U.S. patent application number 11/366784 was filed with the patent office on 2006-09-07 for chemical probe compounds that become fluorescent upon reduction, and methods for their use.
Invention is credited to Ching-Ying Cheung, Diane R. Gray, Stephen T. Yue.
Application Number | 20060199242 11/366784 |
Document ID | / |
Family ID | 36941807 |
Filed Date | 2006-09-07 |
United States Patent
Application |
20060199242 |
Kind Code |
A1 |
Cheung; Ching-Ying ; et
al. |
September 7, 2006 |
Chemical probe compounds that become fluorescent upon reduction,
and methods for their use
Abstract
Chemical stain compounds containing a fluorophore and a
reducible quenching unit are disclosed. The reducible quenching
unit quenches the fluorophore while in its oxidized state. Upon
reduction, the quenching properties of the quenching unit are
diminished or eliminated. The chemical compounds can be used in a
variety of applications, including the detection of bacterial
cells, monitoring the electron transport chain function of
bacterial cells, monitoring the oxidation state of non-biological
systems, and assaying the effectiveness of antibacterial or
antimicrobial agents.
Inventors: |
Cheung; Ching-Ying; (San
Ramon, CA) ; Gray; Diane R.; (Eugene, OR) ;
Yue; Stephen T.; (Eugene, OR) |
Correspondence
Address: |
KOREN ANDERSON;MOLECULAR PROBES, INC.
29851 WILLOW CREEK ROAD
EUGENE
OR
97402-9132
US
|
Family ID: |
36941807 |
Appl. No.: |
11/366784 |
Filed: |
March 1, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60657944 |
Mar 1, 2005 |
|
|
|
Current U.S.
Class: |
435/32 ; 436/62;
546/159; 546/269.7 |
Current CPC
Class: |
C07D 417/14 20130101;
G01N 2021/6432 20130101; C12Q 1/04 20130101; G01N 33/52 20130101;
G01N 33/582 20130101; C12Q 1/18 20130101; C07D 211/62 20130101;
C07D 401/14 20130101; C07D 417/06 20130101; G01N 33/1806 20130101;
C09B 23/04 20130101; C07D 295/112 20130101; G01N 21/643
20130101 |
Class at
Publication: |
435/032 ;
436/062; 546/159; 546/269.7 |
International
Class: |
C12Q 1/18 20060101
C12Q001/18; G01N 33/18 20060101 G01N033/18; C07D 417/02 20060101
C07D417/02 |
Claims
1. A chemical compound having the structure FU-RQU or FU-L-RQU;
where FU is a fluorophore unit, RQU is a reducible quenching unit,
and L is a linker unit.
2. The compound of claim 1, having the structure FU-RQU.
3. The compound of claim 1, having the structure FU-L-RQU.
4. The compound of claim 1, wherein: the reducible quenching unit
has an oxidized state and a reduced state; the reducible quenching
unit in its oxidized state partially or fully quenches the
fluorescence of the fluorophore unit.
5. The compound of claim 1, wherein the fluorophore unit is
fluorescein, BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene;
boron dipyromethene difluoride), a rhodamine, a cyanine, or a
coumarin.
6. The compound of claim 1, wherein the reducible quenching unit is
quinone, naphthaquinone, anthraquinone, quinonemethine, copper
(Cu(II)), sulfate (SO.sub.4.sup.2-), or nitrate
(NO.sub.3.sup.-).
7. The compound of claim 1, wherein the reducible quenching unit is
quinone, naphthaquinone, and anthraquinone.
8. The compound of claim 1, wherein the linker unit is a linear
linker, a branched linker, a cyclic linker, an aromatic linker, a
polycyclic aromatic linker, or an unsaturated linker.
9. The compound of claim 1, wherein the linker unit comprises
carbon atoms, hydrogen, oxygen, sulfur, chlorine, fluorine, iodine,
phosphorous, silicon, nitrogen, other atoms, or combinations
thereof.
10. The compound of claim 1, having the structure FU-polyethylene
glycol-RQU, FU--O--P(.dbd.O)--O-RQU, FU--S(.dbd.O)-RQU, or
FU--S(.dbd.O).sub.2-RQU.
11. A chemical compound having the structure: compound (5) shown in
FIG. 1B; compound (10) shown in FIG. 1D; compound (11) shown in
FIG. 1D; compound (18) shown in FIG. 1G; compound (19) shown in
FIG. 1G; compound (22) shown in FIG. 1I; or compound (24) shown in
FIG. 1J.
12. A kit comprising a chemical compound having the structure
FU-RQU or FU-L-RQU; where FU is a fluorophore unit, RQU is a
reducible quenching unit, and L is a linker unit; and a
solvent.
13. The kit of claim 12, wherein the solvent comprises water, DMSO,
ethanol, methanol, dimethylacetamide, dimethylformamide (DMF),
N-methyl pyrrolidinone (NMP), or mixtures thereof.
14. A method of assaying the oxidative or reductive environment of
a material, the method comprising: providing the material;
contacting the material with at least one chemical compound to form
a test sample, wherein the chemical compound has the structure
FU-RQU or FU-L-RQU; where FU is a fluorophore unit, RQU is a
reducible quenching unit, and L is a linker unit; and determining
the fluorescence of the test sample.
15. The method of claim 14, wherein the material comprises one or
more cells, one or more tissues, or one or more organisms.
16. The method of claim 14, wherein the material comprises one or
more bacterial cells.
17. The method of claim 14, wherein the determining step comprises
irradiating the test sample with light or energy of a suitable
wavelength to excite the chemical compound.
18. A method of assaying the efficacy of an antibacterial compound,
the method comprising: providing at least one bacterial cell;
contacting the bacterial cell with at least one chemical compound
to form a test sample, wherein the chemical compound has the
structure FU-RQU or FU-L-RQU; where FU is a fluorophore unit, RQU
is a reducible quenching unit, and L is a linker unit; determining
the fluorescence of the test sample to provide an initial
fluorescence; contacting the test sample with at least one
antibacterial compound to form a treated sample; determining the
fluorescence of the treated sample to provide a final fluorescence;
and determining the difference between the initial fluorescence and
the final fluorescence to assay the efficacy of the antibacterial
compound.
19. A method of monitoring change in the oxidation state of a
non-biological system, the method comprising: providing a
non-biological system; contacting the non-biological system with at
least one chemical compound to form a test system, wherein the
chemical compound has the structure FU-RQU or FU-L-RQU; where FU is
a fluorophore unit, RQU is a reducible quenching unit, and L is a
linker unit; determining the fluorescence of the test system at a
first time point to provide a first fluorescence; determining the
fluorescence of the test system at a second time point to provide a
second fluorescence; determining the difference between the first
fluorescence and the second fluorescence to monitor change in the
oxidation state of the non- biological system.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority to U.S. Provisional
Patent Application Ser. No. 60/657,944 filed Mar. 1, 2005, the
contents of which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The invention relates to quenched chemical stain compounds
that can be reduced to a fluorescing form, and methods for their
use.
DESCRIPTION OF RELATED ART
[0003] Oxidation and reduction are natural metabolic functions of
living cells. Methods and materials for the detection of the
oxidative states of cells have been desirable for some time, as
they allow researchers to achieve a better understanding of the
conditions within cells or cell compartments. Various methods have
been used to date.
[0004] One approach is to use a chemical probe that is not
fluorescent outside of the cell, but which becomes oxidized to a
fluorescent derivative once it enters the cell. Dihydrorhodamine
and dihydroethidium are examples of such chemicals that oxidize
inside a cell compartment (Haugland, Richard P.; Handbook of
Fluorescent Probes and Research Products; ninth edition; 2002).
These stains are used to gain entry into live cells, and are not
used for measuring redox functions in cells. The stains can be
readily oxidized by molecular oxygen.
[0005] Derivatives of quinones, naphthaquinones, and anthraquinones
have been reported to quench fluorescence when linked to a
fluorophore (Kong, J. C. Y. and Loach, R. A.; J Heterocycl. Chem.
17: 737 (1978); Wasielewski, M. R. and Nienczyk, M. P.; J. Am.
Chem. Soc. 106: 5040 (1984)).
[0006] Israel Patent Application No. 156355 (filed Jun. 9, 2003;
published Jan. 4, 2004) suggests the preparation of compounds
containing a reversible quinone/hydroquinone redox site, a spacer,
and a fluorophore. The compounds do not fluoresce when in the
oxidized state, but become fluorescent once reduced. The compounds
are suggested as being useful as fluorescent redox probes for
chemical, biochemical, and biophysical investigations.
[0007] U.S. Pat. No. 6,057,120 (issued May 2, 2000) describes the
use of redox-active compounds for the determination of an analyte.
The redox pairs have a benzoquinoxaline substructure. The compounds
are suggested to be used to determine the reducing or oxidizing
activities of cells and enzymes.
[0008] 5-Cyano-2,3-ditolyl tetrazolium chloride ("CTC";
commercially available from Polysciences, Inc.; Warrington, PA) is
a monotetrazolium redox dye which produces a fluorescent formazan
("CTF") upon reduction. According to the manufacturer, the dye can
be used in flow cytometry with excitation using a 488 nm laser and
detection in the red color region (Data Sheet #486; September
1999).
[0009] Various species can effect the oxidation of dyes,
complicating the interpretation of experimental results. Examples
of such species include molecular oxygen, superoxide, and hydrogen
peroxide. This oxidation is used in connection with compounds such
as dihydrorhodamine and dihydroethidium, where the neutral reduced
form can enter cells by diffusion, and be subsequently oxidized to
the fluorescent dye. These materials are not generally used to
measure the oxidative function of cells, as atmospheric oxygen can
oxidize them as well.
[0010] Despite advances made to date, there still exists a need for
chemical probes and methods for the monitoring indicators of
metabolic functions within cells, tissues, and other materials.
SUMMARY OF THE INVENTION
[0011] Chemical compound probes containing a fluorophore unit and a
reducible quenching unit are disclosed. The probes can also contain
a linker unit covalently bonded to the fluorophore unit and the
reducible quenching unit. Upon reduction, the reducible quenching
unit exhibits a diminished quenching ability, and fluorescence of
the compound can be detected. The probes can be used in a variety
of applications to monitor the conditions of cells (including
bacteria), tissues, and other materials. Applications include
monitoring of change in oxidation state of a material, change in
electron transport chain function, and change in cellular vitality
after treatment with antibiotics, inhibitors, or other chemical
compounds.
DESCRIPTION OF THE FIGURES
[0012] The following figures form part of the present specification
and are included to further demonstrate certain aspects of the
present invention. The invention may be better understood by
reference to one or more of these figures in combination with the
detailed description of specific embodiments presented herein.
[0013] FIG. 1 shows chemical intermediates and compounds that are
illustrative of embodiments of the invention.
[0014] FIG. 2 shows a titration curve for Gram-positive
Staphylococcus aureus (S. aureus) and Bacillus subtilis (B.
subtilis). The x-axis represents the concentration of compound (18)
in nM, while the y-axis represents FL1 GeoMean fluorescence.
[0015] FIG. 3 shows a titration curve for Gram-negative Escherichia
coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae). The
x-axis represents the concentration of compound (18) in nM, while
the y-axis represents FL1 GeoMean fluorescence.
[0016] FIG. 4 shows growth curve data obtained during a time-course
experiment. The x-axis represents time in hours, while the y-axis
represents S. aureus cells per mL.
[0017] FIG. 5 shows fluorescence data obtained during a time-course
experiment. The x-axis represents time in hours, while the y-axis
represents FL1 GeoMean fluorescence.
[0018] FIG. 6 shows fluorescence data obtained during a time-course
experiment. The x-axis represents time in hours, while the y-axis
represents FL1 GeoMean fluorescence.
DETAILED DESCRIPTION OF THE INVENTION
[0019] While compositions and methods are described in terms of
"comprising" various components or steps (interpreted as meaning
"including, but not limited to"), the compositions and methods can
also "consist essentially of" or "consist of" the various
components and steps, such terminology should be interpreted as
defining essentially closed-member groups.
[0020] Compounds
[0021] One embodiment of the invention is directed towards chemical
compound probes. The chemical compound probe comprises a
fluorophore unit ("probe"), and a reducible quenching unit. The
fluorophore unit and the reducible quenching unit can be covalently
linked or non-covalently linked. The covalent linkage can be direct
with no intervening linker unit, or can be indirect by use of a
linker unit. Non-covalent linkage can be through formation of a
coordination complex, an ionic bond, a pi-pi interaction, van der
waals interaction, or other linkage methods that bring the
fluorophore unit and the reducible quenching unit into a position
whereby the reducible quenching unit can partially or fully quench
the fluorophore unit. Where the chemical compound probe is charged
(either positively or negatively), the embodiment of the invention
also includes salts containing one or more counterions. Examples of
positively charged counterions include alkali metal ions, alkaline
earth metal ions, transition metal ions, ammonium, substituted
ammonium ions, sodium, potassium, lithium, calcium, magnesium, and
ammonium counterions. Examples of negatively charged counterions
include halides (chloride, bromide, iodide), acetate, sulfate,
alkanesulfonate, arylsulfonate, phosphate, perchlorate,
tetrafluoroborate, tetraarylboride, nitrate, anions of aromatic
carboxylic acids, and anions of aliphatic carboxylic acids.
[0022] The chemical compound probes have the advantageous property
of not being fluorescent in the absence of DNA. Once in contact
with DNA, the probes become fluorescent (i.e., they are stains and
not dyes). This property provides for low matrix backgrounds
outside of cells.
[0023] Generally, the chemical compound probes can comprise one of
the following two structures, where FU is the fluorophore unit, L
is a linker unit, and RQU is the reducible quenching unit. FU-RQU;
or FU-L-RQU
[0024] The reducible quenching unit when in its oxidized state
partially or fully quenches the fluorescence of the fluorophore
unit in the probe. This quenching is preferably at least about 50%,
at least about 60%, at least about 70%, at least about 80%, at
least about 90%, at least about 95%, at least about 96%, at least
about 97%, at least about 98%, at least about 99%, and ideally
about 100%. Higher values are more preferred. This reduction in
fluorescence can be measured relative to the fluorescence of the
probe when the reducible quenching unit has been reduced to a
reduced quenching unit (its non-reduced quenching form). The
quenching percentage can be calculated as: ((unquenched
fluorescence minus quenched fluorescence) divided by unquenched
fluorescence) times 100%. The FU, RQU, and optionally the L are
preferably covalently bonded to each other.
[0025] The fluorophore unit can generally comprise any quenchable
fluorophore unit. Examples of such fluorophore units include
fluorescein, BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene;
boron dipyromethene difluoride), rhodamines, cyanines, and
coumarins.
[0026] The reducible quenching unit can generally comprise any unit
that can quench the fluorophore unit when in its oxidized,
non-reduced state, and that partially or fully loses its quenching
ability upon being reduced to its reduced state. Examples of such
reducible quenching units include quinone, naphthaquinone,
anthraquinone, quinonemethine, copper (Cu(II) to Cu(I)), sulfate
(SO.sub.4.sup.2- to SO.sub.3.sup.-), and nitrate (NO.sub.3.sup.- to
NO.sub.2.sup.-). Presently preferred reducible quenching units
include quinone, naphthaquinone, and anthraquinone.
[0027] The linker unit can generally be any linker unit that places
the fluorophore unit and the reducible quenching unit in a suitable
spatial and electronic configuration whereby the reducible
quenching unit can partially or fully quench the fluorophore unit.
The linker unit can be a linear linker, a branched linker, a cyclic
linker, an aromatic linker, a polycyclic aromatic linker, an
unsaturated linker (e.g. containing double and/or triple bonds), or
other linkers. The linker can also contain two or more different
types of linker portions, such as an unsaturated portion and a
cyclic portion. The linker can comprise one or more different types
of atoms. For example, the linker can comprise carbon atoms,
hydrogen, oxygen, sulfur, chlorine, fluorine, iodine, phosphorous,
silicon, nitrogen, other atoms, or combinations thereof. Examples
of such linker units include polyethylene glycol, P0.sub.3
(FU--O--P(.dbd.O)--O-RQU), SO (FU--S(.dbd.O)-RQU), and SO.sub.2
(FU--S(.dbd.O).sub.2-RQU).
[0028] Specific chemical compound probes include: compounds (5),
(10), (11), (18), (19), (22), and (24) shown in FIG. 1.
[0029] Kits
[0030] An additional embodiment of the invention is directed
towards kits containing one or more of the above described chemical
compound probes. The kit can comprise a first container comprising
one or more of the above described chemical compound probes. The
first container can further comprise a solvent. The solvent can
generally be any solvent, and can include aqueous, non-aqueous, or
mixed solvents (e.g. water and DMSO). Examples of solvents are
water, DMSO, ethanol, methanol, dimethylacetamide,
dimethylformamide (DMF), and N-methyl pyrrolidinone (NMP). The
solvent can further comprise a buffer. The kit can further comprise
instruction protocols. The kit can further comprise a positive
control sample. The kit can further comprise a negative control
sample. The kit can further comprise a second container or multiple
additional containers for processing samples.
[0031] The one or more chemical compound probes can be present in
the first container at a "working" concentration, or as a
concentrated stock solution that requires dilution prior to use.
The kit can further comprise a second container comprising a
solvent or buffer solution useful for diluting the stock
solution.
[0032] Methods of Preparation
[0033] An additional embodiment of the invention is directed
towards methods for the preparation of the above described chemical
compound probes. The chemical compound probes can generally be
prepared by the synthetic methods described in the Examples.
[0034] Methods of Use
[0035] An additional embodiment of the invention is directed
towards methods for the use of the above described chemical
compound probes to assay, detect, or monitor the oxidative or
reductive environment of a cell, tissue, or other material. One
probe can be used individually, or multiple probes can be used in
combination. The probes can be used with biological samples (e.g.,
cells, bacterial cells, tissues, organisms), or with non-biological
samples (e.g., electrolytic cells, water samples).
[0036] The methods can comprise providing a material to be assayed,
contacting the material with one or more of the above described
chemical compounds to form a test sample, and determining the
fluorescence of the test sample. The fluorescence can be determined
by irradiating the test sample with light or energy of a suitable
wavelength to excite the chemical compound. Light sources include
LED and laser light sources, such as a 488 nm argon ion laser. The
degree of fluorescence (or lack thereof) can be correlated with the
oxidative or reductive environment of the material. For example, in
an oxidative environment, the reducible quenching unit would be in
its oxidative state, quenching the fluorophore unit, thereby
resulting in little or no fluorescence. In a reducing environment,
the reducible quenching unit would be in its reduced state, not
quenching the fluorophore unit, thereby resulting in fluorescence.
By varying the oxidative or reductive environment of the material,
the degree of detected fluorescence would change.
[0037] The above described chemical compound probes can be used in
a variety of applications, both in vivo and in vitro. The probes
can be used in generally any condition to detect and/or monitor the
oxidative or reductive environment. DNA can be added to the
environment to enhance the response of the probe.
[0038] For example, the probe can be contacted with a target such
as a cell (including bacteria), a collection of cells, a tissue, or
an organism under conditions suitable for uptake of the probe into
the target. If the target has an environment suitable for reduction
of the reducible quenching unit, then the probe can be detected by
its fluorescence properties. The fluorescence of the probe can be
detected at a single point in time, or can be monitored over
multiple points or continuously. The detection can be performed
using instrumentation such as flow cytometry, fluorometers,
epifluorescence microscopes, microplate readers, and fluorescence
readers.
[0039] The uptake can be through diffusion or active uptake by the
target. Alternatively, various chemical or mechanical treatments
may be performed to facilitate uptake. Example treatments include
addition of surfactants, performing electroporation,
microinjection, or addition of peptide or other membrane disruption
agents. Agents such as EDTA or detergents such as Pluronic.RTM.
F127 (Pluronic is a registered trademark of BASF Corporation; Mount
Olive, N.J.) can be used to assist dye entry into Gram-negative
bacteria. Agents such as Tween.RTM. 20 (Tween is a registered
trademark of ICI Americas, Inc.; Bridgewater, N.J.) can be used to
assist dye entry into both Gram-negative and Gram-positive
bacteria.
[0040] Cells can be generally any type of cell. For example, the
cells can be Gram-positive bacterial cells, Gram-negative bacterial
cells, fungal cells, insect cells, fish cells, amphibian cells,
bird cells, reptile cells, or mammalian cells. Bacterial cells are
presently preferred due to their lack of a nucleus. The target can
be a single cell, or a population of cells. The population of cells
can be the same, or a mixture of different cells. The intensity of
the resulting fluorescence of the chemical compound probes is
generally greater in healthy bacterial cells, and lower (or not
changed) in weakened or dead bacterial cells.
[0041] An additional embodiment of the invention is directed
towards methods of evaluating the efficacy of antibacterial
(antibiotic) compounds. A sample of bacteria can be treated with
one or more of the above described chemical compound probes, and
the fluorescence can be measured (the initial fluorescence). The
same or similar sample of bacteria can be treated with a
prospective antibacterial or antibiotic compound. The
antibacterial/antibiotic treated sample is subsequently contacted
with the same one or more chemical compound probes, and the
fluorescence is again measured (the final fluorescence). The two
fluorescence values can be compared, and the difference in
fluorescence can be determined (e.g. final fluorescence minus
initial fluorescence). The magnitude of this difference can be
correlated with the efficacy of the antibacterial or antibiotic
treatment. For example, a small difference in fluorescence would
suggest that the antibacterial or antibiotic treatment was
ineffective. Conversely, a large difference in fluorescence would
suggest that the antibacterial or antibiotic treatment was
effective. This large difference would suggest that the
antibacterial or antibiotic treatment had a substantial effect on
the vitality of the bacteria. Typically, an effective treatment
would be indicated by a reduced increase in fluorescence relative
to a control.
[0042] An additional embodiment of the invention is directed
towards methods of assaying the electron transport system of a
target, and of identifying inhibitors. A target can be treated with
a chemical or other agent suspected of being capable of inhibiting
the target's electron transport system. The treated sample can be
contacted with one or more of the above described chemical compound
probes, and the fluorescence can be measured. The treated sample
can comprise DNA. This fluorescence value can be compared with a
control sample that was not treated with the suspected inhibitor,
but contacted with the chemical compound probe. Alternatively, the
target can be contacted with the chemical compound probe prior to
contacting with the suspected inhibitor. The two fluorescence
values can be compared, and the difference in fluorescence can be
determined (e.g. final fluorescence minus initial fluorescence, or
final fluorescence minus control fluorescence). The magnitude of
this difference can be correlated with the efficacy of the
suspected inhibitor. For example, a small difference in
fluorescence would suggest that the suspected inhibitor was
ineffective at inhibiting the electron transport system of the
target. Conversely, a large difference in fluorescence would
suggest that the suspected inhibitor was effective at inhibiting
the electron transport system of the target. This large difference
would suggest that the suspected inhibitor had a substantial effect
on the vitality of the target. Typically, an effective inhibitor
would be indicated by a higher initial fluorescence and a lower
final fluorescence.
[0043] An additional embodiment of the invention is directed
towards methods of monitoring change in the oxidation state of a
non-biological system. DNA can be added to the environment to
enhance the response of the probe. The system can be a fluid, a
gel, a liquid, a chemical reactor, an environmental sample, or so
on. The system can be contacted with one or more of the above
described chemical compound probes, and the fluorescence can be
measured at a first time point to provide a first fluorescence. A
second fluorescence can be measured after a particular time period
has elapsed, or after subjecting the system to some change or
chemical treatment. The fluorescence can be measured at discrete
times, or can be measured continuously. Additional subsequent
fluorescence values such as a third fluorescence value, fourth
fluorescence value, fifth fluorescence value, sixth fluorescence
value, and so on can be determined. The second or subsequent
fluorescence values can be compared against the first fluorescence
value to monitor change in the oxidation state of the
non-biological system. An increase in the fluorescence indicates
that the system is becoming less oxidizing, and more reducing. A
decrease in the fluorescence indicates that the system is becoming
more oxidizing, and less reducing. The two or more fluorescence
values can be plotted in a graph, prepared as a list or table of
results, or displayed in other conventional manners.
[0044] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the scope of the
invention.
EXAMPLES
Example 1
Preparation of Compound (5)
[0045] A mixture of 40 mg compound (1), 60 mg
2-(2-aminoethylthio)methylanthraquinone, and 45 microliters
triethylamine was stirred in 1 mL dimethylformamide overnight at
room temperature. Product compound (5) was purified by column
chromatography on silica gel with 4:4:3:1 ethyl acetate:
chloroform: methanol: acetic acid.
Example 2
Preparation of Compound (6)
[0046] A mixture of 4.4 g of 2-methyl-1,4-napthoquinone and 11.9 g
of 1-tBoc-piperazine was heated in 90 mL of a
methanol/dichloromethane (1:1, v/v) solvent mixture at about
45.degree. C. for 24 hours. The solvent was evaporated and the
crude product was purified on a silica gel column to yield 1.08 g
of desired intermediate. Trifluoroacetic acid (1 mL) was added to
100 mg of this tBoc-protected piperazine in 5 mL of
dichloromethane, and after one hour at room temperature, all of the
volatile components were removed and the product compound (6) was
used "as is" without further purification.
Example 3
Preparation of Compound (7)
[0047] Triethylamine (0.21 mL) was added to a mixture of 70 mg of
compound (3) and 0.28 mmole of compound (6) in 5 mL of
dichloroethane, and the resulting reaction mixture was heated at
55-60.degree. C. for one hour. The reaction was cooled to room
temperature and volatile components were evaporated. The crude
product compound (7) was purified using silica gel column
chromatography with 2:2:1 ethyl acetate: chloroform: methanol.
Example 4
Preparation of Compound (8)
[0048] A mixture of 1.63 g of the N-hydroxysuccinimidyl ester of
1-tBoc-4-piperidinecarboxylic acid, 0.82 g of 4-aminophenol, and
1.4 mL of triethylamine in 10 ml of dichloromethane was refluxed
for 16 hours. The crude product compound (8) was purified using
silica gel column chromatography with ethyl acetate: hexane.
Example 5
Preparation of Compound (9)
[0049] Lead tetraacetate (0.41 g) was added to 0.27 g of compound
(8) in 2 mL of acetic acid. The resulting mixture was stirred at
room temperature for one hour. About 50 mL of ethyl acetate was
added, followed by 30 mL of water. The organic layer was separated
and dried over anhydrous magnesium sulfate. The crude was purified
using silica gel column chromatography with ethyl acetate: hexane
to obtain 24 mg of compound (9).
Example 6
Preparation of Compound (10)
[0050] A solution of 15 mg of compound (9) was dissolved in 1 mL of
dichloromethane, and 0.5 mL of trifluoroacetic was added. After
stirring at room temperature for 10 minutes, all of the volatile
components were removed under reduced pressure, and the residue was
dissolved in 5 mL of dichloromethane. To this deprotected
intermediate, a solution of 25 mg of compound (1) in 2.5 mL of DMF
was added, followed by 28 uL of triethylamine. The resulting
mixture was stirred at room temperature for several hours. All
volatile components were evaporated, and the product compound (10)
was purified using silica gel column chromatography with ethyl
acetate: chloroform: methanol.
Example 7
Preparation of Compound (11)
[0051] A sample of 3.12 g of 2-cyano-1,4-dimethoxybenzene was
reduced by 1 g of lithium aluminum hydride in 20 mL of THF at
0.degree. C. to yield 3 g of 2-aminomethy- 1,4-dimethoxybenzene.
This was then refluxed in a 1:1 (v/v) mixture of acetic acid and
48% aqueous hydrobromic acid overnight. The volatile components
were removed under reduced pressure, and the crude
2-aminomethyl-1,4-hydroquinone HBr salt was used "as is" without
further purification. Triethylamine (0.4 mL) was added to a mixture
of 50 mg of compound (1) and 0.6 mmole of
2-aminomethyl-1,4-dihydroquinone HBr salt in 2 mL of
dichloroethane, and stirred overnight. The crude dihydroquinone was
then oxidized to the desired quinone compound (11) with ceric
ammonium nitrate in a water/acetonitrile mixture.
Example 8
Preparation of Compound (12)
[0052] Compound (6) (0.3 mmole) was added to a mixture of about 0.2
mmole compound (4) in 2 mL of dichloroethane at room temperature.
Next, about 1 mmole of triethylamine was added. The crude product
compound (12) was purified using silica gel column chromatography
with 5:5:2 ethyl acetate: chloroform: methanol.
Example 9
Preparation of Compound (13)
[0053] A mixture of 2 g of 2,3,5-trimethylquinone and 3.7 g of
1-tBoc-piperazine was heated at 50.degree. C. in 30 mL of a 1:1
(v/v) dichloromethane: methanol mixture overnight. An additional
portion of 3.7 g of 1-tBoc-piperazine was added and heated for an
additional 3 days. Volatile components were evaporated under
reduced pressure, and the crude product was purified using silica
gel column chromatography with 10:9:1 hexane: chloroform: acetic
acid to yield 0.775 of compound (13).
Example 10
Preparation of Compound (14)
[0054] Trifluoroacetic acid (2 mL) was added to 0.775 g of compound
(13) in 5 mL of dichloromethane at room temperature. The mixture
was stirred for 2 hours. All volatile components were evaporated,
and the product compound (14) was used without further
purification.
Example 11
Preparation of Compound (15)
[0055] A mixture of 1 g of 2,3,5-trimethylquinone and 1.6 g of
tBoc-4-aminopiperidine was heated in 20 mL of a 1:1 (v/v)
dichloromethane: methanol mixture overnight. An additional 0.5 g of
the tBoc-4-aminopiperidine was added and heated for an additional 3
days. Volatile components were evaporated under reduced pressure,
and the crude product was purified using silica gel column
chromatography with 10:9:1 hexane: chloroform: acetic acid to yield
0.16 g of compound (15).
Example 12
Preparation of Compound (16)
[0056] Trifluoroacetic acid (1 mL) was added to 0.16 g of compound
(15) in 2 mL of dichloromethane. The mixture was stirred for 4
hours. All volatile components were evaporated, and the product
compound (16) was used without further purification.
Example 13
Preparation of Compound (17)
[0057] A mixture of 24 mg of compound (4), 0.064 mmole of compound
(6), and 0.04 mL of triethylamine was stirred in 2 mL of
dichloroethane at room temperature for 2 hours. At the end of the
period, volatile components were evaporated, and the crude product
compound (17) was purified using silica gel column chromatography
with 2:2:1 ethyl acetate: chloroform: methanol.
Example 14
Preparation of Compound (18)
[0058] A mixture of 0.24 mmole of compound (1), 0.48 mmole of
compound (16), and 0.1 mL of triethylamine in 1 mL of
dichloroethane was stirred at room temperature for several hours.
The reaction was diluted with chloroform and washed with water,
brine, and dried over magnesium sulfate. The crude compound (18)
thus obtained was further purified using silica gel column
chromatography with 2:2:1 ethyl acetate: chloroform: methanol.
Example 15
Preparation of Compound (19)
[0059] A mixture of 0.039 mmole of compound (2), 0.078 mmole of
compound (14), and 0.1 mL of triethylamine was stirred in 2 mL of
dichloromethane at room temperature for several hours. The reaction
was diluted with chloroform and washed with water, brine, and dried
over magnesium sulfate. The crude compound (19) thus obtained was
further purified using silica gel column chromatography with 5:5:2
ethyl acetate: chloroform: methanol.
Example 16
Preparation of Compound (20)
[0060] Triethylamine (0.1 mL) was added to 0.12 mmole of compound
(6) and 50 mg of compound (1) in 2 mL of dichloromethane. The
mixture was stirred at room temperature for 4 hours. The reaction
was diluted with chloroform and washed with water, brine, and dried
over magnesium sulfate. The crude compound (20) thus obtained was
further purified using silica gel column chromatography with 2:2:1
ethyl acetate: chloroform: methanol to yield 28 mg of compound
(20).
Example 17
Preparation of Compound (21)
[0061] To 0.13 g of 2-methyl-1,4-naphthoquinone in 20 mL of a 1:1
(v/v) methanol:dichloromethane mixture, 0.75 mL of
N,N'-dimethylpropanediamine is added and heated at 50.degree. C.
overnight. The volatile components are removed under reduced
pressure and the crude is purified using silica gel column
chromatography with 4:1 chloroform: methanol to yield 60 mg of
compound (21).
Example 18
Preparation of Compound (22)
[0062] A mixture of 22 mg of compound (3), 15.4 mg of compound
(21), and 0.02 mL of triethylamine was stirred in 2 mL of
dichloroethane at 60.degree. C. for several hours. The reaction
mixture was diluted with chloroform, washed with water, brine, and
dried over magnesium sulfate. The crude compound (22) was purified
using silica gel column chromatography with ethyl acetate:
chloroform: methanol.
Example 19
Preparation of Compound (23)
[0063] A mixture of 0.95 g of 2,3,5-trimethylquinone and 6.65 mL of
N,N'-dimethylpropanediamine in a 1:1 (v/v) methanol:
dichloromethane mixture was heated at 50.degree. C. overnight.
Volatile components were evaporated under reduced pressure, and the
crude compound (23) was purified using silica gel column
chromatography with 4:1 chloroform: methanol.
Example 20
Preparation of Compound (24)
[0064] A mixture of 0.115 mmole compound (3), 0.173 mmole of
compound (23), and 50 uL of triethylamine in 5 mL of dichloroethane
was heated at 60.degree. C. for 3 hours. The reaction was diluted
with chloroform and washed with water, brine, and dried over
magnesium sulfate. The crude compound (24) was purified using
silica gel column chromatography with ethyl acetate: chloroform:
methanol.
Example 21
Titration studies
[0065] A titration series was performed on cultured bacteria.
Chemical compound (18) was used at concentrations ranging from 10
nM to 1 uM for S. aureus and B. subtilis (FIG. 2), and 500 nM to 4
uM for E. coli and K pneumoniae (FIG. 3). Low nanomolar
concentrations were found to work well for S. aureus (50-100 nM),
about 500 nM is about optimal for B. subtilis, and about 1 uM are
preferred for the Gram-negative organisms tested.
Example 22
Staining of Gram-positive and Gram-negative Microorganisms
[0066] Gram-positive and Gram-negative bacteria were labeled using
compound (18). S. aureus and B. subtilis were used as examples of
Gram-positive bacteria, and E. coli and K pneumoniae were used as
examples of Gram-negative bacteria. Cells were diluted in buffer to
about 10.sup.6 cells/mL. The bacteria were contacted with compound
18 (500 nM) for 10 minutes in Hank's Balanced Salt Solution (HBSS)
or various other buffers such as PBS. The samples were analyzed by
flow cytometry using a Becton Dickinson FACScan.TM. instrument
(Becton, Dickinson and Company; Franklin Lakes, N.J.) containing a
488 nm argon ion laser. Cells were analyzed directly without
washing, or optionally, were fixed with 1-4% formaldehyde prior to
flow cytometric analysis. TABLE-US-00001 TABLE 1 Staining of S.
aureus Buffer FL1 GeoMean fluorescence Unlabeled 1.02 Sodium
chloride 2171.61 Sodium chloride + 10 mM glucose 2183.44 PBS
1976.41 PBS + 1 mM EDTA 1583.19 PBS + 0.01% Tween-20 2909.97 PBS +
0.1% Pluronic 1685.21 HBSS 2168.52
[0067] TABLE-US-00002 TABLE 2 Staining of B. subtilis Buffer FL1
GeoMean fluorescence Unlabeled 4.92 Sodium chloride 951.93 Sodium
chloride + 10 mM glucose 943.79 PBS 902.15 PBS + 1 mM EDTA 57.75
PBS + 0.01% Tween-20 844.66 PBS + 0.1% Pluronic 470.35 HBSS
902.22
[0068] TABLE-US-00003 TABLE 3 Staining of E. coli Buffer FL1
GeoMean fluorescence Unlabeled 1.07 Sodium chloride 12.43 Sodium
chloride + 10 mM glucose 11.52 PBS 30.25 PBS + 1 mM EDTA 70.92 PBS
+ 0.01% Tween-20 44.48 PBS + 0.1% Pluronic 136.43 HBSS 42.1
[0069] TABLE-US-00004 TABLE 4 Staining of K. pneumoniae Buffer FL1
GeoMean fluorescence Unlabeled 1 Sodium chloride 9.16 Sodium
chloride + 10 mM glucose 9.7 PBS 17.91 PBS + 1 mM EDTA 48.67 PBS +
0.01% Tween-20 29 PBS + 0.1% Pluronic 78.1 HBSS 19.9
[0070] These results confirm that compounds prepared according to
aspects of the instant invention are effective at interacting with,
and detecting both Gram-positive and Gram-negative bacterial cells.
The results also show that the buffer can be chosen to modulate the
observed fluorescence, due to enhancing uptake of the stain
compound into the cells or a capability of the buffer to maintain
the cell vitality.
Example 23
Time Course Treatments
[0071] S. aureus were treated with various antibiotics:
penicillin/streptomycin (100 U/mL), azide (10 mM), CCCP (carbonyl
cyanide m-chlorophenylhydrazone; 10 uM), or antimycin A (20 uM),
and incubated in growth medium. Control samples that were untreated
were also prepared.
[0072] Samples were taken once every hour for six hours and stained
with 500 nM compound (18) for 10 minutes, then analyzed by flow
cytometry using a Becton Dickinson FACSCalibur.TM. instrument
(Becton, Dickinson and Company; Franklin Lakes, N.J.) containing a
488 nm argon ion laser. Not only did the treated cells not
proliferate, but the signal from the stain in treated samples
decreased once the cells entered `log-growth` phase as compared to
the untreated control. FIG. 4 shows the growth curve data, while
FIG. 5 shows the fluorescence signal data. This experiment also
demonstrated that compound (18), when present in broth culture
medium, prevented proliferation of S. aureus.
Example 24
Stability of Fluorescence
[0073] Compound (18) was contacted with cultures of S. aureus, B.
subtilis, E. coli, and K. pneumoniae. The fluorescence of the
samples were monitored for one hour. The signal developed very
quickly for S. aureus and B. subtilis, and stabilized within 10
minutes. The signal was stable for at least one hour (FIG. 6).
Example 25
Compatibility with Fixed Samples
[0074] A culture of S. aureus was grown to log-phase, and washed
and diluted in HBSS to 106 cells per mL. Samples of 1 mL each were
aliquotted to six flow cytometry tubes. CCCP was added to three of
the samples at a final concentration of 10 .mu.M. The samples were
incubated for 1-5 minutes. Compound (18) was added at a final
concentration of 100 nM, and the samples were allowed to sit for 10
minutes. The +/- CCCP tubes were divided into two sets: one set
received 1 mL of 100% ethanol, and the other set received 100 .mu.L
of 10% formaldehyde (methanol-free). The samples were analyzed
using a Becton Dickinson FACSCalibur as described in Example 23.
The results are shown in the following table. TABLE-US-00005 TABLE
5 Fixation assays with compound (18) CCCP Fixation agent
Fluorescence No None 1720 Yes None 110 No Ethanol 135 Yes Ethanol
53 No Formaldehyde 936 Yes Formaldehyde 274
[0075] These results indicate that fixing samples with 1%
formaldehyde maintains the fluorescence signal better than fixing
with 50% ethanol.
Example 26
Electron Transport System Inhibition Assay Using Compound (18)
[0076] S. aureus and E. coli were treated with known inhibitors of
the electron transport system (ETS) to determine if reductase
moieties in the ETS would reduce exemplary stain compounds. The ETS
inhibitors included rotenone (1 mM), antimycin A (20 uM), azide (10
mM), and CCCP (10 uM). The bacteria were diluted in PBS buffer and
treated with the various ETS inhibitors, stained with 500 nM stain
compound (18) for 10 minutes, and then analyzed by flow cytometry
using a Becton Dickinson FACSCalibur.TM. instrument (Becton,
Dickinson and Company; Franklin Lakes, N.J.) containing a 488 nm
argon ion laser. TABLE-US-00006 TABLE 6 ETS assays with compound
(18) Inhibitor S. aureus E. coli Untreated 351.91 85.33 Rotenone
32.75 24.05 Antimycin A 59.41 34.65 Azide 801.54 15.27 CCCP 4.19
150.7
[0077] For S. aureus, the fluorescence signal obtained from the
treated samples were lower than that for the untreated sample,
except for samples treated with azide. For E. coli, the
fluorescence signal obtained from the treated samples were lower
than that for the untreated sample, except for samples treated with
CCCP. Treatment with rotenone or antimycin A demonstrated reduced
fluorescence values with both organisms. Reduced fluorescence
values are indicative of less cellular reductase activity. These
results show that the stain compound (18) can be used to monitor
the vitality of cells treated with various drugs and
inhibitors.
Example 27
Electron Transport System Inhibition Using Compound (19)
[0078] Stain compound (19) was assayed according to the procedure
outlined in Example 23. TABLE-US-00007 TABLE 7 ETS assays with
compound (19) Inhibitor S. aureus E. coli Untreated 248.1 59.78
Rotenone 8.19 20.56 Antimycin A 4.1 12.4 Azide 612.89 25.73 CCCP
46.73 12.59
[0079] With the exception of S. aureus treated with azide, the
fluorescence values for the treated samples were all lower than the
fluorescence values of the untreated samples. These results
demonstrate that compound (19) can be used to monitor the vitality
of cells treated with various drugs and inhibitors.
Example 28
Electron Transport System Inhibition Using Compound (22)
[0080] Stain compound (22) was assayed according to the procedure
outlined in TABLE-US-00008 TABLE 8 ETS assays with compound (22)
Inhibitor S. aureus E. coli Untreated 594.12 90.91 Rotenone 11.74
38.81 Antimycin A 6.16 20.17 Azide 765.59 62.12 CCCP 240.61
63.26
[0081] With the exception of S. aureus treated with azide, the
fluorescence values for the treated samples were all lower than the
fluorescence values of the untreated samples. These results
demonstrate that compound (22) can be used to monitor the vitality
of cells treated with various drugs and inhibitors.
Example 29
Electron Transport System Inhibition Using Compound (24)
[0082] Stain compound (24) was assayed according to the procedure
outlined in Example 23. TABLE-US-00009 TABLE 9 ETS assays with
compound (24) Inhibitor S. aureus E. coli Untreated 853.47 114.71
Rotenone 17.17 44.66 Antimycin A 6.56 26.28 Azide 1272.336 79.45
CCCP 402.19 152.7
[0083] With the exception of S. aureus treated with azide, the
fluorescence values for the treated samples were all lower than the
fluorescence values of the untreated samples. These results
demonstrate that compound (24) can be used to monitor the vitality
of cells treated with various drugs and inhibitors.
[0084] All of the compositions and/or methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and/or methods and in
the steps or in the sequence of steps of the methods described
herein without departing from the concept and scope of the
invention. More specifically, it will be apparent that certain
agents which are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the scope and concept of the invention.
* * * * *