U.S. patent application number 12/337327 was filed with the patent office on 2009-05-07 for novel coumarin derivatives.
This patent application is currently assigned to The University of Tokyo. Invention is credited to Tetsuo NAGANO, Takuji Shoda, Yasuteru Urano.
Application Number | 20090118471 12/337327 |
Document ID | / |
Family ID | 38429170 |
Filed Date | 2009-05-07 |
United States Patent
Application |
20090118471 |
Kind Code |
A1 |
NAGANO; Tetsuo ; et
al. |
May 7, 2009 |
NOVEL COUMARIN DERIVATIVES
Abstract
A compound represented by the following general formula (I) or a
salt thereof [R.sup.1 represents hydrogen atom, or a C.sub.1-12
alkyl group; R.sup.2 represents a C.sub.1-12 alkyl group having one
or more maleimide groups, an aryl group having two or more
maleimide groups, a group represented by the formula (A):
--(CH.sub.2).sub.n--CO--N(R.sup.11)(R.sup.12) (R.sup.11 and
R.sup.12 represent hydrogen atom, a C.sub.1-12 alkyl group, a
C.sub.1-12 alkyl 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):
--(CH.sub.2).sub.m--NH--CO--R.sup.13 (R.sup.13 represents a
C.sub.1-12 alkyl 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); R.sup.3 and R.sup.5 represent hydrogen atom, or
a halogen atom; R.sup.4 represents hydroxyl group, a C.sub.1-12
acyloxy group, a C.sub.1-12 acyloxymethyloxy 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. ##STR00001##
Inventors: |
NAGANO; Tetsuo; (Tokyo,
JP) ; Urano; Yasuteru; (Kanagawa, JP) ; Shoda;
Takuji; (Chiba, JP) |
Correspondence
Address: |
GREENBLUM & BERNSTEIN, P.L.C.
1950 ROLAND CLARKE PLACE
RESTON
VA
20191
US
|
Assignee: |
The University of Tokyo
Tokyo
JP
|
Family ID: |
38429170 |
Appl. No.: |
12/337327 |
Filed: |
December 17, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11470820 |
Sep 7, 2006 |
7482473 |
|
|
12337327 |
|
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Current U.S.
Class: |
530/345 ;
548/524 |
Current CPC
Class: |
C07D 405/12 20130101;
C07D 405/14 20130101 |
Class at
Publication: |
530/345 ;
548/524 |
International
Class: |
C07K 1/107 20060101
C07K001/107; C07D 405/14 20060101 C07D405/14 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 6, 2006 |
JP |
027815/2006 |
Claims
1-10. (canceled)
11. A compound represented by formula (V), or a salt thereof:
##STR00008##
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:
##STR00009##
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:
##STR00010##
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:
##STR00011##
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:
##STR00012##
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.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] 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.
[0002] The present application also claims priority under 35 U.S.C.
.sctn.119 of Japanese Patent Application No. 2006-027815, filed
Feb. 6, 2006.
TECHNICAL FIELD
[0003] 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
[0004] 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.
[0005] 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).
##STR00002##
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.
[0006] 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
[0007] 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.
[0008] 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.
[0009] 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.
[0010] The present invention thus provides a compound represented
by the following general formula (I) or a salt thereof
##STR00003##
[wherein R.sup.1 represents hydrogen atom, or a C.sub.1-12 alkyl
group; R.sup.2 represents a C.sub.1-12 alkyl group having one or
more maleimide groups, an aryl group having two or more maleimide
groups, a group represented by the following formula (A):
--(CH.sub.2).sub.n--CO--N(R.sup.11)(R.sup.12)
(wherein R.sup.11 and R.sup.12 independently represent hydrogen
atom, a C.sub.1-12 alkyl group, a C.sub.1-12 alkyl group having one
or more maleimide groups, or an aryl group having one or more
maleimide groups, provided that at least one of R.sup.11 and
R.sup.12 represents a C.sub.1-12 alkyl 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):
--(CH.sub.2).sub.m--NH--CO--R.sup.13
(wherein R.sup.13 represents a C.sub.1-12 alkyl 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); R.sup.3
and R.sup.5 independently represent hydrogen atom, or a halogen
atom; R.sup.4 represents hydroxyl group, a C.sub.1-12 acyloxy
group, a C.sub.1-12 acyloxymethyloxy group, or an amino group which
may have one or two substituents].
[0011] The aforementioned compound or a salt thereof itself is
substantially non-fluorescent, but when all the maleimide groups
existing in the group represented by R.sup.2 undergo 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.
[0012] According to a preferred embodiment of this invention,
provided is the aforementioned compound of the formula (I) or a
salt thereof, wherein R.sup.1 is hydrogen atom; R.sup.2 is a
C.sub.1-12 alkyl group having one maleimide group, a group
represented by the aforementioned formula (A) (provided that one of
R.sup.11 and R.sup.12 is hydrogen atom, or a C.sub.1-12 alkyl
group, and the other is a C.sub.1-12 alkyl group having one
maleimide group, or an aryl group having one maleimide group), or a
group represented by the aforementioned formula (B) (provided that
R.sup.13 is a C.sub.1-12 alkyl group having one maleimide group, or
an aryl group having one maleimide group); each of R.sup.3 and
R.sup.5 is independently hydrogen atom, or a halogen atom; and
R.sup.4 is hydroxyl group, a C.sub.1-12 acyloxy group, a C.sub.1-12
acyloxymethyloxy group, or an amino group which may have one or two
substituents.
[0013] 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:
##STR00004##
(wherein p represents an integer of 1 to 6, and q represents an
integer of 1 to 6).
[0014] According to another preferred embodiment of the invention,
provided is the aforementioned compound of the formula (I) or a
salt thereof, wherein R.sup.1 is hydrogen atom; R.sup.2 is a
C.sub.1-12 alkyl 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 R.sup.11 and
R.sup.12 is hydrogen atom or a C.sub.1-12 alkyl group, and the
other is a C.sub.1-12 alkyl 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 R.sup.11 and R.sup.12 is independently a C.sub.1-12 alkyl
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 R.sup.11 and R.sup.12 is 2 or more), or a group
represented by the aforementioned formula (B) (provided that
R.sup.13 is a C.sub.1-12 alkyl group having two or more maleimide
groups, or an aryl group having two or more maleimide groups); each
of R.sup.3 and R.sup.5 is independently hydrogen atom or a halogen
atom; and R.sup.4 is hydroxyl group, a C.sub.1-12 acyloxy group, a
C.sub.1-12 acyloxymethyloxy group, or an amino group which may have
one or two substituents.
[0015] According to the most preferred embodiment of the
aforementioned invention, provided is a compound represented by the
following formula (V) or a salt thereof
##STR00005##
[0016] 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.
[0017] 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.
[0018] 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.
[0019] 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.
[0020] Further, the compound represented by the aforementioned
general formula (I) or a salt thereof having two or more maleimide
groups as R.sup.2 has 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
[0021] 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.
[0022] 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
[0023] The meanings of the terms used in this specification are as
follows.
[0024] 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.
[0025] 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.
[0026] 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.
[0027] 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.
[0028] In the compound represented by the aforementioned general
formula (I), the C.sub.1-12 alkyl group represented by R.sup.1 is
preferably methyl group or ethyl group, most preferably methyl
group.
[0029] The C.sub.1-12 alkyl group on which one or more maleimide
groups are substituted, represented by R.sup.2 in the compound
represented by the aforementioned general formula (I), may be any
linear, branched or cyclic C.sub.1-12 alkyl 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.sub.1-12 alkyl group, they may indirectly substitute on
the C.sub.1-12 alkyl group via an amide group, a phenylene group,
or the like, and substitution positions of the maleimide groups on
the C.sub.1-12 alkyl 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.sub.1-6 linear alkyl group with one or more maleimide groups
directly substituted is preferred. When R.sup.2 is a C.sub.1-12
linear alkyl group with one maleimide group directly substituted,
the group is most preferably monomaleimidomethyl group.
[0030] Examples of the aryl group with two or more maleimide groups
substituted represented by R.sup.2 in 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.
[0031] In the compound represented by the aforementioned general
formula (I), R.sup.2 is 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.
[0032] When R.sup.11 or R.sup.12 represents a C.sub.1-12 alkyl
group, this group is preferably ethyl group or methyl group, most
preferably methyl group.
[0033] Examples of the C.sub.1-12 alkyl group having one or more
maleimide groups and represented by R.sup.11 or R.sup.12 include a
linear, branched or cyclic C.sub.1-12 alkyl group or a combination
thereof with one or more maleimide groups substituted, and when
R.sup.11 or R.sup.12 represents an C.sub.1-12 alkyl group with one
maleimide group substituted, the alkyl group is preferably a
C.sub.1-6 linear alkyl group, and more specifically, R.sup.11 or
R.sup.12 is most preferably 2-maleimidoethyl group,
3-maleimidopropyl group, or 5-maleimidopentyl group. When R.sup.11
or R.sup.12 represents a C.sub.1-12 alkyl group with two maleimide
groups substituted, R.sup.11 or R.sup.12 is preferably a
dimaleimide-substituted cyclohexyl group or a
dimaleimide-substituted adamantyl group, most preferably
3,5-dimaleimidocyclohexyl group. When R.sup.11 or R.sup.12
represents a C.sub.1-12 alkyl group with three maleimide groups
substituted, the group is preferably a trimaleimide-substituted
adamantyl group.
[0034] Examples of the aryl group having one or more maleimide
groups represented by R.sup.11 or R.sup.12 include, 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.
[0035] As for the combination of R.sup.11 and R.sup.12, when the
total number of maleimide group existing in the groups represented
by R.sup.11 and R.sup.12 is 1, it is preferred that one of R.sup.11
and R.sup.12 is hydrogen atom, and the other is a linear C.sub.1-12
alkyl group with one maleimide group substituted. It is
particularly preferred that one of R.sup.11 and R.sup.12 is
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 R.sup.11 and R.sup.12 is 2, it is preferred that R.sup.11 and
R.sup.12 independently represent 2-maleimidoethyl group,
3-maleimidopropyl group, or 5-maleimidopentyl group, or one of
R.sup.11 and R.sup.12 is hydrogen atom, and the other is a
dimaleimide-substituted cyclohexyl group, and it is particularly
preferred that one of R.sup.11 and R.sup.12 is hydrogen atom, and
the other is 3,5-dimaleimidocyclohexyl group.
[0036] In the compound represented by the aforementioned general
formula (I), it is also preferred that R.sup.2 is 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.sub.1-12 alkyl group having one or more
maleimide groups represented by R.sup.13 include a linear, branched
or cyclic C.sub.1-12 alkyl group or a combination thereof with one
or more maleimide groups substituted, and when R.sup.13 represents
a C.sub.1-12 alkyl group with one maleimide group substituted, the
alkyl group is preferably a C.sub.1-6 linear alkyl group. More
specifically, R.sup.13 is most preferably 2-maleimidoethyl group,
3-maleimidopropyl group, or 5-maleimidopentyl group. When R.sup.13
represents a C.sub.1-12 alkyl group with two maleimide groups
substituted, R.sup.13 is preferably a dimaleimide-substituted
cyclohexyl group or a dimaleimide-substituted adamantyl group, most
preferably 3,5-dimaleimidocyclohexyl group. When R.sup.13
represents a C.sub.1-12 alkyl group with three maleimide groups
substituted, the group is preferably a trimaleimide-substituted
adamantyl group.
[0037] In the compound represented by the aforementioned general
formula (I), R.sup.2 preferably substitutes at the 3- or
4-position, most preferably at the 4-position, of the coumarin
structure.
[0038] In the compound represented by the aforementioned general
formula (I), although the halogen atom represented by R.sup.3 or
R.sup.5 may be any of fluorine atom, chlorine atom, bromine atom,
and iodine atom, the halogen is preferably fluorine atom or
chlorine atom. When R.sup.3 and R.sup.5 represent 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.
[0039] As the C.sub.1-12 acyloxy group represented by R.sup.4 in
the compound represented by the aforementioned general formula (I),
a C.sub.1-6 linear alkylcarbonyloxy group is preferred, and
methylcarbonyloxy group is more preferred. As the C.sub.1-12
acyloxymethyloxy group represented by R.sup.4, a C.sub.1-6 linear
alkylcarbonyloxymethyloxy group is preferred, and
methylcarbonyloxymethyloxy group is more preferred. The amino group
represented by R.sup.4 may 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.sub.1-6 alkyl
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.
[0040] 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.
[0041] 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.
[0042] 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.
[0043] 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.
[0044] 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.
[0045] 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.
[0046] 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
[0047] 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
[0048] The synthetic schemes for Compounds 1, 2, 3 and 4 are shown
below. In the schemes, Ac represents acetyl group.
##STR00006##
(a) Synthesis of Compound 1 (mal-1)
[0049] 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%).
[0050] m.p. 262.degree. C.
[0051] .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 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.sup.-1) 3382,
3246, 3185, 3094, 2915, 1777, 1713, 1613, 1570, 1520, 1431, 1400,
1318, 1269, 1148, 997, 833, 696.
[0052] HR-MS (ESI-) Calcd for [M-H]-, 270.0403, Found,
270.0370.
(b) Synthesis of Compound 2 (mal-5)
[0053] 4-Aminomethyl-7-hydroxycoumarin hydrochloride (30.6 mg, 0.13
mmol) was dissolved in acetonitrile (2 mL), and triethylamine (40
.mu.L, 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%).
[0054] m.p. 207-210.degree. C.
[0055] .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 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.sup.-1) 3428, 3351, 3088, 2926, 2540, 1772, 1703, 1661, 1622,
1570, 1555, 1514, 1449, 1408, 1370, 1312, 1236, 1146, 995, 974,
835, 693.
[0056] MS (ESI-) 341.07667, [M-H]-.
[0057] HR-MS (ESI-) Calcd for [M-H]-, 341.0774, Found,
341.0749.
(c) Synthesis of Compound 3 (mal-6)
[0058] 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%).
[0059] m.p. 200-201.degree. C.
[0060] .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 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.sup.-1) 3295, 3080, 2928, 1709, 1655, 1613, 1570, 1549, 1449,
1410, 1313, 1267, 1147, 995, 847, 833, 696.
[0061] MS (ESI-) 355.07, [M-H]-.
[0062] HR-MS (ESI-) Calcd for [M-H]-, 355.0930, Found,
355.0888.
(d) Synthesis of Compound 4 (mal-8)
[0063] 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%).
[0064] m.p. 202-206.degree. C.
[0065] .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 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).
[0066] FT-IR (KBr, cm.sup.-1) 3495, 3088, 2934, 2861, 1707, 1611,
1570, 1541, 1447, 1410, 1319, 1265, 1142, 995, 833, 696.
[0067] MS (ESI-) 383.09, [M-H]-.
[0068] HR-MS (ESI-) Calcd for [M-H]-, 383.1243, Found,
383.1254.
Example 2
Synthesis of Compound 8
[0069] The synthetic scheme for Compound 8 is shown below. In the
scheme, Boc represents tert-butoxycarbonyl group.
##STR00007##
(a) Synthesis of Compound 7
[0070] 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).
[0071] 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).sub.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.
[0072] 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%).
[0073] .sup.1H-NMR (300 MHz, DMSO-d.sub.6) .delta. 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).
[0074] MS (ESI-) 530.16, [M-H]-.
(b) Synthesis of Compound 8
[0075] 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%).
[0076] .sup.1H-NMR (300 MHz, Acetone-d.sub.6) .delta. 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
[0077] Each of Compounds 1 to 4 was dissolved in a 100 mmol/L
sodium phosphate buffer (pH=7.4) at a concentration of 10 .mu.mol/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 .mu.mol/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
[0078] Compound 8 at a concentration of 5 .mu.mol/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.
[0079] 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.
* * * * *