U.S. patent application number 15/955332 was filed with the patent office on 2018-12-20 for fluorogenic ph-sensitive dyes and their methods of use.
The applicant listed for this patent is Life Technologies Corporation. Invention is credited to Daniel Beacham, Judith Berlier, Kyle Gee, Shih-Jung Huang, Michael Janes, Aleksey Rukavishnikov, Upinder Singh, Wenjun Zhou.
Application Number | 20180362766 15/955332 |
Document ID | / |
Family ID | 48014358 |
Filed Date | 2018-12-20 |
United States Patent
Application |
20180362766 |
Kind Code |
A1 |
Gee; Kyle ; et al. |
December 20, 2018 |
Fluorogenic pH-Sensitive Dyes and Their Methods of Use
Abstract
Disclosed herein are compounds, compositions, methods and kits
for detecting pH in samples using pH-sensitive fluorescent dyes.
The compounds disclosed herein are novel xanthene-derivative dyes
comprising an aniline moiety with one or more electron donating
groups, which dyes are for detecting pH in samples either in vitro
or in vivo. Also described herein are processes for preparing said
dyes for use in the disclosed compositions, methods and kits.
Inventors: |
Gee; Kyle; (Springfield,
OR) ; Singh; Upinder; (Eugene, OR) ;
Rukavishnikov; Aleksey; (Eugene, OR) ; Beacham;
Daniel; (Eugene, OR) ; Huang; Shih-Jung;
(Eugene, OR) ; Janes; Michael; (Eugene, OR)
; Zhou; Wenjun; (Eugene, OR) ; Berlier;
Judith; (Eugene, OR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Life Technologies Corporation |
Carlsbad |
CA |
US |
|
|
Family ID: |
48014358 |
Appl. No.: |
15/955332 |
Filed: |
April 17, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14402375 |
Nov 20, 2014 |
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PCT/US13/31535 |
Mar 14, 2013 |
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15955332 |
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61653333 |
May 30, 2012 |
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61653616 |
May 31, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/84 20130101;
C07D 311/90 20130101; G01N 33/582 20130101; A61K 49/0041 20130101;
G01N 33/5005 20130101; C09B 11/24 20130101 |
International
Class: |
C09B 11/24 20060101
C09B011/24; G01N 33/84 20060101 G01N033/84; C07D 311/90 20060101
C07D311/90; A61K 49/00 20060101 A61K049/00; G01N 33/58 20060101
G01N033/58; G01N 33/50 20060101 G01N033/50 |
Claims
1-4. (canceled)
5. A pH-sensitive fluorescent dye compound of structural formula
(I): ##STR00061## wherein R.sup.1 is methoxy; R.sup.2 and R.sup.6
are each H; R.sup.3 is --NR'R'', wherein R' and R'', which may be
the same or different, are each independently methyl or ethyl;
R.sup.4 is methyl or ethyl; and R.sup.5is methyl, ethyl,
carboxyalkyl, (CH.sub.2).sub.nCO(O)R, (CH.sub.2).sub.nC(O)R,
(CH.sub.2).sub.nC(O)NHR, (CH.sub.2).sub.nC(O)NRR.sup.c, wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group selected from carboxyl,
carboxylester, amide, maleimide, succinimidyl ester (SE),
sulfodichlorophenol (SDP) ester, sulfotetrafluorophenol (STP)
ester, tetrafluorophenol (TFP) ester, acetoxymethyl (AM) ester,
nitrilotriacetic acid (NTA), aminodextran, DIBO-amine, -L-R.sub.x,
or -L-S.sub.c, wherein L is a linker, R.sub.x is a reactive group,
and S.sub.c is a conjugated substance.
6-9. (canceled)
10. A pH-sensitive fluorescent dye compound of structural formula
(II): ##STR00062## wherein R.sup.1 is methoxy; R.sup.2 and R.sup.6
are each H; R.sup.3 is --NR'R'', wherein R' and R'', which may be
the same or different, are each independently methyl or ethyl;
R.sup.4 is methyl or ethyl; and R.sup.5is methyl, ethyl,
carboxyalkyl, (CH.sub.2).sub.nCO(O)R, (CH.sub.2).sub.nC(O)R,
(CH.sub.2).sub.nC(O)NHR, (CH.sub.2).sub.nC(O)NRR.sup.c, wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sup.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group selected from carboxyl,
carboxylester, amide, maleimide, succinimidyl ester (SE),
sulfodichlorophenol (SDP) ester, sulfotetrafluorophenol (STP)
ester, tetrafluorophenol (TFP) ester, acetoxymethyl (AM) ester,
nitrilotriacetic acid (NTA), aminodextran, DIBO-amine, -L-R.sub.x,
or -L-S.sub.c, wherein L is a linker, R.sub.x is a reactive group,
and S.sub.c is a conjugated substance.
11. A pH-sensitive dye compound selected from the group consisting
of: ##STR00063## ##STR00064## ##STR00065##
12. A composition for determining the pH of a sample, the
composition comprising: a) one or more of the pH-sensitive
fluorescent dye compounds according to claim 5; and b) a carrier,
wherein the one or more pH-sensitive fluorescent dye compounds are
present in an amount effective to detect the pH of the sample.
13. A composition for determining the pH of a sample, the
composition comprising: (a) one or more of the pH-sensitive
fluorescent dye compounds according to claim 5; and (b) an analyte,
wherein the one or more pH-sensitive fluorescent dye compounds are
present in an amount effective to detect the pH of the sample.
14. A method for determining the pH of a sample, the method
comprising: (a) contacting the sample with one or more of the
pH-sensitive fluorescent dye compounds according to claim 5, to
form a contacted sample; (b) incubating the contacted sample for an
appropriate amount of time to form an incubated sample; (c)
illuminating the incubated sample with an appropriate wavelength to
form an illuminated sample; and (d) detecting fluorescent emissions
from the illuminated sample; wherein the fluorescent emissions are
used to determine the pH of the sample.
15. A method for determining the pH of a sample, the method
comprising: (a) contacting the sample with one or more of the
compositions according to claim 12 to form a contacted sample; (b)
incubating the contacted sample for an appropriate amount of time
to form an incubated sample; (c) illuminating the incubated sample
with an appropriate wavelength to form an illuminated sample; and
(d) detecting fluorescent emissions from the illuminated sample;
wherein the fluorescent emissions are used to determine the pH of
the sample.
16. A method for monitoring the pH inside a live cell, the method
comprising: (a) contacting the cell with one or more of the
pH-sensitive fluorescent dye compounds according to claim 5 to form
a contacted cell; (b) incubating the contacted cell for a
sufficient amount of time for the one or more of the dye compounds
or compositions to enter the cell to form a labeled cell; (c)
illuminating the labeled cell with an appropriate wavelength to
form an illuminated cell; and (d) detecting fluorescent emissions
from the illuminated cell; wherein the fluorescent emissions are
used to monitor the pH inside the cell.
17. A method for monitoring the pH inside a live cell, the method
comprising: (a) contacting the cell with one or more of the
compositions according to claim 12 to form a contacted cell; (b)
incubating the contacted cell for a sufficient amount of time for
the dye compound or composition to enter the cell to form a labeled
cell; (c) illuminating the labeled cell with an appropriate
wavelength to form an illuminated cell; and (d) detecting
fluorescent emissions from the illuminated cell; wherein the
fluorescent emissions are used to monitor the pH inside the
cell.
18. A method for detecting phagocytosis of a carrier molecule in
solution, the method comprising: (a) conjugating the carrier
molecule to one or more of the pH-sensitive fluorescent dye
compounds according to claim 5 to form a carrier conjugate; (b)
contacting the carrier conjugate with a cell to form a contacted
cell; (c) incubating the contacted cell to form an incubated
solution; (d) illuminating the incubated solution to form an
illuminated solution; and (e) detecting fluorescent emissions from
the illuminated solution; wherein fluorescent emissions indicate
phagocytosis of the carrier molecule.
19. A method for detecting phagocytosis of a carrier molecule in
solution, the method comprising: (a) conjugating the carrier
molecule to one or more of the compositions according to claim 12
to form a carrier conjugate; (b) contacting the carrier conjugate
with a cell to form a contacted cell; (c) incubating the contacted
cell to form an incubated solution; (d) illuminating the incubated
solution to form an illuminated solution; and (e) detecting
fluorescent emissions from the illuminated solution; wherein
fluorescent emissions indicate phagocytosis of the carrier
molecule.
20. A method for detecting a pH related intracellular process, the
method comprising: (a) contacting one or more of the pH-sensitive
fluorescent dye compounds according to claim 5 with a cell to form
a contacted cell; (b) incubating the contacted cell to form an
incubated solution; (c) illuminating the incubated solution to form
an illuminated solution; and (d) detecting fluorescent emissions
from the illuminated solution; wherein increased fluorescent
emissions indicates activation of the intracellular process.
21. A method for detecting a pH related intracellular process, the
method comprising: (a) contacting one or more of the compositions
according to claim 12 with a cell to form a contacted cell; (b)
incubating the contacted cell to form an incubated solution; (c)
illuminating the incubated solution to form an illuminated
solution; and (d) detecting fluorescent emissions from the
illuminated solution; wherein increased fluorescent emissions
indicates activation of the intracellular process.
22. A method for identifying a target cell in a population of
cells, wherein the target cell is differentially labeled relative
to neighboring cells within the population, the method comprising:
(a) contacting one or more of the pH-sensitive dye compounds
according to claim 5 with the population of cells to form a
contacted cell population; (b) incubating the contacted cell
population for a period of time sufficient for the one or more of
the pH-sensitive dye compounds to enter the target cell, thereby
forming an incubated cell population; and (c) illuminating the
incubated cell population, wherein the target cell is identified by
a differential label relative to neighboring cells within the
population.
23. A method for identifying a target cell in a population of
cells, wherein the target cell is differentially labeled relative
to neighboring cells within the population, the method comprising:
(a) contacting one or more of the compositions according to claim
12 with the population of cells to form a contacted cell
population; (b) incubating the contacted cell population for a
period of time sufficient for the one or more of the compositions
to enter the target cell, thereby forming an incubated cell
population; and (c) illuminating the incubated cell population,
wherein the target cell is identified by a differential label
relative to neighboring cells within the population.
24. A method for diagnosing or detecting a disease in a subject,
the method comprising: (a) contacting a sample obtained from a
subject suspected of having the disease with one or more of the
pH-sensitive dye compounds according to claim 5 to form a contacted
sample; (b) incubating the contacted sample for an appropriate
amount of time to form an incubated sample; (c) illuminating the
incubated sample with an appropriate wavelength to form an
illuminated sample; and (d) detecting fluorescent emissions from
the illuminated sample; wherein the fluorescent emissions are used
to diagnose or detect the disease.
25. A method for diagnosing or detecting a disease in a subject,
the method comprising: (a) contacting a sample obtained from a
subject suspected of having the disease with one or more of the
compositions according to claim 12 to form a contacted sample; (b)
incubating the contacted sample for an appropriate amount of time
to form an incubated sample; (c) illuminating the incubated sample
with an appropriate wavelength to form an illuminated sample; and
(d) detecting fluorescent emissions from the illuminated sample;
wherein the fluorescent emissions are used to diagnose or detect
the disease.
26. The method according to claim 24, wherein the disease is
selected from cancer, immune-related diseases, oxidative
stress-related diseases and diseases associated with the central
nervous system.
27. A kit for determining the pH of a sample comprising: (a) one or
more of the pH-sensitive dye compounds according to claim 5; (b)
one or more containers; and optionally (c) instructions for
determining the pH of the sample.
28. A kit for determining the pH of a sample comprising: (a) one or
more of the compositions according to claim 12; (b) one or more
containers; and optionally (c) instructions for determining the pH
of the sample.
29-30. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. NonProvisional
Application Ser. No. 14/402,375, filed Nov. 20, 2014, which is a
National Stage Application of PCT/US2013/031535, filed Mar. 14,
2013, and claims the benefit of priority to U.S. Provisional
Application Ser. Nos. 61/653,333, filed May 30, 2012 and
61/653,616, filed May 31, 2012, which are herein incorporated by
reference in their entirety.
FIELD
[0002] Novel pH-sensitive fluorescent dyes and assays for use in a
variety of applications including monitoring of intracellular
processes are disclosed.
BACKGROUND
[0003] pH-sensitive fluorescent dyes employed in biological
research and medical diagnostics belong to two groups, each
distinguished by the origin of fluorescent responses to changes in
pH. The first group includes compounds having fluorescence
controlled by the ionization of phenolic hydroxyl groups in a
fluorophore. Examples include fluorescein, carboxyfluorescein,
Oregon Green.RTM., SNARF.RTM., SNAFL.RTM., and HPTS indicators.
[0004] U.S. Patent Publication No. 2006/0051874 describes
fluorescein-like structures incorporated into a fluorescent
detector for monitoring pH of the blood in bank storages. Because
the degree of ionization of these types of molecules increases upon
lowering the acidity of the environment, they become more
fluorescent as pH increases.
[0005] Fluorescent pH sensors of the second group include an amino
group (aliphatic or aromatic) as an indicator moiety along with a
reporter fluorescent dye moiety. When such a molecule absorbs a
photon creating an excited electronic state, the electron of the
amino group's unshared pair transfers to the orbital vacated by
excitation. Such an electron transfer, referred to as Photoinduced
Electron Transfer (PET) prevents the excited molecule from emission
transition, thus the fluorescence of the dye is quenched.
Protonation of the amino group changes the nature and energy of the
pair's orbital and stops the PET. As a result, the fluorescent
reporter moiety responds to a pH change. Because protonation of the
amino group cancels the quenching, the PET-based sensors become
more fluorescent as pH decreases.
[0006] Examples of PET-based pH sensors include LysoSensor.TM.
dyes, which contain a dimethylamino group as an indicator moiety
and CypHer.RTM. 5E dye which has an indolenine indicator group. One
disadvantage of these sensors is that the working range is shifted
to the acidic side because of the low pKa of the indicator amino
group.
[0007] A family of rhodamine-based pH sensors is described in PCT
Publication No. WO 2005/098437 (Smith et al.). The dyes have a
benzene ring substituted ortho to the xanthene moiety by --OH or
--SH (or their deprotonated forms), such that deprotonation to a
negatively charged state quenches the fluorescence and it is only
upon protonation of the negatively charged --O.sup.- or --S.sup.-
to a neutral state that the fluorescence is restored. Typically,
the pH at which this occurs is less than pH 6. WO 2005/098437
purports that the ionized state of the --OH or --SH group is
responsible for the pH response of the dye and that the strong
electron withdrawing properties of the tetramethylrhodamine moiety
in the dyes significantly decreases the pKa of the indicator group,
thus shifting the sensors' working range toward highly acidic pH
values. However, this limits the applicability of the dyes
described in WO 2005/098437 at a physiological pH (e.g., pH 6-7),
especially in biological systems. An additional disadvantage of
these dyes is that their pKa is not tunable. Furthermore, these
compounds have been found by us to be unstable in solution.
[0008] Accordingly, there is a need for additional pH sensitive
fluorescent dyes with improved properties, including in at least
some compounds the ability to detect pH changes in biological
systems. It is an object of the present invention to develop a
novel class of relatively stable fluorescent pH sensors that
fluoresce in the green portion of the UV/VIS spectrum, preferably
with a working range towards neutral and other biologically
relevant pH values that mitigate or remove the disadvantages of the
compounds known in art.
SUMMARY
[0009] Described herein are compounds, compositions, methods and
kits for detecting pH in samples using pH-sensitive fluorescent
dyes, which in one aspect, are characterized by the omission of a
hydroxyl or thiol group as required by WO 2005/098437. In another
aspect, the pH-sensitive fluorescent dyes as disclosed herein allow
for the detection of fluorescent responses to changes in pH in the
green portion of the UV/VIS spectrum. The current disclosure
provides a new family of pH-sensitive green fluorescent dyes,
having significant and unexpected advantages over existing
fluorescent pH-sensors in that the presence of a dialkylamino group
para to the alkoxy substituent results in physiological pKa values
and the pKa's of the dyes provided herein are also tunable.
Therefore, the pH-sensitive fluorescent dyes provided herein may be
modified to suit a particular application or condition to be
analyzed.
[0010] In certain embodiments, novel dye compounds are provided for
use as fluorescent pH sensors, the dye compounds having structural
formula (I):
##STR00001##
wherein
[0011] R.sup.1 is alkoxy or thioalkyl;
[0012] R.sup.2 and R.sup.6, which may be the same or different, are
each independently H, halogen, --OR.sup.a, --SR.sup.a,
--NR.sup.aR.sup.b, or an electron donating group;
[0013] R.sup.3 is --NR'R'', wherein R' and R'', which may be the
same or different, are each independently alkyl or substituted
alkyl;
[0014] R.sup.4 is selected from the group consisting of alkyl and
substituted alkyl;
[0015] R.sup.5 is selected from the group consisting of alkyl;
substituted alkyl; alkenyl; substituted alkenyl; acyl; aryl;
substituted aryl; carboxyalkyl; heteroaryl; substituted heteroaryl;
heterocyclyl; substituted heterocyclyl; alkylcarboxy;
alkylalkoxycarbonyl; alkylaminocarbonyl; alkylaryloxycarbonyl;
alkylheteroaryl; (CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c;wherein n is
an integer from 1 to 6, and R and R.sup.c, which may be the same or
different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group; -L-R.sub.x; and -L-S.sup.c,
wherein L is a linker, R.sub.x is a reactive group, and S.sub.c is
a conjugated substance;
[0016] R.sup.a is H, alkyl, or substituted alkyl; and
[0017] R.sup.b is alkyl or substituted alkyl.
[0018] In certain embodiments, R.sup.1-R.sup.6 are as follows:
[0019] R.sup.1 is alkoxy or thioalkyl;
[0020] R.sup.2 and R.sup.6, which may be the same or different, are
each independently H or halogen;
[0021] R.sup.3 is --NR'R'', wherein R' and R'', which may be the
same or different, are each independently alkyl;
[0022] R.sup.4 is alkyl; and
[0023] R.sup.5 is selected from the group consisting of alkyl;
substituted alkyl; alkenyl; substituted alkenyl; acyl; aryl;
substituted aryl; carboxyalkyl; heteroaryl; substituted heteroaryl;
heterocyclyl; substituted heterocyclyl; alkylcarboxy;
alkylalkoxycarbonyl; alkylaminocarbonyl; alkylaryloxycarbonyl;
alkylheteroaryl; (CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c;wherein n is
an integer from 1 to 6, and R and R.sup.c, which may be the same or
different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group; -L-R.sub.x; and -L-S.sub.c.
[0024] In certain embodiments, R.sup.1-R.sup.6 are as follows:
[0025] R.sup.1 is alkoxy or thioalkyl;
[0026] R.sup.2 and R.sup.6, which may be the same or different, are
each independently H, Cl or F;
[0027] R.sup.3 is --NR'R'', wherein R' and R'', which may be the
same or different, are each independently alkyl;
[0028] R.sup.4 is alkyl; and
[0029] R.sup.5 is selected from the group consisting of alkyl;
substituted alkyl; alkenyl; substituted alkenyl; acyl; aryl;
substituted aryl; carboxyalkyl; heteroaryl; substituted heteroaryl;
heterocyclyl; substituted heterocyclyl; alkylcarboxy;
alkylalkoxycarbonyl; alkylaminocarbonyl; alkylaryloxycarbonyl;
alkylheteroaryl; (CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c; wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group; -L-R.sub.x; and -L-S.sub.c.
[0030] In certain embodiments, R.sup.1-R.sup.6 are as follows:
[0031] R.sup.1 is alkoxy or thioalkyl;
[0032] R.sup.2 and R.sup.6 are each H;
[0033] R.sup.3 is --NR'R'', wherein R' and R'', which may be the
same or different, are each independently alkyl;
[0034] R.sup.4 is alkyl; and
[0035] R.sup.5 is selected from the group consisting of alkyl;
substituted alkyl; alkenyl; substituted alkenyl; acyl; aryl;
substituted aryl; carboxyalkyl; heteroaryl; substituted heteroaryl;
heterocyclyl; substituted heterocyclyl; alkylcarboxy;
alkylalkoxycarbonyl; alkylaminocarbonyl; alkylaryloxycarbonyl;
alkylheteroaryl; (CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c;wherein n is
an integer from 1 to 6, and R and R.sup.c, which may be the same or
different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group; -L-R.sub.x; and -L-S.sub.c,
[0036] In certain embodiments, R.sup.1-R.sup.6 are as follows:
[0037] R.sup.1 is methoxy;
[0038] R.sup.2 and R.sup.6 are each H;
[0039] R.sup.3 is --NR'R'', wherein R' and R'', which may be the
same or different, are each independently methyl or ethyl;
[0040] R.sup.4 is methyl or ethyl; and
[0041] R.sup.5 is methyl; ethyl; carboxyalkyl;
(CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c; wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group selected from carboxyl,
carboxylester, amide, maleimide, succinimidyl ester (SE),
sulfodichlorophenol (SDP) ester, sulfotetrafluorophenol (STP)
ester, tetrafluorophenol (TFP) ester, acetoxymethoxy (AM) ester,
nitrilotriacetic acid (NTA), aminodextran, DIBO-amine; -L-R.sub.x;
or -L-S.sub.c.
[0042] In certain embodiments, novel dye compounds are provided for
use as fluorescent pH sensors, the dye compounds having structural
formula (II):
##STR00002##
wherein
[0043] R.sup.1 is alkoxy or thioalkyl;
[0044] R.sup.2 and R.sup.6, which may be the same or different, are
each independently H, halogen, --OR.sup.a, --SR.sup.a,
--NR.sup.aR.sup.b, or an electron donating group;
[0045] R.sup.3 is --NR'R'', wherein R' and R'', which may be the
same or different, are each independently alkyl or substituted
alkyl;
[0046] R.sup.4 is selected from the group consisting of alkyl and
substituted alkyl;
[0047] R.sup.5 is selected from the group consisting of alkyl;
substituted alkyl; alkenyl; substituted alkenyl; acyl; aryl;
substituted aryl; carboxyalkyl; heteroaryl; substituted heteroaryl;
heterocyclyl; substituted heterocyclyl; alkylcarboxy;
alkylalkoxycarbonyl; alkylaminocarbonyl; alkylaryloxycarbonyl;
alkylheteroaryl; (CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c; wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group; -L-R.sub.x; and -L-S.sub.c,
wherein L is a linker, R.sub.x is a reactive group, and S.sub.c is
a conjugated substance;
[0048] R.sup.a is H, alkyl, or substituted alkyl; and
[0049] R.sup.b is alkyl or substituted alkyl.
[0050] In certain embodiments, R.sup.1-R.sup.6 are as follows:
[0051] R.sup.1 is alkoxy or thioalkyl;
[0052] R.sup.2 and R.sup.6, which may be the same or different, are
each independently H or halogen;
[0053] R.sup.3 is --NR'R'', wherein R' and R'', which may be the
same or different, are each independently alkyl;
[0054] R.sup.4 is alkyl; and
[0055] R.sup.5 is selected from the group consisting of alkyl;
substituted alkyl; alkenyl; substituted alkenyl; acyl; aryl;
substituted aryl; carboxyalkyl; heteroaryl; substituted heteroaryl;
heterocyclyl; substituted heterocyclyl; alkylcarboxy;
alkylalkoxycarbonyl; alkylaminocarbonyl; alkylaryloxycarbonyl;
alkylheteroaryl; (CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c; wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group; -L-R.sub.x; and -L-S.sub.c.
[0056] In certain embodiments, R.sup.1-R.sup.6 are as follows:
[0057] R.sup.1 is alkoxy or thioalkyl;
[0058] R.sup.2 and R.sup.6, which may be the same or different, are
each independently H, Cl or F;
[0059] R.sup.3 is --NR'R'', wherein R' and R'', which may be the
same or different, are each independently alkyl;
[0060] R.sup.4 is alkyl; and
[0061] R.sup.5 is selected from the group consisting of alkyl;
substituted alkyl; alkenyl; substituted alkenyl; acyl; aryl;
substituted aryl; carboxyalkyl; heteroaryl; substituted heteroaryl;
heterocyclyl; substituted heterocyclyl; alkylcarboxy;
alkylalkoxycarbonyl; alkylaminocarbonyl; alkylaryloxycarbonyl;
alkylheteroaryl; (CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c; wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group; -L-R.sub.x; and -L-S.sub.c.
[0062] In certain embodiments, R.sup.1-R.sup.6 are as follows:
[0063] R.sup.1 is alkoxy or thioalkyl;
[0064] R.sup.2 and R.sup.6 are each H;
[0065] R.sup.3 is --NR'R'', wherein R' and R'', which may be the
same or different, are each independently alkyl;
[0066] R.sup.4 is alkyl; and
[0067] R.sup.5 is selected from the group consisting of alkyl;
substituted alkyl; alkenyl; substituted alkenyl; acyl; aryl;
substituted aryl; carboxyalkyl; heteroaryl; substituted heteroaryl;
heterocyclyl; substituted heterocyclyl; alkylcarboxy;
alkylalkoxycarbonyl; alkylaminocarbonyl; alkylaryloxycarbonyl;
alkylheteroaryl; (CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c; wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group; -L-R.sub.x; and -L-S.sub.c.
[0068] In certain embodiments, R.sup.1-R.sup.6 are as follows:
[0069] R.sup.1 is methoxy;
[0070] R.sup.2 and R.sup.6 are each H;
[0071] R.sup.3 is --NR'R'', wherein R' and R'', which may be the
same or different, are each independently methyl or ethyl;
[0072] R.sup.4 is methyl or ethyl; and
[0073] R.sup.5 is methyl; ethyl; carboxyalkyl;
(CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c; wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group selected from carboxyl,
carboxylester, amide, maleimide, succinimidyl ester (SE),
sulfodichlorophenol (SDP) ester, sulfotetrafluorophenol (STP)
ester, tetrafluorophenol (TFP) ester, acetoxymethoxy (AM) ester,
nitrilotriacetic acid (NTA), aminodextran, DIBO-amine; -L-R.sub.x;
or -L-S.sub.c.
[0074] In certain embodiments, compositions are provided for
determining the pH of a sample, the compositions comprising:
[0075] a) one or more of the pH-sensitive fluorescent dye compounds
described herein; and
[0076] b) a carrier,
[0077] wherein the one or more pH-sensitive fluorescent dye
compounds are present in an amount effective to detect the pH of
the sample.
[0078] In certain embodiments, compositions are provided for
determining the pH of a sample, the compositions comprising:
[0079] (a) one or more of the pH-sensitive fluorescent dye
compounds described herein; and
[0080] (b) an analyte,
[0081] wherein the one or more pH-sensitive fluorescent dye
compounds are present in an amount effective to detect the pH of
the sample.
[0082] In certain embodiments, methods are provided for determining
the pH of a sample, the methods comprising:
[0083] (a) contacting the sample with one or more of the
pH-sensitive fluorescent dye compounds described herein to form a
contacted sample;
[0084] (b) incubating the contacted sample for an appropriate
amount of time to form an incubated sample;
[0085] (c) illuminating the incubated sample with an appropriate
wavelength to form an illuminated sample; and
[0086] (d) detecting fluorescent emissions from the illuminated
sample;
[0087] wherein the fluorescent emissions are used to determine the
pH of the sample.
[0088] In certain embodiments, methods are provided for determining
the pH of a sample, the methods comprising:
[0089] (a) contacting the sample with one or more of the
compositions described herein to form a contacted sample;
[0090] (b) incubating the contacted sample for an appropriate
amount of time to form an incubated sample;
[0091] (c) illuminating the incubated sample with an appropriate
wavelength to form an illuminated sample; and
[0092] (d) detecting fluorescent emissions from the illuminated
sample;
[0093] wherein the fluorescent emissions are used to determine the
pH of the sample.
[0094] In certain embodiments, methods are provided for monitoring
the pH inside a live cell, the methods comprising:
[0095] (a) contacting the cell with one or more of the pH-sensitive
fluorescent dye compounds described herein to form a contacted
cell;
[0096] (b) incubating the contacted cell for a sufficient amount of
time for the one or more pH-sensitive fluorescent dye compounds to
enter the cell to form a labeled cell;
[0097] (c) illuminating the labeled cell with an appropriate
wavelength to form an illuminated cell; and
[0098] (d) detecting fluorescent emissions from the illuminated
cell;
[0099] wherein the fluorescent emissions are used to monitor the pH
inside the cell.
[0100] In certain embodiments, methods are provided for monitoring
the pH inside a live cell, the methods comprising:
[0101] (a) contacting the cell with one or more of the compositions
described herein to form a contacted cell;
[0102] (b) incubating the contacted cell for a sufficient amount of
time for the one or more compositions to enter the cell to form a
labeled cell;
[0103] (c) illuminating the labeled cell with an appropriate
wavelength to form an illuminated cell; and
[0104] (d) detecting fluorescent emissions from the illuminated
cell;
[0105] wherein the fluorescent emissions are used to monitor the pH
inside the cell.
[0106] In certain embodiments, methods are provided for detecting
phagocytosis of a carrier molecule in solution, the methods
comprising:
[0107] (a) conjugating the carrier molecule to one or more of the
pH-sensitive fluorescent dye compounds described herein to form a
carrier-dye conjugate;
[0108] (b) contacting the carrier-dye conjugate with a cell to form
a contacted cell;
[0109] (c) incubating the contacted cell to form an incubated
solution;
[0110] (d) illuminating the incubated solution to form an
illuminated solution; and
[0111] (e) detecting fluorescent emissions from the illuminated
solution;
[0112] wherein fluorescent emissions indicate phagocytosis of the
carrier molecule.
[0113] In certain embodiments, methods are provided for detecting
phagocytosis of a carrier molecule in solution, the methods
comprising:
[0114] (a) conjugating the carrier molecule to one or more of the
compositions described herein to form a carrier-dye conjugate;
[0115] (b) contacting the carrier-dye conjugate with a cell to form
a contacted cell;
[0116] (c) incubating the contacted cell to form an incubated
solution;
[0117] (d) illuminating the incubated solution to form an
illuminated solution; and
[0118] (e) detecting fluorescent emissions from the illuminated
solution;
[0119] wherein fluorescent emissions indicate phagocytosis of the
carrier molecule.
[0120] In certain embodiments, methods are provided for detecting a
pH related intracellular process, the methods comprising:
[0121] (a) contacting any one of the pH-sensitive fluorescent dye
compounds described herein with a cell to form a contacted
cell;
[0122] (b) incubating the contacted cell to form an incubated
solution;
[0123] (c) illuminating the incubated solution to form an
illuminated solution; and
[0124] (d) detecting fluorescent emissions from the illuminated
solution;
[0125] wherein increased fluorescent emissions indicates activation
of the intracellular process.
[0126] In certain embodiments, methods are provided for detecting a
pH related intracellular process, the methods comprising:
[0127] (a) contacting any one of the compositions described herein
with a cell to form a contacted cell;
[0128] (b) incubating the contacted cell to form an incubated
solution;
[0129] (c) illuminating the incubated solution to form an
illuminated solution; and
[0130] (d) detecting fluorescent emissions from the illuminated
solution;
[0131] wherein increased fluorescent emissions indicates activation
of the intracellular process.
[0132] In certain embodiments, methods are provided for identifying
a target cell in a population of cells, wherein the target cell is
differentially labeled relative to neighboring cells within the
population, the methods comprising;
[0133] (a) contacting one or more of the pH-sensitive dye compounds
disclosed herein with the population of cells to form a contacted
cell population;
[0134] (b) incubating the contacted cell population for a period of
time sufficient for the one or more of the pH-sensitive dye
compounds to enter the target cell, thereby forming an incubated
cell population; and
[0135] (c) illuminating the incubated cell population, wherein the
target cell is identified by a differential label relative to
neighboring cells within the population.
[0136] In certain embodiments, methods are provided for identifying
a target cell in a population of cells, wherein the target cell is
differentially labeled relative to neighboring cells within the
population, the methods comprising;
[0137] (a) contacting one or more of the compositions disclosed
herein with the population of cells to form a contacted cell
population;
[0138] (b) incubating the contacted cell population for a period of
time sufficient for the one or more of the compositions to enter
the target cell, thereby forming an incubated cell population;
and
[0139] (c) illuminating the incubated cell population, wherein the
target cell is identified by a differential label relative to
neighboring cells within the population.
[0140] In certain embodiments, methods are provided for diagnosing
or detecting a disease in a subject, the method comprising:
[0141] (a) contacting a sample obtained from a subject suspected of
having the disease with one or more of the pH-sensitive dye
compounds disclosed herein to form a contacted sample;
[0142] (b) incubating the contacted sample for an appropriate
amount of time to form an incubated sample;
[0143] (c) illuminating the incubated sample with an appropriate
wavelength to form an illuminated sample; and
[0144] (d) detecting fluorescent emissions from the illuminated
sample;
[0145] wherein the fluorescent emissions are used to diagnose or
detect the disease.
[0146] In certain embodiments, methods are provided for diagnosing
or detecting a disease in a subject, the methods comprising:
[0147] (a) contacting a sample obtained from a subject suspected of
having the disease with one or more of the compositions disclosed
herein to form a contacted sample;
[0148] (b) incubating the contacted sample for an appropriate
amount of time to form an incubated sample;
[0149] (c) illuminating the incubated sample with an appropriate
wavelength to form an illuminated sample; and
[0150] (d) detecting fluorescent emissions from the illuminated
sample;
[0151] wherein the fluorescent emissions are used to diagnose or
detect the disease.
[0152] In certain embodiments, kits are provided for determining
the pH of a sample comprising:
[0153] (a) one or more of the pH-sensitive fluorescent dye
compounds described herein;
[0154] (b) one or more containers; and optionally
[0155] (c) instructions for determining the pH of the sample.
[0156] In certain embodiments, kits are provided for determining
the pH of a sample comprising:
[0157] (a) one or more of the compositions described herein;
[0158] (b) one or more containers; and optionally
[0159] (c) instructions for determining the pH of the sample.
[0160] In certain embodiments processes are provided for
synthesizing a compound of structural formula (I):
##STR00003##
[0161] the process comprising: [0162] a) contacting a compound of
structural formula (VI):
[0162] ##STR00004## [0163] with a compound of structural formula
(IV):
[0163] ##STR00005## [0164] to form a compound of structural formula
(VII):
[0164] ##STR00006## [0165] b) de-allylating the compound of
structural formula (VII), when R.sup.7, R.sup.8, R.sup.9 and
R.sup.10 are each allyll, to form a compound of structural formula
(I),
[0166] wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6
are as previously defined herein.
[0167] In certain embodiments, processes are provided of
synthesizing a compound of structural formula (II):
##STR00007##
[0168] the process comprising: [0169] a) contacting a compound of
structural formula (III):
[0169] ##STR00008## [0170] with a compound of structural formula
(IV):
[0170] ##STR00009## [0171] to form a compound of structural formula
(V):
[0171] ##STR00010## [0172] b) de-allylating the compound of
structural formula (V), when R.sup.7 and R.sup.8 are each allyl, to
form a compound of structural formula (II),
[0173] wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5 and
R.sup.6 are as previously defined herein.
[0174] Other embodiments and illustrative aspects, features and
advantages of the present invention will become apparent from the
following detailed description. It should be understood, however,
that the detailed description and the specific examples, while
indicating preferred embodiments, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the present invention will become apparent to those
skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE FIGURES
[0175] Although the following figures depict various examples of
the invention, the invention is not limited to the examples
depicted in the figures.
[0176] FIG. 1 is a schematic representation of the pH values for
intracellular compartments and organelles.
[0177] FIG. 2 describes cellular uptake of pH-sensitive fluorescent
dye compounds. Panel A is a schematic representation of cellular
uptake of pH-sensitive fluorescent dye-conjugated compounds
including dye-conjugated bacteria for monitoring phagocytosis and
dye-conjugated particles for monitoring endocytosis, using the
compounds and methods according to embodiments disclosed herein.
Panel B is a fluorescence micrograph showing cells that have taken
up a pH-sensitive fluorescent dye-conjugated compound according to
certain embodiments of the present teachings.
[0178] FIG. 3 graphically shows the correlation of pH with
fluorescence for Compound (1). Panel A shows the fluorescence
intensity of Compound (1) as a function of pH. Panel B shows the
pKa of Compound (1).
[0179] FIG. 4 graphically shows the correlation of pH with
fluorescence for Compound (2). Panel A shows the fluorescence
intensity of Compound (2) as a function of pH. Panel B shows the
pKa of Compound (2).
[0180] FIG. 5 graphically shows the correlation of pH with
fluorescence for Compound (3). Panel A shows the fluorescence
intensity of Compound (3) as a function of pH. Panel B shows the
pKa of Compound (3).
[0181] FIG. 6 shows a micrograph of phagocytic uptake of
pH-sensitive fluorescent dye-conjugated E. coli, Staphylococcus,
and zymosan (yeast) bioparticles of RAW cells.
[0182] FIG. 7 graphically shows a dose response curve of
cytochalasin D inhibition of Compound (1)-conjugated bioparticles
internalization in a high-throughput microplate-based assay using
cultured macrophage "MMM" cells.
[0183] FIGS. 8A and 8B show fluorescent pH sensor dye uptake. FIG.
8A shows a series of pH curves for E. coli bioparticles labeled
with Compound (2), Compound (12) and Compound (28), emphasizing
their relative responses to acidification in vitro. FIG. 8B shows a
false-color fluorescence range for E. coli labeled with Compound
(2), Compound (12) or Compound (28) and resuspended in solutions of
varying pH (pH 4-pH 8.5).
[0184] FIG. 9 shows a graphic representation of the relative
fluorescence increase with acidification of 10,000 MW dextran
labeled with Compound (22).
[0185] FIG. 10 shows a graphic representation of the pH response
(relative fluorescence vs. pH) of a goat-anti-mouse (GAM) antibody
labeled with an amine-reactive (maleimide) Compound (24). The
triangle line is Compound (24)-GAM MR 10 DOS 2 and the square line
is Compound (24)-GAM MR 20 DOS 1.8.
[0186] FIG. 11 shows a graphic representation of a pH response of a
Compound (24)-labeled goat-anti-human (GAH) Fab fragment: the solid
line is pH=10, the diamond line is pH=8, the triangle line is pH=6,
and the square line is pH=4.
[0187] FIG. 12 shows internalization of EGF-conjugated to Compound
(24). The left panel shows cells pretreated with EGF and the right
panel shows cells treated with dye-conjugated EGF.
[0188] FIG. 13 shows internalization of Compound (33).
DETAILED DESCRIPTION
[0189] A family of rhodamine-based pH sensors (Smith, et.al; PCT
Publication No. WO 2005/098437, herein incorporated by reference in
its entirety) displays pH-dependency based on the ionization state
of the X substituent (see Scheme I below), namely a hydroxyl or
thiol group, such that deprotonation of the --OH or --SH group to a
negatively charged state quenches the fluorescence. It is only upon
protonation of the negatively charged --O or --S that the
fluorescence is restored as illustrated in Scheme 1:
##STR00011##
[0190] However, we have unexpectedly found that alkoxy
substitutions at the corresponding X position are still capable of
demonstrating a modulated fluorescence in response to a change in
pH. Thus, while not wishing to be bound by a theory, we postulate
that it is the protonation of the nitrogen at the 4 position on the
aryl ring that modulates fluorescence. In any event, the present
invention is predicated on the surprising discovery that the
hitherto indispensable hydroxy or thiol group X may be dispensed
with provided that a substituent nitrogen is retained.
Advantageously, the addition of a dialkylamino group at the R.sup.3
position resulted in a physiological pKa.
[0191] In addition, the pH-sensitive fluorescent dyes provided
herein fluoresce in the green portion of the UV/VIS spectrum and
have different chemical behavior as compared to other pH-sensitive
dyes. It was surprisingly discovered that: 1) the electron
withdrawing power of the xanthene moiety is significantly stronger
in the pH-sensitive fluorescent dyes provided herein, 2) the
electron withdrawing power of the aniline moiety may be modulated
by adding various electron donating groups to the benzene ring, and
3) the addition of electron donating groups, such as dialkylamino
groups at the R.sup.3 position and/or halogen at positions R.sup.2
and/or R.sup.6, may be used to tune the pKa of the pH-sensitive
fluorescent dyes to be at or near physiological pH. Furthermore, by
altering the electron donating groups at positions R.sup.1-R.sup.6,
the pKa of the pH-sensitive fluorescent dye may be modulated to
suit a particular need.
[0192] Further, we have found that the dyes described by Smith et
al. are not stable in solution, most likely as a result of aerobic
oxidation (e.g., oxidation by exposure to ambient air). In
addition, strong electron withdrawing properties of the reporter
tetramethylrhodamine moiety of the dyes described in Smith et al
(supra) significantly decrease the pKa of the indicator group at
the X position of Scheme 1, thus shifting the sensors' working
range towards acidic pH values.
[0193] In certain embodiments, therefore, the present invention
provides pH-sensitive fluorescent dyes having an aniline moiety (of
which the amino group may be substituted or modified as disclosed
herein) wherein the benzene ring of the aniline moiety is free from
hydroxy and thiol substituents ortho to the fluorophore or, in
certain embodiments, free from hydroxy and thiol substituents at
all positions. In particular, these pH-sensitive fluorescent dye
compounds may have, in place of the hydroxy or thiol substituent
required by Smith et al. (supra), a moiety wherein the oxygen or
sulfur of the hydroxy or thiol group has been incorporated into an
ether or thioether linkage, for example as part of an alkoxy group
or furan moiety, or their sulfur analogs. Viewed alternatively, the
pH-sensitive fluorescent dye compounds provided herein which retain
the oxygen or sulfur in etherified form, are pH-sensitive
fluorescent dye compounds which provide increased electronic
density of the molecule through strategic introduction of electron
donating groups (EDG) to the benzene ring resulting in an electron
rich aniline moiety, thereby moving the pKa closer to a
physiological range (e.g., pH 6-8). Accordingly, the benzene ring
may be substituted one or more times (e.g., 1, 2, 3 or 4 times) by
an electron donating group, the electron donating groups being the
same or different. In certain embodiments, the pH-sensitive
fluorescent dye compounds provided herein have the etherified O or
S replaced by another electron donating group. Irrespective of
whether the etherified O or S is replaced by another EDG,
supplementary electron donating groups may be provided on the
benzene ring to further modulate the pKa. In certain embodiments,
the pH-sensitive fluorescent dye compounds provided herein may
comprise two electron donating groups in total on the benzene ring,
in particular two electron donating groups of the type having a
lone pair of electrons available immediately next to the benzene
ring (e.g. alkoxy or dialkylamino, optionally substituted as
described herein). Additionally, modifications may be made to
modulate the quantum yield of the pH-sensitive fluorescent dye
compounds of the present disclosure. Thus, the pH-sensitive
fluorescent dye compounds as disclosed herein have significant
advantages over other PET-based dyes and advantageously provide the
benefit of having improved stability and/or a pKa in the range of
physiological applications. In addition, the pKa of the
pH-sensitive fluorescent dye compounds provided herein may be tuned
to particular pKa's depending on the electron donating group(s)
used on the benzene ring (e.g., on the aniline moiety). In
addition, the pH-sensitive fluorescent dye compounds fluoresce in
the green portion of the UV/VIS spectrum.
[0194] In certain embodiments, the pKa of the amino group on the
aniline moiety is modulated by modifying the amino group into a
more basic nitrogen functional group. This feature may usefully be
adopted as an alternative to replacing the omitted hydroxy or thiol
group with another electron donating group; alternatively,
modification of the amino group into a more basic group may be
combined with substitution of the benzene ring by at least one
electron donating group other than a hydroxy or thiol group.
[0195] Also included herein are embodiments in which the pKa of the
aniline moiety is modulated by modifying the amino group at
position R.sup.3 to --NR'R'', wherein R' and R'', which may be the
same or different, are each independently alkyl or substituted
alkyl (e.g., a dialkylamino group) in order to bring the pKa closer
to the physiological range. In certain embodiments, the
dialkylamino group is dimethylamino (e.g., --N(CH.sub.3).sub.2). In
certain embodiments, the dialkylamino group is diethylamino (e.g.,
--N(CH.sub.2CH.sub.3).sub.2).
[0196] Particular features targeted by the compounds, compositions,
methods and kits described herein include one or more of: (1)
dissociation constant pKa within the physiological range; (2)
greater stability (considered to be towards oxidation); (3)
flexible synthetic methods allowing introduction of pKa-modulating
substituents along with reactive groups; 4) tuneability of the pKa;
and 5) excitation and emission in the green portion of the UV/VIS
spectrum, which is the most common portion of the UV/VIS spectrum
in terms of usage in biological systems and assays. As described
previously herein, the pH-sensitive fluorescent dye compounds of
the present invention, have enhanced stability by dispensing with
the previously indispensable hydroxy or thiol at position X of
Scheme I, and the hydroxy or thiol group is advantageously replaced
by another electron donating group at the same position, such as an
alkoxy or thioalkyl, preferably an alkoxy. Additionally or
alternatively, such other electron donating groups may be
substituted at other positions on the benzene ring. In certain
embodiments, such other electron donating groups include
dialkylamino groups, wherein each alkyl group, which may be the
same or different, are each independently alkyl or substituted
alkyl. In certain embodiments, such other electron donating groups
include halogen, preferably, chloro and fluoro.
[0197] In order to achieve these goals a novel class of the
pH-sensitive compounds was designed, synthesized and tested in
analytical and biological applications. The structures of the
preferred dye compounds include structural formulae (I) and
(II).
[0198] Substituents on an aromatic ring may either donate electrons
to the aromatic ring or withdraw electrons from the aromatic ring
as compared to a hydrogen atom attached to the ring. Substituents
may therefore be classified as electron donating groups or electron
withdrawing groups.
[0199] Many electron donating groups have lone pairs of electrons
on the atom adjacent to the pi (.pi.) system of the aromatic ring.
Alkyl, aromatic and alkenyl groups are examples of electron
donating groups. Electron withdrawing groups are generally those
where the atom adjacent to the aromatic pi system has a formal
positive charge or a .delta. positive charge (for example, due to
being connected to more electronegative atoms). Electron donating
groups have an activating effect with respect to further
substitution of the ring system and tend to direct further
substitution ortho/para, while electron withdrawing groups are
deactivating and tend to direct further substitution meta. The
exception to this is halogen substituents, which, while overall
electron withdrawing and deactivating, tend to direct further
substitution ortho/para due to resonance (lone pair) donation.
Table 1 indicates the relative electron withdrawing and donating
character of some common substituents.
TABLE-US-00001 TABLE 1 Relative electron donating/withdrawing
character of different aromatic ring substituents, ranked from most
electron donating to most electron withdrawing Character
Activating/ Substituent relative to H deactivating Directing
--O.sup.- electron strongly activate ortho/para donating --NR.sub.2
electron strongly activate ortho/para donating --NH.sub.2 electron
strongly activate ortho/para donating --OH electron strongly
activate ortho/para donating --OR electron strongly activate
ortho/para donating --NHC(O)R electron moderately activate
ortho/para donating --OC(O)R electron moderately activate
ortho/para donating --R electron weakly activate ortho/para
donating --Ph electron weakly activate ortho/para donating
--CH.dbd.CR.sub.2 electron weakly activate ortho/para donating --H
reference neutral ortho/para --X (X = halo) electron weakly
deactivate ortho/para withdrawing --C(O)H electron moderately
deactivate meta withdrawing --C(O)R electron moderately deactivate
meta withdrawing --C(O)OR electron moderately deactivate meta
withdrawing --C(O)OH electron moderately deactivate meta
withdrawing --CF.sub.3 electron strongly deactivate meta
withdrawing --CN electron strongly deactivate meta withdrawing
--S(O).sub.2OH electron strongly deactivate meta withdrawing
--N.sup.(+)H.sub.3 electron strongly deactivate meta withdrawing
--N.sup.(+)R.sub.3 electron strongly deactivate meta withdrawing
--N.sup.(+)(O)O.sup.(-) electron strongly deactivate meta
withdrawing
[0200] The symbol R in Table 1 in particular stands for alkyl,
though it may be substituted in any reasonable way which does not
transform the electronic effect of alkyl from donating to
withdrawing or vice-versa. This specification further describes
suitable electron donating groups for phenylic substitution of the
aniline or aniline-like ring described in the specification.
[0201] Definitions:
[0202] To more clearly and concisely describe and point out the
subject matter of the present disclosure, the following definitions
are provided for specific terms, which are used in the following
description and appended claims. Throughout the specification,
exemplification of specific terms should be considered as
non-limiting examples.
[0203] Before describing the present teachings in detail, it is to
be understood that the disclosure is not limited to specific
compositions or process steps, as such may vary. It should be noted
that, as used in this specification and the appended claims, the
singular form "a", "an" and "the" include plural references unless
the context clearly dictates otherwise. Thus, for example,
reference to "a fluorescent pH sensitive dye" includes a plurality
of dyes and reference to "a cell" includes a plurality of cells and
the like. The phrase "and/or" denotes a shorthand way of indicating
that the specific combination is contemplated in combination and
separately, in the alternative. For illustration purposes, but not
as a limitation, "X and/or Y" can mean "X" or "Y" or "X" and
"Y".
[0204] It will be appreciated that there is an implied "about"
prior to the temperatures, concentrations, times, etc. discussed in
the present disclosure, such that slight and insubstantial
deviations are within the scope of the present teachings herein.
Also, the use of "comprise", "comprises", "comprising", "contain",
"contains", "containing", "include", "includes", and "including"
are not intended to be limiting. It is to be understood that both
the foregoing general description and detailed description are
exemplary and explanatory only and are not restrictive of the
teachings.
[0205] Unless specifically noted in the above specification,
embodiments in the above specification that recite "comprising"
various components are also contemplated as "consisting of" or
"consisting essentially of" the recited components; embodiments in
the specification that recite "consisting of" various components
are also contemplated as "comprising" or "consisting essentially
of" the recited components; and embodiments in the specification
that recite "consisting essentially of" various components are also
contemplated as "consisting of" or "comprising" the recited
components (this interchangeability does not apply to the use of
these terms in the claims).
[0206] The term "or combinations thereof" as used herein refers to
all permutations and combinations of the listed terms preceding the
term. For example, "A, B, C, or combinations thereof" is intended
to include at least one of: A, B, C, AB, AC, BC, or ABC, and if
order is important in a particular context, also BA, CA, CB, ACB,
CBA, BCA, BAC, or CAB. Continuing with this example, expressly
included are combinations that contain repeats of one or more item
or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and
so forth. The skilled artisan will understand that typically there
is no limit on the number of items or terms in any combination,
unless otherwise apparent from the context.
[0207] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the desired
subject matter in any way. All literature cited in the
specification, including but not limited to, patents, patent
applications, articles, books and treatises are expressly
incorporated by reference in their entirety for any purpose. In the
event that any of the incorporated literature contradicts any term
defined in this specification, this specification controls. While
the present teachings are described in conjunction with various
embodiments, it is not intended that the present teachings be
limited to such embodiments. On the contrary, the present teachings
encompass various alternatives, modifications, and equivalents, as
will be appreciated by those of skill in the art.
[0208] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosure is related. The
following terms are defined for purposes of the teachings as
described herein.
[0209] "Alkyl" refers to monovalent saturated aliphatic hydrocarbyl
groups having from 1 to 10 carbon atoms and preferably 1 to 6
carbon atoms, e.g. 1, 2, 3, 4, 5 or 6 carbon atoms. This term
includes, by way of example, linear and branched hydrocarbyl groups
such as methyl (CH.sub.3--), ethyl (CH.sub.3CH.sub.2--), n-propyl
(CH.sub.3CH.sub.2CH.sub.2--), isopropyl ((CH.sub.3).sub.2CH--),
n-butyl (CH.sub.3CH.sub.2CH.sub.2CH.sub.2--), isobutyl
((CH.sub.3).sub.2CHCH.sub.2--), sec-butyl
((CH.sub.3)(CH.sub.3CH.sub.2)CH--), t-butyl ((CH.sub.3).sub.3C--),
n-pentyl (CH.sub.3CH.sub.2CH.sub.2CH.sub.2CH.sub.2--), and
neopentyl ((CH.sub.3).sub.3CCH2--).
[0210] "Substituted alkyl" refers to an alkyl group having from 1
to 5, preferably 1 to 3, or more preferably 1 to 2 substituents
selected from the group consisting of alkoxy, substituted alkoxy,
acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl,
aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino,
aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy,
aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy,
substituted aryloxy, arylthio, substituted arylthio, carboxyl,
carboxylalkyl, carboxyl ester, (carboxyl ester)amino, (carboxyl
ester)oxy, cyano, cycloalkyl, substituted cycloalkyl,
cycloalkyloxy, substituted cycloalkyloxy, cycloalkylthio,
substituted cycloalkylthio, cycloalkenyl, substituted cycloalkenyl,
cycloalkenyloxy, substituted cycloalkenyloxy, cycloalkenylthio,
substituted cycloalkenylthio, guanidino, substituted guanidino,
halo, hydroxy, heteroaryl, substituted heteroaryl, heteroaryloxy,
substituted heteroaryloxy, heteroarylthio, substituted
heteroarylthio, heterocyclic, substituted heterocyclic,
heterocyclyloxy, substituted heterocyclyloxy, heterocyclylthio,
substituted heterocyclylthio, nitro, SO.sub.3H, substituted
sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthio, and substituted
alkylthio, wherein said substituents are defined herein. Particular
substituted alkyl groups comprise a reactive group for direct or
indirect linking to a carrier molecule or solid support, for
example, but not limited to, alkyl substituted by carboxyl or a
carboxyl ester (e.g. an activated ester such as an
N-hydroxysuccinimide ester) and alkyl substituted by aminocarbonyl
--CONHR where R is an organic moiety as defined below with
reference to the term "aminocarbonyl", e.g. a C.sub.1-C.sub.10
(e.g. C.sub.1-C.sub.6) alkyl terminally substituted by a reactive
group (R.sub.x) including, but not limited to, carboxyl,
carboxylester, maleimide, succinimidyl ester (SE),
sulfodichlorophenol (SDP) ester, sulfotetrafluorophenol (STP)
ester, tetrafluorophenol (TFP) ester, acetoxymethoxy (AM) ester,
nitrilotriacetic acid (NTA), aminodextran, and DIBO-amine
[0211] "Alkoxy" refers to the group --O-alkyl wherein alkyl is
defined herein. Alkoxy includes, by way of example, methoxy,
ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, sec-butoxy, and
n-pentoxy.
[0212] "Substituted alkoxy" refers to the group --O-(substituted
alkyl), wherein substituted alkyl is defined herein.
[0213] "Acyl" refers to the groups H--C(O)--, alkyl-C(O)--,
substituted alkyl-C(O)--, alkenyl-C(O)--, substituted
alkenyl-C(O)--, alkynyl-C(O)--, substituted alkynyl-C(O)--,
cycloalkyl-C(O)--, substituted cycloalkyl-C(O)--,
cycloalkenyl-C(O)--, substituted cycloalkenyl-C(O)--, aryl-C(O)--,
substituted aryl-C(O)--, heteroaryl-C(O)--, substituted
heteroaryl-C(O)--, heterocyclic-C(O)--, and substituted
heterocyclic-C(O)--, wherein alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
heterocyclic and substituted heterocyclic are as defined herein.
Acyl includes the "acetyl" group CH.sub.3C(O)--.
[0214] "Acylamino" refers to the groups --NRC(O)alkyl,
--NRC(O)substituted alkyl, --NRC(O)cycloalkyl, --NRC(O)substituted
cycloalkyl, --NRC(O)cycloalkenyl, --NRC(O)substituted cycloalkenyl,
--NRC(O)alkenyl, --NRC(O)substituted alkenyl, --NRC(O)alkynyl,
--NRC(O)substituted alkynyl, --NRC(O)aryl, --NRC(O)substituted
aryl, --NRC(O)heteroaryl, --NRC(O)substituted heteroaryl,
--NRC(O)heterocyclic, and --NRC(O)substituted heterocyclic, wherein
R is hydrogen or alkyl and wherein alkyl, substituted alkyl,
alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic and substituted heterocyclic are as
defined herein.
[0215] "Acyloxy" refers to the groups alkyl-C(O)O--, substituted
alkyl-C(O)O--, alkenyl-C(O)O--, substituted alkenyl-C(O)O--,
alkynyl-C(O)O--, substituted alkynyl-C(O)O--, aryl-C(O)O--,
substituted aryl-C(O)O--, cycloalkyl-C(O)O--, substituted
cycloalkyl-C(O)O--, cycloalkenyl-C(O)O--, substituted
cycloalkenyl-C(O)O--, heteroaryl-C(O)O--, substituted
heteroaryl-C(O)O--, heterocyclic-C(O)O--, and substituted
heterocyclic-C(O)O--, wherein alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
heterocyclic, and substituted heterocyclic are as defined
herein.
[0216] "Amino" refers to the group --NH.sub.2.
[0217] "Substituted amino" refers to the group --NR'R'' where R'
and R'' are independently selected from the group consisting of
hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
heteroaryl, substituted heteroaryl, heterocyclic, substituted
heterocyclic, --SO.sub.2-alkyl, --SO.sub.2-- substituted alkyl,
--SO.sub.2-alkenyl, --SO.sub.2-substituted alkenyl,
--SO.sub.2-cycloalkyl, --SO.sub.2-substituted cylcoalkyl,
--SO.sub.2-cycloalkenyl, --SO.sub.2-substituted cylcoalkenyl,
--SO.sub.2-aryl, --SO.sub.2-substituted aryl,
--SO.sub.2-heteroaryl, --SO.sub.2-- substituted heteroaryl,
--SO.sub.2-heterocyclic, and --SO.sub.2-substituted heterocyclic
and wherein R' and R'' are optionally joined, together with the
nitrogen bound thereto to form a heterocyclic or substituted
heterocyclic group, provided that R' and R'' are both not hydrogen,
and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic are as defined herein. When R' is hydrogen and R'' is
alkyl, the substituted amino group is sometimes referred to herein
as alkylamino When R' and R'' are alkyl, the substituted amino
group is sometimes referred to herein as dialkylamino When
referring to a monosubstituted amino, it is meant that either R' or
R'' is hydrogen but not both. When referring to a disubstituted
amino, it is meant that neither R' nor R'' are hydrogen.
[0218] "Aminocarbonyl" refers to the group --C(O)NR'R'' where R'
and R'' are independently selected from the group consisting of
hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic, and where R' and R'' are optionally joined together
with the nitrogen bound thereto to form a heterocyclic or
substituted heterocyclic group, and wherein alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic and substituted heterocyclic are as
defined herein.
[0219] "Aminothiocarbonyl" refers to the group --C(S)NR'R'' where
R' and R'' are independently selected from the group consisting of
hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic, and where R' and R'' are optionally joined together
with the nitrogen bound thereto to form a heterocyclic or
substituted heterocyclic group, and wherein alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic and substituted heterocyclic are as
defined herein.
[0220] "Aminocarbonylamino" refers to the group --NRC(0)NR' R''
where R is hydrogen or alkyl and R' and R'' are independently
selected from the group consisting of hydrogen, alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted
heteroaryl, heterocyclic, and substituted heterocyclic, and where
R' and R'' are optionally joined together with the nitrogen bound
thereto to form a heterocyclic or substituted heterocyclic group,
and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, heterocyclic and substituted
heterocyclic are as defined herein.
[0221] "Aminothiocarbonylamino" refers to the group --NRC(S)NR'R''
where R is hydrogen or alkyl and R' and R'' are independently
selected from the group consisting of hydrogen, alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted
heteroaryl, heterocyclic, and substituted heterocyclic, and where
R' and R'' are optionally joined together with the nitrogen bound
thereto to form a heterocyclic or substituted heterocyclic group,
and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, heterocyclic and substituted
heterocyclic are as defined herein.
[0222] "Aminocarbonyloxy" refers to the group --O--C(O)NR'R'' where
R' and R'' are independently selected from the group consisting of
hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic and where R' and R'' are optionally joined together
with the nitrogen bound thereto to form a heterocyclic or
substituted heterocyclic group, and wherein alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic and substituted heterocyclic are as
defined herein.
[0223] "Aminosulfonyl" refers to the group --SO.sub.2NR'R'' where
R' and R'' are independently selected from the group consisting of
hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic, and where R' and R'' are optionally joined together
with the nitrogen bound thereto to form a heterocyclic or
substituted heterocyclic group, and wherein alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic and substituted heterocyclic are as
defined herein.
[0224] "Aminosulfonyloxy" refers to the group --O--SO.sub.2NR'R''
where R' and R'' are independently selected from the group
consisting of hydrogen, alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, aryl,
substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,
substituted cycloalkenyl, heteroaryl, substituted heteroaryl,
heterocyclic, and substituted heterocyclic, and where R' and R''
are optionally joined together with the nitrogen bound thereto to
form a heterocyclic or substituted heterocyclic group, and wherein
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, heterocyclic and substituted
heterocyclic are as defined herein.
[0225] "Aminosulfonylamino" refers to the group
--NR--SO.sub.2NR'R'' where R is hydrogen or alkyl and R' and R''
are independently selected from the group consisting of hydrogen,
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkyenyl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic, and where R' and R'' are optionally joined together
with the nitrogen bound thereto to form a heterocyclic or
substituted heterocyclic group, and wherein alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkyenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic and substituted heterocyclic are as
defined herein.
[0226] "Amidino" refers to the group --C(.dbd.NR''')R'R'' where R',
R'', and R''' are independently selected from the group consisting
of hydrogen, alkyl, substituted alkyl, alkenyl, substituted
alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and
substituted heterocyclic, and where R' and R'' are optionally
joined together with the nitrogen bound thereto to form a
heterocyclic or substituted heterocyclic group, and wherein alkyl,
substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, heterocyclic and substituted
heterocyclic are as defined herein.
[0227] "Aniline" refers to C.sub.6H.sub.5NH.sub.2, and consists of
a phenyl ring attached to an amino group. As used herein, the amino
group is para to a fluorophore, as is illustrated as follows:
##STR00012##
[0228] wherein X is a fluorophore, preferably a xanthene
derivative, most preferably a rhodamine or rhodol.
[0229] "Aryl" or "Ar" refers to a monovalent aromatic carbocyclic
group of from 6 to 14 carbon atoms having a single ring (e.g.,
phenyl) or multiple condensed rings (e.g., naphthyl or anthryl)
which condensed rings may or may not be aromatic (e.g.,
2-benzoxazolinone, 2H-1,4-benzoxazin-3(4H)-one-7-yl, and the like)
provided that the point of attachment is at an aromatic carbon
atom. Preferred aryl groups include phenyl and naphthyl.
[0230] "Substituted aryl" refers to aryl groups which are
substituted with 1 to 5, preferably 1 to 3, or more preferably 1 to
2 substituents selected from the group consisting of alkyl,
substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, alkoxy, substituted alkoxy, acyl, acylamino,
acyloxy, amino, substituted amino, aminocarbonyl,
aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino,
aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy,
aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy,
substituted aryloxy, arylthio, substituted arylthio, carboxyl,
carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano,
cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted
cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio,
cycloalkenyl, substituted cycloalkenyl, cycloalkenyloxy,
substituted cycloalkenyloxy, cycloalkenylthio, substituted
cycloalkenylthio, guanidino, substituted guanidino, halo, hydroxy,
heteroaryl, substituted heteroaryl, heteroaryloxy, substituted
heteroaryloxy, heteroarylthio, substituted heteroarylthio,
heterocyclic, substituted heterocyclic, heterocyclyloxy,
substituted heterocyclyloxy, heterocyclylthio, substituted
heterocyclylthio, nitro, SO.sub.3H, substituted sulfonyl,
sulfonyloxy, thioacyl, thiol, alkylthio, and substituted alkylthio,
wherein said substituents are defined herein.
[0231] "Aryloxy" refers to the group --O-aryl, where aryl is as
defined herein, that includes, by way of example, phenoxy and
naphthoxy.
[0232] "Substituted aryloxy" refers to the group --O-(substituted
aryl), where substituted aryl is as defined herein.
[0233] "Arylthio" refers to the group --S-aryl, where aryl is as
defined herein.
[0234] "Substituted arylthio" refers to the group --S-(substituted
aryl), where substituted aryl is as defined herein.
[0235] "Alkenyl" refers to alkenyl groups having from 2 to 6 carbon
atoms and preferably 2 to 4 carbon atoms and having at least 1 and
preferably from 1 to 2 sites of alkenyl unsaturation. Such groups
are exemplified, for example, by vinyl, allyl, but-3-en-1-yl, and
propenyl.
[0236] "Substituted alkenyl" refers to alkenyl groups having from 1
to 3 substituents, and preferably 1 to 2 substituents, selected
from the group consisting of alkoxy, substituted alkoxy, acyl,
acylamino, acyloxy, amino, substituted amino, aminocarbonyl,
aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino,
aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy,
aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy,
substituted aryloxy, arylthio, substituted arylthio, carboxyl,
carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano,
cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted
cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio,
cycloalkenyl, substituted cycloalkenyl, cycloalkenyloxy,
substituted cycloalkenyloxy, cycloalkenylthio, substituted
cycloalkenylthio, guanidino, substituted guanidino, halo, hydroxy,
heteroaryl, substituted heteroaryl, heteroaryloxy, substituted
heteroaryloxy, heteroarylthio, substituted heteroarylthio,
heterocyclic, substituted heterocyclic, heterocyclyloxy,
substituted heterocyclyloxy, heterocyclylthio, substituted
heterocyclylthio, nitro, SO.sub.3H, substituted sulfonyl,
sulfonyloxy, thioacyl, thiol, alkylthio, and substituted alkylthio,
wherein said substituents are defined herein and with the proviso
that any hydroxy substitution is not attached to a vinyl
(unsaturated) carbon atom.
[0237] "Alkynyl" refers to alkynyl groups having from 2 to 6 carbon
atoms and preferably 2 to 3 carbon atoms and having at least 1 and
preferably from 1 to 2 sites of alkynyl unsaturation.
[0238] "Substituted alkynyl" refers to alkynyl groups having from 1
to 3 substituents, and preferably 1 to 2 substituents, selected
from the group consisting of alkoxy, substituted alkoxy, acyl,
acylamino, acyloxy, amino, substituted amino, aminocarbonyl,
aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino,
aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy,
aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy,
substituted aryloxy, arylthio, substituted arylthio, carboxyl,
carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano,
cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted
cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio,
cycloalkenyl, substituted cycloalkenyl, cycloalkenyloxy,
substituted cycloalkenyloxy, cycloalkenylthio, substituted
cycloalkenylthio, guanidino, substituted guanidino, halo, hydroxy,
heteroaryl, substituted heteroaryl, heteroaryloxy, substituted
heteroaryloxy, heteroarylthio, substituted heteroarylthio,
heterocyclic, substituted heterocyclic, heterocyclyloxy,
substituted heterocyclyloxy, heterocyclylthio, substituted
heterocyclylthio, nitro, SO.sub.3H, substituted sulfonyl,
sulfonyloxy, thioacyl, thiol, alkylthio, and substituted alkylthio,
wherein said substituents are defined herein and with the proviso
that any hydroxy substitution is not attached to an acetylenic
carbon atom.
[0239] "Carbonyl" refers to the divalent group --C(O)-- which is
equivalent to --C(.dbd.O)--.
[0240] "Carboxyl" or "carboxy" refers to --COOH or salts
thereof.
[0241] "Carboxyl alkyl" or "carboxyalkyl" refers to the group
--(CH.sub.2).sub.nCOOH, where n is 1-6.
[0242] "Carboxyl ester" or "carboxy ester" refers to the groups
--C(O)O-alkyl, --C(O)O-substituted alkyl, --C(O)O-alkenyl,
--C(O)O-substituted alkenyl, --C(O)O-alkynyl, --C(O)O-substituted
alkynyl, --C(O)O-aryl, --C(O)O-substituted aryl,
--C(O)O-cycloalkyl, --C(O)O-substituted cycloalkyl,
--C(O)O-cycloalkenyl, --C(O)O-substituted cycloalkenyl,
--C(O)O-heteroaryl, --C(O)O-substituted heteroaryl,
--C(O)O-heterocyclic, and --C(O)O-substituted heterocyclic, wherein
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic are as defined herein.
[0243] "(Carboxyl ester)amino" refers to the group
--NR--C(O)O-alkyl, substituted --NR--C(O)O-alkyl,
--NR-C(O)O-alkenyl, --NR--C(O)O-substituted alkenyl,
--NR--C(O)O-alkynyl, --NR--C(O)O-substituted alkynyl,
--NR--C(O)O-aryl, --NR--C(O)O-substituted aryl,
--NR--C(O)O-cycloalkyl, --NR--C(O)O-substituted cycloalkyl,
--NR--C(O)O-cycloalkenyl, --NR--C(O)O-substituted cycloalkenyl,
--NR--C(O)O-heteroaryl, --NR--C(O)O-substituted heteroaryl,
--NR--C(O)O-heterocyclic, and --NR--C(O)O-substituted heterocyclic,
wherein R is alkyl or hydrogen, and wherein alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic, and substituted heterocyclic are as
defined herein.
[0244] "(Carboxyl ester)oxy" refers to the group --O--C(O)O-alkyl,
substituted --O--C(O)O-alkyl, --O--C(O)O-alkenyl,
--O--C(O)O-substituted alkenyl, --O--C(O)O-alkynyl,
--O--C(O)O-substituted alkynyl, --O-C(O)O-aryl,
--O--C(O)O-substituted aryl, --O--C(O)O-cycloalkyl,
--O--C(O)O-substituted cycloalkyl, --O--C(O)O-cycloalkenyl,
--O--C(O)O-substituted cycloalkenyl, --O--C(O)O-heteroaryl,
--O--C(O)O-substituted heteroaryl, --O--C(O)O-heterocyclic, and
--O--C(O)O-substituted heterocyclic, wherein alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic, and substituted heterocyclic are as
defined herein.
[0245] "Cyano" refers to the group --CN.
[0246] "Cycloalkyl" refers to cyclic alkyl groups of from 3 to 10
carbon atoms having single or multiple cyclic rings including
fused, bridged, and spiro ring systems. Examples of suitable
cycloalkyl groups include, for instance, adamantyl, cyclopropyl,
cyclobutyl, cyclopentyl, and cyclooctyl.
[0247] "Cycloalkenyl" refers to non-aromatic cyclic alkyl groups of
from 3 to 10 carbon atoms having single or multiple cyclic rings
and having at least one >C.dbd.C<ring unsaturation and
preferably from 1 to 2 sites of >C.dbd.C<ring
unsaturation.
[0248] "Substituted cycloalkyl" and "substituted cycloalkenyl"
refers to a cycloalkyl or cycloalkenyl group having from 1 to 5 or
preferably 1 to 3 substituents selected from the group consisting
of oxo, thione, alkyl, substituted alkyl, alkenyl, substituted
alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy,
acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl,
aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino,
aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy,
aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy,
substituted aryloxy, arylthio, substituted arylthio, carboxyl,
carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano,
cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted
cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio,
cycloalkenyl, substituted cycloalkenyl, cycloalkenyloxy,
substituted cycloalkenyloxy, cycloalkenylthio, substituted
cycloalkenylthio, guanidino, substituted guanidino, halo, hydroxy,
heteroaryl, substituted heteroaryl, heteroaryloxy, substituted
heteroaryloxy, heteroarylthio, substituted heteroarylthio,
heterocyclic, substituted heterocyclic, heterocyclyloxy,
substituted heterocyclyloxy, heterocyclylthio, substituted
heterocyclylthio, nitro, SO.sub.3H, substituted sulfonyl,
sulfonyloxy, thioacyl, thiol, alkylthio, and substituted alkylthio,
wherein said substituents are defined herein.
[0249] "Cycloalkyloxy" refers to --O-cycloalkyl.
[0250] "Substituted cycloalkyloxy" refers to --O-(substituted
cycloalkyl).
[0251] "Cycloalkylthio" refers to --S-cycloalkyl.
[0252] "Substituted cycloalkylthio" refers to --S-(substituted
cycloalkyl).
[0253] "Cycloalkenyloxy" refers to --O-cycloalkenyl.
[0254] "Substituted cycloalkenyloxy" refers to --O-(substituted
cycloalkenyl).
[0255] "Cycloalkenylthio" refers to --S-cycloalkenyl.
[0256] "Substituted cycloalkenylthio" refers to --S-(substituted
cycloalkenyl).
[0257] "Guanidino" refers to the group --NHC(.dbd.NH)NH.sub.2.
[0258] "Substituted guanidino" refers to
--NR.sup.13C(.dbd.NR.sup.13)N(R.sup.13).sub.2 where each R.sup.13
is independently selected from the group consisting of hydrogen,
alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,
substituted heteroaryl, heterocyclic, and substituted heterocyclic
and two R.sup.13 groups attached to a common guanidino nitrogen
atom are optionally joined together with the nitrogen bound thereto
to form a heterocyclic or substituted heterocyclic group, provided
that at least one R.sup.13 is not hydrogen, and wherein said
substituents are as defined herein.
[0259] "H" indicates hydrogen.
[0260] "Halo" or "halogen" refers to fluoro, chloro, bromo and
iodo.
[0261] "Hydroxy" or "hydroxyl" refers to the group --OH.
[0262] "Heteroaryl" refers to an aromatic group of from 1 to 10
carbon atoms and 1 to 4 heteroatoms selected from the group
consisting of oxygen, nitrogen and sulfur within the ring. Such
heteroaryl groups can have a single ring (e.g., pyridinyl or furyl)
or multiple condensed rings (e.g., indolizinyl or benzothienyl)
wherein the condensed rings may or may not be aromatic and/or
contain a heteroatom provided that the point of attachment is
through an atom of the aromatic heteroaryl group. In one
embodiment, the nitrogen and/or the sulfur ring atom(s) of the
heteroaryl group are optionally oxidized to provide for the N-oxide
(N.fwdarw.O), sulfinyl, or sulfonyl moieties. Preferred heteroaryls
include pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl.
[0263] "Substituted heteroaryl" refers to heteroaryl groups that
are substituted with from 1 to 5, preferably 1 to 3, or more
preferably 1 to 2 substituents selected from the group consisting
of the same group of substituents defined for substituted aryl.
[0264] "Heteroaryloxy" refers to --O-heteroaryl.
[0265] "Substituted heteroaryloxy" refers to the group
--O-(substituted heteroaryl).
[0266] "Heteroarylthio" refers to the group --S-heteroaryl.
[0267] "Substituted heteroarylthio" refers to the group
--S-(substituted heteroaryl).
[0268] "Heterocycle" or "heterocyclic" or "heterocycloalkyl" or
"heterocyclyl" refers to a saturated or unsaturated group having a
single ring or multiple condensed rings, including fused bridged
and spiro ring systems, from 1 to 10 carbon atoms and from 1 to 4
hetero atoms selected from the group consisting of nitrogen, sulfur
or oxygen within the ring wherein, in fused ring systems, one or
more the rings can be cycloalkyl, aryl or heteroaryl provided that
the point of attachment is through the non-aromatic ring. In one
embodiment, the nitrogen and/or sulfur atom(s) of the heterocyclic
group are optionally oxidized to provide for the N-oxide, sulfinyl,
sulfonyl moieties.
[0269] "Substituted heterocyclic" or "substituted heterocycloalkyl"
or "substituted heterocyclyl" refers to heterocyclyl groups that
are substituted with from 1 to 5, or preferably 1 to 3 of the same
substituents as defined for substituted cycloalkyl.
[0270] "Heterocyclyloxy" refers to the group --O-heterocyclyl.
[0271] "Substituted heterocyclyloxy" refers to the group
--O-(substituted heterocyclyl).
[0272] "Heterocyclylthio" refers to the group --S-heterocyclyl.
[0273] "Substituted heterocyclylthio" refers to the group
--S-(substituted heterocyclyl).
[0274] Examples of heterocycle and heteroaryls include, but are not
limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine,
pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole,
dihydroindole, indazole, purine, quinolizine, isoquinoline,
quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline,
cinnoline, pteridine, carbazole, carboline, phenanthridine,
acridine, phenanthroline, isothiazole, phenazine, isoxazole,
phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine,
piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline,
4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine,
thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also
referred to as thiamorpholinyl), 1,1-dioxothiomorpholinyl,
piperidinyl, pyrrolidine, and tetrahydrofuranyl.
[0275] "Hydrazinyl" refers to the group --NHNH.sub.2-- or
.dbd.NNH--.
[0276] "Substituted hydrazinyl" refers to a hydrazinyl group,
wherein a non-hydrogen atom, such as an alkyl group, is appended to
one or both of the hydrazinyl amine groups. An example of
substituted hydrazinyl is --N(alkyl)--NH.sub.2 or
.dbd.N.sup.+(alkyl)-NH.sub.2.
[0277] "Nitro" refers to the group --NO.sub.2.
[0278] "Oxo" refers to the atom (.dbd.O) or (--O.sup.-).
[0279] "Spirocyclyl" refers to divalent saturated cyclic group from
3 to 10 carbon atoms having a cycloalkyl or heterocyclyl ring with
a spiro union (the union formed by a single atom which is the only
common member of the rings) as exemplified by the following
structure:
##STR00013##
[0280] "Sulfonyl" refers to the divalent group --S(O).sub.2--.
[0281] "Substituted sulfonyl" refers to the group --SO.sub.2-alkyl,
--SO.sub.2-substituted alkyl, --SO.sub.2-alkenyl, --SO.sub.2--
substituted alkenyl, --SO.sub.2-cycloalkyl, --SO.sub.2-substituted
cylcoalkyl, --SO.sub.2-cycloalkenyl, --SO.sub.2-substituted
cylcoalkenyl, --SO.sub.2-aryl, --SO.sub.2-substituted aryl,
--SO.sub.2-heteroaryl, --SO.sub.2-substituted heteroaryl,
--SO.sub.2-- heterocyclic, --SO.sub.2-substituted heterocyclic,
wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, heterocyclic and substituted
heterocyclic are as defined herein. Substituted sulfonyl includes
groups such as methyl-SO.sub.2--, phenyl-SO.sub.2--, and
4-methylphenyl-SO.sub.2--.
[0282] "Sulfonyloxy" refers to the group --OSO.sub.2-alkyl,
--OSO.sub.2-substituted alkyl, --OSO.sub.2-alkenyl, --OSO.sub.2--
substituted alkenyl, --OSO.sub.2-cycloalkyl,
--OSO.sub.2-substituted cylcoalkyl, --OSO.sub.2-cycloalkenyl,
--OSO.sub.2-- substituted cylcoalkenyl, --OSO.sub.2-aryl,
--OSO.sub.2-substituted aryl, --OSO.sub.2-heteroaryl,
--OSO.sub.2-substituted heteroaryl, --OSO.sub.2-heterocyclic,
--OSO.sub.2-substituted heterocyclic, wherein alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic and substituted heterocyclic are as
defined herein.
[0283] "Thioacyl" refers to the groups H--C(S)--, alkyl-C(S)--,
substituted alkyl-C(S)--, alkenyl-C(S)--, substituted
alkenyl-C(S)--, alkynyl-C(S)--, substituted alkynyl-C(S)--,
cycloalkyl-C(S)--, substituted cycloalkyl-C(S)--,
cycloalkenyl-C(S)--, substituted cycloalkenyl-C(S)--, aryl-C(S)--,
substituted aryl-C(S)--, heteroaryl-C(S)--, substituted
heteroaryl-C(S)--, heterocyclic-C(S)--, and substituted
heterocyclic-C(S)--, wherein alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
heterocyclic and substituted heterocyclic are as defined
herein.
[0284] "Thiol" refers to the group --SH.
[0285] "Thiocarbonyl" refers to the divalent group --C(S)-- which
is equivalent to --C(.dbd.S)--.
[0286] "Thione" refers to the atom (.dbd.S).
[0287] "Alkylthio" refers to the group --S-alkyl wherein alkyl is
as defined herein.
[0288] "Substituted alkylthio" refers to the group --S-(substituted
alkyl), wherein substituted alkyl is as defined herein.
[0289] A dashed line projecting from a substituent, such as:
##STR00014##
indicates the point of attachment to the base molecule. For a fused
ring, dashed lines indicate portions of the base molecule where the
fused ring is attached, such as:
##STR00015##
wherein the full molecule could have the structure:
##STR00016##
[0290] Unless indicated otherwise, the nomenclature of substituents
that are not explicitly defined herein are arrived at by naming the
terminal portion of the functionality followed by the adjacent
functionality toward the point of attachment. For example, the
substituent "arylalkyloxycarbonyl" refers to the group
(aryl)-(alkyl)-O--C(O)--.
[0291] It is understood that in all substituted groups defined
above, polymers arrived at by defining substituents with further
substituents to themselves (e.g., substituted aryl having a
substituted aryl group as a substituent which is itself substituted
with a substituted aryl group, which is further substituted by a
substituted aryl group etc.) are not intended for inclusion herein.
In such cases, the maximum number of such substitutions is three.
For example, serial substitutions of substituted aryl groups with
two other substituted aryl groups are limited to -substituted
aryl-(substituted aryl)-substituted aryl.
[0292] Similarly, it is understood that the above definitions are
not intended to include impermissible substitution patterns (e.g.,
methyl substituted with 5 fluoro groups). Such impermissible
substitution patterns are well known to the skilled artisan.
[0293] The pH-sensitive fluorescent dye compounds disclosed herein
may exist in unsolvated forms as well as solvated forms, including
hydrated forms. These compounds may exist in multiple crystalline
or amorphous forms. In general, all physical forms are equivalent
for the uses described herein and are intended to be within the
scope of the present disclosure. The dye compounds disclosed herein
may possess asymmetric carbon atoms (i.e., chiral centers) or
double bonds; the racemates, diastereomers, geometric isomers and
individual isomers of the compounds described herein are within the
scope of the present disclosure. The dye compounds described herein
may be prepared as a single isomer or as a mixture of isomers.
[0294] Where substituent groups are specified by their conventional
chemical formulae and are written from left to right, they equally
encompass the chemically identical substituents, which would result
from writing the structure from right to left, e.g., --CH.sub.2O--
is intended to also recite --OCH.sub.2--.
[0295] It will be understood that the chemical structures that are
used to define the dye compounds disclosed herein are each
representations of one of the possible resonance structures by
which each given structure can be represented. Further, it will be
understood that by definition, resonance structures are merely a
graphical representation used by those of skill in the art to
represent electron delocalization, and that the present disclosure
is not limited in any way by showing one particular resonance
structure for any given structure.
[0296] Where a disclosed compound includes a conjugated ring
system, resonance stabilization may permit a formal electronic
charge to be distributed over the entire molecule. While a
particular charge may be depicted as localized on a particular ring
system, or a particular heteroatom, it is commonly understood that
a comparable resonance structure can be drawn in which the charge
may be formally localized on an alternative portion of the
compound.
[0297] The term "carrier molecule" as used herein, refers to a
biological or a non-biological component that is or becomes
covalently bonded to a pH-sensitive fluorescent dye compound
disclosed herein. Such components include, but are not limited to,
an amino acid, a peptide, a protein, a polysaccharide, a
nucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a
hapten, a psoralen, a drug, a hormone, a lipid, a lipid assembly, a
synthetic polymer, a polymeric microparticle, a biological cell, a
virus and combinations thereof. Included is one embodiment in which
carrier molecules comprise an organic moiety having at least 4
plural valent atoms and often more than 10 plural valent atoms
(i.e., atoms other than hydrogen and halo), e.g. at least 15 such
atoms, as in the case of moieties having at least 20 such
atoms.
[0298] The term "conjugated substance" or "S.sub.c" refers to a
carrier molecule or solid support.
[0299] The term "detectable response" as used herein refers to an
occurrence of or a change in, a signal that is directly or
indirectly detectable either by observation or by instrumentation.
Typically, the detectable response is an optical response resulting
in a change in the wavelength distribution patterns or intensity of
absorbance or fluorescence or a change in light scatter,
fluorescence lifetime, fluorescence polarization, or a combination
of the above parameters.
[0300] The term "dye" as used herein refers to a compound that
emits light to produce an observable detectable signal.
[0301] The term "electron donating group" or "EDG" refers to a
substituent with lone electron pairs that is adjacent to an
aromatic ring, such as phenyl, and increases electron density on
the ring through a resonance donating effect. Electron donating
groups of the present disclosure include, for example, alkoxy,
substituted alkoxy, amino, substituted amino, alkylthio, acylamino,
(carboxyl ester)oxy, and halogen. Alkoxy is a particular EDG.
Substituted alkoxy is another particular EDG. Also to be mentioned
is dialkylamino A further example is dialkylamino having a
substituted alkyl group. Preferred EDGs are --OCH.sub.3,
--NH.sub.2, --NHCH.sub.3, --N(CH.sub.3).sub.2, and
--N(CH.sub.2CH.sub.3).sub.2, particularly --OCH.sub.3, --NH.sub.2,
--NHCH.sub.3, --N(CH.sub.3).sub.2 and --N(CH.sub.2CH.sub.3).sub.2.
Also to be mentioned are alkoxy, alkythio and dialkylamino, in any
of those instances having an alkyl substituent in which the alkyl
part is substituted by a moiety -L-R.sub.x, -L-S.sub.c, or
-L.sub.R-S.sub.c. The specification also discloses specific
compounds or compound classes which include other EDGs than those
with a lone pair of electrons adjacent an aromatic ring.
[0302] "Fluorescent pH-sensitive dye," "pH-sensitive fluorescent
dye," and "fluorescent pH sensor dye" are equivalent and are used
interchangeably to refer to a compound whose fluorescent spectrum
or intensity is affected by pH.
[0303] The term "fluorophore" or "fluorogenic" as used herein
refers to a composition that is inherently fluorescent or
demonstrates a change in fluorescence upon protonation, or binding
to a biological compound or metal ion, or metabolism by an enzyme.
Preferred fluorophores of the present disclosure include
fluorescent dyes having a high quantum yield in aqueous media.
Exemplary fluorophores include xanthene derivatives, preferably
rhodamines and rhodols. The fluorophores disclosed herein may be
substituted to alter the solubility, spectral properties or
physical properties of the fluorophore.
[0304] The term "linker" or "L", as used herein, refers to a single
covalent bond or a moiety comprising series of stable covalent
bonds, the moiety often incorporating 1-40 plural valent atoms
selected from the group consisting of C, N, O, S and P that
covalently attach the fluorogenic or fluorescent compounds to
another moiety such as a chemically reactive group or a biological
and non-biological component. The number of plural valent atoms in
a linker may be, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,
25, 30 or a larger number up to 40 or more. A linker may be linear
or non-linear; some linkers have pendant side chains or pendant
functional groups, or both. Examples of such pendant moieties are
hydrophilicity modifiers, for example solubilizing groups like,
e.g. sulfo (--SO.sub.3H or --SO.sub.3.sup.31 ). In certain
embodiments, L is composed of any combination of single, double,
triple or aromatic carbon-carbon bonds, carbon-nitrogen bonds,
nitrogen-nitrogen bonds, carbon-oxygen bonds and carbon-sulfur
bonds. Exemplary linking members include a moiety that includes
--C(O)NH--, --C(O)O--, --NH--, --S--, --O--, and the like. Linkers
may, by way of example, consist of a combination of moieties
selected from alkyl; --C(O)NH--; --C(O)O--; --NH--; --S--; --O--;
--C(O)--; --S(O).sub.n-- where n is 0, 1 or 2; --O--; 5- or
6-membered monocyclic rings; and optional pendant functional
groups, for example sulfo, hydroxy and carboxy. The moiety formed
by a linker bonded to a reactive group (R.sub.x) may be designated
-L-R.sub.x. The reactive group may be reacted with a substance
reactive therewith, whereby the linker becomes bonded to a
conjugated substance (S.sub.c) and may be designated -L-Sc, or in
some cases, the linker may contains a residue of a reactive group
(e.g. the carbonyl group of an ester) and may be designated
"-L.sub.R". A "cleavable linker" is a linker that has one or more
cleavable groups that may be broken by the result of a reaction or
condition. The term "cleavable group" refers to a moiety that
allows for release of a portion, e.g., a fluorogenic or fluorescent
moiety, of a conjugate from the remainder of the conjugate by
cleaving a bond linking the released moiety to the remainder of the
conjugate. Such cleavage is either chemical in nature, or
enzymatically mediated. Exemplary enzymatically cleavable groups
include natural amino acids or peptide sequences that end with a
natural amino acid.
[0305] In addition to enzymatically cleavable groups, it is within
the scope of the present invention to include one or more sites
that are cleaved by the action of an agent other than an enzyme.
Exemplary non-enzymatic cleavage agents include, but are not
limited to, acids, bases, light (e.g., nitrobenzyl derivatives,
phenacyl groups, benzoin esters), and heat. Many cleavable groups
are known in the art. See, for example, Jung et al., Biochem.
Biophys. Acta, 761:152-162 (1983); Joshi et al., J. Biol. Chem.,
265:14518-14525 (1990); Zarling et al., J. Immunol., 124:913-920
(1980); Bouizar et al., Eur. J. Biochem., 155:141-147 (1986); Park
et al., J. Biol. Chem., 261:205-210 (1986); Browning et al., J.
Immunol., 143:1859-1867 (1989). Moreover a broad range of
cleavable, bifunctional (both homo- and hetero-bifunctional) spacer
arms are commercially available.
[0306] An exemplary cleavable group, such as an ester, is cleavable
group that may be cleaved by a reagent, e.g., sodium hydroxide,
resulting in a carboxylate-containing fragment and a
hydroxyl-containing product.
[0307] The linker may be used to attach the pH-sensitive
fluorescent dye compound to another component of a conjugate, such
as a targeting moiety (e.g., antibody, ligand, non-covalent
protein-binding group, etc.), an analyte, a biomolecule, a drug and
the like.
[0308] In certain embodiments, compounds are provided in which -L-
is of the formula -L1-(L2).sub.p-(L3).sub.r-wherein:
[0309] p is 0 or 1; r is 0 or 1; L1 is a bond, --CONH--, --COO--,
or a moiety comprising at least two amino acids; L2 is
--(CH.sub.2)--,
--CH.sub.2CH.sub.2O--(CH.sub.2CH.sub.2O).sub.s--CH.sub.2CH.sub.2--,
or alkylene having from 1 to 30 carbon atoms and unsubstituted or
substituted by at least one R.sup.a, e.g. 1, 2, 3, 4, 5 or 6
R.sup.a; L3 is --CONH--(CH.sub.2).sub.t--,
--COO--(CH.sub.2).sub.t-- or a moiety comprising at least two amino
acids, wherein: [0310] r is from 1 to 30, e.g., 1 to 20 as in the
case of 1 to 10, such as 1, 2, 3, 4, 5 or 6; s is 0, 1, 2, 3, 4, 5,
6, 7, 8, 9 or 10, e.g., 1 to 7; t is from 1 to 30, e.g. 1 to 20 as
in the case of 1 to 10, such as 1, 2, 3, 4, 5 or 6; R.sup.a is
sulfo (--SO.sub.3H and/or --SO.sub.3), hydroxy, carboxy or amino,
particularly sulfo.
[0311] In certain embodiments, the total number of carbon atoms
comprised in alkylene moieties in L is no more than 40, e.g., up to
35, 30, 25, 20, 15 or 10. In certain embodiments, there is only a
single one of L1 and L3 which comprises a moiety comprising at
least two amino acids. In certain embodiments, p and r are both 0.
In certain embodiments, p is 1 and r is 0. In certain embodiments,
p and r are both 1.
[0312] In certain embodiments, L1 is a bond, --CONH-- or --COO--.
In certain compounds L1 is a bond. In certain others, L1 is
--CONH--.
[0313] L2, in certain embodiments, is --(CH.sub.2)--, where u is
from 1 to 10, e.g., 1, 2, 3, 4, 5 or 6. In certain embodiments, L2
is
--CH.sub.2CH.sub.2O--(CH.sub.2CH.sub.2O).sub.s-CH.sub.2CH.sub.2--
where s is from 1 to 7. In certain embodiments, L2 is alkylene
having from 1 to 10 carbon atoms, e.g., 1, 2, 3, 4, 5 or 6 carbon
atoms, and which is unsubstituted or substituted by 1, 2, 3, 4, 5
or 6 sulfo groups, e.g., 1 to 4 sulfo groups. For all L2 moieties
mentioned in this paragraph, L1 is --CONH-- in a particular class
of compounds. For all L2 moieties mentioned in this paragraph and
all -L1-L2-combinations mentioned in this paragraph, r is 0 in one
class of compounds.
[0314] In certain embodiments, (r+t) is from 1 to 30, e.g., 1 to 20
as in the case of 1 to 10, such as 1, 2, 3, 4, 5 or 6, for
example.
[0315] Exemplary linkers include, but are not limited to, the
following: a single covalent bond (for example between alkyl and a
carboxy group or ester of a carboxy group, or other reactive
group); aminocarbonyl (for example linking an alkyl group to a
conjugated lipophilic moiety); a PEG-NH--CO-- moiety (for example
linking an alkyl group to an NHS-ester or other reactive group); an
alkylaminocarbonyl group (for example linking an alkyl group to an
NHS-ester, amine or other reactive group); an alkylaminocarbonyl
group having a pendant group comprising sulfo--e.g. a pendant
sulfoalkyl group (for example linking an alkyl group to NHS-ester
or other reactive group or to a lipophilic group); or a single
covalent bond linking an alkyl group to a reactive group such as a
carboxy group or ester thereof.
[0316] The terms "patient," "subject" or "individual" refer to
mammals and includes humans and non-human mammals, such as monkeys,
dogs, cats, pocket pets, horses, cows, pigs or rats.
[0317] The terms "protein" and "polypeptide" are used herein in a
generic sense to include polymers of amino acid residues of any
length. The term "peptide" is used herein to refer to polypeptides
having less than 250 amino acid residues, typically less than 100
amino acid residues. The terms apply to amino acid polymers in
which one or more amino acid residues are an artificial chemical
analogue of a corresponding naturally occurring amino acid, as well
as to naturally occurring amino acid polymers.
[0318] The term "reactive group" (or "R.sub.x"), as used herein,
refers to a group that is capable of reacting with another chemical
group to form a covalent bond, i.e., is covalently reactive under
suitable reaction conditions, and generally represents a point of
attachment for another substance. The reactive group is a moiety,
such as carboxylic acid or succinimidyl ester, on the compounds of
the present disclosure that is capable of chemically reacting with
a functional group on a different compound to form a covalent
linkage. Reactive groups generally include nucleophiles,
electrophiles and photoactivatable groups.
[0319] Exemplary reactive groups include, but not limited to,
olefins, acetylenes, alcohols, phenols, ethers, oxides, halides,
aldehydes, ketones, carboxylic acids, esters, amides, cyanates,
isocyanates, thiocyanates, isothiocyanates, amines, hydrazines,
hydrazones, hydrazides, diazo, diazonium, nitro, nitriles,
mercaptans, sulfides, disulfides, sulfoxides, sulfones, sulfonic
acids, sulfinic acids, acetals, ketals, anhydrides, sulfates,
sulfenic acids isonitriles, amidines, imides, imidates, nitrones,
hydroxylamines, oximes, hydroxamic acids thiohydroxamic acids,
allenes, ortho esters, sulfites, enamines, ynamines, ureas,
pseudoureas, semicarbazides, carbodiimides, carbamates, imines,
azides, azo compounds, azoxy compounds, and nitroso compounds.
Reactive functional groups also include those used to prepare
bioconjugates, e.g., N-hydroxysuccinimide esters, maleimides,
succinimidyl esters (SE), sulfodichlorophenol (SDP) esters,
sulfotetrafluorophenol (STP) esters, tetrafluorophenol (TFP)
esters, acetoxymethoxy (AM) esters, nitrilotriacetic acids (NTA),
aminodextrans, DIBO-amines and the like. Methods to prepare each of
these functional groups are well known in the art and their
application to or modification for a particular purpose is within
the ability of one of skill in the art (see, for example, Sandler
and Karo, eds., Organic Functional Group Preparations, Academic
Press, San Diego, 1989).
[0320] The term "salt" refers to acceptable salts of a compound,
which salts are derived from a variety of organic and inorganic
counter ions well known in the art and include, by way of example
only, sodium, potassium, calcium, magnesium, ammonium, and
tetraalkylammonium; and when the molecule contains a basic
functionality, salts of organic or inorganic acids, such as
hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate,
and oxalate.
[0321] The term "sample," as used herein, refers to any material
that may contain an analyte of interest or cells. Typically, the
sample is a live cell or a biological fluid that comprises
endogenous host cells. Alternatively, the sample may be a buffer
solution or an environmental sample for which pH determination is
needed. The sample may be in an aqueous solution, a viable cell
culture or immobilized on a solid or semi-solid surface such as a
polyacrylamide gel, membrane blot or on a microarray.
[0322] The term "solid support," as used herein, refers to a matrix
or medium that is substantially insoluble in liquid phases and
capable of binding a molecule or particle of interest. Solid
supports suitable for use herein include semi-solid supports and
are not limited to a specific type of support. Useful solid
supports include solid and semi-solid matrixes, such as aerogels
and hydrogels, resins, beads, biochips (including thin film coated
biochips), microfluidic chip, a silicon chip, multi-well plates
(also referred to as microtitre plates or microplates), membranes,
conducting and nonconducting metals, glass (including microscope
slides) and magnetic supports. More specific examples of useful
solid supports include silica gels, polymeric membranes, particles,
derivatized plastic films, glass beads, cotton, plastic beads,
alumina gels, polysaccharides such as Sepharose.RTM.,
poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose,
agar, cellulose, dextran, starch, FICOLL.RTM., heparin, glycogen,
amylopectin, mannan, inulin, nitrocellulose, diazocellulose,
polyvinylchloride, polypropylene, polyethylene (including
poly(ethylene glycol)), nylon, latex bead, magnetic bead,
paramagnetic bead, superparamagnetic bead, starch and the like.
[0323] The terms "stereoisomer" or "stereoisomers" refer to
compounds that differ in the chirality of one or more
stereocenters. Stereoisomers include enantiomers and
diastereomers.
[0324] The term "tautomer" refers to alternate forms of a compound
that differ in the position of a proton, such as enol-keto and
imine-enamine tautomers, or the tautomeric forms of heteroaryl
groups containing a ring atom attached to both a ring --NH-- moiety
and a ring .dbd.N-- moiety such as pyrazoles, imidazoles,
benzimidazoles, triazoles, and tetrazoles.
[0325] The terms "treating" or "treatment" of a disease in a
patient refer to 1) preventing the disease from occurring in a
patient that is predisposed or does not yet display symptoms of the
disease; 2) inhibiting the disease or arresting its development; or
3) ameliorating or causing regression of the disease.
[0326] Dye Compounds and Compositions:
[0327] In general, for ease of understanding the present
disclosure, the pH-sensitive fluorescent dye compounds and
corresponding substituents will first be described in detail,
followed by various methods in which the pH-sensitive fluorescent
dye compounds of the present invention are useful, which is
followed by exemplary methods of use and synthesis of certain novel
pH-sensitive fluorescent dye compounds that are particularly
advantageous for use with the methods provided herein.
[0328] The pH-sensitive fluorescent dye compounds disclosed herein
are useful for monitoring or detecting pH in a sample. For example,
we have found that by introducing an electron donating group (EDG)
into the 4-amino-2-hydroxyphenyl ring of a fluorogenic pH-sensitive
fluorescent dye compound that we were able to tune the fluorescent
properties of the pH-sensitive fluorescent dye compound (See,
structural formulae I and II). In particular we were able to tune
the pKa value and obtain a pH sensitive compound with a pKa value
compatible with live cell intracellular applications. We also found
that replacing the hydroxyl at position R.sup.1 with an alkoxy or
thioalkyl moiety not only increased the stability of the dye
compounds in an aqueous environment but also resulted in a dye
compound that was pH sensitive, an unexpected advantage in view of
the teaching by Smith et al. (supra). Advantageously, the addition
of a dialkylamino group to the aniline moiety at position R.sup.3
resulted in the unexpected advantage of yielding pKa values in the
physiological range. In certain embodiments, the dialkylamino group
is diethylamino In certain embodiments, the dialkylamino group is
dimethylamino
[0329] In certain embodiments, the pKa value is about 6 to about 7.
Without wishing to be bound by a theory, the pKa of the amino group
of the aniline moiety of the compounds of structural formulae I and
II appears to depend on the ability of the aromatic system to share
a lone electron pair on the oxygen atom. This ability is affected
by additional functional groups introduced into the aromatic system
and thus, the pKa is tuned by adding EDG groups to pH-sensitive
dyes comprising an electron rich aniline moiety.
[0330] In certain embodiments, the sample to be analyzed includes
live cells or a biological fluid, including cytosol that comprises
endogenous host cell proteins, buffer solutions and environmental
samples. Therefore, the pH-sensitive fluorescent dye compounds
disclosed herein are useful for monitoring or determining pH
changes and those events directly and indirectly associated with a
change in pH. Monitoring of the pH may also be accomplished in live
cells wherein the present pH-sensitive fluorescent dye compounds
are internalized by live cells through a number of different
mechanisms, including both passive and cell mediated mechanisms.
For example, the present pH-sensitive fluorescent dye compounds may
comprise a lipophilic group such as an acetoxymethoxy (AM) or
acetate ester that allows for entry across the live cell membrane.
Once inside the cells, nonspecific esterases cleave the AM or
acetate ester resulting in a charged molecule that is well retained
in the cell. Alternatively, the present pH-sensitive fluorescent
dye compounds may be conjugated to a carrier molecule that allows
the dye compound to be taken up by live cells. Examples include
internalization during phagocytosis, wherein the pH-sensitive
fluorescent dye compounds are conjugated to bacterial particles or
other proteins (or peptides) that induce phagocytosis by
macrophages or monocytes; or up-take through receptor
internalization when the present pH-sensitive fluorescent dye
compounds are conjugated to a carrier molecule that binds a
receptor and thus induces internalization.
[0331] The pH-sensitive fluorescent dye compounds disclosed herein
function as reporter molecules to confer a detectable signal,
directly or indirectly, to the sample as a result of a change in
pH. This results in the ability to measure and monitor pH changes
in a sample to directly and indirectly detect specific events
associated with a change in pH.
[0332] Where the detectable response is a fluorescence response, it
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. In certain embodiments, the detectable
optical response upon protonation is a change in fluorescence
intensity that is greater than approximately 150% relative to the
same dye compound wherein the aniline moiety is not protonated on
the nitrogen. Preferably, the change in fluorescence intensity is
greater than 5-fold, and more preferably more than 10-fold.
[0333] The pH-sensitive fluorescent dye compounds provided herein
may comprise a fluorophore that may be any rhodamine or rhodol
fluorophore, known to one skilled in the art, whose excitation and
fluorescence is in the green portion of the UV/VIS spectrum.
Preferably, the fluorophore is quenched, or substantially
non-fluorescent, until the nitrogen on the aniline moiety is
protonated.
[0334] .In certain embodiments, the pH-sensitive fluorescent dye
compounds are independently substituted by substituents selected
from the group consisting of hydrogen, halogen, amino, substituted
amino, alkyl, substituted alkyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, alkoxy, sulfo, reactive group
(R.sub.x), conjugated substance (S.sub.c), solid support and
carrier molecule. In another embodiment, the rhodamine and rhodol
fluorophores disclosed herein comprise both substituted and
unsubstituted moieties on the carbon atom of the central ring of
the xanthene by substituents typically found in the xanthene-based
dyes such as phenyl and substituted-phenyl moieties. Most preferred
dyes are rhodamine, rhodol, and derivatives thereof. The choice of
the fluorophore attached to the aniline moiety will determine the
pH-sensitive compound's absorption and fluorescence emission
properties as well as its live cell properties, i.e. ability to
localize within a cell.
[0335] .In certain embodiments, the fluorophore (e.g., rhodamine or
rhodol) is attached to the aniline moiety via a linker. In certain
embodiments, the fluorophore (and reactive group, carrier
molecules, and solid support) comprises a linker that is used to
covalently attach the substituents to the aniline moieties
disclosed herein. The fluorophore (and solid support, carrier
molecule or reactive group) may be directly attached to the
moieties (where the linker is a single bond) or attached through a
series of stable bonds. Preferably the fluorophore is directly
attached by a single covalent bond to the aniline moiety, but may
also be attached via a linker as described below for reactive
group, carrier molecules, and solid support. When the linker is a
series of stable covalent bonds the linker typically incorporates
1-30 nonhydrogen atoms selected from the group consisting of C, N,
O, S and P. When the linker is not a single covalent bond, the
linker may be any combination of stable chemical bonds, optionally
including, single, double, triple or aromatic carbon-carbon bonds,
as well as carbon-nitrogen bonds, nitrogen-nitrogen bonds,
carbon-oxygen bonds, sulfur-sulfur bonds, carbon-sulfur bonds,
phosphorus-oxygen bonds, phosphorus-nitrogen bonds, and
nitrogen-platinum bonds. Typically the linker incorporates less
than 15 nonhydrogen atoms and are composed of any combination of
ether, thioether, thiourea, amine, ester, carboxamide, sulfonamide,
hydrazide bonds and aromatic or heteroaromatic bonds. Typically the
linker is a combination of single carbon-carbon bonds and
carboxamide, sulfonamide or thioether bonds. The bonds of the
linker typically result in the following moieties that may be found
in the linker: ether, thioether, carboxamide, thiourea,
sulfonamide, urea, urethane, hydrazine, alkyl, aryl, heteroaryl,
alkoxy, cycloalkyl and amine moieties. Examples of a linker
include, but are not limited to, substituted or unsubstituted
polymethylene, arylene, alkylarylene, arylenealkyl, or
arylthio.
[0336] In certain embodiments, the linker contains 1-6 carbon
atoms. In certain embodiments, the linker comprises a thioether
linkage. Exemplary linking members include a moiety that includes,
but is not limited to, --C(O)NH--, --C(O)O--, --NH--, --S--, --O--,
and the like. In certain embodiments, the linker is or incorporates
the formula --(CH.sub.2).sub.d(CONH(CH.sub.2).sub.e).sub.z--, or
where d is an integer from 0 to 5, e is an integer from 1 to 5 and
z is 0 or 1. In certain embodiments, the linker is or incorporates
the formula --O(CH.sub.2)--. In certain embodiments, the linker is
or incorporates a phenylene or a 2-carboxy-substituted
phenylene.
[0337] .Any combination of linkers may be used to attach the
carrier molecule, solid support or reactive group and the present
pH-sensitive fluorescent dye compounds together. The linker may
also be substituted to alter the physical properties of the
fluorophore or aniline moiety, such as spectral properties of the
pH-sensitive fluorescent dye compounds.
[0338] Another important feature of the linker is to provide an
adequate space between the carrier molecule, reactive group or
solid support and the aniline moiety or fluorophore so as to
prevent steric hindrance. Therefore, the linker of the pH-sensitive
fluorescent dye compounds disclosed herein is important for (1)
attaching the carrier molecule, reactive group or solid support to
the dye compounds and attaching the fluorophore to the aniline
moiety, (2) providing an adequate space between the carrier
molecule, reactive group or solid support and the dye compound so
as not to sterically hinder the action of the compound and (3) for
altering the physical properties of the dye compounds disclosed
herein.
[0339] The pH sensing or electron rich aniline moiety of the
pH-sensitive fluorescent dye compounds disclosed herein is any
moiety that, when protonated, results in the compound being
fluorescent, whilst the compound is quenched when the aniline
moiety is not in the protonated state. The pH-sensitive fluorescent
dye compounds often have a pKa value in the range of about 2 to
about 10. In certain embodiments the pKa of the pH-sensitive
fluorescent dye compound is about 3 to about 10. In certain
embodiments, the pKa of the pH-sensitive fluorescent dye compound
is about 5 to about 8. In certain embodiments the pKa of the
pH-sensitive fluorescent dye compound is about 6 to about 8. In
certain embodiments the pKa of the pH-sensitive fluorescent dye
compound is about 6 to about 7. In certain embodiment the pKa of
the pH-sensitive fluorescent dye compound is about 6.5. Preferably
the pKa of the pH-sensitive fluorescent dye compounds provided
herein is about 6 to about 7.
[0340] To tune the pKa to about 6 to about 7, electron donating
groups (EDG) were introduced into the aniline moiety on the aryl
group. This combined with the presence of an alkoxy or other like
substituents on the aryl when a --OH or --SH were not present,
unexpectedly resulted in pH-sensitive fluorescent dye compounds
that were stable in an aqueous environment and provided a pKa in
the desired range. As disclosed in and with reference to the
formulae herein, the amino group of the aniline moiety may be
substituted or replaced by another basic moiety of higher pKa.
Advantageously, the addition of a dialkylamino group, wherein each
of the alkyl groups, which may be the same or different, is
independently alkyl or substituted alkyl, to the aniline group at
position R.sup.3 resulted in the unexpected advantage of yielding
pKa values in the physiological range. In certain embodiments, the
dialkylamino group is dimethylamino In certain embodiments, the
dialkylamino group is diethylamino
[0341] In certain embodiments, without wishing to be bound by a
theory, the functioning of the pH-sensitive fluorescent dye
compounds provided herein as a pH indicator is illustrated below in
Scheme 2:
##STR00017##
[0342] wherein X is a fluorophore and R.sup.4 and R.sup.5 are as
described previously herein.
[0343] The EDG is typically at R.sup.3, but may be located at any
position on the aryl group. Electron donating groups of the present
invention include, for example, alkoxy, substituted alkoxy, amino,
substituted amino, dialkylamino, halogen, alkylthio, acylamino, and
(carboxyl ester)oxy. Preferred EDGs are --OCH.sub.3,--NH.sub.2,
--NHCH.sub.3, --N(CH.sub.3).sub.2, and --N(CH.sub.2CH.sub.3).sub.2.
In certain embodiments, Z is O-alkyl. In certain embodiments, Z is
thioalkyl.
[0344] In certain embodiments, novel dye compounds are provided for
use as fluorescent pH sensors, the dye compounds having structural
formula (I):
##STR00018##
[0345] wherein
[0346] R.sup.1 is alkoxy or thioalkyl;
[0347] R.sup.2 and R.sup.6, which may be the same or different, are
each independently H, halogen, --OR.sup.a, --SR.sup.a,
--NR.sup.aR.sup.b, or an electron donating group;
[0348] R.sup.3 is --NR'R'', wherein R' and R'', which may be the
same or different, are each independently alkyl or substituted
alkyl;
[0349] R.sup.4 is selected from the group consisting of alkyl and
substituted alkyl;
[0350] R.sup.5 is selected from the group consisting of alkyl;
substituted alkyl; alkenyl; substituted alkenyl; acyl; aryl;
substituted aryl; carboxyalkyl; heteroaryl; substituted heteroaryl;
heterocyclyl; substituted heterocyclyl; alkylcarboxy;
alkylalkoxycarbonyl; alkylaminocarbonyl; alkylaryloxycarbonyl;
alkylheteroaryl; (CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c; wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sup.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group; -L-R.sub.x; and -L-S.sup.c,
wherein L is a linker, R.sub.x is a reactive group, and S.sub.c is
a conjugated substance;
[0351] R.sup.a is H, alkyl, or substituted alkyl; and
[0352] R.sup.b is alkyl or substituted alkyl.
[0353] In certain embodiments, R.sup.1-R.sup.6 are as follows:
[0354] R.sup.1 is alkoxy or thioalkyl;
[0355] R.sup.2 and R.sup.6, which may be the same or different, are
each independently H or halogen;
[0356] R.sup.3 is --NR'R'', wherein R' and R'', which may be the
same or different, are each independently alkyl;
[0357] R.sup.4 is alkyl; and
[0358] R.sup.5 is selected from the group consisting of alkyl;
substituted alkyl; alkenyl; substituted alkenyl; acyl; aryl;
substituted aryl; carboxyalkyl; heteroaryl; substituted heteroaryl;
heterocyclyl; substituted heterocyclyl; alkylcarboxy;
alkylalkoxycarbonyl; alkylaminocarbonyl; alkylaryloxycarbonyl;
alkylheteroaryl; (CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c; wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group; -L-R.sub.x; and -L-S.sub.c.
[0359] In certain embodiments, R.sup.1-R.sup.6 are as follows:
[0360] R.sup.1 is alkoxy or thioalkyl;
[0361] R.sup.2 and R.sup.6, which may be the same or different, are
each independently H, Cl or F;
[0362] R.sup.3 is --NR'R'', wherein R' and R'', which may be the
same or different, are each independently alkyl;
[0363] R.sup.4 is alkyl; and
[0364] R.sup.5 is selected from the group consisting of alkyl;
substituted alkyl; alkenyl; substituted alkenyl; acyl; aryl;
substituted aryl; carboxyalkyl; heteroaryl; substituted heteroaryl;
heterocyclyl; substituted heterocyclyl; alkylcarboxy;
alkylalkoxycarbonyl; alkylaminocarbonyl; alkylaryloxycarbonyl;
alkylheteroaryl; (CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c; wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group; -L-R.sub.x; and -L-S.sub.c.
[0365] In certain embodiments, R.sup.1-R.sup.6 are as follows:
[0366] R.sup.1 is alkoxy or thioalkyl;
[0367] R.sup.2 and R.sup.6 are each H;
[0368] R.sup.3 is --NR'R'', wherein R' and R'', which may be the
same or different, are each independently alkyl;
[0369] R.sup.4 is alkyl; and
[0370] R.sup.5 is selected from the group consisting of alkyl;
substituted alkyl; alkenyl; substituted alkenyl; acyl; aryl;
substituted aryl; carboxyalkyl; heteroaryl; substituted heteroaryl;
heterocyclyl; substituted heterocyclyl; alkylcarboxy;
alkylalkoxycarbonyl; alkylaminocarbonyl; alkylaryloxycarbonyl;
alkylheteroaryl; (CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c; wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group; -L-R.sub.x; and -L-S.sub.c.
[0371] In certain embodiments, R.sup.1-R.sup.6 are as follows:
[0372] R.sup.1 is methoxy;
[0373] R.sup.2 and R.sup.6 are each H;
[0374] R.sup.3 is --NR'R'', wherein R' and R'', which may be the
same or different, are each independently methyl or ethyl;
[0375] R.sup.4 is methyl or ethyl; and
[0376] R.sup.5 is methyl; ethyl; carboxyalkyl;
(CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c; wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sup.c, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group selected from carboxyl,
carboxylester, amide, maleimide, succinimidyl ester (SE),
sulfodichlorophenol (SDP) ester, sulfotetrafluorophenol (STP)
ester, tetrafluorophenol (TFP) ester, acetoxymethoxy (AM) ester,
nitrilotriacetic acid (NTA), aminodextran, DIBO-amine; -L-R.sub.x;
or -L-S.sub.c.
[0377] In certain embodiments, novel dye compounds are provided for
use as fluorescent pH sensors having structural formula (II):
##STR00019##
[0378] wherein
[0379] R.sup.1 is alkoxy or thioalkyl;
[0380] R.sup.2 and R.sup.6, which may be the same or different, are
each independently H, halogen, --OR.sup.a, 'SR.sup.a,
--NR.sup.aR.sup.b, or an electron donating group;
[0381] R.sup.3 is --NR'R'', wherein R' and R'', which may be the
same or different, are each independently alkyl or substituted
alkyl;
[0382] R.sup.4 is selected from the group consisting of alkyl and
substituted alkyl;
[0383] R.sup.5 is selected from the group consisting of alkyl;
substituted alkyl; alkenyl; substituted alkenyl; acyl; aryl;
substituted aryl; carboxyalkyl; heteroaryl; substituted heteroaryl;
heterocyclyl; substituted heterocyclyl; alkylcarboxy;
alkylalkoxycarbonyl; alkylaminocarbonyl; alkylaryloxycarbonyl;
alkylheteroaryl; (CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c; wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group; -L-R.sub.x; and -L-S.sub.c,
wherein L is a linker, R.sub.x is a reactive group, and S.sub.c is
a conjugated substance;
[0384] R.sup.a is H, alkyl, or substituted alkyl; and
[0385] R.sup.b is alkyl or substituted alkyl.
[0386] In certain embodiments, R.sup.1-R.sup.6 are as follows:
[0387] R.sup.1 is alkoxy or thioalkyl;
[0388] R.sup.2 and R.sup.6, which may be the same or different, are
each independently H or halogen;
[0389] R.sup.3 is --NR'R'', wherein R' and R'', which may be the
same or different, are each independently alkyl;
[0390] R.sup.4 is alkyl; and
[0391] R.sup.5 is selected from the group consisting of alkyl;
substituted alkyl; alkenyl; substituted alkenyl; acyl; aryl;
substituted aryl; carboxyalkyl; heteroaryl; substituted heteroaryl;
heterocyclyl; substituted heterocyclyl; alkylcarboxy;
alkylalkoxycarbonyl; alkylaminocarbonyl; alkylaryloxycarbonyl;
alkylheteroaryl; (CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c;wherein n is
an integer from 1 to 6, and R and R.sup.c, which may be the same or
different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group; -L-R.sub.x; and -L-S.sub.c.
[0392] In certain embodiments, R.sup.1-R.sup.6 are as follows:
[0393] R.sup.1 is alkoxy or thioalkyl;
[0394] R.sup.2 and R.sup.6, which may be the same or different, are
each independently H, Cl or F;
[0395] R.sup.3 is --NR'R'', wherein R' and R'', which may be the
same or different, are each independently alkyl;
[0396] R.sup.4 is alkyl; and
[0397] R.sup.5 is selected from the group consisting of alkyl;
substituted alkyl; alkenyl; substituted alkenyl; acyl; aryl;
substituted aryl; carboxyalkyl; heteroaryl; substituted heteroaryl;
heterocyclyl; substituted heterocyclyl; alkylcarboxy;
alkylalkoxycarbonyl; alkylaminocarbonyl; alkylaryloxycarbonyl;
alkylheteroaryl; (CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c; wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group; -L-R.sub.x; and -L-S.sub.c.
[0398] In certain embodiments, R.sup.1-R.sup.6 are as follows:
[0399] R.sup.1 is alkoxy or thioalkyl;
[0400] R.sup.2 and R.sup.6 are each H;
[0401] R.sup.3 is --NR'R'', wherein R' and R'', which may be the
same or different, are each independently alkyl;
[0402] R.sup.4 is alkyl; and
[0403] R.sup.5 is selected from the group consisting of alkyl;
substituted alkyl; alkenyl; substituted alkenyl; acyl; aryl;
substituted aryl; carboxyalkyl; heteroaryl; substituted heteroaryl;
heterocyclyl; substituted heterocyclyl; alkylcarboxy;
alkylalkoxycarbonyl; alkylaminocarbonyl; alkylaryloxycarbonyl;
alkylheteroaryl; (CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c; wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group; -L-R.sub.x; and -L-S.sub.c.
[0404] In certain embodiments, R'-R.sup.6 are as follows:
[0405] R.sup.1 is methoxy;
[0406] R.sup.2 and R.sup.6 are each H;
[0407] R.sup.3 is --NR'R'', wherein R' and R'', which may be the
same or different, are each independently methyl or ethyl;
[0408] R.sup.4 is methyl or ethyl; and
[0409] R.sup.5 is methyl; ethyl; carboxyalkyl;
(CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c; wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sup.c, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group selected from carboxyl,
carboxylester, amide, maleimide, succinimidyl ester (SE),
sulfodichlorophenol (SDP) ester, sulfotetrafluorophenol (STP)
ester, tetrafluorophenol (TFP) ester, acetoxymethoxy (AM) ester,
nitrilotriacetic acid (NTA), aminodextran, DIBO-amine; -L-R.sub.x;
or -L-S.sub.c.
[0410] In certain embodiments, the EDG is selected from the group
consisting of alkoxy, substituted alkoxy, amino, substituted amino,
halogen, alkylthio, acylamino, and (carboxyl ester)oxy. In certain
embodiments, the EDG is not hydroxy or thiol. In certain
embodiments, the EDG is a dialkylamino group. In certain
embodiments, the dialkylamino group is dimethylamino or
diethylamino
[0411] In certain embodiments, R.sup.1 is --OCH.sub.3 and R.sup.3
is --N(CH.sub.3).sub.2 or --N(CH.sub.2CH.sub.3).sub.2.
[0412] In certain embodiments, R.sup.4 and R.sup.5 are alkyl or
substituted alkyl. In certain embodiments, R.sup.5 is methyl;
ethyl; carboxyalkyl; (CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c; wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group selected from carboxyl,
carboxylester, amide, maleimide, succinimidyl ester (SE),
sulfodichlorophenol (SDP) ester, sulfotetrafluorophenol (STP)
ester, tetrafluorophenol (TFP) ester, acetoxymethoxy (AM) ester,
nitrilotriacetic acid (NTA), aminodextran, DIBO-amine; -L-R.sub.x;
or -L-S.sub.c.
[0413] In certain embodiments of any of the previous embodiments,
the pKa of the pH-sensitive fluorescent dye compound is about 5 to
about 8. In certain embodiments, the pKa of the pH-sensitive
fluorescent dye compound is about 6 to about 8. In certain
embodiments, the pKa of the pH-sensitive fluorescent dye compound
is about 6 to about 7. In certain embodiments, the pKa of the
pH-sensitive fluorescent dye compound is about 6.5. In certain
embodiments, the pKa of the pH-sensitive fluorescent dye compound
is about 3 to about 10.
[0414] The 4-position nitrogen of the aniline or aniline-like ring
of the compounds disclosed herein does of course always have a
permissible valency.
[0415] Reactive Groups:
[0416] In certain embodiments, the pH-sensitive fluorescent dye
compounds provided herein are chemically reactive, and are
substituted by at least one reactive group (R.sub.x). The reactive
group functions as the site of attachment for another moiety, such
as a carrier molecule or a solid support, wherein the reactive
group chemically reacts with an appropriate reactive or functional
group on the carrier molecule or solid support. Thus, in certain
embodiments, the pH-sensitive fluorescent dye compounds provided
herein comprise an aniline moiety, linker, fluorophore, a reactive
group moiety and optionally a carrier molecule and/or a solid
support.
[0417] In certain embodiments, the pH-sensitive fluorescent dye
compounds provided herein further comprise a reactive group which
is a member selected from an acrylamide, an activated ester of a
carboxylic acid, a carboxylic ester, an acyl azide, an acyl
nitrile, an aldehyde, an alkyl halide, an anhydride, an aniline, an
amine, an aryl halide, an azide, an aziridine, a boronate, a
diazoalkane, a haloacetamide, a haloalkyl, a halotriazine, a
hydrazine, an imido ester, an isocyanate, an isothiocyanate, a
maleimide, a phosphoramidite, a photoactivatable group, a reactive
platinum complex, a silyl halide, a sulfonyl halide, and a thiol.
In certain embodiments the reactive group is selected from the
group consisting of carboxylic acid, succinimidyl ester of a
carboxylic acid, hydrazide, amine and a maleimide. The reactive
group may be attached to any appropriate site on the reporter
molecule or the aniline moiety. In certain embodiments, at least
one member selected from R.sup.1, R.sup.2, R.sup.3, R.sup.4,
R.sup.5, and R.sup.6 is a reactive group. Preferably, at least one
of R.sup.3, R.sup.4, R.sup.5, and R.sup.6 is a reactive group, most
preferred is at least one of R.sup.4 or R.sup.5. Alternatively, if
the pH-sensitive fluorescent dye compounds disclosed herein
comprise a carrier molecule or solid support a reactive group may
be covalently attached independently to those substituents,
allowing for further conjugation to a fluorophore, carrier molecule
or solid support.
[0418] These reactive groups are synthesized during the formation
of the pH-sensitive fluorescent dye compounds provided herein and
carrier molecule- and/or solid support-containing compounds to
provide chemically reactive pH-sensitive fluorescent dye compounds.
In this way, pH-sensitive fluorescent dye compounds incorporating a
reactive group may be covalently attached to a wide variety of
carrier molecules or solid supports that contain, or are modified
to contain, functional groups with suitable reactivity, resulting
in chemical attachment of the components. In certain embodiments,
the reactive group of the pH-sensitive fluorescent dye compounds
disclosed herein and the functional group of the carrier molecule
or solid support comprise electrophiles and nucleophiles that can
generate a covalent linkage between them. In certain embodiments,
the reactive group comprises a photoactivatable group, which
becomes chemically reactive only after illumination with light of
an appropriate wavelength. Typically, the conjugation reaction
between the reactive group and the carrier molecule or solid
support results in one or more atoms of the reactive group being
incorporated into a new linkage attaching the pH-sensitive
fluorescent dye compounds disclosed herein to the carrier molecule
or solid support. Selected examples of functional groups and
linkages are shown in Table 2, where the reaction of an
electrophilic group and a nucleophilic group yields a covalent
linkage.
TABLE-US-00002 TABLE 2 Examples of some routes to useful covalent
linkages Electrophilic Group Nucleophilic Group Resulting Covalent
Linkage activated esters* amines/anilines carboxamides acrylamides
thiols thioethers acyl azides** amines/anilines carboxamides acyl
halides amines/anilines carboxamides acyl halides alcohols/phenols
esters acyl nitriles alcohols/phenols esters acyl nitriles
amines/anilines carboxamides aldehydes amines/anilines imines
aldehydes or ketones hydrazines hydrazones aldehydes or ketones
hydroxylamines oximes alkyl halides amines/anilines alkyl amines
alkyl halides carboxylic acids esters alkyl halides thiols
thioethers alkyl halides alcohols/phenols ethers alkyl sulfonates
thiols thioethers alkyl sulfonates carboxylic acids esters alkyl
sulfonates alcohols/phenols ethers anhydrides alcohols/phenols
esters anhydrides amines/anilines carboxamides aryl halides thiols
thiophenols aryl halides amines aryl amines aziridines thiols
thioethers boronates glycols boronate esters carbodiimides
carboxylic acids N-acylureas or anhydrides diazoalkanes carboxylic
acids esters epoxides thiols thioethers haloacetamides thiols
thioethers haloplatinate amino platinum complex haloplatinate
heterocycle platinum complex haloplatinate thiol platinum complex
halotriazines amines/anilines aminotriazines halotriazines
alcohols/phenols triazinyl ethers halotriazines thiols triazinyl
thioethers imido esters amines/anilines amidines isocyanates
amines/anilines ureas isocyanates alcohols/phenols urethanes
isothiocyanates amines/anilines thioureas maleimides thiols
thioethers phosphoramidites alcohols phosphite esters silyl halides
alcohols silyl ethers sulfonate esters amines/anilines alkyl amines
sulfonate esters thiols thioethers sulfonate esters carboxylic
acids esters sulfonate esters alcohols ethers sulfonyl halides
amines/anilines sulfonamides sulfonyl halides phenols/alcohols
sulfonate esters *Activated esters, as understood in the art,
generally have the formula --CO.OMEGA., where .OMEGA. is a suitable
leaving group (e.g., succinimidyloxy (--OC.sub.4H.sub.4O.sub.2),
sulfosuccinimidyloxy (--OC.sub.4H.sub.3O.sub.2--SO.sub.3H),
-1-oxybenzotriazolyl (--OC.sub.6H.sub.4N.sub.3); or an aryloxy
group or aryloxy substituted one or more times by electron
withdrawing substituents such as nitro, fluoro, chloro, cyano, or
trifluoromethyl, or combinations thereof, used to form activated
aryl esters; or a carboxylic acid activated by a carbodiimide to
form an anhydride or mixed anhydride --OCOR.sup.x or
--OCNR.sup.xNHR.sup.y, where R.sup.x and R.sup.y, which may be the
same or different, are C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6
perfluoroalkyl, or C.sub.1-C.sub.6 alkoxy; or cyclohexyl,
3-dimethylaminopropyl, or N-morpholinoethyl). **Acyl azides can
also rearrange to isocyanates.
[0419] The choice of the reactive group used to attach the
pH-sensitive fluorescent dye compounds disclosed herein to the
substance to be conjugated typically depends on the reactive or
functional group on the substance to be conjugated and the type or
length of covalent linkage desired. The types of functional groups
typically present on the organic or inorganic substances
(biomolecule or non-biomolecule) include, but are not limited to,
amines, amides, thiols, alcohols, phenols, aldehydes, ketones,
phosphates, imidazoles, hydrazines, hydroxylamines, disubstituted
amines, halides, epoxides, silyl halides, carboxylate esters,
sulfonate esters, purines, pyrimidines, carboxylic acids, olefinic
bonds, or a combination of these groups. A single type of reactive
site may be available on the substance (typical for polysaccharides
or silica), or a variety of sites may occur (e g , amines, thiols,
alcohols, phenols), as is typical for proteins.
[0420] Typically, the reactive group will react with an amine, a
thiol, an alcohol, an aldehyde, a ketone, or with silica.
Preferably, reactive groups react with an amine or a thiol
functional group, or with silica. In certain embodiments, the
reactive group is an acrylamide, an activated ester of a carboxylic
acid, an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide,
a silyl halide, an anhydride, an aniline, an aryl halide, an azide,
an aziridine, a boronate, a diazoalkane, a haloacetamide, a
halotriazine, a hydrazine (including hydrazides), an imido ester,
an isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a
reactive platinum complex, a sulfonyl halide, or a thiol group. As
used herein, "reactive platinum complex" refers to chemically
reactive platinum complexes such as described in U.S. Pat. No.
5,714,327, herein incorporated by reference in its entirety.
[0421] In certain embodiments, the pH-sensitive fluorescent dye
compounds disclosed herein comprise at least one reactive group
that selectively reacts with an amine group. This amine-reactive
group is selected from the group consisting of succinimidyl ester
(SE), sulfonyl halide, tetrafluorophenyl (TFP) ester,
sulfodichlorophenol (SDP) ester, sulfotetrafluorophenol (STP)
ester, acetoxymethoxy (AM) ester, nitrilotriacetic acid (NTA),
aminodextran, DIBO-amine and iosothiocyanates. Thus, in certain
embodiments, the pH-sensitive fluorescent dye compounds provided
herein form a covalent bond with an amine containing molecule in a
sample. In certain embodiments, the pH-sensitive fluorescent dye
compounds provided herein comprise at least one reactive group that
selectively reacts with a thiol group. This thiol-reactive group is
selected from the group consisting of maleimide, haloalkyl and
haloacetamide (including any reactive groups disclosed in U.S. Pat.
Nos. 5,362,628; 5,352,803 and 5,573,904, all of which are herein
incorporated by reference in their entirety).
[0422] Where the reactive group is an activated ester of a
carboxylic acid, such as a succinimidyl ester of a carboxylic acid,
a sulfonyl halide, tetrafluorophenyl (TFP) ester,
sulfodichlorophenol (SDP) ester, sulfotetrafluorophenol (STP)
ester, acetoxymethoxy (AM) ester, nitrilotriacetic acid (NTA),
aminodextran, DIBO-amine or an isothiocyanate, the resulting
pH-sensitive fluorescent dye compound is particularly useful for
preparing conjugates of carrier molecules such as proteins,
nucleotides, oligonucleotides, or haptens. Where the reactive group
is a maleimide, haloalkyl or haloacetamide (including any reactive
groups disclosed in U.S. Pat. Nos. 5,362,628; 5,352,803 and
5,573,904, all of which are herein incorporated by reference in
their entirety) the resulting compound is particularly useful for
conjugation to thiol-containing substances. Where the reactive
group is a hydrazide, the resulting compound is particularly useful
for conjugation to periodate-oxidized carbohydrates and
glycoproteins, and in addition is an aldehyde-fixable polar tracer
for cell microinjection. Where the reactive group is a silyl
halide, the resulting compound is particularly useful for
conjugation to silica surfaces, particularly where the silica
surface is incorporated into a fiber optic probe subsequently used
for remote ion detection or quantitation.
[0423] In a certain embodiments, the reactive group is a
photoactivatable group such that the group is only converted to a
reactive species after illumination with an appropriate wavelength.
An appropriate wavelength is generally a UV wavelength that is less
than 400 nm. This method provides for specific attachment to only
the target molecules, either in solution or immobilized on a solid
or semi-solid matrix. Photoactivatable reactive groups include,
without limitation, benzophenones, aryl azides and diazirines.
[0424] Preferably, the reactive group is a photoactivatable group,
succinimidyl ester of a carboxylic acid, a haloacetamide,
haloalkyl, a hydrazine, an isothiocyanate, a maleimide group, an
aliphatic amine, a silyl halide, a cadaverine or a psoralen. More
preferably, the reactive group is a succinimidyl ester of a
carboxylic acid, a maleimide, an iodoacetamide, or a silyl halide.
In certain embodiments, the reactive group is a succinimidyl ester
of a carboxylic acid, a sulfonyl halide, a tetrafluorophenyl ester,
an iosothiocyanates or a maleimide. In certain embodiments, the
reactive group is selected from sulfodichlorophenyl (SDP) ester,
sulfotetrafluorophenol (STP) ester, tetrafluorophenol (TFP) ester,
an acetoxymethoxy (AM) ester, and a nitrilotriacetic acid
(NTA).
[0425] Carrier Molecules:
[0426] In certain embodiments, the pH-sensitive fluorescent dye
compounds provided herein are covalently bound to a carrier
molecule. If the pH-sensitive fluorescent dye compound has a
reactive group, then the carrier molecule may alternatively be
linked to the pH-sensitive fluorescent dye compound through the
reactive group. The reactive group may contain both a reactive
functional moiety and a linker, or only the reactive functional
moiety.
[0427] A variety of carrier molecules are useful herein. Exemplary
carrier molecules include antigens, steroids, vitamins, drugs,
haptens, metabolites, toxins, environmental pollutants, amino
acids, peptides, proteins, nucleic acids, nucleic acid polymers,
carbohydrates, lipids, polymers and bacterial particles. In certain
embodiments, at least one member selected from R.sup.1, R.sup.2,
R.sup.3, R.sup.4, R.sup.5, and R.sup.6 is a carrier molecule.
Preferably, at least one of R.sup.3, R.sup.4, R.sup.5, and R.sup.6
is a carrier molecule, most preferred is at least one of R.sup.4 or
R.sup.5.
[0428] In certain embodiments, the carrier molecule comprises an
amino acid, a peptide, a protein, a polysaccharide, a nucleoside, a
nucleotide, an oligonucleotide, a nucleic acid, a hapten, a
psoralen, a drug, a hormone, a lipid, a lipid assembly, a synthetic
polymer, a polymeric microparticle, a biological cell, a virus and
combinations thereof. In certain embodiments, the carrier molecule
is selected from a hapten, a nucleotide, an oligonucleotide, a
nucleic acid polymer, a protein, a peptide or a polysaccharide. In
certain embodiments the carrier molecule is amino acid, a peptide,
a protein, a polysaccharide, a nucleoside, a nucleotide, an
oligonucleotide, a nucleic acid, a hapten, a psoralen, a drug, a
hormone, a lipid, a lipid assembly, a tyramine, a synthetic
polymer, a polymeric microparticle, a biological cell, cellular
components, an ion chelating moiety, an enzymatic substrate or a
virus. In certain embodiments, the carrier molecule is an antibody
or fragment thereof, an antigen, an avidin or streptavidin, a
biotin, a dextran, an IgG binding protein, a fluorescent protein,
agarose, and a non-biological microparticle. In certain
embodiments, carrier molecules may comprise a label or a
fluorescent dye or quencher.
[0429] In certain embodiments, the carrier molecule is an amino
acid (including those that are protected or are substituted by
phosphates, carbohydrates, or C.sub.1 to C.sub.22 carboxylic
acids), or a polymer of amino acids such as a peptide or protein.
In certain embodiments, the carrier molecule contains at least five
amino acids, more preferably 5 to 36 amino acids. Exemplary
peptides include, but are not limited to, neuropeptides, cytokines,
toxins, protease substrates, and protein kinase substrates. Other
exemplary peptides may function as organelle localization peptides,
that is, peptides that serve to target the conjugated compound for
localization within a particular cellular substructure by cellular
transport mechanisms. Preferred protein carrier molecules include
enzymes, antibodies, lectins, glycoproteins, histones, albumins,
lipoproteins, avidin, streptavidin, protein A, protein G,
phycobiliproteins and other fluorescent proteins, hormones, toxins
and growth factors. Typically, the protein carrier molecule is an
antibody, an antibody fragment, avidin, streptavidin, a toxin, a
lectin, a growth factor, bacterial particle or a binding partner
for a cell receptor.
[0430] In certain embodiments, the carrier molecule comprises a
nucleic acid base, nucleoside, nucleotide or a nucleic acid
polymer, optionally containing an additional linker or spacer for
attachment of a fluorophore or other ligand, such as an alkynyl
linkage (U.S. Pat. No. 5,047,519), an aminoallyl linkage (U.S. Pat.
No. 4,711,955) or other linkage. In certain embodiments, the
nucleotide carrier molecule is a nucleoside or a deoxynucleoside or
a dideoxynucleoside.
[0431] Exemplary nucleic acid polymer carrier molecules are single-
or multi-stranded, natural or synthetic DNA or RNA
oligonucleotides, or DNA/RNA hybrids, or incorporating an unusual
linker such as morpholine derivatized phosphates (AntiVirals, Inc.,
Corvallis Oreg.), or peptide nucleic acids such as
N-(2-aminoethyl)glycine units, where the nucleic acid contains
fewer than 50 nucleotides, more typically fewer than 25
nucleotides.
[0432] In certain embodiments, the carrier molecule comprises a
carbohydrate or polyol that is typically a polysaccharide, such as
dextran, FICOLL.RTM., heparin, glycogen, amylopectin, mannan,
inulin, starch, agarose and cellulose, or is a polymer such as a
poly(ethylene glycol). In certain embodiments, the polysaccharide
carrier molecule includes dextran, agarose or FICOLL.RTM..
[0433] In certain embodiments, the carrier molecule comprises a
lipid (typically having 6-25 carbons), including glycolipids,
phospholipids, and sphingolipids. In certain embodiments, the
carrier molecule comprises a lipid vesicle, such as a liposome, or
is a lipoprotein. Some lipophilic substituents are useful for
facilitating transport of the conjugated dye into cells or cellular
organelles.
[0434] In certain embodiments, the carrier molecule is a cell,
cellular system, cellular fragment, or subcellular particles,
including virus particles, bacterial particles, virus components,
biological cells (such as animal cells, plant cells, bacteria, or
yeast), or cellular components. Examples of cellular components
that are useful as carrier molecules include lysosomes, endosomes,
cytoplasm, nuclei, histones, mitochondria, Golgi apparatus,
endoplasmic reticulum and vacuoles.
[0435] In certain embodiments, the carrier molecule non-covalently
associates with organic or inorganic materials. Exemplary
embodiments of the carrier molecule that possess a lipophilic
substituent may be used to target lipid assemblies such as
biological membranes or liposomes by non-covalent incorporation of
the pH-sensitive fluorescent dye compound within the membrane,
e.g., for use as probes for membrane structure or for incorporation
in liposomes, lipoproteins, films, plastics, lipophilic
microspheres or similar materials.
[0436] In certain embodiments, the carrier molecule comprises a
specific binding pair member wherein the pH-sensitive fluorescent
dye compounds provided herein are conjugated to a specific binding
pair member and used to the formation of the bound pair.
Alternatively, the presence of the labeled specific binding pair
member indicates the location of the complementary member of that
specific binding pair; each specific binding pair member having an
area on the surface or in a cavity which specifically binds to, and
is complementary with, a particular spatial and polar organization
of the other. In this instance, the dye compounds disclosed herein
function as a reporter molecule for the specific binding pair.
Exemplary binding pairs are set forth in Table 3.
TABLE-US-00003 TABLE 3 Representative Specific Binding Pairs
Antigen Antibody biotin avidin (or streptavidin or anti-biotin)
IgG* protein A or protein G drug drug receptor folate folate
binding protein toxin toxin receptor carbohydrate lectin or
carbohydrate receptor peptide peptide receptor protein protein
receptor enzyme substrate enzyme DNA (RNA) cDNA (cRNA).sup..dagger.
hormone hormone receptor ion chelator *IgG is an immunoglobulin
.sup..dagger.cDNA and cRNA are the complementary strands used for
hybridization
[0437] Solid Supports:
[0438] In certain embodiments, the pH-sensitive dye compounds
disclosed herein are covalently bonded to a solid support. The
solid support may be attached to the dye compounds either through
the aniline moiety, fluorophore, or through a reactive group, if
present, or through a carrier molecule, if present. Even if a
reactive group and/or a carrier molecule are present, the solid
support may be attached through the aniline moiety or fluorophore.
In certain embodiments, at least one member selected from R.sup.1,
R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 is a solid support.
Preferably, at least one of R.sup.3, R.sup.4, R.sup.5, and R.sup.6
is a solid support, most preferred is at least one of R.sup.4 or
R.sup.5.
[0439] Solid supports suitable for use herein are typically
substantially insoluble in liquid phases. Solid supports for use
herein are not limited to a specific type of support. Rather, a
large number of supports are available and are known to one of
ordinary skill in the art. Thus, useful solid supports include
solid and semi-solid matrixes, such as aerogels and hydrogels,
resins, beads, biochips (including thin film coated biochips),
microfluidic chip, a silicon chip, multi-well plates (also referred
to as microtitre plates or microplates), membranes, conducting and
nonconducting metals, glass (including microscope slides) and
magnetic supports. More specific examples of useful solid supports
include silica gels, polymeric membranes, particles, derivatized
plastic films, glass beads, cotton, plastic beads, alumina gels,
polysaccharides such as Sepharose.RTM., poly(acrylate),
polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose,
dextran, starch, FICOLL.RTM., heparin, glycogen, amylopectin,
mannan, inulin, nitrocellulose, diazocellulose, polyvinylchloride,
polypropylene, polyethylene (including poly(ethylene glycol)),
nylon, latex bead, magnetic bead, paramagnetic bead,
superparamagnetic bead, starch and the like.
[0440] In certain embodiments, the solid support may include a
solid support reactive functional group, including, but not limited
to, hydroxyl, carboxyl, amino, thiol, aldehyde, halogen, nitro,
cyano, amido, urea, carbonate, carbamate, isocyanate, sulfone,
sulfonate, sulfonamide, sulfoxide, etc., for attaching the dye
compounds disclosed herein. Useful reactive groups are disclosed
above and are equally applicable to the solid support reactive
functional groups herein.
[0441] A suitable solid phase support may be selected on the basis
of desired end use and suitability for various synthetic protocols.
For example, where amide bond formation is desirable to attach the
pH-sensitive fluorescent dye compounds disclosed herein to the
solid support, resins generally useful in peptide synthesis may be
employed, such as polystyrene (e.g., PAM-resin obtained from Bachem
Inc., Peninsula Laboratories, etc.), POLYHIPE.TM. resin (obtained
from Aminotech, Canada), polyamide resin (obtained from Peninsula
Laboratories), polystyrene resin grafted with polyethylene glycol
(TentaGel.TM., Rapp Polymere, Tubingen, Germany),
polydimethyl-acrylamide resin (available from Milligen/Biosearch,
California), or PEGA beads (obtained from Polymer
Laboratories).
[0442] Preparation of Conjugates:
[0443] In certain embodiments, conjugates of the pH-sensitive
fluorescent dye compounds disclosed herein are provided. Conjugates
of components (carrier molecules or solid supports), e.g., drugs,
peptides, toxins, nucleotides, phospholipids, proteins and other
organic molecules are prepared by organic synthesis methods using
the pH-sensitive fluorescent dye compounds disclosed herein, are
generally prepared by means well recognized in the art (Haugland,
MOLECULAR PROBES HANDBOOK, supra, (2002)). Preferably, conjugation
to form a covalent bond consists of mixing the pH-sensitive
fluorescent dye compounds disclosed herein in a suitable solvent in
which both the pH-sensitive fluorescent dye compound and the
substance to be conjugated are soluble. The reaction preferably
proceeds spontaneously without added reagents at room temperature
or below. For those reactive compounds that are photoactivated,
conjugation is facilitated by illumination of the reaction mixture
to activate the reactive compound. Chemical modification of
water-insoluble substances, so that a desired compound-conjugate
may be prepared, is preferably performed in an aprotic solvent such
as dimethylformamide, dimethylsulfoxide, acetone, ethyl acetate,
toluene, or chloroform. Similar modification of water-soluble
materials is readily accomplished through the use of the instant
reactive compounds to make them more readily soluble in organic
solvents.
[0444] Preparation of peptide or protein conjugates typically
comprises first dissolving the protein to be conjugated in aqueous
buffer at about 1-10 mg/mL at room temperature or below.
Bicarbonate buffers (pH about 8.3) are especially suitable for
reaction with succinimidyl esters, phosphate buffers (pH about
7.2-8) for reaction with thiol-reactive functional groups and
carbonate or borate buffers (pH about 9) for reaction with
isothiocyanates and dichlorotriazines. The appropriate reactive
compound is then dissolved in a nonhydroxylic solvent (usually DMSO
or DMF) in an amount sufficient to give a suitable degree of
conjugation when added to a solution of the protein to be
conjugated. The appropriate amount of compound for any protein or
other component is conveniently predetermined by experimentation in
which variable amounts of the dye compound are added to the
protein, the conjugate is chromatographically purified to separate
unconjugated dye compound and the dye compound-protein conjugate is
tested in its desired application.
[0445] Following addition of the pH-sensitive fluorescent dye
compound to the component solution, the mixture is incubated for a
suitable period (typically about 1 hour at room temperature to
several hours on ice), the excess pH-sensitive fluorescent dye
compound is removed by gel filtration, dialysis, HPLC, adsorption
on an ion exchange or hydrophobic polymer or other suitable means.
The pH-sensitive fluorescent dye compound-conjugate may be used in
solution or lyophilized. In this way, suitable conjugates may be
prepared from antibodies, antibody fragments, avidins, lectins,
enzymes, proteins A and G, cellular proteins, albumins, histones,
growth factors, hormones, and other proteins.
[0446] Conjugates of polymers, including biopolymers and other
higher molecular weight polymers are typically prepared by means
well recognized in the art (for example, Brinkley et al.,
Bioconjugate Chem., 3:2 (1992)). In these embodiments, a single
type of reactive site may be available, as is typical for
polysaccharides) or multiple types of reactive sites (e g amines,
thiols, alcohols, phenols) may be available, as is typical for
proteins. Selectivity of labeling is best obtained by selection of
an appropriate reactive dye compound. For example, modification of
thiols with a thiol-selective reagent such as a haloacetamide or
maleimide, or modification of amines with an amine-reactive reagent
such as an activated ester, acyl azide, isothiocyanate or
3,5-dichloro-2,4,6-triazine. Partial selectivity may also be
obtained by careful control of the reaction conditions.
[0447] When modifying polymers with the pH-sensitive fluorescent
dye compounds disclosed herein, an excess of pH-sensitive
fluorescent dye compound is typically used, relative to the
expected degree of pH-sensitive fluorescent dye compound
substitution. Any residual, unreacted pH-sensitive fluorescent dye
compound or a pH-sensitive fluorescent dye compound hydrolysis
product is typically removed by dialysis, chromatography or
precipitation. Presence of residual, unconjugated dye may be
detected by thin layer chromatography using a solvent that elutes
the dye away from its conjugate. In all cases it is usually
preferred that the reagents be kept as concentrated as practical so
as to obtain adequate rates of conjugation.
[0448] In certain embodiments, the dye-conjugates disclosed herein
are associated with an additional substance, that binds either to
the fluorophore or the conjugated substance (carrier molecule or
solid support) through noncovalent interaction. In another
exemplary embodiment, the additional substance is an antibody, an
enzyme, a hapten, a lectin, a receptor, an oligonucleotide, a
nucleic acid, a liposome, or a polymer. The additional substance is
optionally used to probe for the location of the dye-conjugate, for
example, as a means of enhancing the signal of the
dye-conjugate.
[0449] In certain embodiments, compositions are provided for
determining the pH of a sample, the compositions comprising:
[0450] a) one or more of the pH-sensitive fluorescent dye compounds
described herein; and
[0451] b) a carrier,
[0452] wherein the one or more of the pH-sensitive fluorescent dye
compounds are present in an amount effective to detect the pH of
the sample.
[0453] In certain embodiments, compositions are provided for
determining the pH of a sample, the compositions comprising:
[0454] (a) one or more of the pH-sensitive fluorescent dye
compounds described herein; and
[0455] (b) an analyte,
[0456] wherein the one or more of the pH-sensitive fluorescent dye
compounds are present in an amount effective to detect the pH of
the sample.
[0457] In certain embodiments, the analyte is a cell and the
pH-sensitive fluorescent dye compound is located inside the cell.
In certain embodiments, the analyte is a protein, lipid or nucleic
acid. In certain embodiments, the pH-sensitive fluorescent dye
compound is conjugated to a carrier molecule.
[0458] Methods:
[0459] In certain embodiments, the pH-sensitive fluorescent dye
compounds, dye-conjugates and compositions provided herein may be
used in methods including, but not limited to, methods to determine
the pH of living cells or cell compartments, to determine a change
in pH to the local environment caused by a cell, and directly and
indirectly detect specific cellular events associated with a change
in pH. In certain embodiments, the methods involve detecting
contamination in cell culture or on agar plates. For the sake of
clarity, the sample may also include material other than live cells
and cell compartments such as, but not limited to, cell culture
medium, biological fluids, diagnostic materials, and bacterial
medium such as agar plates. As used herein, the term "a cell
compartment" refers to one of the many organelles suspended in the
cell cytoplasm. The pH of a cell or cell compartment may be
measured by introducing one or more of the pH-sensitive fluorescent
dye compounds, dye conjugates or compositions provided herein into
a cell or cell compartment, irradiating the dye or dye conjugate
with a suitable light source, and observing the intensity of
fluorescence of the dye or conjugate. The observed fluorescence
intensity may then be used to determine pH by a variety of methods
known in the field, selected according to the method of
accumulation of the dye compound or dye conjugate. For instance,
the observed fluorescence may be compared to a known standard, for
example a calibration curve of fluorescence intensity versus pH, or
to fluorescence intensity measurements indicative of the total
pH-sensitive fluorescent dye compound, dye conjugate, or
composition present. Any conventional fluorimetric equipment may be
used to irradiate the sample, and to measure the resulting
fluorescent response.
[0460] As stated above the sample may comprise live cells,
intracellular fluids, extracellular fluids, biological fluids,
sera, biological fermentation media, environmental sample,
industrial samples, proteins, peptides, buffer solutions,
biological fluids or chemical reactors, blood cells, immune cells,
cultured cells, muscle tissue, neurons, extracellular vesicles;
vascular tissue, blood fluids, saliva, urine, water, soil, waste
water, sea water; pharmaceuticals, foodstuffs or beverages. In
certain embodiments, the sample is immobilized on a polymeric
membrane, within a polymeric gel, on a microparticle, on a
microarray, on a silicon chip, on a glass slide, on a microwell
plate, and on a microfluidic chip.
[0461] The pH-sensitive fluorescent dye compounds disclosed herein
may therefore be used as pH sensors in relation to samples
comprising or suspected of comprising a biological entity or
biological substance. The pH-sensitive fluorescent dye compounds
disclosed herein may be used in assays involving a biological
entity or biological substance. In certain embodiments, the
invention provides for the use of the pH-sensitive fluorescent dye
compounds in a biological assay for the purposes described herein,
particularly as a pH sensor.
[0462] Thus, in certain embodiments, the methods disclosed herein
comprise determining the pH of a sample, wherein the methods
comprise:
[0463] (a) contacting the sample with one or more of the
pH-sensitive fluorescent dye compounds disclosed herein, to form a
contacted sample;
[0464] (b) incubating the contacted sample for an appropriate
amount of time to form an incubated sample;
[0465] (c) illuminating the incubated sample with an appropriate
wavelength to form an illuminated sample; and
[0466] (d) detecting fluorescent emissions from the illuminated
sample;
[0467] wherein the fluorescent emissions are used to determine the
pH of the sample.
[0468] In certain embodiments, the methods disclosed herein
comprise determining the pH of a sample, wherein the methods
comprise:
[0469] (a) contacting the sample with one or more of the
compositions provided herein to form a contacted sample;
[0470] (b) incubating the contacted sample for an appropriate
amount of time to form an incubated sample;
[0471] (c) illuminating the incubated sample with an appropriate
wavelength to form an illuminated sample; and
[0472] (d) detecting fluorescent emissions from the illuminated
sample;
[0473] wherein the fluorescent emissions are used to determine the
pH of the sample.
[0474] In certain embodiments, the pH-sensitive fluorescent dye
compounds disclosed herein are used in cell culture for detection
of contamination. In certain embodiments, the pH-sensitive
fluorescent dye compounds disclosed herein are used in or on agar
plates for the detection of contamination.
[0475] In certain embodiments, a change in the pH inside the cell
corresponds to a cellular process. In certain embodiments, the
pH-sensitive fluorescent dye compound is conjugated to a protein,
nucleic acid or lipid. In certain embodiments, the pH-sensitive
fluorescent dye compound is conjugated to transferrin. In certain
embodiments, the pH-sensitive fluorescent dye compound is
conjugated to a carrier molecule through a succinimidyl ester. In
certain embodiments the pH-sensitive fluorescent dye compound is
conjugated to epithelial growth factor (EGF) or EGF receptor
(EGFR). In certain embodiments the pH-sensitive fluorescent dye
compound is non-fluorescent before entering the cell. More
particularly, the pH-sensitive fluorescent dye compound becomes
fluorescent after entering the cell. In certain embodiments, the
pH-sensitive fluorescent dye compound enters the cell through
phagocytosis. In certain embodiments, the pH-sensitive fluorescent
dye compound enters the cell through receptor-mediated
endocytosis.
[0476] In certain embodiments, methods are provided for monitoring
the pH inside a live cell, the methods comprising:
[0477] (a) contacting the cell with a pH-sensitive fluorescent dye
compound disclosed herein to form a contacted cell;
[0478] (b) incubating the contacted cell for a sufficient amount of
time for the one or more pH-sensitive fluorescent dye compounds to
enter the cell to form a labeled cell;
[0479] (c) illuminating the labeled cell with an appropriate
wavelength to form an illuminated cell; and
[0480] (d) detecting fluorescent emissions from the illuminated
cell;
[0481] wherein the fluorescent emissions are used to monitor the pH
inside the cell.
[0482] In certain embodiments, methods are provided for monitoring
the pH inside a live cell, the methods comprising:
[0483] (a) contacting the cell with one or more of the compositions
provided herein to form a contacted cell;
[0484] (b) incubating the contacted cell for a sufficient amount of
time for the one or more compositions to enter the cell to form a
labeled cell;
[0485] (c) illuminating the labeled cell with an appropriate
wavelength to form an illuminated cell; and
[0486] (d) detecting fluorescent emissions from the illuminated
cell;
[0487] wherein the fluorescent emissions are used to monitor the pH
inside the cell.
[0488] Typically, the pH-sensitive fluorescent dyes and/or dye
conjugates and/or compositions disclosed herein are introduced into
a living cell or cell compartment by mixing with a sample
comprising a cell or cell compartment, and then leaving the mixture
to incubate for a time interval adequate to allow entry of the
pH-sensitive fluorescent dye, dye conjugate, or composition into
the cell or cell compartment. During this time interval, the
pH-sensitive fluorescent dye compound, dye conjugate, or
composition either passively diffuses across the plasma membrane or
is taken up by the cell or cell compartment by a cell mediated
mechanism.
[0489] In the case of conjugates, typically target molecules,
including bacterial particles that induce phagocytosis and specific
binding proteins that bind a cellular receptor and induce receptor
internalization, are generally cell or cell compartment specific,
hence a specific conjugate generally attaches to only one kind of
cell or cell compartment. Once attached to a cell or cell
compartment, the pH-sensitive fluorescent dye conjugate may diffuse
through a membrane of that cell or cell compartment or be
trafficked to a specific cell compartment by receptor-mediated
endocytosis, hence exposing itself to the internal pH of the cell
or cell compartment.
[0490] Advantageously, the pH-sensitive fluorescent dye compounds,
dye conjugates, and compositions disclosed herein allow for a more
accurate determination of pH as compared to existing pH sensor dyes
because the pKa's of the pH-sensitive fluorescent dye compounds,
dye conjugates, and compositions disclosed herein may, by design,
be adjusted by substitution to a variety of pKa values. This is
accomplished by the addition of EDG groups on the aniline moiety
and by substitution at one of the remaining R.sup.1-R.sup.6 with a
group that is not --OH or --SH. Thus, some are tuned to the pH of
the cell or cell compartment of interest, and consequently will be
ideal for measuring the pH of a cell or cell compartment when
accumulated by receptor-mediated endocytosis or any non-passive
accumulation mechanism as well as by passive accumulation. Others
will have a pKa far from the pH of the cell media or extracellular
matrix. The pH-sensitive fluorescent dye compounds disclosed herein
are tuned to match the sample of choice with the understanding that
the dye compounds become fluorescent when the pH of the sample
drops below the pKa of the pH-sensitive fluorescent dye compound(s)
disclosed herein. In certain embodiments, the pKa of the
pH-sensitive fluorescent dye compound may be modified by the
addition of a dialkylamino group at position R.sup.3 advantageously
resulting in a physiological pKa. In certain embodiments, the
dialkylamino group is diethylamino. In certain embodiments, the
dialkylamino group is dimethylamino
[0491] Accumulation will occur passively when one form of the
pH-sensitive fluorescent dye compound, dye conjugate, or
composition with respect to pH (the uncharged form) freely
penetrates the cell or cell compartment of interest and the other
form (a charged form) is non-penetrating. Fluorescence will
approach its equilibrium position provided the form of the
accumulated dye is the fluorescent form and that accumulation to
equilibrium has occurred. The observed fluorescence intensity may
then be used to determine pH according to any of the known methods,
for instance by reference to calibration data, or by comparing the
observed fluorescence intensity to the fluorescence intensity
observed on acidifying the test sample so that all the dye or
conjugate fluoresces, the ratio of the two fluorescence intensities
coupled with the known pKa allowing determination of pH. Passive
accumulation may be achieved by use of a pH-sensitive fluorescent
dye compound that is not attached to a carrier molecule or solid
support or a pH-sensitive fluorescent dye compound that is attached
to a small, relatively hydrophobic target molecule capable of
diffusing through the cell membrane, such as one or more
acetoxymethoxy (AM) ester groups. However, we have found that
pH-sensitive fluorescent dye compounds comprising a reactive group,
such as succinimidyl ester, also appear to passively accumulate in
cells.
[0492] In certain embodiments, methods are provided for identifying
a target cell within a population of cells wherein the target cell
is differentially labeled relative to neighboring cells within the
population, the methods comprising:
[0493] (a) contacting one or more of the pH-sensitive fluorescent
dye compounds disclosed herein with the population of cells to form
a contacted cell population;
[0494] (b) incubating the contacted cell population for a period of
time sufficient for the one or more pH-sensitive fluorescent dye
compounds to enter the target cell, thereby forming an incubated
cell population; and
[0495] (c) illuminating the incubated cell population, wherein the
target cell is identified by a differential label relative to
neighboring cells within the population.
[0496] In certain embodiments, methods are provided for identifying
a target cell within a population of cells wherein the target cell
is differentially labeled relative to neighboring cells within the
population, the methods comprising:
[0497] (a) contacting one or more of the compositions disclosed
herein with the population of cells to form a contacted cell
population;
[0498] (b) incubating the contacted cell population for a period of
time sufficient for the one or more compositions to enter the
target cell, thereby forming an incubated cell population; and
[0499] (c) illuminating the incubated cell population, wherein the
target cell is identified by a differential label relative to
neighboring cells within the population.
[0500] In certain embodiments, the target cell is a neuronal cell.
In certain embodiments, the neuronal cell is identified by
increased fluorescence as compared with neighboring cells. In
certain embodiments, the population of cells is part of a tissue.
More particularly, the tissue is selected from the group consisting
of tumor tissue, epidermal tissue, muscle tissue, bone marrow
tissue, neural tissue, brain tissue, organ tissue, and human biopsy
tissue.
[0501] In certain embodiments, methods are provided for identifying
a first neuron or plurality of neurons in a neural tissue slice, or
a neuronal cell is a heterogeneous mixture comprising neuronal and
non-neuronal cell types. Also provided are methods for detecting
the effect of a neuromodulator on a connection between neurons or a
plurality of neurons forming a circuit; methods for identifying an
inhibitory connection between or on neurons; and methods for
identifying neurons in vivo or in vitro.
[0502] In certain embodiments, healthy neurons are identified in
mixed cultures of living cells or preparations of cells, such as
tissue slices or whole mount. In vivo identification of neurons or
other metabolically active cells such as cardiac and skeletal
myocytes are particularly preferred methods employing the
pH-sensitive dye compounds disclosed herein.
[0503] Non-passive accumulation may occur through cell-mediated
mechanisms such as phagocytosis and endocytosis, typically when a
pH-sensitive fluorescent dye compound disclosed herein comprises a
carrier molecule or solid support that is bound by a cellular
receptor. In this instance, whenever the dye compound provided
herein is accumulated in the cell or cell compartment by a
mechanism that does not rely solely on passive accumulation, the
accuracy of a pH measurement will be highest when the pKa of the
dye compound is near the pH to be measured. In this situation,
without wishing to be bound by a theory, the increased accuracy
available with the pH-sensitive dye compounds disclosed herein may
arise from the fact that the pKa is the pH of the aqueous medium
containing a species when it is 50% protonated and that at this pH
a change in proton intensity will have greatest effect on the
properties of the species. Hence, the greatest change in
fluorescence intensity occurs at the pKa of the pH-sensitive
fluorescent dye, and measurements of absolute fluorescence
intensity at this pH so that the pH-sensitive fluorescent dye
compounds used to analyze a particular cell or cell compartment
embraces the pH of that cell or cell compartment is generally
sufficient.
[0504] In certain embodiments, methods are provided for detecting a
pH related intracellular process, the methods comprising:
[0505] (a) contacting one or more of the pH-sensitive fluorescent
dye compounds disclosed herein with a cell to form a contacted
cell;
[0506] (b) incubating the contacted cell to form an incubated
solution;
[0507] (c) illuminating the incubated solution to form an
illuminated solution; and
[0508] (d) detecting fluorescent emissions from the illuminated
solution;
[0509] wherein increased fluorescent emissions indicates activation
of the intracellular process.
[0510] In certain embodiments, methods are provided for detecting a
pH related intracellular process, the methods comprising:
[0511] (a) contacting one or more of the compositions provided
herein with a cell to form a contacted cell;
[0512] (b) incubating the contacted cell to form an incubated
solution;
[0513] (c) illuminating the incubated solution to form an
illuminated solution; and
[0514] (d) detecting fluorescent emissions from the illuminated
solution;
[0515] wherein increased fluorescent emissions indicates activation
of the intracellular process.
[0516] In certain embodiments, the intracellular process is the
opening of an ion channel. More particular still, the ion channel
is calcium.
[0517] In certain embodiments, the pH-sensitive fluorescent dye
compound is internalized after incubation with the cytosol of the
cell.
[0518] Certain embodiments provide for a no-wash, no-quench assay
for phagocytosis that is based on fluorogenic bioparticles
comprising the pH-sensitive fluorescent dye compounds provided
herein. Current protocols for measuring phagocytosis that use
fluorescent bioparticles, require a trypan blue quenching step and
several washing steps. These steps can introduce significant
variability in the assay. To address this issue, provided herein is
a no-wash phagocytosis kit, using E. coli bioparticles conjugated
to a pH-sensitive fluorescent dye as described herein. These
bioparticle conjugates are weakly fluorescent at extracellular pH.
However, when added to phagocytic J774.2 murine macrophages, they
become ingested into acidic compartments and fluoresce from within
the cells, giving specific signals that meet or exceed the
brightness of the Vybrant.TM. Phagocytosis Assay Kit (Life
Technologies Corporation). Quantitation of the phagocytic index
with these conjugates requires no wash or quenching steps, and
uptake of the bioparticles is potently inhibited by cytochalaisin
D, a known blocker of phagocytosis. The pH-sensitive fluorescent
bioparticles described herein may be used in plate based, as well
as imaging and flow cytometry assays of phagocytosis.
[0519] In certain embodiments, methods are provided for detecting
phagocytosis of a carrier molecule in solution, the methods
comprising:
[0520] (a) conjugating the carrier molecule to one or more of the
pH-sensitive fluorescent dye compounds disclosed herein to form a
carrier-dye conjugate;
[0521] (b) contacting the carrier-dye conjugate with a cell to form
a contacted cell;
[0522] (c) incubating the contacted cell to form an incubated
solution;
[0523] (d) illuminating the incubated solution to form an
illuminated solution; and
[0524] (e) detecting fluorescent emissions from the illuminated
solution;
[0525] wherein fluorescent emissions indicate phagocytosis of the
carrier molecule.
[0526] In certain embodiments, methods are provided for detecting
phagocytosis of a carrier molecule in solution, the methods
comprising:
[0527] (a) conjugating the carrier molecule to one or more of the
compositions disclosed herein to form a carrier-dye conjugate;
[0528] (b) contacting the carrier-dye conjugate with a cell to form
a contacted cell;
[0529] (c) incubating the contacted cell to form an incubated
solution;
[0530] (d) illuminating the incubated solution to form an
illuminated solution; and
[0531] (e) detecting fluorescent emissions from the illuminated
solution;
[0532] wherein fluorescent emissions indicate phagocytosis of the
carrier molecule.
[0533] In certain embodiments, the carrier molecule is an E. coli
bioparticle.
[0534] In certain embodiments, methods are provided for diagnosing
or detecting a disease in a subject, the methods comprising:
[0535] (a) contacting a sample obtained from a subject suspected of
having the disease with one or more of the pH-sensitive fluorescent
dye compounds disclosed herein, to form a contacted sample;
[0536] (b) incubating the contacted sample for an appropriate
amount of time to form an incubated sample;
[0537] (c) illuminating the incubated sample with an appropriate
wavelength to form an illuminated sample; and
[0538] (d) detecting fluorescent emissions from the illuminated
sample;
[0539] wherein the fluorescent emissions are used to diagnose or
detect the disease.
[0540] In certain embodiments, methods are provided for diagnosing
or detecting a disease in a subject, the methods comprising:
[0541] (a) contacting a sample obtained from a subject suspected of
having the disease with one or more of the compositions disclosed
herein, to form a contacted sample;
[0542] (b) incubating the contacted sample for an appropriate
amount of time to form an incubated sample;
[0543] (c) illuminating the incubated sample with an appropriate
wavelength to form an illuminated sample; and
[0544] (d) detecting fluorescent emissions from the illuminated
sample;
[0545] wherein the fluorescent emissions are used to diagnose or
detect the disease.
[0546] In certain embodiments, the disease is associated with the
central nervous system. In certain embodiments, the disease is
Alzheimer's disease (AD). In certain embodiments, the pH-sensitive
fluorescent dye compound is conjugated to a carrier molecule
associated with the disease. In certain embodiments, the
pH-sensitive fluorescent dye compound is conjugated to
.beta.-amyloid or a fragment or thereof. Accordingly, certain
embodiments provide a blood based assay for Alzheimer's disease,
based on the phagocytosis of the pH-sensitive fluorescent dye
compounds disclosed herein conjugated to .beta.-amyloid
protein.
[0547] In certain embodiments, the disease is associated with the
immune system. In certain embodiments, the disease is associated
with inflammation. In certain embodiments, the disease is cancer.
In certain embodiments, the disease is associated with oxidative
stress. In certain embodiments, the pH-sensitive fluorescent dye
compound is conjugated to a carrier molecule associated with the
disease.
[0548] In certain embodiments, methods are provided for detecting a
target molecule capable of modulating a cellular process that
affects the pH or directly affects the pH of a cell. In certain
embodiments, the target molecule is a small molecule. In certain
embodiments, the cell is a neuronal cell. In certain embodiments,
the cell is a cancer cell. In certain embodiments, the cell is an
immune cell.
[0549] In certain embodiments, methods are provided for detecting
any one of the following with a pH-sensitive fluorescent dye
compound as described herein: an antibody, protein, peptide, enzyme
substrate, hormone, lymphokine, metabolite, receptor, antigen,
hapten, lectin, avidin, streptavidin, toxin, carbohydrate,
oligosaccharide, polysaccharide, nucleic acid, derivatized deoxy
nucleic acid, DNA fragment, RNA fragment, derivatized DNA fragment,
derivatized RNA fragment, nucleoside, nucleotide, natural drug,
synthetic drug, virus particle, bacterial particle, virus
component, yeast component, blood cell, blood cell component,
plasma component, serum component, biological cell, neuronal cells,
noncellular blood component, bacteria, bacterial component, natural
or synthetic lipid vesicle, poison, environmental pollutant,
polymer, polymer particle, glass particle, glass surface, plastic
particle, plastic surface, polymer membrane, conductor or
semiconductor comprising detecting a compound disclosed herein
bound to said antibody, protein, peptide, enzyme substrate,
hormone, lymphokine, metabolite, receptor, antigen, hapten, lectin,
avidin, streptavidin, toxin, carbohydrate, oligosaccharide,
polysaccharide, nucleic acid, derivatized deoxy nucleic acid, DNA
fragment, RNA fragment, derivatized DNA fragment, derivatized RNA
fragment, nucleoside, nucleotide, natural drug, synthetic drug,
virus particle, bacterial particle, virus component, yeast
component, blood cell, blood cell component, plasma component,
serum component, biological cell, noncellular blood component,
bacteria, bacterial component, natural or synthetic lipid vesicle,
poison, environmental pollutant, polymer, polymer particle, glass
particle, glass surface, plastic particle, plastic surface, polymer
membrane, conductor or semiconductor.
[0550] In certain embodiments, methods are provided for detecting
acidic or basic conditions comprising contacting a pH-sensitive
fluorescent dye compound as described herein with a composition
suspected of being acidic or basic and detecting the fluorescence
of the pH-sensitive fluorescent dye compound as an indicator of
said acidic or basic conditions. In certain embodiments, the
composition being tested comprises an intracellular
environment.
[0551] Accuracy for the general means of measuring pH may be
further increased by using a plurality of the pH-sensitive
fluorescent dye compounds provided herein having different
fluorescent responses. In certain embodiments, two or more
pH-sensitive fluorescent dye compounds according to the present
invention may be used, optionally bonded to identical carrier
molecules or solid supports, or a pH-sensitive fluorescent dye
compound as disclosed herein and another different dye. In certain
embodiments, the second fluorescent dye has a positive fluorescence
response with increasing pH (i.e., that the intensity of
fluorescence exhibited by the dye or complex increases with
increasing pH). It is preferable that the two or more dyes have
overlapping titration ranges, and more preferably the different
dyes or conjugates have pKa values within about 1 unit of each
other. The intensity of the fluorescence of each dye compound or
dye-conjugate is then measured, and pH determined by calculating
the ratio of the fluorescence intensity of the first compound to
the fluorescence intensity of the second compound and then
comparing the value obtained to a calibration curve.
[0552] In certain embodiments, the pH-sensitive dye compounds may
be used to analyze the kinetics of migration of a species into or
through a cell or cell compartment. This may be done by monitoring
the fluorescence intensity of a pH-sensitive dye compound over a
time interval. Where pH is known, the pH-sensitive dye compound
should be selected so as to have a pKa in the range between the pH
at the starting point and the pH at the end point of the pathway to
be analyzed. In some cases it may be desirable to use a plurality
of pH-sensitive dye compounds having a variety of pKa values, with
each dye or complex tuned to a different portion of the pathway to
be analyzed.
[0553] In certain embodiments, methods are provided for using a
pH-sensitive fluorescent dye compound, dye conjugate, or
composition provided herein for analysis or detection. More
particularly, the detection may be performed by optical means. In
certain embodiments, the fluorescence emission is optionally
detected by visual inspection, or by use of any of the following
devices: CCD cameras, video cameras, photographic film, laser
scanning devices, fluorometers, photodiodes, quantum counters,
epifluorescence microscopes, scanning microscopes, flow cytometers,
fluorescence microplate readers, or by means for amplifying the
signal such as photomultiplier tubes.
[0554] In certain embodiments, the sample or medium in which a
pH-sensitive fluorescent dye compound provided herein is present is
illuminated with a wavelength of light selected to give a
detectable optical response, and observed with a means for
detecting the optical response. Equipment that is useful for
illuminating the pH-sensitive fluorescent dye compounds and
compositions disclosed herein includes, but is not limited to,
hand-held ultraviolet lamps, mercury arc lamps, xenon lamps, lasers
and laser diodes. These illumination sources are optically
integrated into laser scanners, fluorescence microplate readers or
standard or microfluorometers.
[0555] The pH-sensitive fluorescent dye compounds, dye conjugates,
and compositions disclosed herein may, at any time after or during
an assay, be illuminated with a wavelength of light that results in
a detectable optical response, and observed with a means for
detecting the optical response. Upon illumination, such as by an
ultraviolet or visible wavelength emission lamp, an arc lamp, a
laser, or even sunlight or ordinary room light, the fluorescent
compounds, including those bound to the complementary specific
binding pair member, display intense visible absorption as well as
fluorescence emission. Selected equipment that is useful for
illuminating the fluorescent pH-sensitive fluorescent dye compounds
disclosed herein include, but is not limited to, hand-held
ultraviolet lamps, mercury arc lamps, xenon lamps, argon lasers,
laser diodes, and YAG lasers. These illumination sources are
optionally integrated into laser scanners, fluorescence microplate
readers, standard or mini fluorometers, or chromatographic
detectors. This fluorescence emission is optionally detected by
visual inspection, or by use of any of the following devices: CCD
cameras, video cameras, photographic film, laser scanning devices,
fluorometers, photodiodes, quantum counters, epifluorescence
microscopes, scanning microscopes, flow cytometers, fluorescence
microplate readers, or by means for amplifying the signal such as
photomultiplier tubes. Where the sample is examined using a flow
cytometer, a fluorescence microscope or a fluorometer, the
instrument is optionally used to distinguish and discriminate
between the fluorescent compounds disclosed herein and a second
fluorophore with detectably different optical properties, typically
by distinguishing the fluorescence response of the fluorescent
compounds of the invention from that of the second fluorophore.
Where a sample is examined using a flow cytometer, examination of
the sample optionally includes isolation of particles within the
sample based on the fluorescence response by using a sorting
device. In certain embodiments, the illumination source is used to
form a covalent bond between the present pH-sensitive fluorescent
dye compound and an analyte of interest. In this instance the
pH-sensitive fluorescent dye comprises a photoactivatable reactive
group, such as those discussed above.
[0556] Kits:
[0557] In certain embodiments, kits are provided for determining
the pH of a sample comprising:
[0558] (a) one or more of the pH-sensitive fluorescent dye
compounds described herein;
[0559] (b) one or more containers; and optionally
[0560] (c) instructions for determining the pH of the sample.
[0561] In certain embodiments, kits are provided for determining
the pH of a sample comprising:
[0562] (a) one or more of the pH-sensitive fluorescent dye
compositions described herein;
[0563] (b) one or more containers; and optionally
[0564] (c) instructions for determining the pH of the sample.
[0565] In certain embodiments, the kits further comprise one or
more of the following: a buffering agent, a purification medium, a
vial comprising the sample, or an organic solvent.
[0566] As used herein, the term "kit" refers to a packaged set of
related components, typically one or more pH-sensitive fluorescent
dye compounds or compositions. In certain embodiments, the kits
disclosed herein comprise one or more of the pH-sensitive
fluorescent dye compounds described herein, one or more carriers
suitable for in vitro or in vivo applications, and one or more
containers in which to store the one or more pH-sensitive
fluorescent dyes and/or one or more carriers, such as solvents,
buffers, stabilizers, pH adjusting agents, etc. The kit optionally
contains instructions for how to prepare the one or more
pH-sensitive fluorescent dyes or how to prepare a composition
containing the one or more pH-sensitive fluorescent dye, and how to
administer the dye or composition containing the dye. In certain
embodiments, the kit comprises instructions for performing an assay
that detects the pH or pH changes in samples. In certain
embodiments, the assay is an in vitro assay. In certain
embodiments, the assay is an in vivo assay. The kit may further
comprise one or more pieces of equipment to administer the dye
compound, or composition containing the pH-sensitive fluorescent
dye compound including, but not limited to, syringes, pipettes,
pipette bulbs, spatulas, vials, syringe needles, and various
combinations thereof.
[0567] In certain embodiments, the kits provided herein comprise
indicator solutions or indicator "dipsticks", blotters, culture
media, cuvettes, and the like. In certain embodiments, the kits
provided herein comprise indicator cartridges (where a kit
component is bound to a solid support) for use in an automated
detector. In certain embodiments, the kits provided herein further
comprise molecular weight markers, wherein said markers are
selected from phosphorylated and non-phosphorylated polypeptides,
calcium-binding and non-calcium binding polypeptides, sulfonated
and non-sulfonated polypeptides, and sialylated and non-sialylated
polypeptides. In certain embodiments, the kits provided herein
further comprise a member selected from a fixing solution, a
detection reagent, a standard, a wash solution, and combinations
thereof.
[0568] Synthesis and Processes of Preparation of the pH-Sensitive
Fluorescent Dye Compounds:
[0569] In certain embodiments processes are provided for
synthesizing a compound of structural formula (I):
##STR00020##
[0570] the process comprising: [0571] a) contacting a compound of
structural formula (VI):
[0571] ##STR00021## [0572] with a compound of structural formula
(IV):
[0572] ##STR00022## [0573] to form a compound of structural formula
(VII):
[0573] ##STR00023## [0574] b) de-allylating the compound of
structural formula (VII), when R.sup.7, R.sup.8, R.sup.9 and
R.sup.10 are each allyll, to form a compound of structural formula
(I),
[0575] wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6
are as previously defined herein.
[0576] In certain embodiments, processes are provided for
synthesizing a compound of structural formula (II):
##STR00024##
[0577] the process comprising: [0578] a) contacting a compound of
structural formula (III):
[0578] ##STR00025## [0579] with a compound of structural formula
(IV):
[0579] ##STR00026## [0580] to form a compound of structural formula
(V):
[0580] ##STR00027## [0581] b) de-allylating the compound of
structural formula (V), when R.sup.7 and R.sup.8 are each allyl, to
form a compound of structural formula (II), and wherein R.sup.1,
R.sup.2, R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are as previously
defined herein.
[0582] An exemplary reaction scheme is shown in detail below:
##STR00028##
[0583] wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5,
R.sup.6, R.sup.7 and R.sup.8 are as previously defined herein.
[0584] In one illustrative embodiment of such a process, a compound
of structural formula (I) is prepared in Scheme 3:
##STR00029## ##STR00030##
[0585] In another illustrative embodiment of such a process, a
compound of structural formula (I) is prepared in Scheme 4:
##STR00031## ##STR00032##
[0586] In yet another illustrative embodiment of such a process, a
compound of structural formula (II) is prepared in Scheme 5:
##STR00033## ##STR00034##
[0587] In yet another illustrative embodiment of such a process, a
compound of structural formula (I) is prepared in Scheme 6:
##STR00035## ##STR00036##
[0588] In certain embodiments, any one of the above methods of
synthesis further comprises a purifying step. In certain more
particular embodiments, the purifying step comprises at least one
of: column chromatography, trituration, recrystallization,
filtration, or aqueous separation.
[0589] A detailed description of the present teachings having been
provided above, the following examples are given for the purpose of
illustrating the invention and shall not be construed as being a
limitation on the scope of the invention or claims.
EXAMPLES
[0590] Referring to the examples that follow, pH-sensitive
fluorescent dye compounds disclosed herein were synthesized using
the methods described herein, or other methods, which are known in
the art.
[0591] It should be understood that the organic compounds according
to the invention may exhibit the phenomenon of tautomerism. As the
chemical structures within this specification can only represent
one of the possible tautomeric forms, it should be understood that
the invention encompasses any tautomeric form of the drawn
structure.
[0592] Chemical Synthesis of the Fluorescent pH-Sensitive Dye
Compounds Disclosed Herein:
Example 1
Preparation of Compound (5)
##STR00037##
[0594] Compound (5) can be obtained via multistep synthesis from
compound (4), according to U.S. Patent Publication No. 2008/076524,
herein incorporated by reference in its entirety.
Example 2
Preparation of Compounds (7) and (8):
##STR00038##
[0596] Compound (6) (9.00 g, 18.3 mmol, see Wu et al., Org. Lett.
10:1779-1782 (2008), herein incorporated by reference in its
entirety) was treated with DMSO (56 mL) and diallylamine (21.2 g,
219 mmol) at room temperature, and the mixture was heated to
90.degree. C. for 36 hours. The mixture was cooled to room
temperature, and quenched by adding water (250 mL). The product was
extracted with ethyl acetate (200 mL.times.3). The combined organic
layers were washed by water (200 mL.times.2), brine (300 mL), dried
by MgSO.sub.4, and evaporated. The crude product was purified by
chromatography on silica gel column (Biotage, SNAP 340 g,
6.5.times.18 cm, ethyl acetate:hexanes=1:2) to give Compound (7)
(3.30 g, 47%, white solid), and Compound (8) (1.10 g, 20%, pale
yellow solid).
Example 3
Preparation of Compound (9)
##STR00039##
[0598] Compound (7) (149 mg, 0.386 mmol) was treated with anhydrous
DCM (5 mL) and Tf.sub.2O (80 .mu.L, 0.476 mmol), and the mixture
was stirred at room temperature. After 30 min of stirring, Compound
(5) (150 mg, 0.486 mmol) in DCM (2 mL) and DIEA (120 .mu.L, 0.689
mmol) were added. The reaction was stirred at room temperature for
16 hours. Evaporation gave a crude product. The crude product was
purified by chromatography on silica gel column (Biotage, SNAP 25
g, 2.5.times.8 cm, MeOH:CHCl.sub.3=1:10.about.1:4) to give Compound
(9) (200 mg, 62%) as a purple gummy material.
Example 4
Preparation of Compound (1):
##STR00040##
[0600] Compound (9) (200 mg, 0.239 mmol) was treated with MeOH (5
mL) and NaBH.sub.4 (36 mg, 0.956 mmol), and the mixture was stirred
at room temperature for 30 min. The mixture was evaporated, loaded
into a short silica gel column (3.times.5 cm, ethyl
acetate:hexanes=1:4) to remove borane side product. Concentration
gave the dihydro-form of Compound (9). This intermediate was
treated with Pd(PPh.sub.3).sub.4 (30 mg, 0.0259 mmol),
1,3-dimethylbarbituric acid (302 mg, 1.93 mmol) and anhydrous DCM
(5 mL). The new mixture was cooled in dry ice/acetone bath and
evacuated through syringe needle. The flask was filled back with
argon and evacuation/refilling with argon was repeated two more
times. The mixture was heated to 40.degree. C. for 20 hours under
argon atmosphere. DCM was evacuated by aspirator, and the residue
was treated by MeOH (3 mL) and chloranil (120 mg, 0.488 mmol), and
the mixture was stirred at room temperature for 30 min. MeOH was
removed by aspirator, and the crude product was purified by
chromatography on silica gel column (Biotage, SNAP 50 g,
3.5.times.8.5 cm, H.sub.2O:MeCN:HOAc=1:10:0.1.about.1:4:0.1) to
give Compound (1) (69 mg, 50%) as dark brown gummy material.
Example 5
Preparation of Compound (10)
##STR00041##
[0602] A solution of Compound (1) (69 mg, 0.121 mmol) in MeOH (10
mL) was treated with 5 mL of 1 M NaOH, and the mixture was stirred
at room temperature for 2 hours. 1 M HCl (4.8 mL) was added to
quench base. The whole mixture was evaporated to give a crude
product. The crude product was purified by chromatography on silica
gel column (Biotage SNAP 50 g, MeOH:CHCl.sub.3=1:5.about.1:2) to
give Compound (10) (30 mg, 51%) as a dark brown solid.
Example 6
Preparation of Compound (12)
##STR00042##
[0604] A solution of Compound (10) (20 mg, 0.041 mmol) and DMAP
(7.0 mg, 0.057 mmol) in DMF (4 mL) was treated with Compound (11)
(see U.S. Patent Publication No 2009/0004753, herein incorporated
by reference in its entirety) (24 mg, 0.046 mmol), and the mixture
was stirred at room temperature for 1 hour. DMF was removed by pump
to give a crude product. The product was purified by chromatography
on silica gel column (Biotage 25 g, 2.5.times.8 cm,
MeOH:CHCl.sub.3=1:3) to give Compound (12) (15 mg, 53%) as a brown
solid.
Example 7
Preparation of Compounds (14) and (15)
##STR00043##
[0606] A solution of 4-methoxyaniline (Compound (13)) (10.0 g, 81.2
mmol) in DMF (100 mL) was treated with 60% NaH (8.10 g, 203 mmol)
and iodoethane (26.0 mL, 235 mmol). The temperature of the mixture
was kept at 0.degree. C. by ice bath in the first 3 hours of
stirring. The ice bath was removed and the mixture was stirred at
room temperature for another 12 hours. Water (200 mL) was added to
quench this reaction. The product was extracted by ethyl acetate
(200 mL.times.3). The combined organic layers were washed by water
(200 mL.times.5), brine (200 mL), and dried over sodium sulfate.
Filtration and evaporation gave Compound (14) (16.2 g, .about.100%)
as a dark green oil.
[0607] To a solution of N,N-diethyl-4-methoxyaniline (Compound
(14)) (6.0 g, 27 mmol) in water (500 mL) and acetic acid (50 mL)
was added a solution of NaNO.sub.2 (3.8 g, 55 mmol) in water (70
mL) dropwise within 30 min at room temperature. The reaction
mixture was stirred at room temperature for 3 hours. The product
was extracted by ethyl acetate (150 mL.times.3). The combined
organic layers were washed by 1M KOH (100 mL.times.4), brine (100
mL) and dried over anhydrous sodium sulfate. Filtration and
evaporation gave a crude product as dark red oil. The crude product
was purified by chromatography on silica gel column (Biotage, SNAP
100 g, 3.5.times.16 cm, from 15% CHCl.sub.3 in hexanes to 100%
CHCl.sub.3) to give Compound (15) (4.90 g, 81%) as a dark red
oil.
Example 8
Preparation of Compounds (16) and (17)
##STR00044##
[0609] To palladium on activated charcoal (650 mg) was added
ethanol (15 mL) under argon carefully. A solution of Compound (15)
(5.30 g, 23.6 mmol) in ethanol (75 mL) was treated with the
palladium suspension solution by a pipet. The solution was equipped
with a 3-way stopcock attached by a .about.3 L balloon filled with
hydrogen. The solution was evacuated and refilled with hydrogen
carefully three times. The solution was stirred under hydrogen
atmosphere for 18 hours. Filtration through a Celite pad was
performed to remove palladium, and the filtrate was concentrated to
dryness to get a crude product. The crude product was purified by
chromatography on silica gel column (Biotage, SNAP 100 g,
3.5.times.16 cm, ethyl acetate:hexanes=1:4) to give Compound (16)
(4.14 g, 90%, as light brown oil).
[0610] A solution of Compound (16) (4.14 g, 21.3 mmol) in DCM (60
mL) was treated with acetyl chloride (1.80 mL, 25.6 mmol) and
triethylamine (4.44 mL, 32.0 mmol) at 0.degree. C. The mixture was
stirred at 0.degree. C. for 30 min, and quenched by adding MeOH (1
mL). The mixture was diluted by ethyl acetate (300 mL) and the
solution was washed by water (200 mL), brine (150 mL), and dried
over anhydrous sodium sulfate. Evaporation gave pure crude product
of Compound (17) (4.73 g, 94%) as a light brown oil. No further
purification was required.
Example 9
Preparation of Compounds (18) and (19)
##STR00045##
[0612] A solution of Compound (17) (4.73 g, 20.0 mmol) in anhydrous
THF (15 mL) was treated with BH.sub.3-THF (60 mL of 1M solution in
THF, 60 mmol) carefully. The mixture was heated to reflux for 2
hours and cooled to room temperature. MeOH (60 mL) was added to the
solution slowly to quench extra amount of borane complex at room
temperature, and the mixture was heated to reflux for 10 min. The
solution was cooled to room temperature, and evaporated to dryness.
The residue was dissolved in 300 mL of ethyl acetate, and washed by
saturated sodium bicarbonate (150 mL.times.2), brine (100 mL), and
dried over anhydrous sodium sulfate. Evaporation gave pure crude
product of Compound (18) (4.12 g, 93%) as a light brown oil. No
further purification was required.
[0613] A solution of Compound (18) (4.06 g, 18.3 mmol) in 40 mL of
DMF was treated with methyl 4-bromobutanoate (9.23 mL, 73.1 mmol),
NaI (1.36 g, 9.07 mmol), and DIEA (9.44 mL, 54.2 mmol), and the
mixture was heated to 100.degree. C. for 22 hours. The reaction was
cooled to room temperature, added 200 mL of water, and extracted by
ethyl acetate (150 mL.times.3). The combined organic layers were
washed by water (150 mL.times.3), brine (100 mL), dried over
anhydrous sodium sulfate, and evaporated to give a crude product.
The crude product was purified by chromatography on silica gel
column (Biotage, SNAP 100 g, 3.5.times.16 cm, ethyl
acetate:hexanes=1:4) to give Compound (19) (4.60 g, 79%) as light
brown oil.
[0614] Example 10
Preparation of Compound (20)
##STR00046##
[0616] Compound (7) (2.15 g, 5.54 mmol) was treated with anhydrous
DCM (50 mL) and Tf.sub.2O (0.94 mL, 0.5.60 mmol), and the mixture
was stirred at room temperature. After 30 min of stirring, Compound
(19) (2.13 g, 6.65 mmol) in DCM (15 mL) and DIEA (1.25 mL, 7.18
mmol) were added. The reaction was stirred at room temperature for
22 hours. Evaporation gave a crude product. The crude product was
purified by chromatography on silica gel column (Biotage, SNAP 100
g, 3.5.times.16 cm, H.sub.2O:MeCN:HOAc=1:10:0.1.about.1:6:0.1) to
give Compound (20) (2.58 g, 62%) as a purple gummy material.
Example 11
Preparation of Compound (2):
##STR00047##
[0618] Compound (20) (2.58 g, 4.27 mmol) was treated with MeOH (20
mL), THF (10 mL) and NaBH.sub.4 (330 mg, 8.50 mmol), and the
mixture was stirred at room temperature for 30 min. The mixture was
diluted by 300 mL of ethyl acetate, and the combined solution was
washed by water (50 mL) and brine (50 mL), and dried over anhydrous
sodium sulfate. Concentration gave the dihydro-form of Compound
(20). This intermediate was treated with Pd(PPh.sub.3).sub.4 (493
mg, 0.427 mmol), 1,3-dimethylbarbituric acid (3.30 mg, 21.0 mmol)
and anhydrous DCM (30 mL). The new mixture was cooled in dry
ice/acetone bath and evacuated through syringe needle. The flask
was filled back with argon and evacuation/refilling with argon was
repeated two more times. The mixture was heated to 40.degree. C.
for 20 hours under argon atmosphere. DCM was evacuated by
aspirator, and the residue was treated by MeOH (30 mL) and
chloranil (2.10 g, 8.50 mmol), and the mixture was stirred at room
temperature for 30 min. The mixture was loaded into a samplet of
Biotage, and dried under vacuum. The crude product was purified by
chromatography on silica gel column (Biotage, SNAP 340 g,
6.5.times.18 cm, H.sub.2O:MeCN:HOAc=1:10:0.1.about.1:4:0.1) to give
Compound (2) (1.60 g, 62%) as dark brown gummy material.
Example 13
Preparation of Compound (21)
##STR00048##
[0620] A solution of Compound (2) (1.62 g, 2.74 mmol) in MeOH (250
mL) was treated with 130 mL of 1 M NaOH, and the mixture was
stirred at room temperature for 2 hours. Acetic acid (35 mL) was
added to quench base. The whole mixture was evaporated to obtain
.about.130 mL of crude solution. The solution was packed into a
column and purified (Biotage C-18 100 g,
MeOH:water=0:100.about.80:20) to give Compound (21) (788 mg, 56%)
as a dark brown solid.
Example 14
Preparation of Compound (22):
##STR00049##
[0622] A solution of Compound (21) (10 mg, 0.0194 mmol) and DMAP
(3.5 mg, 0.0286 mmol) in DMF (2 mL) was treated with Compound (11)
(12 mg, 0.0232 mmol), and the mixture was stirred at room
temperature for 1 hour. DMF was removed by pump to give a crude
product. The product was purified on silica gel column (Biotage 25
g, 2.5.times.8 cm, MeOH:CHCl.sub.3=1:3) to give Compound (22) (4.6
mg, 32%) as a brown solid.
Example 15
Preparation of Compound (24)
##STR00050##
[0624] A solution of Compound (21) (8 mg, 0.0155 mmol) and DMAP
(3.8 mg, 0.031 mmol) in DMF (3 mL) was treated with Compound (23)
(prepared by the method described in U.S. Patent Publication No.
2009/0004753, herein incorporated by reference in its entirety) (16
mg, 0.031 mmol), and the mixture was stirred at room temperature
for 2 hour. DMF was removed by pump to give a crude product. The
product was purified by HPLC chromatography (C-18,
H.sub.2O:MeCN=1:10.about.1:5) to give Compound (24) (6 mg, 52%) as
a brown solid.
Example 16
Preparation of Compound (26)
##STR00051##
[0626] A solution of Compound (21) (8 mg, 0.0155 mmol) and DMAP
(3.8 mg, 0.031 mmol) in DMF (3 mL) was treated with Compound (25)
(prepared by the method of US 2009/0004753, herein incorporated by
reference in its entirety) (13 mg, 0.031 mmol), and the mixture was
stirred at room temperature for 2 hour. DMF was removed by pump to
give a crude product. The product was purified by chromatography on
silica gel column (Biotage 25 g, 2.5.times.8 cm,
H.sub.2O:MeCN=1:10.about.1:5) to give Compound (26) (5.5 mg, 53%)
as a brown solid.
Example 17
Preparation of Compound (25)
##STR00052##
[0628] A solution of Compound (8) (170 mg, 0.553 mmol) in DMF (5
mL) was treated with 60% NaH (33.0 mg, 0.825 mmol) and allyl
bromide (134 mg, 1.11 mmol) at 0.degree. C. The mixture was allowed
warm to room temperature and was stirred overnight (.about.16
hours). The reaction was quenched by water (20 mL). The solution
was extracted by ethyl acetate (30 mL.times.3). The combined
organic layers were washed by water (30 mL.times.3), brine (50 mL),
dried over magnesium sulfate and evaporated to give Compound (25)
(210 mg, .about.100%) as pale yellow solid. No further purification
was required.
Example 18
Preparation of Compound (29)
##STR00053##
[0630] Compound (25) (210 mg, 0.605 mmol) was treated with
anhydrous DCM (8 mL) and Tf.sub.2O (121 .mu.L, 0.719 mmol), and the
mixture was stirred at room temperature. After 30 min of stirring,
Compound (5) (239 mg, 0.774 mmol) in DCM (6 mL) and DIEA (160
.mu.L, 0.910 mmol) were added. The reaction was stirred at room
temperature for 20 hours. Evaporation gave a crude product. The
crude product was purified by chromatography on silica gel column
(Biotage, SNAP 50 g, 3.5.times.8.5 cm,
MeOH:CHCl.sub.3=1:10.about.1:4) to give Compound (29) (412 mg, 87%)
as a gray gummy material.
Example 19
Preparation of Compound (3)
##STR00054##
[0632] Compound (29) (390 mg, 0.495 mmol) was treated with MeOH (5
mL) and NaBH.sub.4 (77 mg, 2.04 mmol), and the mixture was stirred
at room temperature for 30 min. The mixture was evaporated, loaded
into a short silica gel column (3.times.5 cm, ethyl
acetate:hexanes=1:4) to remove borane side product. Concentration
gave the dihydro-form of Compound (26). This intermediate was
treated with Pd(PPh.sub.3).sub.4 (55 mg, 0.0476 mmol),
1,3-dimethylbarbituric acid (303 mg, 1.94 mmol) and anhydrous DCM
(5 mL). The new mixture was cooled in dry ice/acetone bath and
evacuated through syringe needle. The flask was filled back with
argon and evacuation/refilling with argon was repeated two more
times. The mixture was heated to 40.degree. C. for 16 hours under
argon atmosphere. DCM was evacuated by aspirator, and the residue
was treated by MeOH (3 mL) and chloranil (242 mg, 0.984 mmol), and
the mixture was stirred at room temperature for 30 min. MeOH was
removed by aspirator, and the crude product was purified by
chromatography on silica gel column (Biotage, SNAP 50 g,
3.5.times.8.5 cm, MeOH:CHCl.sub.3=1:10.about.1:4) to give Compound
(3) (135 mg, 53%) as dark brown gummy material.
Example 20
Preparation of Compound (27)
##STR00055##
[0634] A solution of Compound (3) (33 mg, 0.0638 mmol) in MeOH (6
mL) was treated with 3 mL of 1 M NaOH, and the mixture was stirred
at room temperature for 2 hours. 1 M HCl (2.8 mL) was added to
quench base. The whole mixture was evaporated to dryness. The crude
product was purified by chromatography on silica gel column
(Biotage 25 g, 2.5.times.8 cm, MeOH:CHCl.sub.3=1:3) to give
Compound (27) (18 mg, 58%) as a dark brown solid.
Example 21
Preparation of Compound (28)
##STR00056##
[0636] A solution of Compound (27) (18 mg, 0.0368 mmol) and DMAP
(7.0 mg, 0.0573 mmol) in DMF (2 mL) was treated with Compound (11)
(26 mg, 0.0442 mmol), and the mixture was stirred at room
temperature for 1 hour. DMF was removed by pump to give a crude
product. The product was purified by chromatography on silica gel
column (Biotage 25 g, 2.5.times.8 cm, MeOH:CHCl.sub.3=1:3) to give
Compound (28) (14 mg, 53%) as a brown solid.
Example 22
Preparation of Compound (30)
##STR00057##
[0638] A solution of Compound (24, STP ester) (7.0 mg, 0.00940
mmol) and Compound (29) (7 mg, 0.0186 mmol) in DMF (2 mL) was
treated with 13 .mu.L of triethylamine (0.094 mmol), and the
mixture was stirred at room temperature for 1 hour. All volatile
materials were removed by vacuum pump overnight. The crude mixture
was treated with 1 mL of 1 M TEAA buffer, and was purified by
chromatography on C-18 (Biotage SNAP 12 g,
MeOH:H.sub.2O=0:100.about.60:40) to give Compound (30) (3.9 mg,
56%) as a dark brown material.
Example 23
Preparation of Compound (31)
##STR00058##
[0640] A solution of Compound (29) (80 mg, 0.213 mmol) and
Boc.sub.2O (56 mg, 0.255 mmol) in DMF (1 mL) was treated with 93
.mu.L of DIEA (0.532 mmol), and the mixture was stirred at room
temperature. After 5-hour stirring, the mixture was treated with
bromomethyl acetate (83 .mu.L, 0.848 mmol) and more DIEA (186
.mu.L, 1.06 mmol), and the mixture was stirred at room temperature
overnight (>15 hours). All volatile materials were removed by
vacuum pump. The residue was dissolved in ethyl acetate (30 mL),
washed by water (25 mL), brine (25 mL), and dried over MgSO.sub.4.
Evaporation gave the crude product Compound (31) as colorless oil
(.about.120 mg). No further purification was necessary.
Example 24
Preparation of Compound (33)
##STR00059##
[0642] Compound (31) (20 mg, 0.0346 mmol) was treated with DCM (2
mL) and TFA (0.4 mL), and the mixture was stirred at 0.degree. C.
for 1 hour. All volatile materials were removed by house vacuum and
the residue was dried by vacuum pump for 2 hours. The resulting
crude amine (Compound (32)) was treated with Compound (24, STP
ester) (10 mg, 0.0134 mmol), DMF (1 mL) and DIEA (12 .mu.L, 0.067
mmol) sequentially. The mixture was stirred at room temperature for
1 hour. The reaction mixture was dried carefully by vacuum pump,
and purified by chromatography on silica gel column (Biotage, SNAP
10 g, MeOH:ethyl acetate:CHCl.sub.3=1:4:4.about.1:2:2) to give
Compound (33) (5.1 mg, 31%) as a dark brown material.
Example 25
Preparation of Compound (35)
##STR00060##
[0644] A solution of Compound (24, STP ester) (10.0 mg, 0.0134
mmol) and DIBO amine (Compound (34), Molecular Probes/C10411) (5.0
mg, 0.0156 mmol) in DMF (1 mL) was treated with 10 .mu.L of
triethylamine (0.072 mmol), and the mixture was stirred at room
temperature for 1 hour. All volatile materials were removed by
vacuum pump overnight. The crude reaction mixture was dried
carefully by vacuum pump, and purified by chromatography on silica
gel column (Biotage, SNAP 10 g, MeOH:CHCl.sub.3=1:10.about.1:6) to
give Compound (35) (11.0 mg, 77%) as a dark brown material.
Example 26
Preparation of EGF Conjugate of Compound (24)
[0645] 0.6 mg of a 5 mg/ml stock solution of epithelial growth
factor (EGF) (120 .mu.L, 0.098 .mu.mol) was added into a 2 mL vial.
1.13 mg of Compound (24) STP ester (1.52 .mu.mol) was dissolved in
200 .mu.L of DMSO. 27 .mu.L of this solution was added into the
vial with EGF. The dye to protein molar ratio was .about.2. 50
.mu.L of Triethylamine in 0.5 mL of DMSO was dissolved and 6 .mu.L
of triethylamine solution was added into the EGF solution. The
reaction vessel was covered and stirred for .about.24 hours. The
reaction was quenched with 1.5 M hydroxylamine (pH 8.0) and the
mixture was stirred for .about.30 minutes at room temperature. The
conjugate was purified on a P-2F column (15.times.1 cm) with PBS
(pH 7.2). The concentration of the conjugate was measured by
determining the A511 nm/A280 nm (60 .mu.g/ml) and degree of
substitution (DOS=1) by absorbance. BSA powder was added to the
conjugate solution (final concentration of 1%) and stirred to
gently dissolve. The EGF conjugate was frozen in dry ice for at
least 1 hour and lyophilized for at least 18 hours.
[0646] Biological Application Examples of the Fluorescent
pH-Sensitive Dye Compounds
Example 27
Correlation Between pH and Fluorescence of the pH-Sensitive Dye
Compounds Provided Herein
[0647] The fluorogenic nature of the pH-sensitive fluorescent dye
compounds described herein makes them very useful for studying a
variety of internalization processes that occur in cells, such as
phagocytosis and endocytosis. This is because upon internalization,
there is a drop in pH inside the phagosome or endosome which
results in an increase of fluorescence from the pH-sensitive
fluorescent dye compounds. Conjugation of the pH-sensitive
fluorescent dye compounds to biomolecules of interest provide for
convenient assays of internalization of these molecules. Examples
include transferrin, EGF and low-density lipoprotein (LDL) for
studying receptor-mediated endocytosis, and labeled bioparticles,
such as E. coli, Staphylococcus, and zymosan for studying
phagocytosis. Assays using these fluorogenic bioconjugates offer
significant advantages over existing techniques due to the fact
that the pH-sensitive fluorescent dye compounds are relatively
non-fluorescent at the neutral pH outside the cell. This reduces or
eliminates the need for wash steps and quencher dyes normally
needed to reduce background signal from bioconjugates outside of
the cells.
[0648] The study of the fluorescent response of the pH-sensitive
fluorescent dye compounds to changes in pH was performed in aqueous
buffers (in concentrations around 10 .mu.mol/mL). The results of
the titrations are shown in FIGS. 3-5.
Example 28
Intracellular Uptake of the Fluorescent pH-sensitive Dye
Compounds
[0649] FIG. 2, panel B shows cellular internalization of an
exemplary pH-sensitive fluorescent dye compound. Cells were imaged
using standard fluorescent illumination and microscopy.
[0650] RAW macrophage cells were plated onto poly-D-lysine coated
glass dishes and cultured under normal conditions. To demonstrate
phagocytic uptake of the fluorescent pH-sensitive dye compound
(Compound (1))-labeled E. coli, Staphylococcus and zymosan (yeast)
bioparticles, culture medium was removed and the cells were washed
in pH-neutral buffer before the labeled bioparticles were added.
Cells were incubated for 90 minutes at 37.degree. C. and imaged
with a standard FITC filter set (see, FIG. 6).
[0651] FIG. 7 shows a dose-response curve of cytochalasin D
inhibition of Compound (1)-labeled bioparticle internalization in a
high-throughput microplate-based assay. These results demonstrated
that an extracellular quenching agent "BackDrop" had a minimal
effect on the magnitude of specific internalization and background
fluorescence in the assay, which may be carried out with no washes
or extracellular quenching to achieve a physiologically relevant
and functional measurement of the phagocytic index of cultured
macrophage "MMM" cells.
[0652] FIG. 8A shows a series of pH curves for E. coli bioparticles
labeled with Compound (2), Compound (12) and Compound (28),
emphasizing their relative responses to acidification in vitro.
Briefly, E. coli labeled with the indicated dye species were
resuspended at 100 .mu.g/mL at pH 4-pH 8.5. 100 mL of the
suspension was dispensed into a 96-well plate for imaging on a
Hamamatsu FDSS 6000 and the wells were imaged with standard FITC
illumination. The fluorescence range is pseudocolored red for
brightest signal (acidic pH) to blue (for alkaline pH) (see, FIG.
8B).
[0653] In FIG. 9, 10,000 MW dextran was labeled with Compound (22)
and resuspended at 1 .mu.g/mL in aqueous solution, buffered to the
pH indicated on the plot. Samples were placed in a fluorimeter and
excited at 488 nm, while scanning emission across the wavelengths
shown. Plots show the relative fluorescence increase with
acidification.
Example 29
Reaction of Goat Anti-Mouse IgG (GAM) and Compound (24)
[0654] 0.100 mL (1.0 mg) of a 10.0 mg/mL solution of GAM in 10 mM
potassium phosphate, 150 mM sodium chloride buffer (PBS) was
measured into reaction tubes and the pH raised to >8.0 with 10
.mu.L 1 M sodium bicarbonate, pH 9.0. The GAM solution was reacted
with a 2.5, 5, 10, 15, or 20-fold molar excess of Compound (24) at
10 mg/mL in anhydrous DMSO for 1 h at room temperature. The
dye-protein conjugates were separated from free dye by size
exclusion chromatography using 5-0.75.times.20 cm columns packed
with BioRad.TM. Bio-Gel.RTM. P-30 fine in PBS and eluted with same.
The initial protein-containing band from each column was collected.
Absorbance spectra were obtained on a Perkin-Elmer Lambda 35 UV/Vis
spectrometer, diluting the samples in guanidine-HCL. The degree of
substitution (DOS) measured ranged from 0.8-3.2 respectively.
Example 30
Antibody Labeling Using pH-Sensitive Fluorescent Dye Compounds
[0655] A freshly prepared 10 mM DMSO solution of Thiol reactive
Compound (24)-maleimide conjugate was added to an IgG solution (6
mg/mL) in PBS buffer at pH 7.0-7.5 in sufficient amount to give
10-20 moles of dye for each IgG molecule. The reaction was allowed
to proceed for approximately 2 hours at which time the reaction
mixture was poured on to a pre-packed Sephadex G-25 column. The
column was eluted with PBS buffer to collect purified conjugated
IgG. TLC analysis of a small aliquot (.about.5 .mu.L) of the
purified IgG indicated no free dye in the conjugate solution. The
degree of labeling (DOL) was determined through a typical
absorbance reading.
TABLE-US-00004 TABLE 4 Compound (24)-labeled IgG Properties
Compound Moles of dye/IgG molecule Observed DOL 24 10 2 24 20
1.8
[0656] Labeled IgGs were collected from the column and checked for
purity via thin layer chromatography, to insure that all free dye
was separated from the labeled IgG. These samples were diluted 1:10
to final concentrations of 10-100 .mu.g/mL into a series of pH
standard solutions from pH 4 to pH 9 and transferred into a 96 well
microplate and scanned for fluorescence in a Molecular Devices
FlexStation 384 plate reader. As can be seen in FIG. 10, relative
fluorescence units (RFU) on the y-axis demonstrate the pH
activation of fluorescent signal from the IgG conjugates,
increasing signal with decreasing pH. pKa (midpoint) values near
6.8 of the conjugated antibodies track closely with the signal from
the pH-sensitive fluorescent dyes.
Example 31
Reaction of Fab Fragment of Goat Anti-Human IgG (GAH) and Compound
(24)
[0657] 0.123 mL (1.0 mg) of an 8.18 mg/mL solution of GAH in 10 mM
potassium phosphate, 150 mM sodium chloride buffer (PBS) was
measured into a reaction tube and the pH raised to >8.0 with
12.5 .mu.L 1 M sodium bicarbonate, pH 9.0. The GAH solution was
reacted with a 12-fold molar excess of Compound (24) at 10 mg/mL in
anhydrous DMSO for 1 h at room temperature. The dye-protein
conjugate was separated from free dye by size exclusion
chromatography using a 0.75.times.20 cm column packed with
BioRad.TM. Bio-Gel.RTM. P-30 fine in PBS and eluted with same. The
initial protein-containing band from the column was collected. The
absorbance spectrum was obtained on a Perkin-Elmer Lambda 35 UV/Vis
spectrometer, diluting the sample in guanidine-HCL. The degree of
substitution (DOS) measured was 1.4. The fluorescence emission
spectra were obtained using a Perkin-Elmer LS 55Fluorescence
Spectrometer, excited at 475 nm. FIG. 11 shows a pH response
Compound (24)-labeled goat-anti-human (GAH) Fab fragment. The solid
line shows the pH response at pH 10, the diamond line shows the pH
response at pH 8, the triangle line shows the pH response at pH 6,
and the square line shows the pH response at pH 4.
Example 32
Live Cell Phagocytosis/Endocytosis with Dye Conjugates
[0658] A 431 cells were grown in complete medium on 35 mm
poly-D-lysine coated glass bottom culture dishes from MatTek. On
the day of the assay, cells were rinsed once with LCIS +1% BSA
(Live Cell imaging solution, Cat# A14291DJ) and placed at
37.degree. C. Control dishes received LCIS; EGF pretreatment dishes
received 10 .mu.g/mL unlabeled EGF in LCIS. Cells were incubated at
37.degree. C. for 30 minutes. Cells were then cooled to 4.degree.
C. on ice for 10 minutes. Compound (24)-labeled EGF was added to
the dishes 1:10 at 5 .mu.g/mL from a 50 .mu.g/mL stock in LCIS.
These dishes were incubated on ice for 30 minutes, then washed
2.times. with cold LCIS and allowed to warm to 37.degree. C. for 60
minutes before imaging with standard TRITC and FITC filter sets on
a DeltaVision Core microscope. Cells pretreated with unlabeled EGF
showed no signal owing to the occlusion of dye-labeled EGF binding
sites and internalization of EGF receptors by excess unlabeled EGF.
Specific signal from untreated plates was from dye-labeled EGF
internalization. All images were matched for gain and exposure
times. FIG. 12 shows internalization of EGF conjugated to Compound
(24). The left panel shows cells pretreated with EGF and the right
panel shows cells treated with dye-conjugated EGF.
[0659] pH-sensitive fluorescent dye compounds were made up in LCIS
from 5 and 10 mM DMSO stocks respectively, and loaded for imaging
as follows: 10 mL loading buffer, Compound (33): 10 .mu.L Compound
(33) from a 10 mM DMSO stock was added to 100 .mu.L Powerload and
mixed. 10 mL LCIS was added to this solution for final
concentration of dye compound-AM ester of 10 .mu.M.
[0660] Cells were cultured on 35 mm MatTek glass bottom dishes. For
loading, cells were rinsed 1.times. with LCIS and replaced with
loading buffer described above. Cells were incubated at 37.degree.
C. for 60 minutes, then rinsed 2.times. with LCIS and imaged with
standard TRITC or FITC filter sets. (see FIG. 13).
Example 33
Labeling of Transferrin with pH Sensing Dyes
[0661] All materials are from Life Technologies Corp. (Carlsbad,
Calif.) unless otherwise stated. Dissolve transferrin from human
serum (Sigma, T4132) in 0.1 M NaHCO.sub.3, pH 8.3, to a
concentration of 10 mg/mL. Make a 10 mg/mL solution of the
succinimidyl ester of the dye in dry DMSO and sonicate briefly to
aid in dissolution of the dye. Add a 10 to 30-fold molar excess of
the reactive dye solution to the transferrin solution dropwise
while stirring. Note that the volume of dye added depends on the
specific dye and the amount of transferrin to be labeled. Protect
the reaction vessel from light and stir for .about.1 hour at room
temperature. Purify the conjugate on a P-30M gel filtration column
(BioRad, 150-4150) in PBS, pH 7.2. Centrifuge the conjugate at
19,000 rpm for 20 minutes to remove aggregates, if present.
Determine the degree of labeling by measuring A560 nm/A280 nm.
Example 34
Monitoring Cytosolic Acidification Associated with Ion Channel or
Transporter Activation
[0662] A cytosolically localized version of the pH-sensitive
fluorescent dye compound is be a useful indication of proton influx
through ion channels or transporters. This may be used for
screening of antagonists, agonists, and other modulators of
channel/transporter function.
Example 35
Receptor Internalization Assay
[0663] The .beta.-2-Adrenergic Receptor (.beta.2AR) is modified to
incorporate an epitope tag (VSV-G tag) at the N-terminus. A clonal,
stable HEK 293 cell line is established which expresses this
receptor (approximately 1.8 pmol/mg cell homogenate). Anti-VSV-G
antibody labeled with a pH-sensitive fluorescent dye compound
described herein is used to monitor agonist-mediated receptor
internalization in these live cells. The assay is performed in the
presence and absence of a specific agonist, isoproterenol.
[0664] a) Isoproterenol-induced receptor internalization in
VSV-G-B2 Adrenergic cells. For HEK 293 cells it is preferable to
coat plates with poly-D-lysine (Sigma P-6407, 5 mg in 50 ml sterile
PBS) prior to seeding the cells. 30-80 .mu.l/well is added and
maintained at room temperature for 45 minutes. The coating solution
is then aspirated, washed 4.times. (or more) with 100 .mu.l sterile
PBS. Plates can be treated in advance and stored at 4.degree. C.
for up to a week (with the final PBS wash still in the wells).
Cultured cells can be seeded directly into the wells without first
drying the plates. Cultured cells are diluted to
.about.1.6.times.105 cells/ml in complete MEM media (Sigma M2279)
containing 200 .mu.g/ml G418. 100 .mu.l of cell suspension is
pipetted into each assay well of a poly-D-lysine treated 96-well
Packard Viewplate (cell density=16000 cells per well). Plates are
then incubated 24-48 hours at 37.degree. C. with 5% CO.sub.2. 250
.mu.g lyophilized dye compound-labeled anti-VSV-G antibody
(PA45407) is reconstituted with 0.5 ml sterile deionized water and
mixed thoroughly (stock concentration 0.5 mg/ml). The mixture is
centrifuged to remove any precipitate. The dye compound-labeled
anti-VSV-G antibody is further diluted to a concentration of 2.5-5
.mu.g/ml using serum-free, phenol red free MEM media. Hoechst 33342
nuclear stain may be added to the 2.5-5 .mu.g/ml antibody solution
to a final concentration of 5 .mu.M. Media is subsequently removed
from the cells and 100 .mu.l antibody and Hoechst solution is added
to each well. The solution is then incubated at room temperature
for 15 minutes. 3 .mu.M working dilution of isoproterenol agonist
(from 10 mM stock in sterile water; Sigma 15627) is added to the
solution and then 50 .mu.l is added to required wells, giving 1
.mu.M final concentration. The wells are incubated at 37.degree. C.
for 30 minutes (in a CO.sub.2 incubator or on the IN Cell Analyzer
3000). The cells are imaged on an IN Cell Analyzer 3000, IN Cell
Analyzer 1000 or a confocal microscope.
[0665] b) Internalization of Compound-labeled anti-VSV-G antibody.
HEK 293 cells expressing a VSV-G-.beta.2-Adrenergic Receptor are
preincubated with anti-VSV-G antibody-dye compound conjugate and
stimulated with 1 .mu.M isoproterenol. The cells are imaged using
an IN Cell Analyzer 1000. Quantification of the agonist-mediated
response is achieved using a granularity algorithm, which defines
grains as distinct focal regions within a cell that have pronounced
intensity differences from the region of the cell immediately
surrounding the grains. The operator can adjust a variety of
parameters to control what size and intensity of grain will be
counted and analyzed.
[0666] c) Internalization of Compound-labeled anti-VSV-G antibody.
HEK VSV-G-.beta.2-Adrenergic Receptor cells are preincubated with
compound labeled anti-VSV-G antibody and increasing concentrations
of isoproterenol (0-1 .mu.M) are then added to the cells. After 30
minutes at 37.degree. C., agonist mediated internalization is
analyzed by measuring the increase in compound fluorescence using
an IN Cell Analyzer 1000 and the granularity analysis
algorithm.
Example 36
Detection of Neuronal Cells with a pH-Sensitive Fluorescent Dye
Compound
[0667] Astroglial feeder layers are established for one week in
culture on glass bottomed culture dishes, 35 mm diameter, coated
with poly-L-lysine. Neurons from embryonic day 18 rat hippocampi
are dissociated in culture medium, and seeded onto the feeder
layers at a density of 25-35,000 cells per milliliter, two
milliliters per dish, and allowed to grow in neuronal culture
medium plus mitotic inhibitors to prevent glial proliferation.
[0668] Cells are pre-stained for 15 minutes with 200 ng/mL Hoescht
to visualize DNA in the nuclei, and 50 ng/mL calcien AM ester to
visualize cytoplasm by adding 1000.times. DMSO stocks of these
compounds to the cells in complete medium, and then returning them
to the cell culture incubator for fifteen minutes at 37.degree. C.
Cells are removed, and the medium is gently poured off. The cells
are immediately placed in 5 .mu.M pH-sensitive fluorescent dye
compound, diluted from a 1 mM DMSO stock into normal saline plus 20
mM HEPES and 20 mM glucose, final pH set to 7.4 with NaOH. The
cells are incubated in labeling solution for ten minutes at room
temperature, and then gently washed twice with saline (above) minus
dye for imaging.
Example 37
Phagocytosis of .beta.1 Amyloid Conjugates
[0669] 1 mg of beta amyloid 1-42 is labeled with a pH-sensitive
fluorescent dye compound to yield a dye-beta amyloid conjugate,
which is purified by gel filtration to yield a solution of
approximately 200 ng/mL with a degree of labeling between 1 and 2
dye molecules per beta amyloid molecule.
[0670] 2 mL of J774A.1 cells are seeded onto 35 mm, poly-D-lysine
coated glass bottom culture dishes at a density of 35,000 cells per
mL one day in advance of the study, in serum-free OptiMem culture
medium. The dye-beta amyloid conjugate is filtered through a 0.2
micron syringe filter, and 20 microliters of the solution is added
to the cells. The culture is returned to the incubator (37.degree.
C., 5% CO.sub.2) for overnight incubation, and imaged on the
following day.
Example 38
Copper-Less Click Reactions Using DIBO-Containing pH-Sensitive
Fluorescent Dye Compounds
[0671] a) Live Cell Labeling: Mammalian cells are grown in an
appropriate medium at 37.degree. C. in 5% CO.sub.2. Supplement the
growth medium with an azide-derivatized metabolite (e.g.,
Click-iT.RTM. ManNAz, Life Technologies, Catalog No. C33366) and
grow the cells for 2 to 3 days. Wash the cells two times with D-PBS
(Life Technologies, Catalog No. 14190-144) containing 1% fetal
bovine serum (FBS). Label the azide-modified macromolecules at room
temperature in the dark for 1 hour with about 5 to 30 .mu.M
Compound (35) in D-PBS containing 1% FBS. Wash the cells four times
with D-PBS containing 1% FB S. Fix the cells with 4% formaldehyde
in D-PBS for 15 minutes at room temperature. Wash the cells with
D-PBS. Optionally, counterstain the cells with an appropriate
counterstain, such as Hoechst 33342 and wash the cells. Image the
cells.
[0672] b) Protein Labeling: Introduce azide into proteins, e.g.,
using GalNAz in antibodies using the Click-iT.RTM. O-GlcNAc
Enzymatic Labeling System (Life Technologies, Catalog No. C33368).
Modify the protein-bound azide with Compound (35). Incubate the
protein in TBS with about 5 to 10 .mu.M Compound (35) for at least
1 hour at room temperature. Remove the excess label. Analyze the
modified protein.
* * * * *