U.S. patent application number 12/040753 was filed with the patent office on 2008-10-16 for fluorescent ion indicators and their applications.
This patent application is currently assigned to ABD Bioquest, Inc.. Invention is credited to Zhenjun Diwu, Jianjun He, Jinfang Liao.
Application Number | 20080254498 12/040753 |
Document ID | / |
Family ID | 39854060 |
Filed Date | 2008-10-16 |
United States Patent
Application |
20080254498 |
Kind Code |
A1 |
Diwu; Zhenjun ; et
al. |
October 16, 2008 |
FLUORESCENT ION INDICATORS AND THEIR APPLICATIONS
Abstract
Fluorescent dyes useful for preparing fluorescent metal ion
indicators, the fluorescent indicators themselves, and the use of
the fluorescent indicators for the detection, discrimination and
quantification of metal cations.
Inventors: |
Diwu; Zhenjun; (Sunnyvale,
CA) ; He; Jianjun; (Sunnyvale, CA) ; Liao;
Jinfang; (Foster City, CA) |
Correspondence
Address: |
KOLISCH HARTWELL, P.C.
200 PACIFIC BUILDING, 520 SW YAMHILL STREET
PORTLAND
OR
97204
US
|
Assignee: |
ABD Bioquest, Inc.
Sunnyvale
CA
|
Family ID: |
39854060 |
Appl. No.: |
12/040753 |
Filed: |
February 29, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60923452 |
Apr 13, 2007 |
|
|
|
Current U.S.
Class: |
435/29 ; 549/265;
549/388 |
Current CPC
Class: |
C07C 2603/24 20170501;
C07C 229/24 20130101; C07D 311/90 20130101; G01N 33/84 20130101;
C07D 219/06 20130101; C07D 493/10 20130101; G01N 33/533
20130101 |
Class at
Publication: |
435/29 ; 549/388;
549/265 |
International
Class: |
C12Q 1/02 20060101
C12Q001/02; C07D 311/82 20060101 C07D311/82; C07D 493/10 20060101
C07D493/10 |
Claims
1. A compound having the formula: ##STR00100## where A and B are
independently hydrogen, alkyl, cycloalkyl, or aryl; or A and B
taken in combination are cycloalkyl or aryl; R.sup.3, R.sup.4, j,
k, m, n and V are independently hydrogen, halogen, carboxy, alkoxy,
aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso,
cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic
acid, aryl, or heteroaryl; or alkyl, or alkoxy that is itself
optionally substituted one or more times by halogen, amino,
hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or
heteroaryl; each R.sup.5 is independently hydrogen, alkyl having
1-9 carbons, acetoxymethyl, or a biologically compatible salt; T
and U are independently hydrogen, alkyl having 1-12 carbons, alkoxy
having 1-12 carbons, aryloxy, amino, halogen, cyano, carboxy,
acetoxymethylcarbonyl, carboxyalkyl, carbonyl, sulfonyl,
phosphonyl, boronic acid, aryl or heteroaryl; provided that at
least one of T and U is not hydrogen.
2. The compound of claim 1 where at least one of T and U is fluoro,
methyl, methoxy, carboxymethyl, cyano, carboxy or boronic acid.
3. A compound having the formula: ##STR00101## where A and B are
independently hydrogen, alkyl, cycloalkyl, or aryl; or A and B
taken in combination are cycloalkyl or aryl; R.sup.3, R.sup.4, j,
k, m, n and V are independently hydrogen, halogen, carboxy, alkoxy,
aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso,
cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic
acid, aryl, or heteroaryl; or alkyl, or alkoxy that is itself
optionally substituted one or more times by halogen, amino,
hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or
heteroaryl; T and U are independently selected from hydrogen, an
alkyl having 1-2 carbons, alkoxy having 1-12 carbons, aryloxy,
amino, halogen, cyano, carboxy, acetoxymethylcarbonyl,
carboxyalkyl, carbonyl, sulfonyl, phosphonyl, boronic acid, aryl or
heteroaryl; and Z is --OC(.dbd.O)R.sup.5,
OCH.sub.2OC(.dbd.O)R.sup.5, NH.sub.2, NHR.sup.5, NR.sup.5R.sup.6,
NHCH.sub.2C(.dbd.O)R.sup.5 or N(CH.sub.2C(.dbd.O)R.sup.5).sub.2
where R.sup.5 and R.sup.6 are independently methyl or alkyl of 1-10
carbon atoms.
4. The compound of claim 3 where at least one of T and U is
hydrogen, fluoro, methyl, methoxy, cyano or
acetoxymethylcarbonyl.
5. A compound having the formula: ##STR00102## where A and B are
independently hydrogen, alkyl, cycloalkyl, or aryl; or A and B
taken in combination are cycloalkyl or aryl; R.sup.1-R.sup.6, j, k,
m, n and V are independently hydrogen, halogen, carboxy, alkoxy,
aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso,
cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic
acid, aryl or heteroaryl; or alkyl, or alkoxy that is itself
optionally substituted one or more times by halogen, amino,
hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or
heteroaryl; R.sup.7 and R.sup.8 are independently hydrogen or alkyl
having 1-12 carbons or carboxyalkyl; and T and U are independently
hydrogen, alkyl having 1-12 carbons, alkoxy having 1-12 carbons,
aryloxy, amino, halogen, cyano, carboxy, carboxyalkyl, carbonyl,
sulfonyl, phosphonyl, boronic acid, aryl or heteroaryl.
6. A compound having the formula: ##STR00103## where A and B are
independently hydrogen, alkyl, cycloalkyl, or aryl; or A and B
taken in combination are cycloalkyl or aryl; R.sup.1-R.sup.6, j, k,
m, n and V are independently hydrogen, halogen, carboxy, alkoxy,
aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso,
cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic
acid, aryl or heteroaryl; or alkyl, or alkoxy that is itself
optionally substituted one or more times by halogen, amino,
hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or
heteroaryl; Y is NR.sup.9 or CR.sup.10R.sup.11; X and Z are
independently selected from O or NR.sup.12R.sup.13; where each of
R.sup.9, R.sup.10, R.sup.11, R.sup.12 and R.sup.13 are
independently hydrogen or alkyl having 1-12 carbons or
carboxyalkyl. T and U are independently hydrogen, alkyl having 1-12
carbons, alkoxy having 1-12 carbons, aryloxy, amino, halogen,
cyano, carboxy, carboxyalkyl, carbonyl, sulfonyl, phosphonyl,
boronic acid, aryl or heteroaryl.
7. The compound of claim 6, where Y is dimethylmethylene and both
of X and Z are oxygen.
8. A compound having the formula: ##STR00104## where A and B are
independently hydrogen, alkyl, cycloalkyl, or aryl; or A and B
taken in combination are cycloalkyl or aryl; Y is O, S, Se,
CR.sup.9R.sup.10, or NR.sup.11; R.sup.1-R.sup.11, j, k, m, n and V
are independently hydrogen, halogen, carboxy, alkoxy, aryloxy,
thiol, alkylthiol, arylthiol, azido, nitro, nitroso, cyano, amino,
hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or
heteroaryl; or alkyl, or alkoxy that is itself optionally
substituted one or more times by halogen, amino, hydroxy,
phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or heteroaryl; Z
is an acetyl, acyl having 1-9 carbons, acetoxymethyl or
acyloxymethyl having 1-9 carbons.
9. The compound of claim 8 where Y is oxygen, and R.sup.2 and
R.sup.5 are independently hydrogen, fluoro or chloro.
10. The compound of claim 8 where Z is acetyl or acetoxymethyl.
11. A compound having the formula: ##STR00105## where A and B are
independently hydrogen, alkyl, cycloalkyl, or aryl; or A and B
taken in combination are cycloalkyl or aryl; X and Y are
independently H, halogen, OH, OR.sup.7, OC(.dbd.O)R.sup.7,
NH.sub.2, NHR.sup.7, NR.sup.7R.sup.8, or X and Y taken in
combination are O; R.sup.1-R.sup.6 are independently hydrogen,
halogen, carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol,
azido, nitro, nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl,
carbonyl, boronic acid, aryl or heteroaryl; or alkyl, or alkoxy
that is itself optionally substituted one or more times by halogen,
amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl
or heteroaryl; R.sup.7 and R.sup.8 are independently alkyl, aryl or
heteroaryl; and Z is hydrogen, an alkyl, an aryl or a salt.
12. The compound of claim 11 where X and Y are independently
hydrogen, halogen, hydroxy, methoxy, ethoxy, t-butoxy or
phenylmethoxy.
13. A method of monitoring intracellular calcium using a compound
of any one of claims 3-4 and 8-10, comprising: a) adding the
compound to a sample containing a cell; b) incubating the sample
for a time sufficient for the compound to be loaded into the cell
and an indicator compound to be generated intracellularly; c)
illuminating the sample at a wavelength that generates a
fluorescence response from the indicator compound; d) detecting a
fluorescence response from the indicator compound; e) correlating
the fluorescence response with the presence of intracellular
calcium.
14. The method of claim 13, further comprising: stimulating the
cell; monitoring changes in the intensity of the fluorescence
response from the indicator compound; and correlating the changes
in fluorescence intensity with changes in intracellular calcium
levels.
15. The method of claim 13, further comprising adding a
cell-impermeant and non-fluorescent dye to the sample.
16. The method of claim 15, where the cell-impermeant and
non-fluorescent dye is a water-soluble azo dye.
17. The method of claim 13, further comprising adding probenecid or
a probenecid derivative to the sample.
18. A kit for performing a calcium assay, comprising at least one
compound according to one of claims 3-4 and 8-10 and a
non-fluorescent and cell-impermeant quencher dye.
19. The kit of claim 18, where the non-fluorescent and
cell-impermeant quencher dye is present in a mixed composition with
the compound, or the cell-impermeant quencher dye is provided in a
container distinct from the compound.
20. A kit for performing a calcium assay, comprising at least one
compound according to one of claims 3-4 and 8-10 and a probenecid
or a probenecid derivative.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C.
.sctn.119(e) to U.S. provisional patent application Ser. No.
60/923,452, filed Apr. 13, 2007, which is hereby incorporated by
reference.
BACKGROUND
[0002] Metal ions play important roles in many biological systems.
Cells utilize metal ions for a wide variety of functions, such as
regulating enzyme activities, protein structures, cellular
signaling, as catalysts, as templates for polymer formation and as
regulatory elements for gene transcription. Metal ions can also
have a deleterious effect when present in excess of bodily
requirements or capacity to excrete. A large number of natural and
synthetic materials are known to selectively or non-selectively
bind to or chelate metal ions. Ion chelators are commonly used in
solution for in vivo control of ionic concentrations and
detoxification of excess metals, and as in vitro buffers. Ion
chelators can be used as optical indicators of ions when bound to a
fluorophore, and may be useful in the analysis of cellular
microenvironments or dynamic properties of proteins, membranes and
nucleic acids. For example, Ca.sup.2+ ions play an important role
in many biological events, and so the determination of
intracellular Ca.sup.2+ is an important biological application.
[0003] Fluorescent indicators utilizing a polycarboxylate BAPTA
chelator have been predominantly used for intracellular calcium
detections (see for example U.S. Pat. No. 4,603,209; U.S. Pat. No.
5,049,673; U.S. Pat. No. 4,849,362; U.S. Pat. No. 5,453,517; U.S.
Pat. No. 5,501,980; U.S. Pat. No. 5,459,276; U.S. Pat. No.
5,501,980; U.S. Pat. No. 5,459,276; and U.S. Pat. No. 5,516,911;
each of which is hereby incorporated by reference). Xanthene-based
fluorescent calcium indicators (such as Fluo-3, Fluo-4 and Rhod-2
as represented by Formula 1) are the most common fluorescent
indicators used in biological assays. However, these existing
xanthene-based calcium indicators typically have low fluorescence
quantum yields, resulting in low detection sensitivity).
Furthermore their corresponding acetoxymethyl esters may not
readily penetrate the membranes of live cells (thus requiring
higher temperatures to achieve optimal dye loading), and once
inside the cells, they exhibit a slow conversion to the
corresponding BAPTA free acid.
TABLE-US-00001 Formula 1 ##STR00001## Indicator X Z R.sup.2 R.sup.5
Fluo-3 O O Cl Cl Fluo-4 O O F F Rhod-2 N(Me).sub.2 N(Me).sub.2 H
H
[0004] In view of the existing drawbacks for currently used
xanthene-based fluorescent calcium indicators, what is needed are
improved compositions and methods that offer sensitive detection of
small variations in calcium concentrations, with a rapid response
and a strong fluorescence signal. Also needed are fluorescent
indicators that can be readily loaded into live cells. In addition,
compositions and methods that are less susceptible to the effects
of external changes (such as temperature) are preferred for high
throughput screening and high content analysis.
[0005] The present application is directed to a family of
fluorescent dyes that are useful for preparing fluorescent metal
ion indicators. The indicators include a fluorophore and a
ionophore, and are useful for the detection, discrimination and
quantification of metal cations.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] FIG. 1. A synthetic scheme for the preparation of selected
BAPTA aldehyde compounds, where. R.sub.x and R.sub.y represent one
or more substituents of each ring.
[0007] FIG. 2. A synthetic scheme for the preparation of BAPTA
anhydride compounds and xanthene-substituted BAPTA compounds, where
R.sub.x, Ry and Rz represent one or more substituents of each
ring.
[0008] FIG. 3. A synthetic scheme for the preparation of selected
BAPTA acid compounds and their derivatives, where R.sub.x and
R.sub.y represent one or more substituents of each ring.
[0009] FIG. 4. A synthetic scheme for the preparation of selected
BAPTA bromide compounds (Method A), where R.sub.x and R.sub.y
represent one or more substituents of each ring.
[0010] FIG. 5. An alternative synthetic scheme for the preparation
of BAPTA bromide compounds (Method B), where R.sub.x and R.sub.y
represent one or more substituents of each ring.
[0011] FIG. 6. A synthetic scheme for the preparation of selected
fluorescein-based ion indicators (Method A), where R.sub.x, R.sub.y
and R.sub.z represent one or more substituents of each ring.
[0012] FIG. 7. An alternative synthetic scheme for the preparation
of selected fluorescein-based ion indicators (Method B), where
R.sub.x, R.sub.y and R.sub.z represent one or more substituents of
each ring.
[0013] FIG. 8. A synthetic scheme for the preparation of selected
rhodamine-based ion indicators (Method A), where R.sub.x, R.sub.y
and R.sub.z represent one or more substituents of each ring.
[0014] FIG. 9. An alternative synthetic scheme for the preparation
of selected rhodamine-based ion indicators (Method B), where
R.sub.x, R.sub.y and R.sub.z represent one or more substituents of
each ring.
[0015] FIG. 10. A synthetic scheme for the preparation of selected
rhodol-based ion indicators (Method A), where R.sub.x, R.sub.y and
R.sub.z represent one or more substituents of each ring.
[0016] FIG. 11. An alternative synthetic scheme for the preparation
of selected rhodol-based ion indicators (Method B), where R.sub.x,
R.sub.y and R.sub.z represent one or more substituents of each
ring.
[0017] FIG. 12. Another alternative synthetic scheme for the
preparation of selected rhodol-based ion indicators (Method C),
where R.sub.x, R.sub.y and R.sub.z represent one or more
substituents of each ring.
[0018] FIG. 13. The absorption spectra of Compound 284 in the
presence of 0.5 mM Ca.sup.2+ and in the absence of Ca.sup.2+ (as
described in Example 28).
[0019] FIG. 14. The calcium-dependent fluorescence spectra of
Compound 284 in the presence of 0.5 mM Ca.sup.2+ and in the absence
of Ca.sup.2+ ion with fluorescence excitation at 460 nm, as
described in Example 28.
[0020] FIG. 15. Intracellular Ca.sup.2+ response of the fluorescent
indicator Fluo-3 AM when measured by a fluorescence microscope, as
described in Example 29.
[0021] FIG. 16. Intracellular Ca.sup.2+ response of the fluorescent
indicator Fluo-4 AM when measured by a fluorescence microscope, as
described in Example 29.
[0022] FIG. 17. Intracellular Ca.sup.2+ response of the fluorescent
indicator Compound 365 when measured by a fluorescence microscope,
as described in Example 29.
[0023] FIG. 18. The intracellular Ca.sup.2+ response of selected
fluorescent calcium indicators as measured by a fluorescence
microplate reader that is equipped with an automated liquid
handling system, as described in Example 30.
DEFINITIONS
[0024] The following definitions are set forth to illustrate and
define the meaning and scope of the various terms used to describe
the invention herein.
[0025] The term "organic substituent", as used herein, refers to a
carbon-containing organic radical that incorporates straight,
branched chain or cyclic radicals having up to 50 carbons, unless
the chain length or ring size is limited thereto. The organic
substituent may include one or more elements of unsaturation, such
as carbon-carbon double or triple bonds. Organic substituents may
include alkyl, alkylene, alkenyl, alkenylene and alkynyl moieties,
among others.
[0026] The term "alkyl," as used herein, by itself or as part of
another group, refers to straight, branched chain or cyclic
radicals having up to 50 carbons, unless the chain length or ring
size is limited thereto, such as methyl, ethyl, propyl,
cyclopropanyl, isopropyl, butyl, t-butyl, isobutyl, pentyl, hexyl,
cyclohexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl,
2,2,4-trimethylpentyl, nonyl, and decyl, among others.
[0027] The term "alkylene," as employed herein, by itself or as
part of another group, refers to straight, branched chain or cyclic
divalent radicals having up to 50 carbons, unless the chain length
or ring size is limited thereto. Typical examples include methylene
(--CH.sub.2--), ethylene (--CH.sub.2CH.sub.2--), hexylene,
heptylene, octylene, nonylene, and decylene, among others.
[0028] The term "alkenyl," as used herein, by itself or as part of
another group, means a straight, branched chain or cyclic radical
having 2-50 carbon atoms and one or more carbon-carbon double
bonds, unless the chain length or ring size is limited thereto,
such as ethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl,
1-butenyl, and 2-butenyl, among others. The alkenyl chain may be 2
to 10 carbon atoms in length. Alternatively, the alkenyl chain may
be 2 to 4 carbon atoms in length.
[0029] The term "alkenylene," as used herein, by itself or as part
of another group, means straight, branched chain or cyclic divalent
radical having 2-50 carbon atoms, unless the chain length or ring
size is limited thereto, said straight, branched chain or cyclic
radical containing at least one carbon-carbon double bond. Typical
examples include ethenylene (--CH.dbd.CH--), propenylene
(--CH.dbd.CHCH.sub.2-- and --CH.sub.2CH.dbd.CH--), n-butenylene,
and 3-methyl-2-pentenylene, hexenylene, heptenylene, octenylene,
nonenylene, and decenylene, among others.
[0030] The term "alkynyl," as used herein, by itself or as part of
another group, means a straight, branched chain or cyclic radical
of 2-50 carbon atoms, unless the chain length or ring size is
limited thereto, having at least one carbon-carbon triple bond
between two of the carbon atoms in the chain, such as acetylenyl,
1-propynyl, and 2-propynyl, among others. The alkynyl chain may be
2 to 10 carbon atoms in length. Alternatively, the alkynyl chain
may be from 2 to 4 carbon atoms in length.
[0031] The term "alkynylene" as used herein, by itself or as part
of another group, means a straight, branched chain or cyclic
divalent radical having 2-50 carbon atoms, unless the chain length
or ring size is limited thereto, that contains at least one
carbon-carbon triple bond. Typical examples include ethynylene
(--C.ident.C--), propynylene (--C.ident.CCH.sub.2-- and
--CH.sub.2C.ident.C--), n-butynylene, 4-methyl-2-pentynylene,
1-butynylene, 2-butynylene, 3-butynylene, 4-butynylene,
pentynylene, hexynylene, heptynylene, octynylene, nonynylene, and
decynylene, among others.
[0032] The term "alkoxy" as used herein, by itself or as part of
another group, refers to any of the above radicals linked via an
oxygen atom. Typical examples include methoxy, ethoxy,
isopropyloxy, sec-butyloxy, n-butyloxy, t-butyloxy, n-pentyloxy,
2-methylbutyloxy, 3-methylbutyloxy, n-hexyloxy, and
2-ethylbutyloxy, among others. Alkoxy also may include PEG groups
(--OCH.sub.2CH.sub.2O--) or alkyl moieties that contain more than
one oxygen atom.
[0033] The term "aryl," as employed herein, by itself or as part of
another group, refers to an aryl or aromatic ring system containing
1 to 4 unsaturated rings (each ring containing 6 conjugated carbon
atoms and no heteroatoms) that are optionally vised to each other
or bonded to each other by carbon-carbon single bonds, that is
optionally further substituted as described below. Examples of aryl
ring systems include, but are not limited to, substituted or
unsubstituted derivatives of phenyl, biphenyl, o-, m-, or
p-terphenyl, 1-naphthyl, 2-naphthyl, 1-, 2-, or 9-anthryl, 1-, 2-,
3-, 4-, or 9-phenanthrenyl and 1-, 2- or 4-pyrenyl. Aryl
substituents may include phenyl, substituted phenyl, naphthyl or
substituted naphthyl.
[0034] The term "heteroaryl," as employed herein, by itself or as
part of another group, refers to groups having 5 to 14 ring atoms;
6, 10 or 14 .pi. electrons shared in a cyclic array; and containing
carbon atoms and 1, 2, 3, or 4 oxygen, nitrogen or sulfur
heteroatoms (where examples of heteroaryl groups are: thienyl,
benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furyl,
pyranyl, isobenzofuranyl, benzoxazolyl, chromenyl, xanthenyl,
phenoxathiinyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl,
pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl,
isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl,
4H-quinolizinyl, isoquinolyl, quinolyl, phthalazinyl,
naphthyridinyl, quinazolinyl, cinnolinyl, pteridinyl, carbazolyl,
phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl,
phenazinyl, isothiazolyl, phenothiazinyl, isoxazolyl, furazanyl,
phenoxazinyl, and tetrazolyl groups).
[0035] Any aryl or heteroaryl ring system is unsubstituted or
optionally and independently substituted by any synthetically
accessible and chemically stable combination of substituents, such
as H, halogen, cyano, sulfo, alkali or ammonium salt of sulfo,
nitro, carboxy, alkyl, perfluoroalkyl, alkoxy, alkylthio, amino,
monoalkylamino, dialkylamino or alkylamido, the alkyl portions of
which having 18 or fewer carbons.
[0036] The terms "halogen" or "halo" as employed herein, by itself
or as part of another group, refers to chlorine, bromine, fluorine
or iodine.
[0037] The terms "AM ester" or "AM" as employed herein, by itself
or as part of another group, refers to an acetoxymethyl ester of a
carboxylic acid.
[0038] The terms "amino" or "amine" include NH.sub.2,
"monoalkylamine" or "monoalkylamino," and "dialkylamine" or
"dialkylamino". The terms "monoalkylamine" and "monoalkylamino,"
"dialkylamine" and "dialkylamino as employed herein, by itself or
as part of another group, refers to the group NH.sub.2 where one
hydrogen has been replaced by an alkyl group, as defined above.
[0039] The terms "dialkylamine" and "dialkylamino" as employed
herein, by itself or as part of another group, refers to the group
NH.sub.2 where both hydrogens have been replaced by alkyl groups,
as defined above.
[0040] The term "hydroxyalkyl," as employed herein, by itself or as
part of another group, refers to an alkyl group where one or more
hydrogens thereof are substituted by one or more hydroxyl
moieties.
[0041] The term "haloalkyl," as employed herein, by itself or as
part of another group, refers to an alkyl group where one or more
hydrogens thereof are substituted by one or more halo moieties.
Typical examples include chloromethyl, fluoromethyl,
difluoromethyl, trifluoromethyl, trichloroethyl, trifluoroethyl,
fluoropropyl, and bromobutyl, among others.
[0042] The term "haloalkenyl," as employed herein, by itself or as
part of another group, refers to an alkenyl group where one or more
hydrogens thereof are substituted by one or more halo moieties.
[0043] The term "haloalkynyl," as employed herein, by itself or as
part of another group, refers to an alkynyl group where one or more
hydrogens thereof are substituted by one or more halo moieties.
[0044] The term "carboxyalkyl," as employed herein, by itself or as
part of another group, refers to an alkyl group where one or more
hydrogens thereof are substituted by one or more carboxylic acid
moieties.
[0045] The term "heteroatom" as used herein, by itself or as part
of another group, means an oxygen atom ("O"), a sulfur atom ("S")
or a nitrogen atom ("N"). It will be recognized that when the
heteroatom is nitrogen, it may form an NR.sub.1R.sub.2 moiety,
where R.sub.1 and R.sub.2 are, independently from one another,
hydrogen or alkyl, or together with the nitrogen to which they are
bound, form a saturated or unsaturated 5-, 6-, or 7-membered
ring.
[0046] The term "chelator", "chelate", "chelating group",
"ionophore", or "ionophoric moiety" as used herein, by itself or as
part of another group, refers to a chemical moiety that binds to,
or complexes with, one or more metal ions, such as lithium,
calcium, sodium, magnesium, potassium, and/or other biologically
important metal ions. The binding affinity of a chelator for a
particular metal ion can be determined by measuring the
dissociation constant between that chelator and that ion. Chelators
may include one or more chemical moieties that bind to, or complex
with, a cation or anion. Examples of suitable chelators include
1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA),
bipyridyl (bipy); terpyridyl (terpy); ethylenediaminetetraacetic
acid (EDTA); crown ethers; aza-crown ethers; succinic acid; citric
acid; salicylic acids; histidines; imidazoles;
ethyleneglycol-bis-(beta-aminoethyl ether) N,N'-tetraacetic acid
(EGTA); nitroloacetic acid; acetylacetonate (acac); sulfate;
dithiocarbamates; carboxylates; alkyldiamines; ethylenediamine
(en); diethylenetriamine (dien); nitrate; nitro; nitroso; glyme;
diglyme; bis(acetylacetonate)ethylenediamine (acacen);
1,4,7,10-tetraazacyclododecanetetraacetic acid (DOTA),
1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A),
1-oxa-4,7,10-triazacyclododecane-triacetic acid (OTTA),
1,4,7-triazacyclononanetriacetic acid (NOTA),
1,4,8,11-tetraazacyclotetra-decanetetraacetic acid (TETA),
DOTA-N-(2-aminoethyl) amide; DOTA-N-(2-aminophenethyl) amide; and
1,4,8,11-tetraazacyclotetradecane, among others.
[0047] The term "BAPTA" or
"1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid" as used
herein, by itself or as part of another group, refers to the
following ring structure or its derivatives, such as esters,
amides, carbamates and so on:
##STR00002##
[0048] The term "fluorophore or fluorophore moiety" as used herein,
by itself or as part of another group, means a molecule or a
portion of a molecule which exhibits fluorescence. By fluorescence
is meant that the molecule or portion of a molecule can absorb
excitation energy having a given wavelength and emit energy at a
different wavelength. The intensity and wavelength of the emitted
energy depend on the fluorophore, the chemical environment of the
fluorophore, and the specific excitation energy used. Exemplary
fluorophores include, but are not limited to, fluoresceins,
rhodamines, coumarins, oxazines, cyanines, pyrenes, and other
polycyclic aromatic molecules.
[0049] The term "xanthene", or "xanthene derivative", as used
herein, by itself or as part of another group, means any compounds
or substituents that contain one or more of the following fused
ring structures or its derivatives:
##STR00003##
[0050] The term "fluorescein" as used herein, by itself or as part
of another group, means any compounds or substituents that contain
one or more of the following fused ring structures or its
derivatives:
##STR00004##
[0051] The term "rhodamine" as used herein, by itself or as part of
another group, means any compounds or substituents that contain one
or more of the following fused ring structures or its
derivatives:
##STR00005##
[0052] The term "rhodol" as used herein, by itself or as part of
another group, means any compounds or substituents that contain one
or more of the following fused ring structures or its
derivatives:
##STR00006##
[0053] The term "substituted," as used herein, refers to the formal
replacement of a hydrogen on a chemical moiety or functional group
with an alternative radical. Where a compound, chemical moiety or
functional group is described as substituted, the alternative
radical substituent moiety is generally selected from the group
consisting of hydroxy, oxo, nitro, trifluoromethyl, halogen,
alkoxy, alkylenedioxy, aminoalkyl, aminoalkoxy, amino,
monoalkylamino, dialkylamino, alkylcarbonylamino,
alkoxycarbonylamino, alkoxycarbonyl, carboxy, hydroxyalkoxy,
alkoxyalkoxy, monoalkylaminoalkoxy,
dialkylaminoalkoxymono(carboxyalkyl)amino. bis(carboxyalkyl)amino,
alkoxycarbonyl, alkynylcarbonyl, alkylsulfonyl, alkenylsulfonyl,
alkynylsulfonyl, arylsulfonyl, alkylsulfonyl, alkylsulfinyl,
alkylsulfonamido, arylsulfonamido, alkylsulfonamido, carboxyalkoxy,
carboxyalkyl, carboxyalkylamino, cyano, trifluoromethoxy,
perfluoroethoxy, guanidine, amidino, oxyguanidino, alkylimino,
formylimino, acyl nitrile, acyl azide, acetyl azide,
dichlorotriazene, isothiocyante, sulfonyl halide, sulfosuccinimidyl
ester, isocyante, acyl halide, aldehyde, haloacetamide, maleimido,
aziridinyl, alkylthio (disulfide), acrylo, haloalkylcarbonyl,
boronate, hydrazide, semicarbazide, carbohydrazide, arylalkyl,
heteroarylalkyl, cycloalkylalkyl, cycloalkenylalkyl,
cycloheteroalkylalkyl, and cycloheteroalkenylalkyl.
[0054] The term "indicator compound" refers to the compounds of the
invention, specifically to those compounds having utility as
fluorescent metal ion indicators, as well as their acylated or
otherwise protected precursor compounds, such as the acetoxymethyl
ester derivatives suitable for adding to samples containing
biological cells.
[0055] The term "screening" refers to the testing and/or evaluation
of a multiplicity of molecules or compounds for a selected property
or therapeutic utility. Screening is typically a repetitive, or
iterative, process. A multiplicity of candidate molecules may be
screened for their ability to bind to a target molecule which is
capable of denaturing and/or unfolding. For example, a multiplicity
of candidate molecules may be evaluated for their ability to bind
to a target molecule (e.g., a protein receptor) in a thermal shift
assay. If none of a selected subset of molecules from the
multiplicity of candidate molecules (for example, a combinatorial
library) binds to the target molecule, then a different subset may
be tested for binding in the thermal shift assay.
[0056] The term "high-throughput", as used herein, encompasses
screening activity in which human intervention is minimized, and
automation is maximized. For example, high-throughput screening may
include any of a variety of automated processes, including for
example the automation of pipetting, mixing, and/or heating, the
software-controlled generation of thermal unfolding information,
and the software-controlled comparisons of thermal unfolding
information. Alternatively, a high-throughput method is one in
which hundreds of compounds can be screened per 24 hour period by a
single individual operating a single suitable apparatus.
Description of the Preferred Embodiments
[0057] The present application is directed to Fluorescent dyes
useful for preparing fluorescent metal ion indicators, the
fluorescent indicators themselves, and the use of the fluorescent
indicators for the detection, discrimination and quantification of
metal cations.
[0058] In one aspect of the invention, the compounds of the
invention may be described by Formula 2, below:
##STR00007##
[0059] Substituents R.sup.1-R.sup.6 are independently H, halogen,
carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido,
nitro, nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl,
carbonyl, boronic acid, aryl, or heteroaryl; or alkyl, or alkoxy
that is itself optionally substituted one or more times by halogen,
amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl
or heteroaryl.
[0060] The heteroatom Y is independently selected from O, S, Se,
NR.sup.9 and CR.sup.10R.sup.11. The X and Z substituents are
independently selected from O and NR.sup.12R.sup.13, where each
R.sup.9, R.sup.10, R.sup.11, R.sup.12 and R.sup.13 is independently
H, an alkyl having 1-12 carbons, or carboxyalkyl.
[0061] The T and U substituents are independently selected from
alkyl having 1-12 carbons, alkoxy having 1-12 carbons, aryloxy,
amino, halogen, cyano, carboxy, carboxyalkyl, carbonyl, sulfonyl,
phosphonyl, boronic acid, aryl, and heteroaryl.
[0062] The V and W are independently selected from OR.sup.14,
SR.sup.15 or NR.sup.12R.sup.13, such that at least one of V or W,
in combination with NR.sup.7R.sup.8, forms a metal chelator, where
each R.sup.7, R.sup.8, and R.sup.12-R.sup.15 are independently H,
an alkyl having 1-12 carbons, carboxyalkyl, alkoxy or aryloxy.
[0063] In one aspect of the invention X and Z are both O. In
another aspect of the invention, X and Y are O, and Z is
NR.sup.12R.sup.13. In yet another aspect of the invention, X, Y and
Z are each O. Careful selection of the nature of the X, Y, and Z
heteroatoms allows the spectral properties of the indicators to be
tuned through the selection of the appropriate xanthene dye
[0064] The compound of the invention may include exactly two
fluorophores, which may be the same or different, and which may
each be independently bound to the chelator by a covalent linkage
L, or may be fused to the chelator moiety. Where the compound of
the invention includes two fluorophores, the two fluorophores may
result in an indicator compound that exhibits ratiometric spectral
properties (such as Indo-1 or Fura-2).
[0065] The compounds of the present invention are xanthene-based
metal ion indicators. The existing xanthene-based BAPTA calcium
indicators are either fluorescein--(where X, Y and Z are O) or
rhodamine--(where X and Z are N while Y is O) based structures such
as Fluo-3, Fluo-4 and Rhod-2. The spectral properties of the
existing xanthene-based ion indicators may be modulated by
selecting substituents R.sup.1-R.sup.6, while the chelating
properties of the indicator may be adjusted by selecting and/or
modifying substituents j, k, m and n on the phenyl ring that is not
conjugated to the xanthene ring.
[0066] The substituents T and U can play unexpectedly important
roles in determining both the spectral properties and the chelating
properties of the indicator compounds. Another unexpected discovery
is that substituents R.sup.1, R.sup.2, R.sup.5 and R.sup.6 play
important roles in controlling the cell loading and intracellular
esterase-induced hydrolysis rate of acetoxymethyl (AM) esters of
xanthene-based fluorescent BAPTA indicators. For example, the
acetoxymethyl (AM) esters of xanthene-based BAPTA indicators are
much more readily loaded into live cells when R.sup.1, R.sup.2,
R.sup.5 and R.sup.6 are all hydrogen. The compounds of the present
invention provide sensitive and selective xanthene-based
fluorescent indicators for optical measurement of ion
concentrations in cells. Furthermore, substituents T and U can be
selected to provide the optimized spectral responses of
xanthene-based fluorescent ion indicators for selective measurement
of ions in cells. Careful selection of the R.sup.1, R.sup.2,
R.sup.5 and R.sup.6 groups of acetoxymethyl (AM) esters of
xanthene-based BAPTA indicators may result in optimal cell-loading
properties.
[0067] In one aspect of the invention, the compounds of the
invention can be described by Formula 3, below.
##STR00008##
[0068] In the embodiment of formula 3, heteroatom Y is
independently selected from O, S, Se, NR.sup.9 and
CR.sup.10R.sup.11. Substituents X and Z are independently selected
from O and NR.sup.12R.sup.13 where each R.sup.9, R.sup.10,
R.sup.11, R.sup.12 and R.sup.13 are independently H or an alkyl
having 1-12 carbons or carboxyalkyl. Substituents T and U are
independently selected from an alkyl having 1-12 carbons, alkoxy
having 1-12 carbons, aryloxy, amino, halogen, cyano, carboxy,
carboxyalkyl, carbonyl, sulfonyl, phosphonyl, boronic acid, aryl
and heteroaryl. R.sup.3, R.sup.4, j, k, m, n and V are
independently H, halogen, carboxy, alkoxy, aryloxy, thiol,
alkylthiol, arylthiol, azido, nitro, nitroso, cyano, amino,
hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or
heteroaryl; or alkyl, alkoxy that is itself optionally substituted
one or more times by halogen, amino, hydroxy, phosphonyl, sulfonyl,
carbonyl, boronic acid, aryl or heteroaryl.
[0069] In another aspect of the invention, the compounds of the
invention can be described by Formula 4, below.
##STR00009##
[0070] In this embodiment of the invention, the heteroatom Y is
independently selected from O, S, Se, NR.sup.9 and
CR.sup.10R.sup.11. Z is acyl having less than 10 carbon atom or
--CH.sub.2OAc. R.sup.9, R.sup.10, R.sup.11, R.sup.12 and R.sup.13
are independently H or alkyl having 1-12 carbons, or carboxyalkyl.
T and U are independently selected from hydrogen, alkyl having 1-12
carbons, alkoxy having 1-12 carbons, aryloxy, amino, halogen,
cyano, carboxy, carboxyalkyl, carbonyl, sulfonyl, phosphonyl,
boronic acid, aryl, and heteroaryl. R.sup.3, R.sup.4, j, k, m, n
and V are independently H, halogen, carboxy, alkoxy, aryloxy,
thiol, alkylthiol, arylthiol, azido, nitro, nitroso, cyano, amino,
hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or
heteroaryl; or alkyl or alkoxy that is itself optionally
substituted one or more times by halogen, amino, hydroxy,
phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or
heteroaryl.
[0071] In yet another aspect of the invention, the compounds of the
invention can be described by Formula 5, below.
##STR00010##
[0072] In this embodiment, the heteroatom Y is independently
selected from O, S, Se, NR.sup.9 and CR.sup.10R.sup.11. X and Z are
independently selected from O or NR.sup.12R.sup.13, where each
R.sup.9, R.sup.10, R.sup.11, R.sup.12 and R.sup.13 is independently
H or an alkyl having 1-12 carbons or carboxyalkyl. T and U are
independently selected from an alkyl having 1-12 carbons, alkoxy
having 1-12 carbons, aryloxy, amino, halogen, cyano, carboxy,
carboxyalkyl, carbonyl, sulfonyl, phosphonyl, boronic acid, aryl
and heteroaryl. R.sup.1-R.sup.6, j, k, m, n and V are independently
H, halogen, carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol,
azido, nitro, nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl,
carbonyl, boronic acid, aryl or heteroaryl; or alkyl or alkoxy that
is itself optionally substituted one or more times by halogen,
amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl
or heteroaryl. In this embodiment, the fluorophore moiety is
typically a xanthene.
[0073] In yet another aspect of the invention, the compounds of the
invention are fluorescent indicators which can be described by
Formula 6, below.
##STR00011##
[0074] In this embodiment, the substituents T and U are
independently selected from alkyl having 1-12 carbons, alkoxy
having 1-12 carbons, aryloxy, amino, halogen, cyano, carboxy,
carboxyalkyl, carbonyl, sulfonyl, phosphonyl, boronic acid, aryl or
heteroaryl. R.sup.1-R.sup.6, j, k, m, n and V are independently H,
halogen, carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol,
azido, nitro, nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl,
carbonyl, boronic acid, aryl or heteroaryl; or alkyl, alkoxy that
is itself optionally substituted one or more times by halogen,
amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl
or heteroaryl.
[0075] The disclosed indicator compounds typically exhibit low
fluorescence quantum efficiency in the absence of metal ions.
However, in the presence of increasing metal ion concentration the
fluorescence quantum efficiency rises dramatically. For example,
selected indicators of this family exhibit a fluorescence signal
increase of over 100-times between zero and a saturating calcium
concentration.
[0076] In yet another aspect of the invention, the compounds of the
invention are fluorescent indicators which can be described by
Formula 7, below.
##STR00012##
[0077] In this embodiment, substituents T and U are independently
selected from alkyl having 1-12 carbons, alkoxy having 1-12
carbons, aryloxy, amino, halogen, cyano, carboxy, carboxyalkyl,
carbonyl, sulfonyl, phosphonyl, boronic acid, aryl and heteroaryl.
R.sup.1-R.sup.6, j, k, m, n and V are independently H, halogen,
carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido,
nitro, nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl,
carbonyl, boronic acid, aryl or heteroaryl; or alkyl, or alkoxy
that is itself optionally substituted one or more times by halogen,
amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl
or heteroaryl. R.sup.12 and R.sup.13 are independently H or alkyl
having 1-12 carbons or carboxyalkyl.
[0078] In yet another aspect of the invention, the compounds of the
invention are fluorescent indicators which can be described by
Formula 8, below.
##STR00013##
[0079] In this embodiment, substituents T and U are independently
selected from alkyl having 1-12 carbons, alkoxy having 1-12
carbons, aryloxy, amino, halogen, cyano, carboxy, carboxyalkyl,
carbonyl, sulfonyl, phosphonyl, boronic acid, aryl and heteroaryl.
R.sup.1-R.sup.6, j, k, m, n and V are independently H, halogen,
carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido,
nitro, nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl,
carbonyl, boronic acid, aryl or heteroaryl; or alkyl, or alkoxy
that is itself optionally substituted one or more times by halogen,
amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl
or heteroaryl. X and Z, which may be same or different, are
independently selected from NR.sup.12R.sup.13, where R.sup.12 and
R.sup.13 are independently H or alkyl having 1-12 carbons or
carboxyalkyl.
[0080] In yet another aspect of the invention, the compounds of the
invention are fluorescent indicators which can be described by
Formula 9, below.
##STR00014##
[0081] In this embodiment, substituents R.sup.1-R.sup.6, j, k, m,
n, T, U and V are independently H, halogen, carboxy, alkoxy,
aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso,
cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic
acid, aryl or heteroaryl; or alkyl, or alkoxy that is itself
optionally substituted one or more times by halogen, amino,
hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or
heteroaryl.
[0082] In yet another aspect of the invention, the compounds of the
invention are fluorescent indicators which can be described by
Formula 10, below.
##STR00015##
[0083] In this embodiment, substituents R.sup.1-R.sup.6, j, k, m,
n, T, U and V are independently H, halogen, carboxy, alkoxy,
aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso,
cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic
acid, aryl or heteroaryl; or alkyl, or alkoxy that is itself
optionally substituted one or more times by halogen, amino,
hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or
heteroaryl.
[0084] In another aspect of the invention, the compounds of the
invention are fluorescent indicators having Formula 11.
##STR00016##
[0085] In this embodiment, substituents R.sup.1-R.sup.6, j, k, m,
n, T, U and V are independently H, halogen, carboxy, alkoxy,
aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso,
cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic
acid, aryl or heteroaryl; or alkyl, or alkoxy that is itself
optionally substituted one or more times by halogen, amino,
hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or
heteroaryl.
[0086] In yet another aspect of the invention, the compounds of the
invention are fluorescent indicators which can be described by
Formula 12, below.
##STR00017##
[0087] In this embodiment, substituents R.sup.1-R.sup.6, j, k, m,
n, T, U and V are independently H, halogen, carboxy, alkoxy,
aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso,
cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic
acid, aryl or heteroaryl; or alkyl, or alkoxy that is itself
optionally substituted one or more times by halogen, amino,
hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or
heteroaryl.
[0088] In yet another aspect of the invention, the compounds of the
invention are fluorescent indicators which can be described by
Formula 13, below.
##STR00018##
[0089] In this embodiment, substituents R.sup.1-R.sup.6, j, k, m,
n, T, U and V are independently H, halogen, carboxy, alkoxy,
aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso,
cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic
acid, aryl or heteroaryl; or alkyl, or alkoxy that is itself
optionally substituted one or more times by halogen, amino,
hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or
heteroaryl.
[0090] In yet another aspect of the invention, the compounds of the
invention are fluorescent indicators which can be described by
Formula 14, below.
##STR00019##
[0091] In this embodiment, R.sup.1-R.sup.6, j, k, m, n, T, U and V
are independently H, halogen, carboxy, alkoxy, aryloxy, thiol,
alkylthiol, arylthiol, azido, nitro, nitroso, cyano, amino,
hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or
heteroaryl; or alkyl, alkoxy that is itself optionally substituted
one or more times by halogen, amino, hydroxy, phosphonyl, sulfonyl,
carbonyl, boronic acid, aryl or heteroaryl.
[0092] In yet another aspect of the invention, the compounds of the
invention are fluorescent indicators which can be described by
Formula 15.
##STR00020##
[0093] In this embodiment, substituents R.sup.1-R.sup.6, j, k, m,
n, T, U and V are independently H, halogen, carboxy, alkoxy,
aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso,
cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic
acid, aryl or heteroaryl; or alkyl, alkoxy that is itself
optionally substituted one or more times by halogen, amino,
hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or
heteroaryl.
[0094] In yet another aspect of the invention, the compounds of the
invention are fluorescent indicators which can be described by
Formula 16, below.
##STR00021##
[0095] In this embodiment, substituents R.sup.1-R.sup.6, j, k, m,
n, T, U and V are independently H, halogen, carboxy, alkoxy,
aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso,
cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic
acid, aryl or heteroaryl; or alkyl, or alkoxy that is itself
optionally substituted one or more times by halogen, amino,
hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or
heteroaryl.
[0096] In yet another aspect of the invention, the compounds of the
invention are fluorescent indicators which can be described by
Formula 17, below.
##STR00022##
[0097] In this embodiment, substituents R.sup.1-R.sup.6, j, k, m,
n, T, U and V are independently H, halogen, carboxy, alkoxy,
aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso,
cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic
acid, aryl or heteroaryl; or alkyl, or alkoxy that is itself
optionally substituted one or more times by halogen, amino,
hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or
heteroaryl.
[0098] In yet another aspect of the invention, the compounds of the
invention are fluorescent indicators which may be described by
Formula 18.
##STR00023##
[0099] In this embodiment, substituents R.sup.1-R.sup.6, j, k, m,
n, T, U and V are independently H, halogen, carboxy, alkoxy,
aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso,
cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic
acid, aryl or heteroaryl; or alkyl, or alkoxy that is itself
optionally substituted one or more times by halogen, amino,
hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or
heteroaryl.
[0100] In yet another aspect of the invention, the compounds of the
invention are fluorescent indicators which can be described by
Formula 19, below.
##STR00024##
[0101] In this embodiment, substituents R.sup.1-R.sup.6, j, k, m,
n, T, U and V are independently H, halogen, carboxy, alkoxy,
aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso,
cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic
acid, aryl or heteroaryl; or alkyl, or alkoxy that is itself
optionally substituted one or more times by halogen, amino,
hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or
heteroaryl.
[0102] In yet another aspect of the invention, the compounds of the
invention are fluorescent indicators which can be described by
Formula 20, below.
##STR00025##
[0103] In this embodiment, substituents R.sup.1-R.sup.6, j, k, m,
n, T, U and V are independently H, halogen, carboxy, alkoxy,
aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso,
cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic
acid, aryl or heteroaryl; or alkyl, or alkoxy that is itself
optionally substituted one or more times by halogen, amino,
hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or
heteroaryl.
[0104] In yet another aspect of the invention, the compounds of the
invention are fluorescent indicators which can be described by
Formula 21, below.
##STR00026##
[0105] In this embodiment, substituents R.sup.1-R.sup.6, j, k, m,
n, T, U and V are independently H, halogen, carboxy, alkoxy,
aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso,
cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic
acid, aryl or heteroaryl; or alkyl, or alkoxy that is itself
optionally substituted one or more times by halogen, amino,
hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or
heteroaryl.
[0106] In yet another aspect of the invention, the compounds of the
invention are fluorescent indicators which can be described by
Formula 22, below.
##STR00027##
[0107] In this embodiment, substituents R.sup.1-R.sup.6, j, k, m,
n, T, U and V are independently H, halogen, carboxy, alkoxy,
aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso,
cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic
acid, aryl or heteroaryl; or alkyl, or alkoxy that is itself
optionally substituted one or more times by halogen, amino,
hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or
heteroaryl.
[0108] In yet another aspect of the invention, the compounds of the
invention are fluorescent indicators which can be described by
Formula 23, below.
##STR00028##
[0109] In this embodiment, substituents R.sup.1-R.sup.6, j; k, m,
n, T, U and V are independently H, halogen, carboxy, alkoxy,
aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso,
cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic
acid, aryl or heteroaryl; or alkyl, or alkoxy that is itself
optionally substituted one or more times by halogen, amino,
hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or
heteroaryl.
[0110] In yet another aspect of the invention, the compounds of the
invention are fluorescent indicators which can be described by
Formula 24, below.
##STR00029##
[0111] In this embodiment, heteroatom Y is NR.sup.9 or
CR.sup.11R.sup.12, where R.sup.9, R.sup.11 and R.sup.12 are
independently alkyl having 1-12 carbons, carboxyalkyl having 1-12
carbons, alkoxy having 1-12 carbons, a polyethylene glycol (PEG)
moiety, aryloxy; or alkyl, or alkoxy that is itself optionally
substituted one or more times by halogen, amino, hydroxy,
phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or heteroaryl.
Substituents T and U are independently selected from H, alkyl
having 1-12 carbons, alkoxy having 1-12 carbons, aryloxy, amino,
halogen, cyano, carboxy, carboxyalkyl, carbonyl, sulfonyl,
phosphonyl, boronic acid, aryl and heteroaryl. R.sup.1-R.sup.6, j,
k, m, n and V are independently H, halogen, carboxy, alkoxy,
aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso,
cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic
acid, aryl or heteroaryl; or alkyl, or alkoxy that is itself
optionally substituted one or more times by halogen, amino,
hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or
heteroaryl. X and Z, which may be the same or different, are
independently selected from O or NR.sup.12R.sup.13, where R.sup.12
and R.sup.13 are independently H or an alkyl having 1-12 carbons or
carboxyalkyl having 2-12 carbons. In this embodiment, Y is
typically N-alkyl where the alkyl group has 1-12 carbon atoms or
.dbd.C(alkyl).sub.2, where each alkyl independently has 1-6
carbons. More, Preferably Y is NMe or C(Me).sub.2.
[0112] In yet another aspect of the invention, the compounds of the
invention are fluorescent indicators which can be described by
Formula 25, below.
##STR00030##
[0113] In this embodiment, substituents T and U are independently
selected from H, alkyl having 1-12 carbons, alkoxy having 1-12
carbons, aryloxy, amino, halogen, cyano, carboxy, carboxyalkyl,
carbonyl, sulfonyl, phosphonyl, boronic acid, aryl and heteroaryl.
R.sup.1-R.sup.6, j, k, m, n and V are independently H, halogen,
carboxy, alkoxy, aryloxy, thio, alkylthiol, arylthiol, azido,
nitro, nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl,
carbonyl, boronic acid, aryl or heteroaryl; or alkyl, or alkoxy
that is itself optionally substituted one or more times by halogen,
amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl
or heteroaryl. R.sup.11 and R.sup.12 are independently an alkyl
having 1-12 carbons or carboxyalkyl, alkoxy having 1-12 carbons,
PEG chain, aryloxy; or alkyl, alkoxy that is itself optionally
substituted one or more times by halogen, amino, hydroxy,
phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or heteroaryl. X
and Z, which may be the same or different, are independently
selected from O and NR.sup.13R.sup.14, where R.sup.13 and R.sup.14
are independently H or alkyl having 1-12 carbons, or carboxyalkyl
having 2-12 carbons. In this embodiment, R.sup.13 and R.sup.14 are
typically lower alkyl or lower alkoxy having 1-12 carbon atoms.
Preferably, R.sup.13 and R.sup.14 are methyl or ethyl.
[0114] In yet another aspect of the invention, the compounds of the
invention are fluorescent indicators which can be described by
Formula 26, below.
##STR00031##
[0115] In this embodiment, substituents T and U are independently
selected from H, alkyl having 1-12 carbons, alkoxy having 1-12
carbons, aryloxy, amino, halogen, cyano, carboxy, carboxyalkyl,
carbonyl, sulfonyl, phosphonyl, boronic acid, aryl and heteroaryl.
R.sup.1-R.sup.6, j, k, m, n and V are independently H, halogen,
carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido,
nitro, nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl,
carbonyl, boronic acid, aryl or heteroaryl; or alkyl, or alkoxy
that is itself optionally substituted one or more times by halogen,
amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl
or heteroaryl. R.sup.23 is H or an alkyl having 1-12 carbons or
carboxyalkyl, alkoxy, aryloxy, amino, alkylamino or arylamino. In
this embodiment, R.sup.23 is typically lower alkyl or lower alkoxy
having 1-12 carbon atoms. Preferably R.sup.23 is methyl or methoxy.
More preferably R.sup.23 is methyl.
[0116] In yet another aspect of the invention, the compounds of the
invention are fluorescent indicators which can be described by
Formula 27, below.
##STR00032##
[0117] In this embodiment, substituents R.sup.1-R.sup.6, j, k, m,
n, T, U and V are independently H, halogen, carboxy, alkoxy,
aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso,
cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic
acid, aryl or heteroaryl; or alkyl, or alkoxy that is itself
optionally substituted one or more times by halogen, amino,
hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or
heteroaryl. R.sup.23 is independently H or an alkyl having 1-12
carbons or carboxyalkyl, alkoxy, aryloxy, amino, alkylamino or
arylamino. In this embodiment, R.sup.23 is typically lower alkyl or
lower alkoxy having 1-12 carbon atoms. Preferably R.sup.23 is
methyl or methoxy. More preferably R.sup.23 is methyl.
[0118] In yet another aspect of the invention, the compounds of the
invention are fluorescent indicators which can be described by
Formula 28, below.
##STR00033##
[0119] In this embodiment, substituents R.sup.1-R.sup.6, j, k, m,
n, T, U and V are independently H, halogen, carboxy, alkoxy,
aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso,
cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic
acid, aryl or heteroaryl; or alkyl, or alkoxy that is itself
optionally substituted one or more times by halogen, amino,
hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or
heteroaryl. R.sup.23 is independently H or alkyl having 1-12
carbons or carboxyalkyl, alkoxy, aryloxy, amino, alkylamino or
arylamino. In this embodiment, R.sup.23 is typically lower alkyl or
lower alkoxy of 1-12 carbon atoms. Preferably R.sup.23 is methyl or
methoxy. More preferably R.sup.23 is methyl.
[0120] In yet another aspect of the invention, the compound of the
invention can be described by Formula 29, below.
##STR00034##
[0121] In this embodiment, Y is independently selected from O, S,
Se, NR.sup.9 and CR.sup.10R.sup.11; X and Z are independently
selected from O and NR.sup.12R.sup.13, where each R.sup.9,
R.sup.10, R.sup.11, R.sup.12 and R.sup.13 is independently H or
alkyl having 1-12 carbons or carboxyalkyl. T and U are
independently selected from alkyl having 1-12 carbons, alkoxy
having 1-12 carbons, aryloxy, amino, halogen, cyano, carboxy,
carboxyalkyl, carbonyl, sulfonyl, phosphonyl, boronic acid, aryl
and heteroaryl. R.sup.1-R.sup.6 and V are independently H, halogen,
carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido,
nitro, nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl,
carbonyl, boronic acid, aryl or heteroaryl; or alkyl, or alkoxy
that is itself optionally substituted one or more times by halogen,
amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl
or heteroaryl. In this embodiment, the fluorophore moiety is
typically a xanthene.
[0122] In yet another aspect of the invention, the compound of the
invention can be described by Formula 30, below.
##STR00035##
[0123] In this embodiment, Y is independently selected from O, S,
Se, NR.sup.9 and CR.sup.10R.sup.11. X and Z are independently
selected from O and NR.sup.12R.sup.13, where each R.sup.9,
R.sup.10, R.sup.11, R.sup.12 and R.sup.13 is independently H or
alkyl having 1-12 carbons or carboxyalkyl. T and U are
independently selected from an alkyl having 1-12 carbons, alkoxy
having 1-12 carbons, aryloxy, amino, halogen, cyano, carboxy,
carboxyalkyl, carbonyl, sulfonyl, phosphonyl, boronic acid, aryl
and heteroaryl. R.sup.1-R.sup.6 and V are independently H, halogen,
carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido,
nitro, nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl,
carbonyl, boronic acid, aryl or heteroaryl; or alkyl, or alkoxy
that is itself optionally substituted one or more times by halogen,
amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl
or heteroaryl. In this embodiment, preferably the fluorophore
moiety is a xanthene. R.sup.20 is typically alky or aryl.
[0124] In yet another aspect of the invention, the compound of the
invention can be described by Formula 31, below.
##STR00036##
[0125] In this embodiment, Y is independently selected from O, S,
Se, NR.sup.9 and CR.sup.10R.sup.11, X and Z are independently
selected from O and NR.sup.12R.sup.13, where each R.sup.9,
R.sup.10, R.sup.11, R.sup.12 and R.sup.13 are independently H or an
alkyl having 1-12 carbons or carboxyalkyl. T and U are
independently selected from an alkyl having 1-12 carbons, alkoxy
having 1-12 carbons, aryloxy, amino, halogen, cyano, carboxy,
carboxyalkyl, carbonyl, sulfonyl, phosphonyl, boronic acid, aryl or
heteroaryl. R.sup.1-R.sup.6 and V are independently H, halogen,
carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido,
nitro, nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl,
carbonyl, boronic acid, aryl or heteroaryl; or alkyl, alkoxy that
is itself optionally substituted one or more times by halogen,
amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl
or heteroaryl. In this embodiment, preferably the fluorophore
moiety is a xanthene.
[0126] In yet another aspect of the invention, the compound of the
invention can be described by Formula 32, below.
##STR00037##
[0127] In this embodiment, Y is independently selected from O, S,
Se, NR.sup.9 and CR.sup.10R.sup.11 X and Z are independently
selected from O and NR.sup.12R.sup.13, where each R.sup.9,
R.sup.10, R.sup.11, R.sup.12 and R.sup.13 are independently H,
alkyl having 1-12 carbons, or carboxyalkyl. T and U are
independently selected from an alkyl having 1-12 carbons, alkoxy
having 1-12 carbons, aryloxy, amino, halogen, cyano, carboxy,
carboxyalkyl, carbonyl, sulfonyl, phosphonyl, boronic acid, aryl,
and heteroaryl. R.sup.1-R.sup.6 and V are independently H, halogen,
carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido,
nitro, nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl,
carbonyl, boronic acid, aryl or heteroaryl; or alkyl, or alkoxy
that is itself optionally substituted one or more times by halogen,
amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl,
or heteroaryl. R.sup.20 and R.sup.21 are independently H or alkyl
having 1-12 carbons, or carboxyalkyl. Typically the fluorophore
moiety is a xanthene.
[0128] In yet another aspect of the invention, the compounds of the
invention further include the alkyl ester derivatives of any of the
compounds described by Formulas 2 to 32, in order to facilitate the
delivery of fluorescent metal ion indicators into live cells. The
acetoxymethyl (AM) esters of the disclosed fluorescent indicators
are preferably used for applications that include the detection of
ions in live cells.
[0129] The AM esters of the invention can be described by Formula
33, below.
##STR00038##
In the above formula, the "Fluorescent Ion Indicator" moiety
corresponds to a compound of Formula 2 to 32, n is an integer from
1 to 10, and R.sup.23 is H or an alkyl having 1-12 carbons or
carboxyalkyl, alkoxy, aryloxy, amino, alkylamino or arylamino. In
this embodiment, R.sup.23 is typically lower alkyl or lower alkoxy
having 1-12 carbon atoms. Preferably R.sup.23 is methyl or methoxy.
More preferably R.sup.23 is methyl.
[0130] The fluorophore moiety can be any compound described by any
of Formulas 2-32 that exhibits an absorption maximum beyond 450 nm,
that is bound to a chelator by a covalent linkage L, or that is
fused to a chelator. The covalent linkage L may be a single
covalent bond, or a suitable combination of stable chemical bonds,
as described in greater detail below. The covalent linkage binding
the fluorophore moiety to the chelator is typically a single bond,
but optionally incorporates 1-20 nonhydrogen atoms selected from
the group consisting of C, N, O, P, and S.
[0131] As described above, where the fluorophore moiety is a
xanthene, the resulting compound may be a fluorescein, a rhodol
(U.S. Pat. No. 5,227,487, hereby incorporated by reference), or a
rhodamine. As used herein, fluorescein includes benzo- or
dibenzofluoresceins, seminaphthofluoresceins, or
naphthofluoresceins. Similarly, as used herein rhodol includes
seminaphthorhodafluors (U.S. Pat. No. 4,945,171, hereby
incorporated by reference). Fluorinated xanthene dyes have been
described previously as possessing particularly useful fluorescence
properties (U.S. Pat. No. 6,162,931, hereby incorporated by
reference).
[0132] Alternatively, the fluorophore moiety is a xanthene that is
bound via a covalent linkage L that is a single covalent bond at
the 9-position of the xanthene. Preferred xanthenes include
derivatives of 3H-xanthen-6-ol-3-one bound at the 9-position,
derivatives of 6-amnino-3H-xanthen-3-one bound at the 9-position,
or derivatives of 6-amino-3H-xanthen-3-imine bound at the
9-position.
[0133] In one aspect of the invention, the fluorophore moiety has
an absorption maximum beyond 480 nm. In a particularly useful
embodiment, the fluorophore moiety absorbs at or near 488 nm to 514
nm, and so is particularly suitable for excitation by the output of
an argon-ion laser excitation source, or near 546 nm, and so is
particularly suitable for excitation by a mercury arc lamp.
[0134] The fluorophore moiety is typically selected to confer its
fluorescence properties on the indicator compound it is
incorporated into. That is, the resulting indicator compound
exhibits a detectable optical response when excited by energy
having a wavelength at which that fluorophore absorbs As used
herein, a detectable optical response means a change in, or
occurrence of, an optical property that is detectable either by
observation or instrumentally, such a change in absorption
(excitation) wavelength, fluorescence emission wavelength,
fluorescence emission intensity, fluorescence polarization, or
fluorescence lifetime, among others.
[0135] In addition, the compounds of the invention preferably
exhibit a detectable change in the optical response upon binding a
target metal ion. Where the detectable response is a fluorescence
response, the detectable change is typically a change in
fluorescence, such as a change in the intensity, excitation or
emission wavelength distribution of fluorescence, fluorescence
lifetime, fluorescence polarization, or a combination thereof.
Preferably, the change in optical response upon binding the target
metal ion is a change in fluorescence intensity that is greater
than approximately 50-fold, more preferably greater than 100-fold.
In another aspect, the change in optical response up on binding the
target metal ion is a shift in the wavelength of maximal excitation
or emission. Typically the shift in wavelength is greater than
about 20 nm, preferably greater than about 30 nm.
[0136] Selected specific compounds of the invention are provided in
Table 2.
TABLE-US-00002 TABLE 2 Selected embodiment of the compounds of the
invention: Cpd. Method of no. Structure synthesis 256 ##STR00039##
Example 7 258 ##STR00040## Example 8 275 ##STR00041## Example 11
280 ##STR00042## Example 12 282 ##STR00043## Example 13 284
##STR00044## Example 14 286 ##STR00045## Example 15 288
##STR00046## Example 16 290 ##STR00047## Example 17 292
##STR00048## Example 18 294 ##STR00049## Example 19 296
##STR00050## Example 20 298 ##STR00051## Example 21 300
##STR00052## Example 22 302 ##STR00053## Example 23 304
##STR00054## Example 24 306 ##STR00055## Example 25 308
##STR00056## Example 26 310 ##STR00057## Example 27 350
##STR00058## Figure 8 351 ##STR00059## Figure 8 orFigure 9 352
##STR00060## Figure 10 orFigure 11 orFigure 12 353 ##STR00061##
Figure 6 352 ##STR00062## Figure 7 353 ##STR00063## Figure 6 354
##STR00064## Figure 7 355 ##STR00065## Figure 7 356 ##STR00066##
Figure 6 357 ##STR00067## Figure 9 358 ##STR00068## Figure 7 359
##STR00069## Figure 7 360 ##STR00070## Figure 6 orFigure 7 365
##STR00071## Figure 6 orFigure 7 366 ##STR00072## Figure 2
Synthesis
[0137] The compounds of the invention may be prepared using any
suitable synthetic scheme. The methodology used to prepare the
compounds of the invention may involve two components. The first
component may involve the formation of the chelator, while the
second may involve the modification of the chelator by forming a
reactive functional group, covalently attaching a conjugate, or
covalently attaching a fluorophore moiety to form the desired
indicator compound. Although these synthetic components are
typically performed in the order given, they may be carried out in
any other suitable sequence. For example, a portion of the chelator
may be derivatized with a fluorescent dye prior to formation of the
complete chelator ring. The representative synthetic methods are
summarized in FIGS. 1-12. The appropriate methods may be used to
synthesize the desired compounds of the invention.
[0138] As the metal binding ability of the resulting chelators may
be significantly influenced by the nature of the amine
substituents, careful selection of the alkylating agent may be
necessary to prepare a reporter for a particular target ion. BAPTA
chelators are typically selective for calcium ion. Where the
chelator nitrogens are alkylated by methyl bromoacetate, the
resulting bis-aza-crown ether is typically selective for sodium
ions. If the alkylating agent is 2-picolyl chloride, the resulting
crown ether is typically selective for zinc ions. Selection of an
alkylating agent that incorporates a precursor to a reactive
functional group is useful for producing chemically reactive
compounds of the invention, as well as acting as a useful
intermediate for preparing conjugates, as described above.
[0139] The syntheses of chelating groups selective for different
metal ions has been well described in the literature (U.S. Pat. No.
4,603,209; U.S. Pat. No. 5,049,673; U.S. Pat. No. 4,849,362; U.S.
Pat. No. 5,453,517; U.S. Pat. No. 5,501,980; U.S. Pat. No.
5,459,276; U.S. Pat. No. 5,501,980; U.S. Pat. No. 5,459,276; U.S.
Pat. No. 5,516,911; U.S. Application No. 2002/0164616; each of
which is incorporated by reference). These methods can be readily
adapted to prepare chelator intermediates useful for the synthesis
of the compounds of the invention.
[0140] Synthesis of conventional xanthene dyes such as
fluoresceins, rhodamines and rhodols typically involves the
condensation of two equivalents of resorcinol (for fluoresceins),
aminophenol (for rhodamines) or a mixture of a resorcinol and an
aminophenol (for rhodols) with a carbonyl-containing moiety such as
a phthalic acid derivative or benzaldehyde. However, in the
synthesis of the xanthene indicators of the invention, the desired
resorcinol or aminophenol is condensed with a chelator intermediate
that contains a carbonyl group, yielding either the reduced
xanthene (where the chelator contains an aldehyde) or the oxidized
xanthene (where the chelator intermediate ether contains a
carboxylic acid, anhydride or acyl halide) bound directly to the
chelating moiety. This synthetic method is illustrated in FIGS. 7,
9 and 11.
[0141] An oxidation step is typically required after condensation
of a formyl-substituted chelator with the fluorophore precursors.
Optionally, the dihydro condensation product may be isolated and
subsequently oxidized with air or by standard chemical oxidants,
such as DDQ or chloranil. For some fluorophores, the oxidation
reaction is enhanced by acidic reaction conditions. These mild
oxidation reaction conditions tolerate a wide variety of
substituents on the fluorophore and/or crown ether of the resulting
indicators. These carbonyl-derived methods are well described in
the literature (K. R. Gee, Z. Zhou, W. Qian and R. Kennedy, J. Am.
Chem. Soc. 2002, 124, 776; J. P. Bacci, A. M. Kerarney and D. L.
Van Vranken, J. Org. Chem. 2005, 70, 9051; U.S. Application No.
2002/0164616; each of which is incorporated by reference).
[0142] Unsymmetrical xanthene dyes are typically constructed using
statistical methods, using a 1:1 mixture of the desired resorcinols
or aminophenols in the condensation reaction, and purifying the
desired product from the resulting statistical mixture of products
using methods known in the art. This synthetic method is
represented by FIG. 11. In addition, unsymmetrical xanthene dyes
can be prepared from benzophenone intermediate as shown in FIG.
12.
[0143] Alternatively the fluorescent indicators of the invention
can be prepared via the condensation of properly protected
xanthones with a chelator anion, typically prepared from the
corresponding chelator bromide or iodide. This organometallic
chemistry is also well described in the literature (C. Chen, R. Yeh
and D. S. Lawrence, J. Am. Chem. Soc. 2002, 124, 3840; U.S. Pat.
No. 5,049,673); Y. Urano, M. Kamiya, K. Kanda, T. Ueno, K. Hirose
and T. Nagano, J. Am. Chem. Soc. 2005, 127, 4888; each of which is
incorporated by reference) and can be readily adapted to synthesize
the compounds of the invention (see FIGS. 6, 8 and 10).
[0144] Post-condensation modifications of both the chelator and the
fluorophore moiety are typically analogous to known methods of
indicator modification. For example, the reduction of nitro
substituents to amino groups, the conversion of carboxy
substituents to cyano groups, and the preparation of esters of
carboxylic acids, including acetoxymethyl esters (see FIGS. 6-11).
Additionally, a given salt or counterion of the indicators of the
invention may be readily converted to other salts by treatment with
ion-exchange resins, selective precipitation, and basification, as
is well-known in the art.
[0145] Post-condensation modifications of xanthylium dyes are well
known. For instance, the xanthenone portion of the dye can be
halogenated by treatment with an appropriate halogenating agent,
such as liquid bromine. Xanthenes containing unsaturated fused
rings can be hydrogenated to the saturated derivatives.
[0146] The reduced and oxidized versions of the xanthene indicators
are freely interconvertible by well-known oxidation or reduction
reagents, including borohydrides, aluminum hydrides,
hydrogen/catalyst, and dithionites. Care must be exercised to
select an oxidation or reducing agent that is compatible with the
chelator used. A variety of oxidizing agents mediate the oxidation
of dihydroxanthenes, including molecular oxygen in the presence or
absence of a catalyst, nitric oxide, peroxynitrite, dichromate,
triphenylcarbenium and chloranil. The dihydroxanthenes may also be
oxidized electrochemically, or by enzyme action, including the use
of horseradish peroxidase in combination with peroxides or by
nitric oxide.
[0147] Rather than condensing the fluorophore moiety precursors
directly with substituted chelators, the preformed fluorophore
moiety may be covalently bound to the chelator via a conventional
cross-linking reaction. A wide variety of chemically reactive or
potentially chemically reactive and fluorescent fluorescein,
rhodamine, rhodol, benzoxanthenes, dibenzoxanthene and other
xanthene oxygen heterocycles that absorb maximally beyond about 490
nm are commercially available as described by Haugland, HANDBOOK OF
FLUORESCENT PROBES AND RESEARCH CHEMICALS (7th ed., 1999), as
described above, or in other literature references. The nature of
the bond that links fluorophore moiety to the chelator appears to
have an effect on the optical response of the fluorophore moiety to
ion binding, sometimes a significant effect. Acceptability of the
linking chemistry can be determined by titration of the resultant
indicator with the ion of interest over the target range of
response.
Applications of the Fluorescent Indicators of the Invention
[0148] The indicators disclosed herein possess particular utility
for the detection and/or quantification of metal ions in a sample
of interest. Such indicators may be useful for measuring ions in
extracellular spaces; in vesicles; in vascular tissue of plants and
animals; biological fluids such as blood and urine; in fermentation
media; in environmental samples such as water, soil, waste water
and seawater; and in chemical reactors. Optical indicators for ions
are important for qualitative and quantitative determination of
ions, particularly in living cells. Fluorescent indicators for
metal cations also permit the continuous or intermittent optical
determination of these ions in living cells, and in solutions
containing the ions.
[0149] In effecting such determination, the substance to be
determined, or analyte, which contains the ion of interest is
contacted with a fluorescent indicator as disclosed above.
Complexation of the metal ion in the chelator of the indicator
results in a detectable change in the fluorescence properties of
the indicator. Detection and optionally quantification of the
detectable change permits the ion of interest to be detected and
optionally quantified.
[0150] Upon binding the target ion in the chelating moiety of the
indicator, the optical properties of the attached fluorophore are
generally affected in a detectable way, and this change may be
correlated with the presence of the ion according to a defined
standard. Compounds having relatively long wavelength excitation
and emission bands can be used with a variety of optical devices
and require no specialized (quartz) optics, such as are required by
indicators that are excited or that emit at shorter wavelengths.
These indicators are suitable for use in fluorescence microscopy,
flow cytometry, fluorescence microplate readers, or any other
application that currently utilize fluorescent metal ion
indicators.
[0151] This determination method may be based on the so-called "PET
effect", or the transfer, induced by photons, of electrons
(photoinduced electron transfer=PET) from the ionophoric moiety or
ionophore, respectively, to the fluorophore moiety or fluorophore,
respectively, which leads to a decrease in the (relative)
fluorescence intensity and the fluorescence decay time of the
fluorophore. Absorption and emission wavelengths, however, are not
significantly affected in the process (J. R. Lakowicz in "Topics in
Fluorescence Spectroscopy", Volume 4: Probe Design and Chemical
Sensing; Plenum Press, New York & London (1994)).
[0152] By the binding of ions to the ionophore, the PET effect may
be partly or completely inhibited, so that there is an increase in
the fluorescence of the fluorophore moiety. Hence, the
concentration or the activity of the ion to be determined can be
deduced by measuring the change in fluorescence properties, i.e.
fluorescence intensity and/or fluorescence decay time.
[0153] A variety of fluorescent indicators that are useful for the
detection of biologically relevant soluble free metal ions (such as
Ca.sup.2+, Mg.sup.2+ and Zn.sup.2+) have been described that
utilize oxygen-containing anionic or polyanionic chelators to bind
to metal ions. In general, a useful property for metal ion
indicators is selectivity, or the ability to detect and/or quantify
a selected metal ion in the presence of other metal ions.
Discrimination of Ca.sup.2+, Na.sup.+ and K.sup.+ ions in the
presence of other metal ions is particularly advantageous in
certain biological or environmental samples. For most biological
applications, it is useful that the indicators be effective in
aqueous solutions. It is also beneficial if the indicator absorbs
and emits light in the visible spectrum where biological materials
typically have low intrinsic absorbance or fluorescence.
[0154] Optical methods using fluorescence detection of metal ions
permit measurement of the entire course of ion flux in a single
cell as well as in groups of cells. The advantages of monitoring
transport by fluorescence techniques include the high level of
sensitivity of these methods, temporal resolution, modest demand
for biological material, lack of radioactivity, and the ability to
continuously monitor ion transport to obtain kinetic information
(Eidelman, O. Cabantchik, Z. I. Biochim. Biophys. Acta, 1989, 988,
319-334). The general principle of monitoring transport by
fluorescence is based on having compartment-dependent variations in
fluorescence properties associated with translocation of
compounds.
[0155] Optical methods were developed initially for measuring
Ca.sup.2+ ion flux (U.S. Pat. No. 5,049,673, hereby incorporated by
reference; Scarpa, A. Methods of Enzymology, 1979, 56, 301 Academic
Press, Orlando, Fla.; Tsien, R. Y. Biochemistry, 1980, 19, 2396;
Grynkiewicz, G., Poenic, M., Tsien, R. Y. J. Biol Chem., 260, 3440)
and have been modified for high-throughput assays (U.S. Pat. No.
6,057,114, hereby incorporated by reference). The flux of Ca.sup.2+
ion is typically performed using calcium-sensitive fluorescent dyes
such as Fluo-3, Fluo-4, Calcium Green, and others. (Molecular
Probes Inc., Handbook of Fluorescent probes and research chemicals,
7th edition, chapter 1, Eugene, Oreg.).
[0156] In particular, fluorescent indicators utilizing a
polycarboxylate BAPTA chelator have been previously described. A
determination method utilizing aza-cryptands as the chelator moiety
and using xanthenes and coumarins as fluorophores has also been
described (U.S. Pat. No. 5,439,828 and US Patent Application
20020164616; each hereby incorporated by reference). These
aza-cryptand may, depending on their structure, exhibit selectivity
for lithium, sodium or potassium ions. Some fluorescent indicators
selective for Li.sup.+, Na.sup.+ and K.sup.+ in aqueous or organic
solution have also been described, based on the chemical
modification of crown ethers (U.S. Pat. No. 5,134,232; U.S. Pat.
No. 5,405,975, each hereby incorporated by reference). ion.
[0157] The desired indicator compound is generally prepared for use
as a detection reagent by dissolving the indicator in solution at a
concentration that is optimal for detection of the indicator at the
expected concentration of the target ion. Modifications that are
designed to enhance permeability of the indicator through the
membranes of live cells, such as functionalization of carboxylic
acid moieties using acetoxymethyl esters and acetates, may require
the indicator to be predissolved in an organic solvent such as
dimethylsulfoxide (DMSO) before addition to a cell suspension,
where the indicators may then readily enter the cells.
Intracellular enzymes then cleave the esters, generating more polar
acids and phenols which are then well-retained inside the cells.
For applications where permeability of cell-membranes is required,
the indicators of the invention are typically substituted by only
one fluorophore.
[0158] The specific indicator used in a particular assay or
experiment may be selected based on the desired affinity for the
target ion as determined by the expected concentration range in the
sample, the desired spectral properties, and the desired
selectivity. Initially, the suitability of a material as an
indicator of ion concentration is commonly tested by mixing a
constant amount of the indicating reagent with a measured amount of
the target ion under the expected experimental conditions.
[0159] Where the binding of an ion in the metal ion-binding moiety
of the indicator results in a detectable change in spectral
properties of the indicator compound, that indicator may be used
for the detection and/or quantification of that ion (the target
ion). Although the change in spectral properties may include for
example a change in absorption intensity or wavelength, preferably
the change in spectral properties is a detectable fluorescence
response. Preferred indicators display a high selectivity, that is,
they show a sufficient rejection of non-target ions. The
interference of a non-target ion is tested by a comparable
titration of the indicator with that ion. In one aspect of the
invention, the target ions for the indicators of the present
invention are selected from Ca.sup.2+, Na.sup.+ and K.sup.+.
[0160] A detectable fluorescence response, as used herein, is a
change in a fluorescence property of the indicator that is capable
of being perceived, either by direct visual observation or
instrumentally, the presence or magnitude of which is a function of
the presence and/or concentration of a target metal ion in the test
sample. This change in a fluorescence property is typically a
change in fluorescence quantum yield, fluorescence polarization,
fluorescence lifetime, a shift in excitation or emission
wavelength, among others, or a combination of one or more of such
changes in fluorescence properties. The detectable change in a
given spectral property is generally an increase or a decrease.
However, spectral changes that result in an enhancement of
fluorescence intensity and/or a shift in the wavelength of
fluorescence emission or excitation may also be useful. The change
in fluorescence on ion binding may be due to conformational or
electronic changes in the indicator that may occur in either the
excited or ground state of the fluorophore, due to changes in
electron density at the ion binding site, due to quenching of
fluorescence by the bound target metal ion, or due to any
combination of these or other effects.
[0161] A typical indicator for a specific target ion is an
indicator that exhibits at least a 50-fold change in net
fluorescence emission intensity (either an increase or decrease),
or at least a 1 nanosecond difference in fluorescence lifetime
(either shorter or longer). In one aspect of the invention, the
indicator exhibits a 50-fold or greater change in net fluorescence
emission intensity, and/or a 100% change in fluorescence lifetime
in the presence of the target ion. In an alternative aspect of the
invention, the indicator exhibits a shift in excitation or emission
wavelength of at least 10 nm. (either to shorter or longer
wavelength), more preferably exhibiting a wavelength shift of 25 nm
or greater.
[0162] The spectral response of a selected indicator to a specific
metal ion is a function of the characteristics of the indicator in
the presence and absence of the target ion. For example, binding to
a metal ion may alter the relative electron densities of the
fluorophore and the metal binding site. Additionally, or in the
alternative, some metal ions may quench fluorescence emission when
in close proximity to a fluorophore (heavy atom quenching). In one
embodiment of the invention, the indicator is essentially
nonfluorescent or exhibits low fluorescence in target ion-free
solution and exhibits an increase in fluorescence intensity or
fluorescence lifetime (or both) upon target metal ion binding. In
yet another embodiment of the invention, the fluorescence intensity
remains approximately the same but there is a shift in the
excitation or emission spectrum, or both, upon metal ion
binding.
[0163] As the optical response of the indicating reagent is
typically determined by changes in fluorescence, the threshold of
detection of the target ion will be dependent upon the sensitivity
of the equipment used for its detection.
[0164] If the optical response of the indicator will be determined
using fluorescence measurements, the sample of interest is
typically stained with indicator concentrations of 10.sup.-9 M to
10.sup.-3 M. The most useful range of analyte concentration
includes about one log unit above and below the dissociation
constant of the ion-indicator complex. This dissociation constant
may be determined by titration of the indicator with known
concentrations of the target ion, usually over the range of
virtually zero concentration to approximately 100 mM of the target
ion, depending on which ion is to be measured and which indicator
is being used. The dissociation constant may be affected by the
presence of other ions, particularly ions that have similar ionic
radii and charge. It may also be affected by other conditions such
as ionic strength, pH, temperature, viscosity, presence of organic
solvents and incorporation of the sensor in a membrane or polymeric
matrix, or conjugation or binding of the sensor to a protein or
other biological molecule. Any or all of these effects are readily
determined, and can be taken into account when calibrating a
selected indicator.
[0165] The indicator is typically combined with a sample in a way
that will facilitate detection of the target ion concentration in
the sample. The sample is generally a fluid or liquid suspension
that is known or suspected to contain the target ion.
Representative samples include intracellular fluids from cells such
as in blood cells, cultured cells, muscle tissue, neurons and the
like; extracellular fluids in areas immediately outside of cells;
fluids in vesicles; fluids in vascular tissue of plants and
animals; biological fluids such as blood, saliva, and urine;
biological fermentation media; environmental samples such as water,
soil, waste water and sea water; industrial samples such as
pharmaceuticals, foodstuffs and beverages; and samples from
chemical reactors. Detection and quantitation of the target ion in
a sample can help characterize the identity of an unknown sample,
or facilitate quality control of a sample of known origin.
[0166] In one embodiment of the invention, the sample includes
cells, and the indicator is combined with the sample in such a way
that the indicator is added within the sample cells. By selection
of the appropriate chelating moiety, fluorophore, and the
substituents thereon, indicators may be prepared that will
selectively localize in a desired organelle, and provide
measurements of the target ion in those organelles. Conjugates of
the indicators of the invention with organelle-targeting peptides
may be used to localize the indicator to the selected organelle,
facilitating measurement of target ion presence or concentration
within the organelle (as described in U.S. Pat. No. 5,773,227,
hereby incorporated by reference). Alternatively, selection of a
lipophilic fluorophore, or a fluorophore having predominantly
lipophilic substituents may result in localization of the indicator
in lipophilic environments in the cell, such as cell membranes.
Selection of cationic indicators will typically result in
localization of the indicator in mitochondria.
[0167] In one embodiment of the invention, the indicator compound
of the invention optionally further includes a metal ion. In
another embodiment, the compounds of the invention, in any of the
embodiments described above, are associated, either covalently or
noncovalently, with a surface such as a microfluidic chip, a
silicon chip, a microscope slide, a microplate well, or another
solid or semisolid matrix, and is combined with the sample of
interest as it flows over the surface. In this embodiment, the
detectable optical response may therefore be detected on the matrix
surface itself, typically by use of instrumental detection. This
embodiment of the invention may be particularly suited to
high-throughput screening using automated methods.
[0168] The fluorescence response of the indicator to the target ion
may be detected by various means that include without limitation
measuring fluorescence changes with fluorometers, fluorescence
microscopes, laser scanners, flow cytometers, and microfluidic
devices, as well as by cameras and other imaging equipment. These
measurements may be made remotely by incorporation of the
fluorescent ion sensor as part of a fiber optic probe. The
indicator may be covalently attached to the fiber optic probe
material, typically glass or functionalized glass (e.g.,
aminopropyl glass) or the indicator may be attached to the fiber
optic probe via an intermediate polymer, such as polyacrylamide.
The indicator solution is alternatively incorporated non-covalently
within a fiber optic probe, as long as there is a means whereby the
target ion may come into contact with the indicator solution. More
preferably, the BAPTA indicators of the invention are used with a
fluorescence microplate reader that is equipped with an automated
liquid handling system such as FLIPR, FLEXSTATION and FDSS.
[0169] In another aspect of the invention, the fluorescent ion
indicators of the invention may be used in combination with one or
more non-fluorescent dyes that are not substantially cell-permeable
in order to reduce the background fluorescence analogous to the
methods described in U.S. Pat. No. 6,420,183, hereby incorporated
by reference. Non-fluorescent dyes and dye mixtures that have large
water solubilities and minimal effects on the physiology of the
cells are preferred for this application. More preferably are
water-soluble azo dyes (such as trypan blue), which have been used
in cell-based assays for many years (H. W. Davis, R. W. Sauter.
Histochemistry, 1977, 54, 177; W. E. Hathaway, L. A. Newby, J. H.
Githens, Blood, 1964, 23, 517; C. W. Adams, O. B. Bayliss, R. S.
Morgan, Atherosclerosis, 1977, 27, 353).
[0170] The screening methods described herein can be performed with
cells growing in or deposited on solid surfaces. A common technique
is to use a microwell plate where the fluorescence measurements are
performing using a commercially available fluorescent plate reader.
These methods lend themselves to use in high throughput screening
using both automated and semi-automated systems.
[0171] Using the indicators of the present invention, the
measurement of fluorescence intensity can provide a sensitive
method for monitoring changes in intracellular ion concentrations.
Thus, fluorescence measurements at appropriate excitation and
emission wavelengths provide a fluorescence readout which is
sensitive to the changes in the ion concentrations.
[0172] In one embodiment, the invention includes a) adding a
compound as described above to a sample containing a cell; b)
incubating the sample for a time sufficient for the compound to be
loaded into the cell and an indicator compound to be generated
intracellularly; c) illuminating the sample at a wavelength that
generates a fluorescence response from the indicator compound; d)
detecting a fluorescence response from the indicator compound; and
e) correlating the fluorescence response with the presence of
intracellular calcium.
[0173] In one aspect of the invention, the disclosed method is
useful for screening potential therapeutic drugs, for example drugs
which may affect ion concentrations in biological cells. These
methods may include measuring ion concentrations as described above
in the presence and absence (as a control measurement) of the test
sample. Control measurements are usually performed with a sample
containing all components of the test sample except for the
putative drug being screened. Detection of a change in ion
concentration in the presence of the test agent relative to the
control indicates that the test agent is active. Ion concentrations
can also be determined in the presence or absence of a
pharmacologic agent of known activity (i.e., a standard agent) or
putative activity (i.e., a test agent). A difference in ion
concentration as detected by the methods disclosed herein allows
one to compare the activity of the test agent to that of a standard
agent of known activity. It will be recognized that many
combinations and permutations of drug screening protocols are known
to one of skill in the art and they may be readily adapted to use
with the method of ion concentration measurement disclosed herein
to identify compounds which affect ion concentrations.
[0174] In yet another aspect of the invention, the disclosed method
may facilitate the screening of test samples in order to identify
one or more compounds that are capable of modulating the activity
of an ion channel, pump or exchanger in a membrane, and the method
further includes stimulating the cell, monitoring changes in the
intensity of the fluorescence response from the indicator compound,
and correlating the changes in fluorescence intensity with changes
in intracellular calcium levels.
[0175] An additional method may be used to evaluate the efficacy of
a stimulus that generates a target ion response, including (a)
loading a first set and a second set of cells with the ion
indicators of the invention which monitor ion concentrations; (b)
optionally, exposing both the first and second set of cells to a
stimulus which modulates the ion channel, pump or exchanger; (c)
exposing the first set of cells to the test sample; (d) measuring
the ion concentrations in the first and second sets of cells; and
(e) relating the difference in ion concentrations between the first
and second sets of cells to the ability of a compound in the test
sample to modulate the activity of an ion channel, pump or
exchanger in cells. In one aspect of the recited method, the method
may include the addition of probenecid or a probenecid derivative
to the sample.
[0176] One or more of the methods disclosed herein may be enhanced
by the addition of a cell-impermeant and non-fluorescent dye to the
sample, such that the dye remains in the extracellular solution,
and acts as an acceptor dye for energy transfer from the indicator
compound, thereby decreasing background signal from the sample
solution. In one aspect of the method, the cell-impermeant and
non-fluorescent dye is a water-soluble azo dye.
[0177] Ion channels of particular interest may include, but are not
limited to, sodium, calcium, potassium, nonspecific cation, and
chloride ion channels, each of which may be constitutively open,
voltage-gated, ligand-gated, or controlled by intracellular
signaling pathways.
[0178] Biological cells of potential interest for screening
application may include, but are not limited to, primary cultures
of mammalian cells, cells dissociated from mammalian tissue, either
immediately or after primary culture. Cell types may include, but
are not limited to white blood cells (e.g. leukocytes),
hepatocytes, pancreatic beta-cells, neurons, smooth muscle cells,
intestinal epithelial cells, cardiac myocytes, glial cells, and the
like. The disclosed method may also include the use of recombinant
cells into which ion transporters, ion channels, pumps and
exchangers have been inserted and expressed by genetic engineering.
Many cDNA sequences for such transporters have been cloned (see
U.S. Pat. No. 5,380,836 for a cloned sodium channel, hereby
incorporated by reference) and methods for their expression in cell
lines of interest are within the knowledge of one of skill in the
art (see, U.S. Pat. No. 5,436,128, hereby incorporated by
reference). Representative cultured cell lines derived from humans
and other mammals include LM cells, HEK-293 (human embryonic kidney
cells), 3T3 fibroblasts, COS cells, CHO cells, RAT1 and HepG2
cells, Hela cells, U.sub.2OS cells and Jurkat cells etc.
Assay Kits
[0179] Due to the advantageous properties and the simplicity of use
of the disclosed ion indicator compounds, they possess particular
utility in the formulation of a kit for the complexation,
detection, or quantification of selected target ions. An exemplary
kit may include one or more compounds or compositions of the
invention in any of the embodiments described above, either present
as a pure compound, in a suitable carrier composition, or dissolved
in an appropriate stock solution. The kit may further include
instructions for the use of the indicator compound to complex or
detect a desired target ion. The kit may further include one or
more additional components, such as an additional detection
reagent.
[0180] The indicator of the invention may be present in the kit
associated with a surface, such as a chip, microplate well, or
other solid or semi-solid matrix.
[0181] The additional kit components may be selected from, without
limitation, calibration standards of a target ion, ionophores,
fluorescence standards, aqueous buffers, surfactants and organic
solvents. The additional kit components may be present as pure
compositions, or as aqueous solutions that incorporate one or more
additional kit components. Any or all of the kit components
optionally further comprise buffers.
[0182] In one aspect of the disclosed kit, the kit includes at
least one indicator compound as described above, and a
non-fluorescent and cell-impermeant quencher dye. The
non-fluorescent and cell-impermeant quencher dye is optionally
present with the indicator compound in a combined composition, such
as a mixed powder or a solution. Alternatively, or in addition, the
cell-impermeant quencher dye is present in a container distinct
from the indicator compound.
[0183] The examples provided below illustrate selected aspects of
the invention. They are not intended to limit or define the entire
scope of the invention.
EXAMPLES
Example 1
Preparation of Compound 205
##STR00073##
[0185] Compound 205 is analogously prepared according to the
procedure of U.S. Application No. 2002/0164616, hereby incorporated
by reference. A mixture of Compound 200 (15 g) and
1-bromo-2-chloroethane (50 g) is dissolved in DMF at room
temperature. To the reaction mixture K.sub.2CO.sub.3 is added with
stirring. The reaction mixture is stirred at room temperature for
4-6 days. The reaction mixture is poured into water, and the
resulted solid is collected. The dried solid is purified on a
silica gel column using a gradient of hexanes/ethyl acetate to give
a light yellow solid.
Example 2
Preparation of Compound 215
##STR00074##
[0187] Compound 215 is prepared analogously to the procedure of
U.S. Pat. No. 5,049,673 and U.S. Application No. 2002/0164616
(hereby incorporated by reference). The mixture of Compound 205 (20
g) and 5-methyl-2-nitrophenol (20 g) is dissolved in DMF at room
temperature. To the reaction mixture K.sub.2CO.sub.3 is added, and
the reaction mixture is stirred at 140-160.degree. C. for 12-24 h.
The reaction mixture is cooled, and poured into water, and resulted
solid is collected. The dried solid is purified on a silica gel
column using a gradient of hexanes/ethyl acetate to give a very
light yellow solid.
Example 3
Preparation of Compound 225
##STR00075##
[0189] Compound 225 is prepared analogously to the procedure of
U.S. Pat. No. 5,049,673 and U.S. Application No. 2002/0164616, each
hereby incorporated by reference. Compound 215 is dissolved in DMF
at room temperature. To the solution 10% palladium on carbon is
added. The reaction mixture is hydrogenated at 40-45 psi for 3-4 h.
The reaction mixture is filtered through diatomaceous earth to
remove the catalyst that is washed with DMF. The combined DMF
solution is poured into water. The formed solid is collected by
filtration, and washed with water. The dried solid is purified on a
silica gel column using a gradient of chloroform/ethyl acetate to
give an off-white solid.
Example 4
Preparation of Compound 235
##STR00076##
[0191] Compound 235 is prepared analogously to the procedure of
U.S. Pat. No. 5,049,673. Compound 225 (25 g) is dissolved in DMF at
room temperature. To the reaction mixture (iPr).sub.2NEt (100 mL)
is added with stirring, and then methyl bromoacetate (50 mL) is
added with stirring. The reaction mixture is heated at
70-90.degree. C. for 24-36 h. The concentrated DMF solution is
poured into water. The formed solid is collected by filtration, and
washed with water. The dried solid is purified on a silica gel
column using a gradient of chloroform/ethyl acetate to give an
off-white solid.
Example 5
Preparation of Compound 245
##STR00077##
[0193] Compound 245 is prepared analogously to the procedure of
U.S. Application No. 2002/0164616. DMF (50 mL) is cooled ice in
water bath. To the DMF is added POCl.sub.3 dropwise. The resulted
solution is stirred at room temperature for 1-2 h, and cooled to
5-10.degree. C. To the POCl.sub.3/DMF mixture is dropwise added a
solution of compound 235 (10 g) in DMF (100 mL) over 40-45 min. The
reaction mixture is heated at 40-45.degree. C. for 12-24 h. The
resulted mixture is cooled to room temperature, and concentrated in
vacuo, and poured into ice/water. The suspension is filtered to
collect the solid that is washed with water. The dried solid is
purified on a silica gel column to give of off-white solid using a
gradient of chloroform/ethyl acetate.
Example 6
Preparation of Compound 252
##STR00078##
[0195] Compound 252 is prepared analogously to the procedures of
U.S. Application No. 2002/0164616; K. R. Gee, Z. L. Zhou, W. J.
Qian, R. Kennedy, J. Am. Chem. Soc. 2002, 124, 776; V. V. Martin,
A. Rothe, Z. Diwu and K. Gee, Bioorg. Med. Chem. Lett. 2004, 14,
5313; and J. P. Bacci, A. M. Kearney and D. L. Van Vranken, J. Org.
Chem. 2005, 70, 9051). The mixture of aldehyde 245 (2 g) and
4-chlororesorcinol (1.5 g) in MeSO.sub.3H (30 mL) is stirred
overnight, and then poured into NaOAc solution. The precipitated
solid is filtered, washed with water and dried to give the dihydro
form of Compound 252 that is directly used in the next step without
additional purification. The mixture of the crude dihydro form of
Compound 252 and chloranil in MeOH is heated at reflux for 12 to 24
h, then cooled to room temperature, filtered (to remove excess
oxidizer), and evaporated. The residue is concentrated and purified
on a silica gel column using a gradient of chloroform/methanol.
Example 7
Preparation of Compound 256
##STR00079##
[0197] Compound 252 (100 mg) is suspended in 1:1 methanol/water (10
mL). To the suspension LiOH (150 mg) is added slowly while cooled
in ice/water bath, and stirred at room temperature for 12-24 h. The
reaction mixture is diluted with water (200 mL), and neutralized
with concentrated HCl (2-3 mL). The mixture is filtered to collect
the precipitate. The solid is redissolved in methanol, and further
purified by HPLC to give Compound 256.
Example 8
Preparation of Compound 258
##STR00080##
[0199] Compound 256 (50 mg) is dissolved in anhydrous DMF (3 mL) at
room temperature. To the solution BrCH.sub.2OAc (70 .mu.L) in
anhydrous DMF (2 mL) is slowly added while stirring in a water
bath. To the resulted mixture iPr.sub.2Net (130 .mu.L) is added
slowly. The resulted mixture is stirred for 24-36 h. The reaction
mixture is poured into ice/water. The suspension is filtered to
collect the solid that is washed with water. The dried solid is
purified on a silica gel column to give an off-white solid using a
gradient of chloroform/ethyl acetate.
Example 9
Preparation of Compound 265
##STR00081##
[0201] Compound 265 is prepared analogously to the procedure of
U.S. Pat. No. 5,049,673. Compound 260 (10 g, prepared analogous to
the procedure of Compound 235), 1,8-bis(dimethylamino)naphthalene
(30 g), anhydrous sodium iodide (2 g), tert-butyl bromoacetate (50
g) and DMF (100 mL) is stirred with heating at 70-90.degree. C. for
18 hours. The concentrated DMF solution is poured into water. The
formed solid is collected by filtration, and washed with water. The
dried solid is purified on a silica gel column to give an off-white
solid.
Example 10
Preparation of Compound 268
##STR00082##
[0203] Compound 268 is prepared analogously to the procedure of
U.S. Pat. No. 5,049,673. Compound 265 (10 g) is dissolved in
dichloromethane (100 mL) and cooled to -78.degree. C. Pyridine (0.2
mL) is added and the mixture is stirred while bromine (3 g) in
dichloromethane (20 mL) is added. The mixture is allowed to warm up
to room temperature and then evaporated in vacuo. The residue is
purified on a silica gel column using a gradient of
chloroform/ethyl acetate to give an off-white solid.
Example 11
Preparation of Compound 275
##STR00083##
[0205] Compound 275 is prepared analogously to the procedure of
U.S. Pat. No. 5,049,673. Compound 268 (150 mg) is dissolved in
2-methyl-tetrahydrofuran (5 mL) and stirred at -150.degree. C. in a
liquid nitrogen-isopentane bath. Tertiary butyllithium (6
equivalents) in hexane is added and the metallation monitored by
thin layer chromatography of small samples quenched into water.
Compound 271 (100 mg, see Parham, W. E., and Bradscher, C. K., Acc.
Chem. Res., 1982, 15, 300; U.S. Pat. No. 5,049,673), dissolved in
tetrahydrofuran, is added dropwise to the reaction mixture.
Stirring is continued for another 30 minutes. The reaction mixture
is quenched with water in tetrahydrofuran and then allowed to warm
up to room temperature, and extracted twice with ethyl acetate. The
combined organic extracts are washed with brine and evaporated to
dryness. The residue is then stirred with acetic acid to convert
all the leuco-base into the desired dye. Evaporation of the acetic
acid in vacuo leaves a gummy residue which is purified by column
chromatography on silica gel using a gradient of chloroform/ethyl
acetate/methanol to give pure Compound 272.
[0206] Compound 272 (10 mg) is dissolved in acetic acid (1 mL) and
BF.sub.3 etherate (0.1 mL) is added. The resulting solution is
stirred at room temperature overnight. The solution is then
evaporated in vacuo. The crude product is further purified by
HPLC.
Example 12
Preparation of Compound 280
[0207] Compound 280 is prepared analogously to the procedure of
Compound 256.
##STR00084##
Example 13
Preparation of Compound 282
[0208] Compound 282 is prepared analogously to the procedure of
Compound 256.
##STR00085##
Example 14
Preparation of Compound 284
##STR00086##
[0210] Compound 284A (20 g, Shaanxi Zhendi Chemical Biology, Ltd.)
is converted to Compound 284C (22g) analogously to the protocol of
Compound 215.
[0211] Compound 284C (12 g) is dissolved in ethanol. To the ethanol
solution is added 23 g stannous chloride hydrate. The reaction
mixture is heated at reflux until Compound 284C is completely
consumed, cooled to room temperature, and poured into ice water.
The reaction mixture is neutralized with sodium carbonate to have
pH=6-7, and filtered to collect the solid that is further purified
on a silica gel column eluted with a gradient of
chloroform/methanol to give pure Compound 284D.
[0212] Compound 284D (10 g) is converted to Compound 284E
analogously according to the protocol of Compound 235.
[0213] Compound 284E is dissolved in DMF at room temperature. To
the solution palladium on carbon is added. The reaction mixture is
hydrogenated until Compound 284E is completely consumed. The
reaction mixture is filtered through diatomaceous earth to remove
the catalyst which is washed with DMF. The combined DMF solution is
poured into water. The formed solid is collected by filtration, and
washed with water. The dried solid is purified on a silica gel
column using a gradient of chloroform/ethyl acetate to give
Compound 284F as an off-white solid.
[0214] Phthalic acid 284F (6 g) is added to the solution of
resorcinol (3 g) in methanesulfonic acid (10 mL). The resulting
mixture is heated under dry nitrogen at 70-80.degree. C. until
Compound 284F is completely consumed. The cooled mixture is poured
into ice water followed by filtration. The filtrate containing
Compound 284 and its isomer 284G is dried, and purified on a silica
gel column eluted with a gradient of water/acetonitrile to give the
mixture of Compound 284 and its isomer 284G. The mixture of
Compounds 284 and 284G is further purified by HPLC using C18 column
and a gradient of 1% TFA acetonitrile-1% TFA buffer to give the
pure Compound 284.
Example 15
Preparation of Compound 286
[0215] Compound 286 is prepared analogously to the procedure of
Compound 284 or FIG. 2.
##STR00087##
Example 16
Preparation of Compound 288
[0216] Compound 288 is prepared analogously to the procedure of
Compound 256.
##STR00088##
Example 17
Preparation of Compound 290
[0217] Compound 290 is prepared analogously to the procedure of
Compound 256.
##STR00089##
Example 18
Preparation of Compound 292
[0218] Compound 292 is prepared analogously to the procedure of
Compound 256 or 275.
##STR00090##
Example 19
Preparation of Compound 294
[0219] Compound 294 is prepared analogously to the procedure of
Compound 258.
##STR00091##
Example 20
Preparation of Compound 296
[0220] Compound 296 is prepared analogously to the procedure of
Compound 258.
##STR00092##
Example 21
Preparation of Compound 298
[0221] Compound 298 is prepared analogously to the procedure of
Compound 258.
##STR00093##
Example 22
Preparation of Compound 300
[0222] Compound 300 is prepared from the reaction of Compound 286
with acetic anhydride analogously to the procedure of U.S. Pat. No.
6,162,931.
##STR00094##
Example 23
Preparation of Compound 302
[0223] Compound 302 is prepared analogously to the procedure of
Compound 258.
##STR00095##
Example 24
Preparation of Compound 304
[0224] Compound 304 is prepared analogously to the procedure of
Compound 258 by using a large excess of bromomethylacetate and
base.
##STR00096##
Example 25
Preparation of Compound 306
##STR00097##
[0226] Compound 284 (350 mg) is heated at 80.degree. C. with
Ac.sub.2O (5 mL) and pyridine (0.1 mL) until Compound 284 is
completely consumed (10 to 30 min). The solution is cooled to room
temperature. The reaction mixture is poured into ice water, and
carefully adjusted to pH=4-5. The aqueous mixture is titrated with
dioxane to give a precipitate that is collected by filtration. The
resulting mixture is first air-dried, and further vacuum-dried in a
desiccator with P.sub.2O.sub.5 for 12 h to yield crude Compound 306
B that is directly used for next step reaction.
[0227] The crude Compound 306B is converted into Compound 306
analogously to the procedure of Compound 258.
Example 26
Preparation of Compound 308
[0228] Compound 308 is prepared analogous to the procedure of
Compound 275.
##STR00098##
Example 27
Preparation of Compound 310
[0229] Compound 310 is prepared analogous to the procedure of
Compound 275.
##STR00099##
Example 28
Spectral Properties of the Fluorescent Indicators
[0230] The absorbance and fluorescence properties of a
representative indicator in the presence and absence of Ca.sup.2+
are shown in FIGS. 13 and 14, using Compound 284. The calcium
binding has little effect on the absorption spectra, as shown in
FIG. 13. However, the indicator compounds of the present invention
demonstrate fluorescence that is strongly enhanced by Ca.sup.2+
binding, as shown in FIG. 14. Additionally, Ca.sup.2+ binding has
little effect on the wavelengths of peak excitation or emission.
Specifically, 200 .mu.L of 5 .mu.M of compound 284 in 100 mM KCl
with 30 mM Tris buffer in the presence and absence of 0.5 mM
calcium is measured for absorption spectra using Spectra Max while
the fluorescence spectra (excitation at 460 nm) is measured with
Gemini fluorescence microplate reader. The indicators of the
invention demonstrate substantially similar spectral responses to
calcium binding.
Example 29
Calcium Responses of the Fluorescent Indicators Measured Using a
Fluorescence Microscope
[0231] Cells expressing a GPCR of interest that signals through
calcium are pre-loaded with a selected indicator that has been
functionalized with acetoxy methyl ester groups (or AM esters),
such as for example Compounds 258, 294, 296, 298, 302, 304 and 306.
Specifically, HEK-293 cells are plated at 50,000 cells per 100
.mu.L per well in DMEM with 5% FBS and 1% L-glutamine in a 96-well
black wall/clear bottom Costar plate, incubated in 5% CO.sub.2,
37.degree. C. incubator overnight. The Growth medium is removed,
and 100 .mu.L/well of 1-8 .mu.M Fluo-3, AM, Fluo-4 AM, Compounds
258, 294, 296, 298, 302, 304, 306 or 365 in Hanks and HEPES buffer
(HHBS) is added into the cells, incubated in 5% CO.sub.2,
37.degree. C. incubator for 1 hr. The cells are washed with 200
.mu.L HHBS buffer twice, and then replaced with 100 .mu.L HHBS.
Images are taken using fluorescence microscope (Olympus, IX 71)
with FITC filter at 20 ms exposure time. The indicators of the
invention remain substantially photostable and permit fluorescence
imaging of the cells.
[0232] Representative fluorescence images for the indicators Fluo-3
AM (where R.sup.2 and R.sup.5 are chloro) and Fluo-4 AM (where
R.sup.2 and R.sup.5 are fluoro) are provided in FIGS. 15 and 16,
respectively. The fluorescence image for cells loaded with Compound
365 is provided in FIG. 17. Compound 365 is loaded into cells much
faster than either Fluo-3 AM or Fluo-4 AM. In addition, Compound
365 is brighter than both Fluo-4 AM and Fluo-3 AM.
Example 30
Calcium Responses of the Fluorescent Indicators Measured Using a
Microplate Reader Equipped with an Automated Liquid Handling
System
[0233] Calcium flux assays are preferred methods in drug discovery
for screening G protein coupled receptors (GPCR). The fluorescent
indicators of the invention provide a homogeneous
fluorescence-based assay for detecting the intracellular calcium
mobilization. Cells expressing a GPCR of interest that signals
through calcium are pre-loaded with the indicator AM esters (such
as Fluo-3 AM, Fluo-4 AM, Compounds 258, 294, 296, 298, 302, 304,
306 and 365) which can cross cell membrane. Once inside the cell,
the lipophilic blocking groups are cleaved by non-specific cell
esterase, resulting in a negatively charged fluorescein dye that is
well-retained in cells, and its fluorescence is greatly enhanced
upon binding to calcium. When the sample cells are stimulated with
screening compounds, the receptor triggers a release of
intracellular calcium, which then greatly increases the
fluorescence of the intracellular indicators. The combination of
long wavelength fluorescence properties, high sensitivity, and
often a >100 times increase in fluorescence upon binding with
calcium make the disclosed indicators well-suited for measurement
of cellular calcium.
[0234] Specifically, CHO cells stably transfected with muscarinic
receptor 1 are plated at 60,000 cells per 100 .mu.l per well in F12
with 5% FBS and 1% L-glutamine in a 96-well black wall/clear bottom
Costar plate, incubated in 5% CO.sub.2, 37.degree. C. incubator
overnight. The growth medium is removed and the cells are incubated
with 100 .mu.L/well of 1-8 .mu.M Fluo-3 AM, Fluo-4 AM, and one of
Compounds 258, 294, 296, 298, 302, 304, 306 or 365 in Hanks and
HEPES buffer with 2.5 mM probenecid for 1 hour at room temperature.
Carbachol (50 .mu.l/well) is added by NOVOstar (BMG LabTech) or
FLIPR (Molecular Devices) to achieve the final desired
concentration. A representative comparison is shown in FIG. 18.
[0235] Compound 365 (in which substituents R.sup.1, R.sup.2,
R.sup.5 and R.sup.6 are all hydrogen) is loaded into cells much
faster than Fluo-3, AM (in which R.sup.2 and R.sup.5 are chloro)
and Fluo-4 AM (in which R.sup.2 and R.sup.5 are fluoro). In
addition, Compound 365 (Curve A) demonstrates 2 times the
fluorescence intensity of Fluo-4 AM (Curve B) and 4 times the
fluorescence intensity of Fluo-3 AM (Curve C).
[0236] Although the present invention has been shown and described
with reference to the foregoing operational principles and
preferred embodiments, it will be apparent to those skilled in the
art that various changes in form and detail may be made without
departing from the spirit and scope of the invention. The present
invention is intended to embrace all such alternatives,
modifications and variances that fall within the scope of the
appended claims.
* * * * *