence as the fluorophore was quenched. On the other hand, only when all the maleimide groups underwent addition reactions with nucleophilic agents such as thiol groups the compound was highly fluorescent. On the basis of these findings, the inventors of the present invention found that when the aforementioned coumarin derivative into which a substituent having two or more maleimide groups were introduced reacted with a peptide having two or more neighboring cysteine residues, the compound provided fluorescent adduct only when all the maleimide groups underwent addition reactions with respective thiol groups of the cysteine residues, and that the compound was useful as a reagent for selectively labeling a peptide having two or more neighboring cysteine residues in the same molecule. The present invention was accomplished on the basis of the aforementioned findings.
The present invention thus provides a compound represented by the following general formula (I) or a salt thereof
[wherein R¹represents hydrogen atom, or a C_1-12alkyl group; R²represents a C_1-12alkyl group having one or more maleimide groups, an aryl group having two or more maleimide groups, a group represented by the following formula (A):
—(CH₂)_n—CO—N(R¹¹)(R¹²)
(wherein R¹¹and R¹²independently represent hydrogen atom, a C_1-12alkyl group, a C_1-12alkyl group having one or more maleimide groups, or an aryl group having one or more maleimide groups, provided that at least one of R¹¹and R¹²represents a C_1-12alkyl group having one or more maleimide groups, or an aryl group having one or more maleimide groups, and n represents an integer of 1 to 6), or a group represented by the following formula (B):
—(CH₂)_m—NH—CO—R¹³
(wherein R¹³represents a C_1-12alkyl group having one or more maleimide groups, or an aryl group having one or more maleimide groups, and m represents an integer of 1 to 6); R³and R⁵independently represent hydrogen atom, or a halogen atom; R⁴represents hydroxyl group, a C_1-12acyloxy group, a C_1-12acyloxymethyloxy group, or an amino group which may have one or two substituents].
The aforementioned compound or a salt thereof itself is substantially non-fluorescent, but when all the maleimide groups existing in the group represented by R²undergo addition reactions with thiol groups of a compound or biological substance containing the thiol group (for example, cysteine, peptide or protein containing a cysteine residue, etc.), the resulting adduct has highly fluorescent property.
According to a preferred embodiment of this invention, provided is the aforementioned compound of the formula (I) or a salt thereof, wherein R¹is hydrogen atom; R²is a C_1-12alkyl group having one maleimide group, a group represented by the aforementioned formula (A) (provided that one of R¹¹and R¹²is hydrogen atom, or a C_1-12alkyl group, and the other is a C_1-12alkyl group having one maleimide group, or an aryl group having one maleimide group), or a group represented by the aforementioned formula (B) (provided that R¹³is a C_1-12alkyl group having one maleimide group, or an aryl group having one maleimide group); each of R³and R⁵is independently hydrogen atom, or a halogen atom; and R⁴is hydroxyl group, a C_1-12acyloxy group, a C_1-12acyloxymethyloxy group, or an amino group which may have one or two substituents.
According to the most preferred embodiment of the aforementioned invention, provided is a compound represented by the following formula (III) or (IV), or a salt thereof:
(wherein p represents an integer of 1 to 6, and q represents an integer of 1 to 6).
According to another preferred embodiment of the invention, provided is the aforementioned compound of the formula (I) or a salt thereof, wherein R¹is hydrogen atom; R²is a C_1-12alkyl group having two or more maleimide groups, an aryl group having two or more maleimide groups, a group represented by the aforementioned formula (A) (provided that one of R¹¹and R¹²is hydrogen atom or a C_1-12alkyl group, and the other is a C_1-12alkyl group having two or more maleimide groups, or an aryl group having two or more maleimide groups), a group represented by the aforementioned formula (A) (provided that each of R¹¹and R¹²is independently a C_1-12alkyl group having one or more maleimide groups, or an aryl group having one or more maleimide groups, and the total number of the maleimide groups existing in R¹¹and R¹²is 2 or more), or a group represented by the aforementioned formula (B) (provided that R¹³is a C_1-12alkyl group having two or more maleimide groups, or an aryl group having two or more maleimide groups); each of R³and R⁵is independently hydrogen atom or a halogen atom; and R⁴is hydroxyl group, a C_1-12acyloxy group, a C_1-12acyloxymethyloxy group, or an amino group which may have one or two substituents.
According to the most preferred embodiment of the aforementioned invention, provided is a compound represented by the following formula (V) or a salt thereof
From another aspect, the present invention provides a compound represented by the aforementioned general formula (I) or a salt thereof, which is used for fluorescent labeling of a compound having a thiol group, or a peptide or protein containing a cysteine residue, and a fluorescent labeling agent used for fluorescent labeling of a compound having a thiol group, or a peptide or protein containing a cysteine residue, which comprises the compound represented by the aforementioned general formula (I) or a salt thereof. The compound represented by the aforementioned general formula (I) or a salt thereof undergoes addition reaction with a thiol group of a cysteine residue to provide a highly fluorescent adduct. As a result, a compound having the thiol group, or a peptide or protein containing the cysteine residue can be fluorescently labeled by the fluorescent adduct.
From a still further aspect, the present invention provides the aforementioned compound or a salt thereof having two or more maleimide groups, which is used for fluorescent labeling of a peptide or protein containing two or more neighboring cysteine residues in the same molecule. When not all the maleimide groups of the compound represented by the aforementioned general formula (I) or a salt thereof having two or more maleimide groups undergo addition reactions with thiol groups, the compound or a salt thereof is substantially non-fluorescent, on the other hand only when all the maleimide groups undergo addition reactions with thiol groups, the compound affords a highly fluorescent adduct. Therefore, when the compound represented by the aforementioned general formula (I) or a salt thereof having two or more maleimide groups reacts with a peptide or protein containing two or more neighboring cysteine residues in the same molecule, all of the maleimide groups very quickly undergo the addition reactions with thiol groups, and as a result, a highly fluorescent adduct is provided. By utilizing this property, a peptide or protein containing two or more neighboring cysteine residues in the same molecule can be selectively fluorescent labeled by using the aforementioned compound or a salt thereof.
The present invention further provides a method for fluorescently labeling a compound having a thiol group, or a peptide or protein containing a cysteine residue, which comprises the step of reacting a compound represented by the aforementioned general formula (I) or a salt thereof with a compound having a thiol group, or a peptide or protein containing a cysteine residue.
The compound represented by the aforementioned general formula (I) or a salt thereof has a property that the compound or a salt, per se, is substantially non-fluorescent, but provides a highly fluorescent adduct only when the compound reacts with a thiol group (for example, a thiol group of a low molecular weight compound such as cysteine, or a peptide or protein containing a cysteine residue). Therefore, the compound represented by the aforementioned general formula (I) or a salt thereof according to the present invention can be used as a fluorescent labeling agent for a substance having thiol group.
Further, the compound represented by the aforementioned general formula (I) or a salt thereof having two or more maleimide groups as R²has a property that the compound or a salt thereof, per se, is substantially non-fluorescent, and only when all of the maleimide groups react with thiol groups, the compound provides a highly fluorescent adduct. Therefore, by using this compound or a salt thereof, a peptide or protein containing neighboring cysteine residues in the same molecule, in a number not smaller than that of the maleimide groups, can be selectively fluorescent labeled.

BRIEF EXPLANATION OF THE DRAWINGS

FIG. 1 shows optical characteristics of Compounds 1, 2, 3, and 4, and optical characteristics of cystein adducts of Compounds 1, 2, 3, and 4.

FIG. 2 shows results of measurement results of changes in fluorescence intensity over time observed when a substance having thiol group(s) (N-acetylcysteine, or a peptide containing two neighboring cysteine residues in the same molecule) was reacted with Compound 8.

BEST MODE FOR CARRYING OUT THE INVENTION

The meanings of the terms used in this specification are as follows.
The “alkyl group” or an alkyl moiety of a substituent containing the alkyl moiety (for example, alkoxyl group and the like) means a linear, branched, or cyclic saturated hydrocarbon group, or a combination thereof. More specifically, examples of the alkyl group include, methyl group, ethyl group, n-propyl group, isopropyl group, cyclopropyl group, n-butyl group, sec-butyl group, isobutyl group, tert-butyl group, cyclopropylmethyl group, n-pentyl group, n-hexyl group, adamantyl group, and the like.
The “aryl group” means a monocyclic or condensed polycyclic aromatic hydrocarbon group or a monocyclic or condensed polycyclic aromatic heterocyclic group, and is preferably a monocyclic to tricyclic aromatic hydrocarbon group, or a monocyclic to tricyclic aromatic heterocyclic group, more preferably a monocyclic or bicyclic aromatic hydrocarbon group. Specific examples include phenyl group, naphthyl group, and the like.
An acyl moiety of a substituent containing the acyl moiety (for example, acyloxy group and the like) may be either an aliphatic acyl group or an aromatic acyl group, and hydrocarbon moiety of the aliphatic acyl group is a linear, branched, or cyclic hydrocarbon group, or a combination thereof, and may contain one or more unsaturated bonds. As an aromatic group of the aromatic acyl group, those explained for the aforementioned aryl group can be used.
The peptide includes dipeptides, tripeptides, oligopeptides (containing several to about 20 amino acid residues), polypeptides (containing more than 20 amino acid residues), and the like, and include those containing natural amino acid residues as well as non-naturally occurring amino acid residues.
In the compound represented by the aforementioned general formula (I), the C_1-12alkyl group represented by R¹is preferably methyl group or ethyl group, most preferably methyl group.
The C_1-12alkyl group on which one or more maleimide groups are substituted, represented by R²in the compound represented by the aforementioned general formula (I), may be any linear, branched or cyclic C_1-12alkyl group or a combination thereof on which one or more maleimide groups are substituted (although the maleimide groups may preferably substitute directly on the C_1-12alkyl group, they may indirectly substitute on the C_1-12alkyl group via an amide group, a phenylene group, or the like, and substitution positions of the maleimide groups on the C_1-12alkyl group may be any substitutable positions, and are not particularly limited: the same shall apply to the term “substituted with maleimide groups” used in this specification). A C_1-6linear alkyl group with one or more maleimide groups directly substituted is preferred. When R²is a C_1-12linear alkyl group with one maleimide group directly substituted, the group is most preferably monomaleimidomethyl group.
Examples of the aryl group with two or more maleimide groups substituted represented by R²in the compound represented by the aforementioned general formula (I) include a phenyl group with two or three maleimide groups substituted, a monocyclic aromatic heterocyclic group with two or three maleimide groups (for example, triazenyl group) substituted, and the like, and a phenyl group with two or three maleimide groups substituted is preferred.
In the compound represented by the aforementioned general formula (I), R²is also preferably group represented by the formula (A). In the group represented by the formula (A), n represents an integer of 1 to 6, preferably an integer of 1 to 3, particularly preferably 1.
When R¹¹or R¹²represents a C_1-12alkyl group, this group is preferably ethyl group or methyl group, most preferably methyl group.
Examples of the C_1-12alkyl group having one or more maleimide groups and represented by R¹¹or R¹²include a linear, branched or cyclic C_1-12alkyl group or a combination thereof with one or more maleimide groups substituted, and when R¹¹or R¹²represents an C_1-12alkyl group with one maleimide group substituted, the alkyl group is preferably a C_1-6linear alkyl group, and more specifically, R¹¹or R¹²is most preferably 2-maleimidoethyl group, 3-maleimidopropyl group, or 5-maleimidopentyl group. When R¹¹or R¹²represents a C_1-12alkyl group with two maleimide groups substituted, R¹¹or R¹²is preferably a dimaleimide-substituted cyclohexyl group or a dimaleimide-substituted adamantyl group, most preferably 3,5-dimaleimidocyclohexyl group. When R¹¹or R¹²represents a C_1-12alkyl group with three maleimide groups substituted, the group is preferably a trimaleimide-substituted adamantyl group.
Examples of the aryl group having one or more maleimide groups represented by R¹¹or R¹²include, for example, a phenyl group with one to three maleimide groups substituted, a monocyclic aromatic heterocyclic group with one to three maleimide groups (for example, triazinyl group) substituted, and the like, and a phenyl group with one to three maleimide groups substituted is preferred.
As for the combination of R¹¹and R¹², when the total number of maleimide group existing in the groups represented by R¹¹and R¹²is 1, it is preferred that one of R¹¹and R¹²is hydrogen atom, and the other is a linear C_1-12alkyl group with one maleimide group substituted. It is particularly preferred that one of R¹¹and R¹²is hydrogen atom, and the other is 2-maleimidoethyl group, 3-maleimidopropyl group, or 5-maleimidopentyl group. When the total number of the maleimide groups existing in the groups represented by R¹¹and R¹²is 2, it is preferred that R¹¹and R¹²independently represent 2-maleimidoethyl group, 3-maleimidopropyl group, or 5-maleimidopentyl group, or one of R¹¹and R¹²is hydrogen atom, and the other is a dimaleimide-substituted cyclohexyl group, and it is particularly preferred that one of R¹¹and R¹²is hydrogen atom, and the other is 3,5-dimaleimidocyclohexyl group.
In the compound represented by the aforementioned general formula (I), it is also preferred that R²is a group represented by the formula (B). In the formula (B), m represents an integer of 1 to 6, preferably an integer of 1 to 3, most preferably 1. Examples of the C_1-12alkyl group having one or more maleimide groups represented by R¹³include a linear, branched or cyclic C_1-12alkyl group or a combination thereof with one or more maleimide groups substituted, and when R¹³represents a C_1-12alkyl group with one maleimide group substituted, the alkyl group is preferably a C_1-6linear alkyl group. More specifically, R¹³is most preferably 2-maleimidoethyl group, 3-maleimidopropyl group, or 5-maleimidopentyl group. When R¹³represents a C_1-12alkyl group with two maleimide groups substituted, R¹³is preferably a dimaleimide-substituted cyclohexyl group or a dimaleimide-substituted adamantyl group, most preferably 3,5-dimaleimidocyclohexyl group. When R¹³represents a C_1-12alkyl group with three maleimide groups substituted, the group is preferably a trimaleimide-substituted adamantyl group.
In the compound represented by the aforementioned general formula (I), R²preferably substitutes at the 3- or 4-position, most preferably at the 4-position, of the coumarin structure.
In the compound represented by the aforementioned general formula (I), although the halogen atom represented by R³or R⁵may be any of fluorine atom, chlorine atom, bromine atom, and iodine atom, the halogen is preferably fluorine atom or chlorine atom. When R³and R⁵represent fluorine atom or chlorine atom, pH stability of the fluorescent tag introduced into a peptide, oligopeptide, polypeptide or protein containing a cysteine residue by a reaction with the compound of the present invention may be improved.
As the C_1-12acyloxy group represented by R⁴in the compound represented by the aforementioned general formula (I), a C_1-6linear alkylcarbonyloxy group is preferred, and methylcarbonyloxy group is more preferred. As the C_1-12acyloxymethyloxy group represented by R⁴, a C_1-6linear alkylcarbonyloxymethyloxy group is preferred, and methylcarbonyloxymethyloxy group is more preferred. The amino group represented by R⁴may have a substituent, more specifically, one or two substituents, and the group may be a primary to tertiary amino group. Examples of the substituent include a C_1-6alkyl group, an acyl group, and the like, and more specifically, methyl group, ethyl group, and the like are preferred. Examples of the amino group having a substituent include, for example, methylamino group, dimethylamino group, diethylamino group, methylethylamino group, acetylamino group, and the like, and dimethylamino group, diethylamino group, and the like are preferred.
The compound of the present invention represented by the aforementioned general formula (I) can exist as an acid addition salt or base addition salt. Examples of the acid addition salt include, for example, mineral acid salts such as hydrochlorides, sulfates and nitrates, organic acid salts such as methanesulfonates, p-toluenesulfonates, oxalates, citrates and tartrates, and the like, and examples of the base addition salt include metal salts such as sodium salts, potassium salts, calcium salts and magnesium salts, ammonium salts, organic amine salts such as triethylamine salts, and the like. Besides these examples, the compound may form a salt with an amino acid such as glycine. The compound of the present invention or a salt thereof may exist also as a hydrate or a solvate, and any of these substances fall within the scope of the present invention.
The compound of the present invention represented by the aforementioned general formula (I) may have one or more asymmetric carbons depending on types of substituents, and stereoisomers including optical isomers based on one or more asymmetric carbons, diastereoisomers based on two or more asymmetric carbons, and the like as well as arbitrary mixtures of stereoisomers, racemates, and the like fall within the scope of the present invention.
The method for preparing typical examples of the compound of the present invention are specifically described in the examples of this specification. Therefore, those skilled in the art can prepare any compounds of the present invention represented by the aforementioned general formula (I) by properly choosing reaction starting materials, reaction conditions, reagents, and the like on the basis of these explanations, and by adding modifications and alterations to these methods if necessary.
The compound represented by the aforementioned general formula (I) or a salt thereof according to the present invention, per se, is substantially non-fluorescent, on the other hand when the compound of the present invention or a salt thereof reacts with a thiol group of a compound having the thiol group or of a protein or a peptide containing a cysteine residue to afford an adduct, the adduct is highly fluorescent. Therefore, the compound of the present invention can be used as a fluorescent labeling agent for a low molecular weight compound having a thiol group such as cysteine or a peptide or a protein containing a cysteine residue.
Moreover, when the compound of the present invention represented by the aforementioned general formula (I) or a salt thereof has two or more maleimide groups, the compound or a salt thereof has a property that it becomes a fluorescent adduct only when all of the maleimide groups undergo addition reactions with thiol groups. Therefore, if the compound of the present invention or a salt thereof having two or more maleimide groups in the same molecule is reacted with a substance having two or more neighboring cysteine residues in the same molecule, a fluorescent adduct is provided, whereas when the compound is reacted with a substance not having neighboring two or more cysteine residues, no fluorescent adduct is produced. More correctly, the compound of the present invention or a salt thereof provides a fluorescent adduct only when it reacts with a substance containing neighboring cysteine residues in a number not smaller than that of maleimide groups contained in the compound or a salt thereof. For example, when the compound or a salt thereof has three or more maleimide groups, it provides a fluorescent adduct only when it reacts with a substance containing three or more neighboring cysteine residues.
Therefore, by using the compound of the present invention or a salt thereof having two or more maleimide groups, a substance containing neighboring cysteine residues can be selectively labeled with fluorescence. For example, a system for specifically monitoring expression of a protein can be constructed, in which a peptide consisting about ten amino acid residues including two or three cysteine residues is prepared by using a molecular biological technique and then the peptide is introduced as a recognition tag into a protein as a target for observation, and the resulting protein is reacted with the compound of the present invention or a salt thereof having two or three maleimide groups, and thereby observation of intracellular localization, dynamic behaviors, and the like of the protein as the target for observation can be achieved.
From the entire disclosure of the specification, it can be understood by those skilled in the art that the term “neighboring” used in the present invention means not only a distance (interval) of two or more thiol groups in the primary structure, but also a spatial distance (interval) of two or more thiol groups, so long as turning on and off of fluorescence can be attained by the reaction of the compound of the present invention and thiol groups. More specifically, the term “neighboring” used in the present invention includes not only neighboring in the primary structure, but also neighboring in the secondary structure or tertiary structure. The methods for use specifically explained above are given only for exemplification, and ways of use of the compound of the present invention or a salt thereof are not limited to the aforementioned embodiments of uses.

EXAMPLES

The present invention will be explained more specifically with reference to examples. However, the scope of the present invention is not limited to the following examples.

Example 1

Synthesis of Compounds 1, 2, 3 and 4

The synthetic schemes for Compounds 1, 2, 3 and 4 are shown below. In the schemes, Ac represents acetyl group.
(a) Synthesis of Compound 1 (mal-1)
4-Aminomethyl-7-hydroxycoumarin hydrochloride (32.4 mg, 0.14 mmol) was dissolved in acetic acid (10 mL), and maleic anhydride (17.6 mg, 0.18 mmol) was added to the solution, followed by stirring overnight under reflux by heating. Acetic acid was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (3% methanol/dichloromethane) to obtain Compound 1 (20.2 mg, 0.074 mmol, colorless powder, yield: 53%).
m.p. 262° C.
¹H NMR (300 MHz, DMSO-d₆) δ 4.78 (2H, s), 5.92 (1H, s), 6.74 (1H, d, J=2.4 Hz), 6.82 (1H, dd, J=2.4, 8.8 Hz), 7.13 (2H, s), 7.71 (1H, d, J=8.8 Hz). FT-IR (KBr, cm⁻¹) 3382, 3246, 3185, 3094, 2915, 1777, 1713, 1613, 1570, 1520, 1431, 1400, 1318, 1269, 1148, 997, 833, 696.
HR-MS (ESI−) Calcd for [M-H]−, 270.0403, Found, 270.0370.
(b) Synthesis of Compound 2 (mal-5)
4-Aminomethyl-7-hydroxycoumarin hydrochloride (30.6 mg, 0.13 mmol) was dissolved in acetonitrile (2 mL), and triethylamine (40 μL, 29.2 mg, 0.29 mmol) was added to the solution, followed by stirring on an ice bath. Then, N-succinimidyl 3-maleimidopropionate (37.5 mg, 0.14 mmol) was added to the mixture, and the mixture was stirred on the ice bath. After that N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (WSCD, 42.5 mg, 0.22 mmol) dissolved in dimethylformamide (5 mL) and a small amount of hydroxybenzotriazole (HOBt) were further added to the reaction mixture, and the mixture was further stirred. After the reaction was completed, the solvent was evaporated under reduced pressure, and saturated citric acid was poured to the residue. The mixture was extracted with ethyl acetate, and washed with saturated brine. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (3% methanol/dichloromethane) to obtain Compound 2 (24.2 mg, 0.071 mmol, colorless powder, yield: 54%).
m.p. 207-210° C.
¹H NMR (300 MHz, DMSO-d₆) δ 3.28 (2H, m), 3.64 (2H, t, J=6.8 Hz), 4.39 (2H, d, J=4.7 Hz), 5.98 (1H, s), 6.72 (1H, d, J=2.4 Hz), 6.78 (1H, dd, J=2.4, 8.3 Hz), 6.98 (2H, s), 7.61 (1H, d, J=8.3 Hz), 8.52 (1H, m), 10.6 (1H, s). FT-IR (KBr, cm⁻¹) 3428, 3351, 3088, 2926, 2540, 1772, 1703, 1661, 1622, 1570, 1555, 1514, 1449, 1408, 1370, 1312, 1236, 1146, 995, 974, 835, 693.
MS (ESI−) 341.07667, [M-H]−.
HR-MS (ESI−) Calcd for [M-H]−, 341.0774, Found, 341.0749.
(c) Synthesis of Compound 3 (mal-6)
4-Aminomethyl-7-hydroxycoumarin hydrochloride (31.9 mg, 0.14 mmol) was dissolved in dimethylformamide (2 mL), and the solution was stirred on an ice bath. To the solution N-succinimidyl 4-maleimidobutyrate (43.1 mg, 0.15 mmol) was added, and the mixture was stirred at room temperature for 4 hours. After the reaction was completed, the solvent was evaporated under reduced pressure, and saturated citric acid was poured to the residue. The mixture was extracted with ethyl acetate, and washed with saturated brine. After the solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (2% methanol/dichloromethane) to obtain Compound 3 (7.9 mg, 0.022 mmol, colorless powder, yield: 16%).
m.p. 200-201° C.
¹H NMR (300 MHz, DMSO-d₆) δ 1.75 (2H, m), 2.20 (2H, m), 3.41 (2H, m), 4.39 (2H, d, J=5.3 Hz), 6.01 (1H, s), 6.71 (1H, d, J=2.2 Hz), 6.77 (1H, dd, J=2.2, 8.7 Hz), 7.00 (2H, s), 7.62 (1H, d, J=8.8 Hz), 8.36 (1H, t, J=5.3 Hz). FT-IR (KBr, cm⁻¹) 3295, 3080, 2928, 1709, 1655, 1613, 1570, 1549, 1449, 1410, 1313, 1267, 1147, 995, 847, 833, 696.
MS (ESI−) 355.07, [M-H]−.
HR-MS (ESI−) Calcd for [M-H]−, 355.0930, Found, 355.0888.
(d) Synthesis of Compound 4 (mal-8)
4-Aminomethyl-7-hydroxycoumarin hydrochloride (27.1 mg, 0.12 mmol) was dissolved in dimethylformamide (2 mL), and the solution was stirred on an ice bath. The mixture was added with N-succinimidyl 4-maleimidohexanoate (40.0 mg, 0.13 mmol), and stirred as it was at room temperature for 5 hours. After the reaction was completed, the solvent was evaporated under reduced pressure, and saturated citric acid was poured to the residue. The mixture was extracted with ethyl acetate, and subjected to a washing operation using saturated brine. After the solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (3% methanol/dichloromethane) to obtain Compound 4 (14.0 mg, 0.037 mmol, colorless powder, yield: 30%).
m.p. 202-206° C.
¹H NMR (300 MHz, DMSO-d₆) δ 1.18-1.25 (2H, m), 1.43-1.57 (4H, m), 2.18 (2H, t, J=7.7 Hz), 3.37-3.90 (2H, m), 4.41 (2H, d, J=5.0 Hz), 5.97 (1H, s), 6.71 (1H, d, J=2.0 Hz), 6.77 (1H, dd, J=2.2, 8.8 Hz), 6.99 (2H, s), 7.62 (1H, d, J=8.8 Hz), 8.35 (1H, t, J=5.0 Hz).
FT-IR (KBr, cm⁻¹) 3495, 3088, 2934, 2861, 1707, 1611, 1570, 1541, 1447, 1410, 1319, 1265, 1142, 995, 833, 696.
MS (ESI−) 383.09, [M-H]−.
HR-MS (ESI−) Calcd for [M-H]−, 383.1243, Found, 383.1254.

Example 2

Synthesis of Compound 8

The synthetic scheme for Compound 8 is shown below. In the scheme, Boc represents tert-butoxycarbonyl group.

(a) Synthesis of Compound 7

cis-1,3,5-Cyclohexanetriamine trihydrobromide (Compound 5) was synthesized by referring to the methods described in Bioorg. Med. Chem. Lett., 6, 7, 807 (1996) and J. Biol. Inorg. Chem., 6, 4, 367 (2001).
cis-1,3,5-Cyclohexanetriamine trihydrobromide (Compound 5, 0.14 g, 0.38 mmol) was dissolved in methanol (5 mL). The solution was added with triethylamine (0.34 mL, 0.25 g, 2.5 mmol), then added with di-tert-butyl dicarbonate ((BOC)₂O, 0.17 g, 0.78 mmol) dissolved in methanol (5 mL) on an ice bath, and the mixture was stirred overnight. After the reaction was completed, the solvent was evaporated under reduced pressure, and dichloromethane was poured to the residue. The mixture was extracted with 1 N acetic acid, made alkaline with sodium hydroxide, and extracted with dichloromethane. The solvent was evaporated under reduced pressure to obtain Compound 6 (35.3 mg, colorless powder) as a crude product. This product was used for the next reaction without any treatment.
7-Hydroxycoumarin-4-acetic acid (66.4 mg, 0.30 mmol) was dissolved in dimethylformamide (2 mL) on an ice bath. The solution was added with N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (WSCD, 0.18 g, 0.94 mmol) and 1-hydroxybenzotriazole (HOBt, 0.16 g, 1.2 mmol), and the resulting mixture was stirred for 30 minutes. Then Compound 6 (crude product, 0.10 g) was added to the solution, and stirred overnight at room temperature. After the reaction was completed, the reaction mixture, into which saturated citric acid solution was poured, was extracted with ethyl acetate, and washed with saturated brine. The solvent was evaporated under reduced pressure, and the residue was purified by reverse phase HPLC (4% acetonitrile/water to 76% acetonitrile/water containing 0.1% trifluoroacetic acid (30 minutes)) to obtain Compound 7 (18.7 mg, 0.035 mmol, colorless powder, yield: 11.7%).
¹H-NMR (300 MHz, DMSO-d₆) δ 0.96-1.08 (3H, m), 1.78-1.81 (3H, m), 2.56 (2H, m), 3.58 (3H, m), 6.12 (1H, s), 6.70 (1H, d, J=2.2 Hz), 6.77 (1H, dd, J=2.2; 8.8 Hz), 6.87 (2H, d, J=7.9 Hz), 7.57 (1H, d, J=8.8 Hz), 8.19 (1H, d, J=7.5 Hz).
MS (ESI−) 530.16, [M-H]−.

(b) Synthesis of Compound 8

Compound 7 (8.6 mg, 0.016 mmol) was suspended in dichloromethane (3 mL) on an ice bath. The suspension was added with anisole (0.1 mL), and the mixture was added with trifluoroacetic acid (3 mL) by using a dropping funnel, and stirred for 45 minutes. Then, the solvent was evaporated under reduced pressure. The residue was dissolved in acetic acid (2 mL), and the mixture was added with maleic anhydride (8.6 mg, 0.088 mmol), and stirred overnight under reflux by heating. After the reaction was completed, the solvent was evaporated under reduced pressure, and the residue was purified by reverse phase HPLC (16% acetonitrile/water containing 0.1% TFA) to obtain Compound 8 (1.8 mg, 0.0037 mmol, colorless powder, yield: 23%).
¹H-NMR (300 MHz, Acetone-d₆) δ 1.29 (1H, s), 1.66-1.70 (1H, m), 2.10-2.16 (2H, m), 2.70-2.81 (2H, m), 3.71 (2H, s), 3.91-4.02 (1H, m), 4.12 (2H, tt, J=3.7; 8.8 Hz), 6.19 (1H, s), 6.73 (1H, d, J=2.4 Hz), 6.81 (4H, s), 6.82 (1H, dd, J=2.4; 8.7 Hz), 7.50 (1H, d, J=7.4 Hz), 7.67 (1H, d, J=8.7 Hz). HR-MS (ESI−) Calcd for [M-H]−, 490.1250, Found, 490.1237.

Example 3

Reactions of Compounds 1, 2, 3 and 4 and Cysteine

Each of Compounds 1 to 4 was dissolved in a 100 mmol/L sodium phosphate buffer (pH=7.4) at a concentration of 10 μmol/L (containing 0.1% dimethyl sulfoxide), and the absorption spectrum and the fluorescence spectrum (excitation wavelength: 328 nm) were measured. Then, L-cysteine was added to the solution at a final concentration of 100 μmol/L, and the absorption spectrum and the fluorescence spectrum (excitation wavelength: 328 nm) were measured. Fluorescence quantum yield of each compound was calculated on the basis of that of quinine sulfate in a 0.1 mol/L sulfuric acid solution, which was taken as 0.577. The results are shown in FIG. 1. Fluorescence quantum yield of Compound 1 (mal-1), Compound 2 (mal-5), Compound 3 (mal-6), and Compound 4 (mal-8) provided by the present invention is fluorescence quantum yields lower than 0.05 or less, and hardly emitted fluorescence before addition reaction with a thiol group of cysteine. However, after addition reaction with a thiol group of cysteine, their fluorescence quantum yields become 0.52 to 0.75, and thus to give significantly fluorescent adducts.

Example 4

Reaction of Compound 8 and Cysteine

Compound 8 at a concentration of 5 μmol/L was added with N-acetylcysteine (NAC), Ac-AECACRA-OH peptide, or Ac-AECAACRA-OH peptide (C-terminus is carboxyl group, N-terminus is acetyl group (Ac), and amino acids are represented by the single letter codes), and fluorescence intensity was measured. For the reaction mixture, a 100 mmol/L sodium phosphate buffer (pH=7.4, containing 0.5% dimethyl sulfoxide as a cosolvent) was used. The results are shown in FIG. 2.
Compound 8 was substantially non-fluorescent even after the reaction with one molecule of cysteine. However, it efficiently reacted with the peptides containing two neighboring cysteine residues in the same molecule to give intensely fluorescent adducts. Therefore, it was demonstrated that the compound of the present invention having two or more maleimide groups was capable of reacting with the peptides containing neighboring cysteine residues and selectively labeling the substances with fluorescence. Moreover, it was confirmed that by introducing a side chain containing such a peptide into an target protein, the target protein was specifically labeled with fluorescence.

Claims

1-10. (canceled)

11. A compound represented by formula (V), or a salt thereof:

12. A method for fluorescent labeling a compound having a thiol group, or a peptide, or a protein containing a cysteine residue comprising the compound or a salt thereof according to claim 11.

13. A fluorescent labeling agent for fluorescent labeling of a compound having a thiol group, or a peptide or protein containing a cysteine residue, which contains the compound or a salt thereof according to claim 11.

14. A fluorescent labeling agent for fluorescent labeling of a compound having two or more cysteine residues, which contains the compound or a salt thereof according to claim 11.

15. A compound represented by formula (VI), or a salt thereof:

16. A method for fluorescent labeling a compound having a thiol group, or a peptide, or a protein containing a cysteine residue comprising the compound or a salt thereof according to claim 15.

17. A fluorescent labeling agent for fluorescent labeling of a compound having a thiol group, or a peptide or protein containing a cysteine residue, which contains the compound or a salt thereof according to claim 15.

18. A fluorescent labeling agent for fluorescent labeling of a compound having two or more cysteine residues, which contains the compound or a salt thereof according to claim 15.

19. A compound represented by formula (VII), or a salt thereof:

20. A method for fluorescent labeling a compound having a thiol group, or a peptide, or a protein containing a cysteine residue comprising the compound or a salt thereof according to claim 19.

21. A fluorescent labeling agent for fluorescent labeling of a compound having a thiol group, or a peptide or protein containing a cysteine residue, which contains the compound or a salt thereof according to claim 19.

22. A fluorescent labeling agent for fluorescent labeling of a compound having two or more cysteine residues, which contains the compound or a salt thereof according to claim 19.

23. A compound represented by formula (VIII), or a salt thereof:

24. A method for fluorescent labeling a compound having a thiol group, or a peptide, or a protein containing a cysteine residue comprising the compound or a salt thereof according to claim 23.

25. A fluorescent labeling agent for fluorescent labeling of a compound having a thiol group, or a peptide or protein containing a cysteine residue, which contains the compound or a salt thereof according to claim 23.

26. A fluorescent labeling agent for fluorescent labeling of a compound having two or more cysteine residues, which contains the compound or a salt thereof according to claim 23.

27. A compound represented by formula (IX), or a salt thereof:

28. A method for fluorescent labeling a compound having a thiol group, or a peptide, or a protein containing a cysteine residue comprising the compound or a salt thereof according to claim 27.

29. A fluorescent labeling agent for fluorescent labeling of a compound having a thiol group, or a peptide or protein containing a cysteine residue, which contains the compound or a salt thereof according to claim 27.

30. A fluorescent labeling agent for fluorescent labeling of a compound having two or more cysteine residues, which contains the compound or a salt thereof according to claim 27.