U.S. patent number RE40,572 [Application Number 11/808,125] was granted by the patent office on 2008-11-11 for fluorescent probe for the quantitation of zinc.
This patent grant is currently assigned to Tetsuo Nagano, Sekisui Medical Co., Ltd.. Invention is credited to Tomoya Hirano, Kazuya Kikuchi, Tetsuo Nagano.
United States Patent |
RE40,572 |
Nagano , et al. |
November 11, 2008 |
**Please see images for:
( Certificate of Correction ) ** |
Fluorescent probe for the quantitation of zinc
Abstract
A compound represented by general formula (IA) or a salt thereof
useful as a fluorescent probe for zinc: wherein R.sup.1 and R.sup.2
represent a hydrogen atom or a group represented by formula (A),
wherein X.sup.1, X.sup.2, X.sup.3, and X.sup.4 represent a hydrogen
atom, an alkyl group, a 2-pyridylmethyl group, or a protective
group for an amino group, and m and n represent 0 or 1 provided
that R.sup.1 and R.sup.2 do not simultaneously represent hydrogen
atoms; R.sup.3 and R.sup.4 represent a hydrogen atom or a halogen
atom; and R.sup.5 and R.sup.6 represent a hydrogen atom, an
alkylcarbonyl group, or an alkylcarbonyloxymethyl group, and
R.sup.7 represents a hydrogen atom or an alkyl group.
##STR00001##
Inventors: |
Nagano; Tetsuo (Tokyo 167-0032,
JP), Kikuchi; Kazuya (Mino, JP), Hirano;
Tomoya (Tokyo, JP) |
Assignee: |
Sekisui Medical Co., Ltd.
(Tokyo, JP)
Nagano; Tetsuo (Tokyo, JP)
|
Family
ID: |
18572607 |
Appl.
No.: |
11/808,125 |
Filed: |
February 28, 2001 |
PCT
Filed: |
February 28, 2001 |
PCT No.: |
PCT/JP01/01503 |
371(c)(1),(2),(4) Date: |
February 10, 2003 |
PCT
Pub. No.: |
WO01/62755 |
PCT
Pub. Date: |
August 30, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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Reissue of: |
10203658 |
Feb 10, 2003 |
06903226 |
Jul 7, 2005 |
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Foreign Application Priority Data
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Feb 28, 2000 [JP] |
|
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2000-50869 |
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Current U.S.
Class: |
549/391 |
Current CPC
Class: |
C07D
311/82 (20130101); C07D 493/10 (20130101); C09B
11/245 (20130101); C09B 11/28 (20130101); C09B
11/24 (20130101); Y02P 20/55 (20151101) |
Current International
Class: |
C07D
311/78 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
T Hirano et al., "Highly Zinc-Selective Fluorescent Sensor
Molecules Suitable for Biological Applications", J. Am. Chem. Soc.,
vol. 122, No. 49, pp. 12399-12400 (2000). cited by other .
G. Walkup et al., "A New Cell-Permeable Fluorescent Probe for
Zn.sup.2+", J. Am. Chem. Soc., vol. 122, pp. 5644-5645 (2000).
cited by other .
J.G. Reyes et al., "A Fluorescence Method to Determine Picomole
Amounts of ZN (II) in Biological Systems", Biol. Res., vol. 27, pp.
49-56 (1994). cited by other .
M. Tsuda et al., "Expression of Zinc Transporter Gene, ZnT-1, is
Induced after Transient Forebrain Ischemia in the Gerbil",
Neurosci., vol. 17, No. 17, pp. 6678-6684 (1994). cited by other
.
T. Koike et al., "A Novel Biomimetic Zinc (II)--Fluorophore,
Dansylamidoethyl--Pendant Macrocyclic Tetraamine
1,4,7,10-Tetraazacyclododecane (Cyclen)", J. Am. Chem. Soc., vol.
118, pp. 12696-12703, 1996. cited by other .
Saibou Kougaku (Cell Technology), 17, pp. 584-595, 1998. cited by
other .
Tanpakushitsu Kakusan Kouso (Protein, Nucleic Acid and Enzyme),
extra No., 42, pp. 171-176, 1997. cited by other .
Tetsuji Kametani, Nankodo Co., Ltd., p. 215, 1997. cited by other
.
R. Hauglang, Handbook of Fluorescent Probes and Research Chemicals,
6th Edition pp. 503 and 531-540. cited by other .
T.W. Greene, Protective Groups in Organic Synthesis, John Wiley
& Sons, Inc. pp. v-xxi and 369-405. cited by other .
H. Kojima et al., "Fluorescent Indicators for Imaging Nitric Oxide
Production", Angew. Chem., Int. Ed. vol. 38, No. 21, pp. 3209-3212
(1999). cited by other .
H. Kojima et al., "Detection and Imaging of Nitric Oxide with Novel
Fluorescent Indicators: Diaminofluoresceins", Anal. Chem. vol. 70,
No. 13, pp. 2446-2453 (1998). cited by other .
J. Wagner et al., "Synthesis of Five Enantiomerically Pure Haptens
Designed for In Vitro Evolution of Antibodies with Peptidase
Activity", Bioorganic & Medicinal Chemistry, vol. 4, No. 6, pp.
901-916 (1996). cited by other .
Bioorg. Khim., vol. 21, No. 10, pp. 795-801 (1995). cited by other
.
S. Zhu et al., "Study of Bulk Morphology of Polymer Latex Films
using Laser Confocal Fluorescence Microscopy" Sci. China, Ser. B:
Chem vol. 41, No. 5, pp. 549-555 (1998). cited by other .
H. Godwin et al., "A Fluorescent Zinc-Probe Based on Metal-Induced
Folding", J. Am. Chem. Soc., vol. 118, pp. 6514-6515 (1996). cited
by other .
English language abstract of JP 2000-239272. cited by other .
U.S. Appl. No. 10/204,417, filed Aug. 28, 2002 (National Stage of
PCT/JP01/01504 filed Feb. 28, 2001) having the title "Agent for
Measurement of Reactive Oxygen" (Applicants: Tetsuo Nagano et al.).
cited by other .
U.S. Appl. No. 10/204,418, filed Aug. 28, 2002 (National Stage of
PCT/JP01/01502 filed Feb. 28, 2001) having the title "Method for
Measurement by Using Longlived Excitation Fluorescence"
(Applicants: Tetsuo Nagano et al.). cited by other.
|
Primary Examiner: Solola; Taofiq A
Attorney, Agent or Firm: Greenblum & Bernstein,
P.L.C.
Parent Case Text
This application is a 371 of PCT/JP01/01503 filed Feb. 28, 2001.
Claims
What is claimed is:
1. A compound represented by general formula (IB) or a salt
thereof: ##STR00016## wherein R.sup.1 and R.sup.2 independently
represent a hydrogen atom or a group represented by formula (A):
##STR00017## wherein X.sup.1, X.sup.2, X.sup.3, and X.sup.4
independently represent a hydrogen atom, an alkyl group, a
2-pyridylmethyl group, or a protective group for an amino group,
and m and n independently represent 0 or 1, .Iadd.and at least one
of m and n is 1, .Iaddend.provided that R.sup.1 and R.sup.2 do not
simultaneously represent hydrogen atoms; R.sup.3 and R.sup.4
independently represent a hydrogen atom or a halogen atom; R.sup.6
represents a hydrogen atom, an alkylcarbonyl group, or an
alkylcarbonyloxymethyl group, and R.sup.7 represents a hydrogen
atom or an alkyl group.
2. A compound represented by general formula (II) or a salt
thereof: ##STR00018## wherein R.sup.13 and R.sup.14 independently
represent a hydrogen atom or a halogen atom; R.sup.17 represents a
hydrogen atom or an alkyl group; and R.sup.18 represents a hydrogen
atom or a protective group for an amino group.
3. The compound or a salt thereof according to claim 2, wherein
R.sup.17 and R.sup.18 independently represent hydrogen atoms.
4. The compound or a salt thereof according to claim 2, wherein
both of R.sup.13 and R.sup.14 are hydrogen atoms, or both of
R.sup.13 and R.sup.14 are chlorine atoms.
5. A compound represented by general formula (IIIB) or a salt
thereof: ##STR00019## wherein R.sup.21 and R.sup.22 independently
represent a hydrogen atom or a group represented by formula (B):
##STR00020## wherein X.sup.11, X.sup.12, X.sup.13, and X.sup.14
independently represent a hydrogen atom, an alkyl group, a
2-pyridylmethyl group, or a protective group for an amino group,
and p and q are independently 0 or 1, provided that R.sup.21 and
R.sup.22 do not simultaneously represent hydrogen atoms; Y
represents --CO--NH-- or --NH--CO--; R.sup.23 and R.sup.24
independently represent a hydrogen atom or a halogen atom; R.sup.26
represents a hydrogen atom, an alkylcarbonyl group, or an
alkylcarbonyloxymethyl group; and R.sup.27 represents a hydrogen
atom or an alkyl group.
6. A fluorescent probe for zinc which comprises a compound
according to claim 1, provided that a compound is excluded wherein
a protective group for an amino group is introduced, or a salt
thereof.
7. A zinc complex which is formed by a compound according to claim
1, provided that a compound is excluded wherein a protective group
for an amino group is introduced, or a salt thereof together with a
zinc ion.
8. A method for measuring a zinc ion which comprises (a) reacting a
compound according to claim 1, provided that a compound is excluded
wherein a protective group for an amino group is introduced, or a
salt thereof with a zinc ion to form a zinc complex; and measuring
fluorescence intensity of the zinc complex.
9. The compound or a salt thereof according to claim 3, wherein
both of R.sup.13 and R.sup.14 are hydrogen atoms, or both of
R.sup.13 and R.sup.14 are chlorine atoms.
Description
TECHNICAL FIELD
The present invention relates to a fluorescent probe for zinc that
emits fluorescence by specifically trapping a zinc ion.
BACKGROUND ART
Zinc is an essential metallic element that is present in the human
body in the largest amount next to iron. Most zinc ions in cells
strongly couple to proteins and are involved in the maintenance of
structure or in the expression of function of the protein. Various
reports have been also made on the physiological role of free zinc
ions, which are present in the cell in a very small quantity
(generally at a level of .mu.M or lower). In particular, zinc ions
are considered to be significantly involved in one type of cell
death, i.e., apoptosis, and it is reported that zinc ions
accelerate senile plaque formation in Alzheimer's disease.
A compound (a fluorescent probe for zinc), which specifically traps
a zinc ion to form a complex and emits fluorescence upon the
formation of the complex, has been conventionally used to measure
zinc ions in tissue. For example, TSQ (Reyes, J. G., et al., Biol.
Res., 27, 49, 1994), Zinquin ethyl ester (Tsuda, M. et al.,
Neurosci., 17, 6678, 1997), Dansylaminoethylcyclen (Koike, T. et
al., J. Am. Chem. Soc., 118, 12686, 1996), and Newport Green (a
catalog of Molecular Probe: "Handbook of Fluorescent Probes and
Research Chemicals" 6th Edition by Richard P. Haugland pp. 531-540)
have been used practically as fluorescent probes for zinc.
##STR00002##
The measurement using TSQ, Zinquin, or Dansylaminoethylcyclen,
however, requires the use of a short wavelength excitation light
(an excitation wavelength of 367 nm, 368 nm, and 323 nm,
respectively). Accordingly, when these fluorescent probes for zinc
are used for measurement in living systems, the short wavelength
excitation light may cause damages of cells (Saibou Kougaku (Cell
Technology), 17, pp. 584-595, 1998). A problem also arises that the
measurement may be readily influenced by autofluorescence generated
from cell systems, per se (fluorescence emitted by NADH or flavin).
Further, Dansylaminoethylcyclen has a drawback in that the
fluorescence intensity is significantly varied depending on
different environments in which the agent exists at the time of
measurement, e.g., differences in environments such as a type of a
solvent, or extracellular, intracellular, or intramembrane water
solubility or lipophilicity or the like
(Tanpakushitsu*Kakusan*Kouso (Protein, Nucleic Acid and Enzyme),
extra number, 42, pp. 171-176, 1997). TSQ has a problem in that
even distribution in the whole cell is difficult due to its high
lipophilicity. Newport Green has low affinity for zinc ions and
fails to achieve practical measurement sensitivity, although the
agent enables measurement with a long wavelength excitation light.
Therefore, the development of a fluorescent probe for zinc has been
desired that can measure zinc ions with high sensitivity without
damaging cells.
DISCLOSURE OF THE INVENTION
An object of the present invention is to provide a compound or a
salt thereof which can be used as a highly sensitive fluorescent
probe for zinc. More specifically, the object of the present
invention is to provide a compound usable as a fluorescent probe
for zinc, which can specifically trap zinc ions and has an
excellent fluorescence intensity of a complex after the trap, and
which can measure fluorescence with a long wavelength excitation
light. Another object of the present invention is to provide a
fluorescent probe for zinc comprising a compound having the above
characteristics and a method for measuring zinc ions by using said
fluorescent probe for zinc.
The inventors of the present invention have conducted various
studies to achieve the foregoing objects. As a result, they found
that a compound having a cyclic amine or a polyamine as a
substituent has high specificity with zinc ions, and by trapping
zinc ions, the compound forms a complex which emits strong
fluorescence with a excitation light in longer wavelength range
(Japanese Patent Application No. (Hei) 11-40325). The inventors
have further conducts studies and found that a compounds
represented by general formula (I) can form a complex with zinc
very rapidly and can emit strong fluorescence. They also found that
the compounds represented by general formula (I) can react with
zinc ions for a split second in the living organism to form a
fluorescent complex when they are used as a fluorescent probe for
zinc, thereby zinc in the living organism can be measured with very
high accuracy and sensitivity. The present invention was achieved
on the basis of these findings.
The present invention thus provides a compound represented by
general formula (IA) or (IB) or a salt thereof: ##STR00003##
wherein R.sup.1 and R.sup.2 independently represent a hydrogen atom
or a group represented by formula (A): ##STR00004## wherein
X.sup.1, X.sup.2, X.sup.3, and X.sup.4 independently represent a
hydrogen atom, an alkyl group, a 2-pyridylmethyl group, or a
protective group for an amine group, and m and n independently
represent 0 or 1, provided that R.sup.1 and R.sup.2 do not
simultaneously represent hydrogen atoms; R.sup.3 and R.sup.4
independently represent a hydrogen atom or a halogen atom; R.sup.5
and R.sup.6 independently represent a hydrogen atom, an
alkylcarbonyl group, or an alkylcarbonyloxymethyl group, and
R.sup.7 represents a hydrogen atom or an alkyl group.
As a preferred embodiment of the present invention, provided is a
compound represented by general formula (II) or a salt thereof:
##STR00005## wherein R.sup.13 and R.sup.14 independently represent
a hydrogen atom or a halogen atom; R.sup.17 represents a hydrogen
atom or an alkyl group; and R.sup.18 represents a hydrogen atom or
a protective group for an amino group. According to a preferred
embodiment of the aforementioned invention, provided is the
aforementioned compound or a salt thereof in which R.sup.17 and
R.sup.18 independently represent hydrogen atoms. According to more
preferred embodiment, provided is the aforementioned compound or a
salt thereof in which a substituted amino group on the benzene ring
binds in m-position or p-position relative to the group represented
by --COOR.sup.17.
Further, the present invention provides a compound represented by
general formula (IIIA) or (IIIB) or a salt thereof: ##STR00006##
wherein R.sup.21 and R.sup.22 independently represent a hydrogen
atom or a group represented by formula (B): ##STR00007## wherein
X.sup.11, X.sup.12, X.sup.13, and X.sup.14 independently represent
a hydrogen atom, an alkyl group, a 2-pyridylmethyl group, or a
protective group for an amino group, and p and q are independently
0 or 1, provided that R.sup.21 and R.sup.22 do not simultaneously
represent hydrogen atoms; Y represents --CO--NH-- or --NH--CO--;
R.sup.23 and R.sup.24 independently represent a hydrogen atom or a
halogen atom; R.sup.25 and R.sup.26 independently represent a
hydrogen atom, an alkylcarbonyl group, or an alkylcarbonyloxymethyl
group; and R.sup.27 represents a hydrogen atom or an alkyl group.
According to a preferred embodiment of the invention, provides is
the aforementioned compound in which Y on the benzene ring binds in
m-position relative to the group represented by --COOR.sup.27 (the
corresponding carbonyl group when a lactone ring is formed).
From another aspect, the present invention provides a fluorescent
probe for zinc which comprises a compound represented by the
general formulas (I), (II), or (III) (excluding the compound
wherein a protective group for an amino group is introduced) or a
salt thereof; and a zinc complex constituted by a compound
represented by the general formula (I), (II), or (III) (excluding
the compound wherein a protective group for an amino group is
introduced) or a salt thereof together with a zinc ion. The
aforementioned fluorescent probe for zinc can be used for measuring
zinc ions in tissues or cells.
From further aspect of the present invention, provided are a method
for measuring zinc ions wherein a compound represented by the
general formula (I), (II), or (III) (excluding the compound wherein
a protective group for an amino group is introduced) or a salt
thereof is used as a fluorescent probe for zinc; a method for
measuring zinc ions which comprises the steps of: (a) reacting a
compound represented by the general formula (I), (II), or (III)
(excluding the compound wherein a protective group for an amino
group is introduced) or a salt thereof with zinc ions; and (b)
measuring fluorescence intensity of the zinc complex produced in
the above step; and the use of a compound represented by the
general formula (I), (II), or (III) (excluding the compound wherein
a protective group for an amino group is introduced) or a salt
thereof as a fluorescent probe for zinc.
The compound represented by the general formula (I), (II), or (III)
(limited to the compound wherein a protective group for an amino
group is introduced) is useful as a synthetic intermediate for the
aforementioned fluorescent probe for zinc.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows that the fluorescent probe for zinc according to the
present invention (Compound 6) has excellent zinc ion
selectivity.
FIG. 2 shows that the fluorescent probe for zinc according to the
present invention (Compound 12) has excellent zinc ion
selectivity.
FIG. 3 shows that the fluorescent probe for Zinc according to the
present invention (Compound 21) has excellent zinc ion
selectivity.
FIG. 4 shows results of a comparison of changes with time in
fluorescence intensity between the fluorescent probes for zinc
according to the present invention (Compound 6 and Compound 12) and
ACF-1 having a cyclic polyamine moiety.
FIG. 5 shows a correlation between fluorescence intensity of the
fluorescent probes for zinc according to the present invention
(Compound 6 and Compound 12) and zinc ion concentration.
FIG. 6 shows changes in fluorescence intensity of Compound 12,
Compound 21, and zinc complexes thereof with relation to pH
changes.
FIG. 7 shows a result of investigation on changes in fluorescence
intensity by ischemic stimulus using a rat hippocampal slice.
FIG. 8 shows a result of investigation on changes in fluorescence
intensity in each of regions by ischemic stimulus using a rat
hippocampal slice.
BEST MODE FOR CARRYING OUT THE INVENTION
All the disclosures in the specification and claims of Japanese
Patent Application No. 2000-50869 are incorporated herein by
reference.
"An alkyl group" or an alkyl moiety of a substituent containing the
alkyl moiety (for example, an alkylcarbonyl group or an
alkylcarbonyloxymethyl group) used in the specification means, for
example, a linear, branched, or cyclic alkyl group, or an alkyl
group comprising a combination thereof having 1 to 12 carbon atoms,
preferably 1 to 6 carbon atoms, and more preferably 1 to 4 carbon
atoms. More specifically, a lower alkyl group (an alkyl group
having 1 to 6 carbon atoms) is preferred as an alkyl group.
Examples of the lower alkyl groups 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, and n-hexyl group. When "a
halogen atom" is referred to, the term means any of a fluorine
atom, a chlorine atom, a bromine atom, or an iodine atom, and
preferably means a fluorine atom, a chlorine atom, or a bromine
atom.
Types of protective groups for amino groups are not particularly
limited. For example, a p-nitrobenzenesulfonic acid group, a
trifluoroacetyl group, and a trialkylsily group can be suitably
used. As for the protective groups for amino groups, reference can
be made to, for example, "Protective Groups in Organic Synthesis,"
(T. W. Greene, John Wiley & Sons, Inc. (1981)).
In the general formulas (IA) and (IB), the positions of R.sup.1 and
R.sup.2 substituted on the benzene ring are not particularly
limited. When R.sup.2 is a hydrogen atom, R.sup.1 may preferably
bind in Meta-position or para-position relative to the group
represented by --COOR.sup.7 (or corresponding carbonyl group when a
lactone ring is formed). The position of an amino group substituted
on the benzene ring in general formula (II) is not particularly
limited. Meta-Position or para-position relative to the group
represented by --COOR.sup.17 is preferred. In the general formulas
(IIIA) and (IIIB), the position of Y substituted on the benzene
ring is not particularly limited. Y may preferably bind in
meta-position relative to the group represented by --COOR.sup.27
(or corresponding carbonyl group when a lactone ring is
formed).
In the compounds represented by the general formulas (IA) and (IB),
either of R.sup.1 and R.sup.2 is preferably a hydrogen atom and the
other is preferably a group represented by formula (A). In the
group represented by the formula (A), from X.sup.1 to X.sup.4,
preferably X.sup.1 and X.sup.2, independently represent a
2-pyridylmethyl group. In the compounds represented by the general
formulas (IA) and (IB), preferably, m is 0, n is 1, and X.sup.4 is
hydrogen atom. In the above particular compounds, both of X.sup.1
and X.sup.2 are preferably 2-pyridylmethyl group. R.sup.5 and
R.sup.6 are preferably hydrogen atoms, and R.sup.5 and R.sup.6 are
preferably acetyl groups or acetoxymethyl groups for imaging
application. It is preferred that both of R.sup.3 and R.sup.4 are
hydrogen atoms or chlorine atoms. R.sup.7 is preferably a hydrogen
atom.
In the compound represented by general formula (II), both of
R.sup.13 and R.sup.14 are preferably hydrogen atoms or chlorine
atoms. R.sup.17 and R.sup.18 are preferably hydrogen atoms.
In the compounds represented by the general formulas (IIIA) and
(IIIB), either of R.sup.21 and R.sup.22 is preferably a hydrogen
atom and the other is preferably a group represented by formula
(B). In the group represented by the formula (B), from X.sup.11 to
X.sup.14, preferably X.sup.11 and X.sup.12, independently represent
a 2-pyridylmethyl group. In the compounds represented by the
general formulas (IIIA) and (IIIB), preferably, p is 0, q is 1, and
X.sup.14 is a hydrogen atom. In the above particular compounds,
both of X.sup.11 and X.sup.12 are preferably 2-pyridylmethyl
groups. Both of R.sup.23 and R.sup.24 are preferably hydrogen atoms
or chlorine atoms. R.sup.25 and R.sup.26 are preferably hydrogen
atoms, and R.sup.25 and R.sup.26 are preferably acetyl groups or
acetoxymethyl groups for imaging application. R.sup.27 is
preferably a hydrogen atom.
The compounds of the present invention represented by the general
formulas (I) to (III) can exist as acid addition salts or base
addition salts. Examples of the acid addition salts include:
mineral acid salts such as hydrochloride, sulfate, and nitrate; and
organic acid salts such as methanesulfonate, p-toluenesulfonate,
oxalate, citrate, and tartrate. Examples of the base addition salts
include: metal salts such as sodium salts, potassium salts, calcium
salts, and magnesium salts; ammonium salts; and organic amine salts
such as triethylamine salts. In addition, salts of amino acids such
as glycine may be formed. The compounds or salts thereof according
to the present invention may exit as hydrates or solvates, and
these substances fall within the scope of the present
invention.
the compounds of the present invention represented by general
formulas (IA), (IB), (II), (IIIA), and (IIIB) may have one or more
asymmetric carbons depending on the types of the substituents.
Stereoisomers such as optically active substances based on one or
more asymmetric carbons and diastereoisomers based on two or more
asymmetric carbons, as well as any mixtures of the stereoisomers,
racemates and the like fall within the scope of the present
invention. When R.sup.7, R.sup.17, or R.sup.27 is hydrogen atom, a
carboxyl group may form a lactone, and such structural isomers also
fall within the scope of the present invention. A compound
represented by general formula (IA) in which R.sup.5 is a hydrogen
atom and a compound represented by general formula (IB) in which
R.sup.7 is a hydrogen atom are tautomers, and similarly, a compound
represented by general formula (IIIA) in which R.sup.25 is a
hydrogen atom and a compound represented by general formula (IIIB)
in which R.sup.27 is a hydrogen atom are tautomers. One of ordinary
skill in the art would readily recognize the existence of the
tautomers as explained above, and therefore, it should be
understood that any of these tautomers fall within the scope of the
present invention.
Methods for preparing typical compounds of the present invention
are shown in the following schemes. The preparation methods shown
in the schemes are more specifically detailed in the examples of
the specification. Accordingly, one or ordinary skill in the art
can prepare any of the compounds according to the present invention
represented by the general formula by suitably choosing starting
reaction materials, reaction conditions, reagents and the like
based on these explanations, and optionally modifying and altering
these methods. 4-Aminofluorescein, 5-aminofluorescein, and
6-aminofluorescein, which can be used as starting compounds, can be
prepared by methods described in, for example, "Yuuki Gousei Kagaku
(Synthetic Organic Chemistry) IX," (Tetsuji Kametani, Nankodo Co.,
Ltd., p. 215 (1977)). ##STR00008## ##STR00009## ##STR00010##
##STR00011## ##STR00012## ##STR00013##
The compound represented by the general formula (III) can be
prepared by, for example, a method shows in the following scheme by
using a commercially available compound and the like as a reagent
and a starting reaction material. ##STR00014##
The compounds of the present invention represented by the general
formulas (I), (II), and (III) (excluding a compound having a
protective group for an amino group) or salts thereof are useful as
fluorescent probes for zinc. The compounds of the present invention
represented by the general formulas (I), (II), or (III) or salts
thereof, per se, do not emit strong fluorescence, whilst they come
to emit strong fluorescence after the formation of zinc complexes
by trapping zinc ions. The above compounds or salts thereof are
featured that they can specifically trap zinc ions and form the
complex very rapidly. In addition, the formed zinc complexes is
featured to emit strong fluorescence under a long wavelength
excitation light which does not cause any damage to living tissues
or cells. Accordingly, the compounds of the present invention
represented by the general formula (I), (II), or (III) or salts
thereof are very useful as a fluorescent probes for zinc for
measurement of zinc ions in living cells or living tissues under a
physiological condition. The term "measurement" used in the
specification should be construed in its broadest sense, including
quantitative and qualitative measurement.
The method for using the fluorescent probe for zinc according to
the present invention is not particularly limited, and the probe
can be used in the same manner as conventional zinc probes. In
general, a substance selected from the group consisting of the
compounds represented by the general formula (I) and salts thereof
is dissolved in an aqueous medium such as physiological saline or a
buffered solution, or in a mixture of the aqueous medium and a
water-miscible organic solvent such as ethanol, actone, ethylene
glycol, dimethysulfoxide, and dimethylformamide, and then the
resultant solution is added to a suitable buffered solution
containing cells or tissues and a fluorescence spectrum can be
measured.
For example, the zinc complexes of Compound 6 and Compound 12 shown
in the above scheme have the excitation wavelengths of 491 nm and
492 nm, and the fluorescence wavelengths of 513 nm and 514 nm,
respectively. When the compound is used at a concentration of about
1 to 10 .mu.M, zinc ions with a concentration of 10 .mu.M or below
can be measured. The fluorescent probe for zinc according to the
present invention may be combined with a suitable additive to use
in the form of a composition. For example, the fluorescent probe
for zinc can be combined with additives such as a buffering agent,
a solubilizing agent, and a pH modifier.
Compound 22, for example, has lipophilicity such a degree that it
can easily permeate cell membranes. After Compound 22 permeates
cell membranes, the compound is hydrolyzed by an esterase present
in the cytoplasm, thereby Compound 12 is produced. Compound 12 can
hardly permeate cell membranes due to its water-solubility, and for
this reason, Compound 12 can be retained intracellularly for a
prolonged period of time. Accordingly, Compound 22 is very useful
for measurement of zinc ions existing in an individual cell.
EXAMPLES
The present invention will be more specifically explained with
reference to the following examples. However, the scope of the
present invention is not limited to these examples. The compound
number in the examples correspond to those used in the above
schemes.
Example 1
Synthesis of Compound 6
Cesium carbonate (5.2 g, 16 mmol) was added to a solution of
4-aminofluorescein (1) (2.5 g, 7.2 mmol) dissolved in 50 ml of
dimethylformamide. Subsequently, pivaloyl anhydride (3.1 ml, 15
mmol) was added to this solution, and the mixture was stirred at
room temperature for 1 hour. The reaction solution was filtered
using a Kiriyama funnel, and dimethylformamide was evaporated under
reduced pressure. The residue was purified by column chromatography
on silica gel to obtain 3.6 g of Compound 2 (white solid, yield:
97%).
.sup.1H-NMR (CDCl.sub.3, 300 MHz): 7.19 (m, 1H), 7.02 (d, 2H,
J=2.4), 6.93-6.94 (m, 2H), 6.88 (d, 2H, J=8.7), 6.77 (dd, 2H,
J=8.7, 2.4), 4.06 (br, 2H), 1.34 (s, 18H)
MS (FAB): 516 (M.sup.++1)
m.p.: 206-208.degree. C. (recrystallized from methanol)
Compound 2 (1.0 g, 2.0 mmol) was dissolved in 15 ml of pyridine and
the solution was added with 4-nitrobenzenesulfonyl chloride (1.2 g,
5.3 mmol), and the mixture was then stirred at room temperature for
6 hours. Pyridine was evaporated under reduced pressure, and the
residue was dissolved in 25 ml of ethyl acetate. The ethyl acetate
solution was washed with 2N hydrochloric acid and saturated brine,
and was then dried over sodium sulfate. After ethyl acetate was
evaporated under reduced pressure, purification was carried out by
column chromatography on silica gel to obtain 1.2 g of Compound 3
(white solid, yield: 88%).
.sup.1H-NMR (CDCl.sub.3, 300 MHz): 8.33 (d, 2H, J=9.0), 8.05 (d,
2H, J=9.0), 7.69 (d, 1H, J=2.2), 7.45 (dd, 1H, J=8.2, 2.2), 7.07
(d, 1H, J=8.2), 7.06-7.04 (m, 2H), 6.77-6.74 (m, 4H), 1.36 (s,
18H)
MS (FAB): 701(M.sup.++1)
m.p.: 245-247.degree. C. (recrystallized from ethyl
acetate+n-hexane)
Cesium carbonate (0.48 g, 1.5 mmol) and 1,2-dibromoethane (1.3 ml,
14 mmol) were added to a solution of Compound 3 (0.97 g, 1.4 mmol)
dissolved in 25 ml of dimethylformamide, and the mixture was
stirred at 60.degree. C. for 20 hours. Dimethylformamide was
evaporated under reduced pressure and dissolved in 50 ml of ethyl
acetate. The ethyl acetate solution was washed with water and
saturated brine, and then dried over sodium sulfate. After ethyl
acetate was evaporated under reduced pressure, purification was
carried out by column chromatography on silica gel to obtain 0.78 g
of Compound 4 (white solid, yield: 70%).
.sup.1H-NMR (CDCl.sub.3, 300 MHz): 8.38 (d, 2H, J=9.0), 7.86 (d,
2H, J=9.0), 7.76 (d, 1H, J=2.0), 7.45 (dd, 1H, J=8.0, 2.0), 7.17
(d, 1H, J=8.0), 7.08 (m, 2H), 6.85-6.84 (m, 4H), 4.01 (t, 2H,
J=6.8), 3.45 (t, 2H, J=6.8), 1.37 (s, 18H)
MS (FAB): 807, 809 (M.sup.++1)
m.p.: 280-281.degree. C. (recrystallized from acetonitrile)
Compound 4 (0.10 g, 0.13 mmol) was suspended in 4 ml of
acetonitrile, and the suspension was added with potassium iodide
(55 mg, 0.33 mmol), potassium carbonate (43 mg, 0.31 mmol), and
2,2'-dipicolylamine (78 mg, 0.39 mmol), and then the mixture was
refluxed for 14 hours. After acetonitrile was evaporated under
reduced pressure, the product was dissolved in an aqueous solution
of 2N sodium carbonate, followed by extraction with methylene
chloride. The methylene chloride layer was washed with saturated
brine and was then dried over sodium sulfate. Methylene chloride
was evaporated under reduced pressure, followed by purification by
column chromatography on silica gel to obtain 80 mg of Compound 5
(light yellow oil, yield: 69%).
.sup.1H-NMR (CDCl.sub.3, 300 MHz): 8.47-8.45 (m, 2H), 8.32 (d, 2H,
J=9.0), 7.77 (d, 2H, J=9.0), 7.69-7.61 (m, 3H), 7.61 (d, 2H,
J=7.9), 7.27-7.23 (m, 1H), 7.14 (m, 2H), 7.07 (d, 2H, J=2.2), 6.99
(d, 1H, J=8.0), 6.82 (dd, 2H, J=8.6, 2.2), 6.72 (d, 2H, J=8.6),
3.82 (s, 4H), 3.82 (m, 2H), 2.72 (t, 2H, J=6.4), 1.37 (s, 18H)
MS (FAB): 926 (M.sup.++1)
Potassium carbonate (26 mg, 0.19 mmol) and thiophenol (12 .mu.l,
0.12 mmol) were added to a solution of Compound 5 (34 mg, 37
.mu.mol) dissolved in 4 ml of dimethylformamide, and the mixture
was stirred at room temperature for 3 hours. A solution of
potassium hydroxide (70 mg, 1.2 mmol), dissolved in 1 ml of
methanol and 1 ml of water, was added to the reaction mixture, and
the mixture was stirred at room temperature for 20 hours. After 2
ml of 2N hydrochloric acid was added to the mixture, the solvent
was evaporated under reduced pressure. The product was suspended in
10 ml of ethanol and filtered, and then the ethanol was evaporated
under reduced pressure. The residue was purified by reversed-phase
HPLC to obtain 15 mg of Compound 6 (brown solid, yield: 70%).
.sup.1H-NMR (CD.sub.3OD, 300 MHz): 8.61-8.59 (m, 2H), 8.04-7.98 (m,
2H), 7.63 (d, 2H, J=7.9), 7.51-7.46 (m, 2H), 7.14 (d, 1H, J=2.0),
7.02 (d, 2H, J=9.0), 6.95-6.87 (m, 4H), 6.79 (dd, 2H, J=9.0, 2.4),
4.46 (s, 4H), 3.50 (t, 2H, J=6.0), 3.25 (m, 2H)
MS (FAB): 573 (M.sup.++1)
Example 2
Synthesis of Compound 12
Compound 8 (4.4 g) was obtained from 5-aminofluorescein (7) (3.5 g,
10 mmol) in the same manner as that of the synthesis of Compound 2
(white solid, yield: 84%).
.sup.1H-NMR (CDCl.sub.3, 300 MHz): 7.77 (d, 1H, J=7.9 ), 7.01 (d,
2H, J=2.0), 6.95 (d, 2H, J=8.6), 6.80-6.75 (m, 3H), 6.22 (d, 1H,
J=1.7), 4.21 (br, 2H), 1.36 (s, 18H)
MS (FAB): 516 (M.sup.++1)
m.p.: 161-163.degree. C. (recrystallized from methanol)
Compound 9 (4.1 g) was obtained from Compound 8 (3.6 g, 6.9 mmol)
in the same manner as that of the synthesis of Compound 3 (white
solid, yield: 84%).
.sup.1H-NMR (CDCl.sub.3, 300 MHz): 8.61 (br, 1H), 8.20 (d, 2H,
J=9.0), 7.88 (d, 1H, J=8.3), 7.81 (d, 2H, J=9.0), 7.33-7.29 (m,
1H), 7.05 (d, 2H, J=2.2), 6.84 (d, 1H, J=1.8), 6.74 (dd, 2H, J=8.6,
2.2), 6.69 (d, 2H, J=8.6), 1.38 (s, 18H)
MS (FAB): 701 (M.sup.++1)
m.p.: 189-191.degree. C. (recrystallized from ethyl
acetate+n-hexane)
Compound 10 (0.35 g) was obtained from Compound 9 (0.51 g, 0.73
mmol) in the same manner as that of the synthesis of Compound 4
(white solid, yield: 60%).
.sup.1H-NMR (CDCl.sub.3, 300 MHz): 8.11 (d, 2H, J=9.0), 8.10-8.09
(m, 1H), 7.71 (dd, 1H, J=8.2, 1.8), 7.56 (d, 2H, J=9.0), 7.02 (d,
2H, J=2.2), 6.86 (dd, 2H, J=8.6, 2.2), 6.79 (d, 2H, J=8.6), 6.43
(d, 1H, J=1.8), 3.85 (t, 2H, J=6.6), 3.40 (t, 2H, J=6.6), 1.38 (s,
18H)
MS (FAB): 807, 809 (M.sup.++1)
m.p.: 268-269.degree. C. (recrystallized from acetonitrile)
Compound 11 (0.27 g) was obtained from Compound 10 (0.31 g, 0.38
mmol) in the same manner as that of the synthesis of Compound 5
(light yellow solid, yield: 75%).
.sup.1H-NMR (CDCl.sub.3, 300 MHz): 8.45-8.42 (m, 2H), 8.06 (d, 2H,
J=9.0), 7.96 (d, 1H, J=8.3), 7.64-7.59 (m, 2H), 7.52 (d, 2H,
J=9.0), 7.53-7.50 (m, 1H), 7.33 (d, 2H, J=7.7), 7.17 (d, 2H), 7.00
(d, 2H, J=2.2), 6.78 (dd, 2H, J=8.6, 2.2), 6.64 (d, 2H, J=8.6),
6.48 (d, 1H, J=1.3), 3.71 (s, 4H), 3.67 (t, 2H, J=6.2), 2.67 (t,
2H, J=6.2), 1.37 (s, 18H)
MS (FAB): 926 (M.sup.++1)
m.p.: 146-148.degree. C. (recrystallized from methanol)
Compound 12 (6.6 mg) was obtained from Compound 11 (20 mg, 22
.mu.mol) in the same manner as that of the synthesis of Compound 6
(brown solid, yield: 53%).
.sup.1H-NMR (CD.sub.3OD, 300 MHz): 8.44-8.42 (m, 2H), 7.94-7.88 (m,
2H), 7.60 (d, 1H, J=8.4), 7.49 (d, 2H, J=7.9), 7.45-7.41 (m, 2H),
6.71 (br, 1H), 6.65 (d, 2H, J=2.4), 6.61 (d, 2H, J=8.8), 6.51 (dd,
2H, J=8.8, 2.4), 6.02 (d, 1H, J=1.8), 4.30 (s, 4H), 3.28 (t, 2H,
J=6.0), 3.03 (t, 2H, J=6.0)
MS (FAB): 573 (M.sup.++1)
Example 3
Synthesis of Compound 15
4-Nitrophthalic anhydride (13) (16 g, 84 mmol) and
4-chlororesorcinol (14) (24 g, 0.17 mol) were dissolved in 250 ml
of methanesulfonic acid, and the mixture was stirred under argon at
80.degree. C. for 60 hours. The mixture was cooled to room
temperature and then added in small portions to 1.4 L of ice water.
The precipitated solid was collected by filtration to obtain 37 g
of Compound 15 (quantitative yield).
Synthesis of Compound 16
Compound (15) (20 g, 45 mmol) was suspended in 700 ml of water, and
the suspension was added with sodium sulfate nonahydrate (54 g,
0.23 mol) and sodium hydrosulfide n-hydrate (20 g, 0.25 mol, about
70% of sodium hydrosulfide), and the mixture was refluxed under
argon for 20 hours. After cooled to room temperature, the mixture
was added with hydrochloric acid to adjust pH at 3 to 4. The
precipitated solid was collected by filtration to obtain 19 g of
Compound (16) (quantitative yield).
Synthesis of Compound 17
Compound (17) (3.9g) was obtained from Compound (16) (4.4 g, 11
mmol) in the same manner as that of the synthesis of Compound (2)
(yield: 62%).
MS (FAB): 584, 586, 588 (M.sup.++1)
Synthesis of Compounds 18 and 18'
Compound (18) (1.9 g) and 1.8 g of Compound (18') were obtained
from Compound (17) (3.8 g, 6.5 mmol) in the same manner as that of
the synthesis of Compound (3) (yield: 38% for Compound (18); 35%
for Compound (18')).
Compound (18):
.sup.1H-NMR (CDCl.sub.3, 300 MHz): 8.38 (d, 2H, J=8.7), 8.07 (d,
2H, J=8.7), 7.72 (d, 1H, J=2.1), 7.48 (dd, 1H, J=8.1, 2.1), 7.12
(d, 1H, J=8.1), 7.11 (s, 2H), 6.77 (s, 2H), 1.40 (s, 18H)
MS (FAB): 769, 771, 773 (M.sup.++1)
Compound (18'):
.sup.1N-NMR (CDCl.sub.3, 300 MHz): 8.26 (d, 2H, J=8.6), 7.93 (d,
1H, J=8.4), 7.84 (d, 2H, J=8.6), 7.27 (dd, 1H, J=8.4, 2.0), 7.13
(s, 2H), 6.99 (d, 1H, J=2.0), 6.75 (s, 2H), 1.42 (s, 18H)
MS (FAB): 769, 771, 773 (M.sup.++1)
Synthesis of Compound 19
Compound (19) (1.2 g) was obtained from Compound (18) (1.5 g, 2.0
mmol) in the same manner as that of the synthesis of Compound (4)
(yield: 66%).
.sup.1H-NMR (CDCl.sub.3, 300 MHz): 8.39 (d, 2H, J=9.0), 7.85 (d,
2H, J=9.0), 7.79 (d, 1H, J=2.0), 7.51 (dd, 1H, J=8.2, 2.0), 7.18
(d, 1H, J=8.2), 7.14 (s, 2H), 6.89(s, 2H), 4.06 (t, 2H, J=6.8),
3.50 (t, 2H, J=6.8), 1.40 (s, 18H)
MS (FAB): 875, 877, 879, 881 (M.sup.++1)
Synthesis of Compound 19'
Compound (19') (0.70 g) was obtained from Compound (18') (1.5 g,
2.0 mmol) in the same manner as that of the synthesis of Compound
(4) (yield: 40%).
.sup.1H-NMR (CDCl.sub.3, 300 MHz): 8.19 (d, 2H, J=9.0), 8.13 (d,
1H, J=8.3), 7.70 (brd, 1H), 7.62 (d, 2H, J=9.0), 7.11 (s, 2H), 6.84
(s, 2H), 6.63 (d, 1H, J=1.8), 3.94(t, 2H, J=6.4), 3.46 (t, 2H,
J=6.4), 1.41 (s, 18H)
MS (FAB): 875, 877, 879, 881 (M.sup.++1)
Synthesis of Compound 20
Compound (20) (0.56 g) was obtained from Compound (19) (1.0 g, 1.1
mmol) in the same manner as that of the synthesis of Compound (5)
(yield: 49%).
.sup.1H-NMR (CDCl.sub.3, 300 MHz): 8.50-8.47 (m, 2H), 8.33 (d, 2H,
J=8.7), 7.76 (d, 2H, J=8.7), 7.70-7.60 (m, 3H), 7.46 (d, 2H,
J=7.9), 7.32 (brd, 1H, J=8.3), 7.16-7.12 (m, 2H), 7.14 (s, 2H),
7.00 (d, 1H, J=8.3), 6.79 (s, 2H), 3.87 (t, 2H, J=6.0), 3.83 (s,
4H), 2.76 (t, 2H, J=6.0), 1.41 (s, 18H)
MS (FAB): 994, 996, 998 (M.sup.++1)
Synthesis of Compound 20 '
Compound (20') (75 mg) was obtained from Compound (19') (0.20 g,
0.23 mmol) in the same manner as that of the synthesis of Compound
(5) (yield: 33%).
.sup.1H-NMR (CDCl.sub.3, 300 MHz): 8.43-8.41 (m, 2H), 8.17 (d, 2H,
J=9.0), 7.97 (d, 1H, J=8.3), 7.63-7.57 (m, 2H), 7.56 (d, 2H,
J=9.0), 7.49 (brd, 1H, J=8.3), 7.31 (d, 2H, J=7.7), 7.16-7.12 (m,
2H), 7.08 (s, 2H), 6.79 (s, 2H), 6.72 (d, 2H, J=1.1), 3.74 (t, 2H,
J=6.2), 3.71 (s, 4H), 2.74 (t, 2H, J=6.2), 1.40 (s, 18H)
MS (FAB): 994, 996, 998 (M.sup.++1)
Synthesis of Compound 21
Compound (21) (98 mg) was obtained from Compound (20) (0.26 g, 0.26
mmol) in the same manner as that of the synthesis of Compound (6)
(yield: 35%).
.sup.1H-NMR (CD.sub.3OD, 300 MHz): 8.56 (brd, 2H, J=4.8), 7.98-7.91
(m, 2H), 7.57 (d, 2H, J=7.9), 7.46-7.41 (m, 2H), 6.94-6.81 (m, 3H),
6.73 (s, 2H), 6.56 (s, 2H), 4.48 (s, 4H), 3.50 (t, 2H, J=5.5), 3.29
(t, 2H, J=5.5)
MS (FAB): 641, 643, 645 (M.sup.++1)
Synthesis of Compound 21'
Compound (21') (58 mg) was obtained from Compound (20') (0.20 g,
0.20 mmol) in the same manner as that of the synthesis of Compound
(6) (yield: 26%).
.sup.1H-NMR (CD.sub.3OD, 300 MHz): 8.45-8.43 (m, 2H), 7.93-7.88 (m,
2H), 7.58 (d, 1H, J=8.6), 7.50 (d, 2H, J=7.9), 7.45-7.41 (m, 2H),
6.72 (s, 2H), 6.73-6.88 (m, 1H), 6.58 (s, 2H), 6.01 (d, 1H, J=1.8),
4.30 (s, 4H), 3.27 (t, 2H, J=5.7), 3.06 (t, 2H, J=5.7)
MS (FAB): 641, 643, 645 (M.sup.++1)
Synthesis of Compound 22
Compound (12) (140 mg, 0.13 mmol) was suspended in 10 ml of
acetonitrile, and the suspension was added with cesium carbonate
(0.19 g, 0.30 mmol), and then with 28 .mu.l of acetic anhydride
portionwise. After the mixture was stirred at room temperature for
1 hour, the reaction mixture was filtered. The solvent was
evaporated under reduced pressure, followed by purification by
column chromatography on silica gel to obtain 79 mg of Compound
(22) (yield: 94%).
.sup.1H-NMR (CDCl.sub.3, 300 MHz): 8.51-8.49 (m, 2H), 7.73 (d, 1H,
J=8.4), 7.60-7.54 (m, 2H), 7.31 (d, 2H, J=7.7), 7.15-7.11 (m, 2H),
7.05 (d, 2H, J=2.2), 6.96 (d, 2H, J=8.6), 6.80 (dd, 2H, J=2.2,
8.6), 6.77-6.74 (m, 1H), 6.48 (br, 1H), 6.02 (d, 1H, J=1.7), 3.86
(s, 4H), 3.06 (br, 2H), 2.82 (t, 2H, J=5.1), 2.31 (s, 6H)
Example 4
Compound 6 obtained in Example 1 and Compound 12 obtained in
Example 2 were used to evaluate selectivity for zinc ions. 5 .mu.M
of Compound 6 or Compound 12 was added in 100 mM HEPES buffer (pH
7.5) containing various metal ions (5 .mu.M or 5 mM). The
fluorescence intensity was measured at the excitation wavelength of
491 nm and the fluorescence wavelength of 513 nm for Compound 6,
and the excitation wavelength of 492 nm and the fluorescence
wavelength of 514 nm for Compound 12. The results are shown in FIG.
1 (Compound 6) and FIG. 2 (Compound 12). 1 .mu.M of Compound 21 was
added in 100 mM HEPES buffer (pH 7.5) containing various metal ions
(1 .mu.M or 5 mM), and the fluorescence intensity was measured at
the excitation wavelength of 505 nm and the fluorescence wavelength
of 522 nm. The results are shown in FIG. 3.
In the figures, the fluorescence intensities on the ordinate axis
are shown as numerical values with addition of each metal ion
relative to the fluorescence intensity without addition of metal
ion which is taken as 1. It is clearly understood that Compound 6
and Compound 12 of the present invention have extremely high
selectivity for zinc ions, and the compound give absolutely no
increase of fluorescence intensity even in the presence of sodium
ions, potassium ions, calcium ions, and magnesium ions at high
concentration (5 mM), which exist in a living organism in large
amounts. It is also clearly understood that these metal ions do not
affect the increase in fluorescence intensity by zinc ions.
Compound 21 exhibited high selectively for zinc. In particular, the
addition of sodium, potassium, calcium, and magnesium at high
concentration (5 mM), which are metal ions present abundant in
living organisms, gives almost no increase in fluorescence
intensity. These metal ions did not affect the increase in
fluorescence intensity caused by zinc.
Example 5
Zinc ion (final concentration 5 .mu.M or 50 .mu.M) was added in 100
mM HEPES (pH 7.5) containing 5 .mu.M Compound 6, Compound 12, or
ACF-1 (a compound having a cyclic polyamine moiety described as
Compound (20) in Example 1 in Japanese Patent Applications No.(Hei)
11-40325) to measure fluorescence intensity. The fluorescence
intensity was measured at the excitation wavelength of 491 nm and
the fluorescence wavelength of 513 nm for Compound 6, the
excitation wavelength of 492 nm and the fluorescence wavelength of
514 nm for Compound 12, and the excitation wavelength of 495 nm and
the fluorescence wavelength of 515 nm for ACF-1. The results are
shown in FIG. 4. In the figure, the ordinate axis represents
relative fluorescence intensity. As clearly indicated the results,
the fluorescence intensity is not instantly increased by ACF-1,
whilst fluorescence intensities were instantly increased by
Compound 6 and Compound 12 of the present invention. Accordingly,
the use of the compound according to the present invention enables
very quick detection of zinc, and also enables the detection of
rapid change in the concentration of zinc. ##STR00015##
Example 6
Zinc ions at various concentrations were added in 100 mM HEPES
buffer (pH 7.5) containing 5 .mu.M Compound 6, Compound 12, ACF-1,
or Newport Green (Handbook of Fluorescent Probes and Research
Chemicals, 6th Edition by Richard P. Haugland, pp. 531-540), and
changes in fluorescence intensity were measured. The fluorescence
intensity was measured at the excitation wavelength of 491 nm and
the fluorescence wavelength of 513 nm for Compound 6, the
excitation wavelength of 492 nm and the fluorescence wavelength of
514 nm for Compound 12, the excitation wavelength of 495 nm and the
fluorescence wavelength of 515 nm for ACF-1, and the excitation
wavelength of 505 nm and the fluorescence wavelength of 530 nm for
Newport Green. The results are shown in FIG. 5. In the figure, the
fluorescence intensities on the ordinate axis are shown as
numerical values with addition of each metal ion relative to the
fluorescence intensity without addition of metal ion which is taken
as 1. Compound 6 and Compound 12 of the present invention exhibited
a high detection sensitivity. In particular, the detection
sensitivity of Compound 12 was very high, which verifies an optimum
combination of the chelater moiety and the fluorescence-emitting
moiety of the compound.
Example 7
Changes in fluorescence intensity of Compound 12, Compound 21, and
zinc complexes thereof were investigated with relation of pH
changes. The fluorescence intensity was measured at the excitation
wavelength of 492 nm and the fluorescence wavelength of 514 nm for
Compound 12, and the excitation wavelength of 505 nm and the
fluorescence wavelength of 522 nm for Compound 21. The results are
shown in FIG. 6.
Buffers used are as follows. 100 mM Cl.sub.2CHCOOH--Cl.sub.2CHCOONa
buffer (pH 2.0) 100 mM ClCH.sub.2COOH--ClCH.sub.2COONa buffer (pH
3.0) 100 mM AcOH-AcONa buffer (pH 4.0, 4.5, 5.0) 100 mM MES buffer
(pH 5.5, 6.0, 6.5) 100 mM HEPES buffer (pH 7.0, 7.5, 8.0) 100 mM
CHES buffer (pH 8.5)
The fluorescence intensity of Compound 21 was more stable than
Compound 12 at pH of around 7.4 which is an intercellular pH, which
indicates that the probe is hardly influenced by intercellular pH
changes.
Example 8
The change in fluorescence intentiy by ischemic stimulus was
investigated using a rat hippocampal slice.
10 .mu.M of Compound 22 was added to a rat hippocampal slice and
incubated, and then the slice was subjected to ischemic stimulus
for 10 minutes (2 to 12 minutes in the drawing) to observe a change
with time in fluorescent images. The results are shown in FIG.
7.
For preparation of the hippocampal slice, Ringer's solutions having
the following formulations were used.
(1) Ringer's solution Formulation: 124 mM NaCl, 1.25 mM
NaH.sub.2PO.sub.4, 2.5 mM KCl, 2 mM CaCl.sub.2, 26 mM NaHCO.sub.3,
1 mM Mg Cl.sub.2, 10 mM glucose
(2) Ringer's solution for ischemia Formulation: 124 mM NACl, 1.25
mM NaH.sub.2PO.sub.4, 2.5 mM KCl, 2 mM CaCl.sub.2, 26 mM
NaHCO.sub.3, 1 mM MgCl.sub.2, 10 mM 2-deoxyglucose
(3) Chlorine-Ringer's solution Formulation: 124 mM choline, 1.25 mM
NaH.sub.2PO.sub.4, 2.5 mM KCl, 0.5 mM CaCl.sub.2, 26 mM
NaHCO.sub.3, 2.5 mM MgCl.sub.2, 10 mM glucose
The Ringer's solutions used for the preparation and measurement of
the slices were kept under constant bubbling of 95% O.sub.2/5%
CO.sub.2.
A Wistar rat (200 to 250 g, male) was anesthetized with ether.
After decapitation, the whole brain was rapidly extirpated and put
into the ice-cooled chorline-Ringer's solution, and allowed to
stand for 10 minutes. After the brain was cut into left and right
hemispheriums or Shale loaded with the ice-cooled choline-Ringer's
solution and the sorbet-like choline-Ringer's solution, the
interbrain was removed and the hippocampus was taken out using a
spatula. The hippocampus was placed on agar and fixed to the agar
with pins, and sliced in the width of 300 .mu.m using a rotary
slicer. The sliced hippocampus was put into the Ringer's solution
heated to 30.degree. C. and allowed to stand for 30 minutes to 1
hour. The sliced hippocampus was kept in the Ringer's solution at
room temperature until it was put to use.
Subsequently, a 10 mM solution of Compound 22 dissolved in DMSO was
diluted to 10 .mu.M with the Ringer's solution. The sliced
hippocampus was put into the resulting solution and incubated under
a shaded condition at room temperature for 1.5 hour. After the
sliced hippocampus was put into the fresh Ringer's solution and
washed for about 30 minutes to 1 hour and 30 minutes, and then
transferred into a chamber and subjected to measurement. The warmed
Ringer's solution was circulated (flow rate of 2 to 3 ml/minute) in
the chamber to constantly keep the temperature at 33 to 34.degree.
C. Measurement was carried out using an inverted microscope
(Olympus IX-70, objective lens: 4x, dichroic mirror: 505 nm).
Ischemic stimulus was carried out by exchanging the Ringer's
solution circulated in the chamber in the following manner.
Ringer's solution (95% O.sub.2+5% CO.sub.2 bubbling): 2 minutes (01
00.00 to 02 00. 00 in the figure).fwdarw.Ringer's solution for
ischemia (95% N.sub.2+5% CO.sub.2 bubbling): 10 minutes (03 00. 00
to 12 00. 00 in the figure).fwdarw.Ringer's solution (95%
O.sub.2+5% CO.sub.2 bubbling): 4 minutes (13 00. 00 to 16 00. 00 in
the figure).
As a result, in the CA1 region, where cell death was reported to be
caused by ischemic stimulus, the fluorescence intensity was found
to be increased about 3 minutes after the initiation of ischemic
stimulus.
Further, the fluorescence intensity was most remarkably increased
in the CA1 region (1, 2, 3 in the drawing) after ischemic stimulus
as shown in FIG. 8. The fluorescence intensity was also increased
in the CA3 region (site 4 in the figure) and the dentate gyrus
(sites 6 and 7 in the figure). The ordinate axis of the graphs
shows fluorescence intensity at the time of initiation of
measurement (0.00 sec) which is taken as 1.00.
INDUSTRIAL APPLICABILITY
The compound of the present invention is useful as a fluorescent
probe for measurement of zinc. More specifically, the compound of
the present invention is characterized to form a complex with zinc
very quickly and detection sensitivity is very high. Accordingly,
the compound of the present invention is very useful as an agent
for accurately measuring rapid changes in concentration of zinc
ions in a living organism.
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