WO2014088512A1 - Ratiometric fluorescent dye for the detection of glutathione in cell and tissue - Google Patents

Abstract

The present invention relates to the use of a BODIPY fluorescence dye as a probe for the selective detection of a biological analyte in cellular extracts as well as live cells and tissues. Specifically, the quantitative measurement and visualization of glutathione in cells and tissues are disclosed.

Description

RATIOMETRIC FLUORESCENT DYE FOR THE DETECTION OF GLUTATHIONE IN CELL AND TISSUE

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 61/732,674, filed on December 3, 2012. The entire teachings of the above application are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Biothiols such as cysteine (Cys), homocysteine (Hey), and glutathione (GSH) are of crucial importance in cellular processes, especially in preventing the damage of cellular components caused by reactive oxygen species.¹ This function is controlled by the equilibrium between oxidized species containing disulfides and reduced species containing thiols. Levels of biothiols such as glutathione in cells and organisms is closely related to aging and many diseases, such as cancer, AIDS, cystic fibrosis and neurodegenerative diseases.³ Furthermore, when present in cancer cells, glutathione is known to confer resistance to chemotherapeutic drugs.⁴ Hence, monitoring the biothiol level in biosystems allows insight into disease status and biothiol function and activity.

Synthetically complicated and highly conjugated pro-fluorescent probes have been utilized for the detection of reactive oxygen species, and a panel of fluorescent probes have also been applied to the detection of intracellular components such as glutathione.⁵ However, these probes have been largely limited to in vitro studies without application to disease or clinical context. Therefore, there is a need to develop methods that identify crucial biological analytes such as glutathione with high selectivity and specificity. In particular, such methods need to be explored in cells and tissues within a clinical context. °

SUMMARY OF THE INVENTION

The present invention relates to the use of a fluorescence dye of a BODIPY- based structure as a ratiometric probe for detection of biological analytes.

Specifically, methods disclosed herein employ chemosensors based on a BODIPY- scaffold having the advantage of being highly tunable and attainable by

straightforward synthetic methods. A BODIPY-based compound is disclosed as a glutathione probe, called Glutathione Green. Glutathione Green shows red-to-green emission color change upon binding to GSH with good sensitivity and selectivity over other biological analytes. The invention further relates to a method for the quantitative measurement of a biological analyte in cell extracts and direct visualization of a biological analyte in live cells and tissue sections, specifically the measurement and visualization of glutathione.

Accordingly, the present invention provides methods for detection of a biological analyte in a cellular extract or in a live cell, comprising contacting a cellular extract or live cell with a compound of Formula (I):

wherein the furyl ring is independently substituted by R at the 4-position, the 5- position or the 4- and 5-positions and R is independently selected from (Ci-C₆)alkyl, (C₆-Cio)aryl, (C₄-Ci₀)heteroaryl, or (C₂-C₆)alkenyl, wherein each R is optionally and independently substituted with one or more substituents selected from a halogen, hydroxyl, nitro, cyano, amino, -B(OH)₂, (Ci-C6)alkyl, halo(Ci-C₆)alkyl,

hydroxy(C(-C₆)alkyl, (C₁-C₆)alkoxy, or (C(,-C{o)aryl or a pharmaceutically acceptable salt thereof, thereby forming an incubation media; incubating the incubation media under conditions sufficient to enable detection of a biological analyte by fluorescence, if present in the incubation media; and detecting a fluorescence signal in the incubated media, wherein a change in the fluorescence signal as compared to a fluorescence signal of the compound of Formula (I) not in the presence of the cellular extract or the live cell is indicative of the presence of the biological analyte in the cellular extract or live cell.

In particular embodiments of the invention, R is selected from (Ci-C )alkyl or (C -C|₀)aryl, wherein R is optionally and independently substituted with one or more substituents selected from halogen , hydroxyl, or (Ci-C6)alkoxy. In particular embodiments, the compound of Formula (I) has the structure of Formula (II):

The present invention provides methods for the detection of the biological analyte glutathione, and further provides methods of quantitative measurement of the biological analyte.

The invention further relates to methods for visualizing a biological analyte in a tissue or a live cell, comprising contacting a tissue or a live cell with the compound of Formula (I), thereby forming an incubation media, incubating the media, and imaging the incubated media to visualize the biological analyte in the live cell or tissue by fluorescence spectroscopy.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing will be apparent from the following more particular description of example embodiments of the invention, as illustrated in the accompanying drawings.

FIG. la shows the chemical structure of 9 BODIPY-based glutathione probes. FIG. lb shows the fluorescent response of the 9 probes at 520nm after incubation with glutathione for 30 minutes in HEPES buffer (20 mM, pH=7.4) under excitation at 470 nm.

FIGs. 2a and 2b show time dependent absorbance (FIG. 2a) and fluorescence

(FIG. 2b) spectra of Glutathione Green (10 μΜ) with glutathione (GSH, at 5 mM) in DMSO/HEPES buffer (1 :4 v/v, 20 mM, pH = 7.4). FIG. 2c shows a picture of Glutathione Green solution (10 μΜ) with 5 mM GSH (right) and without GSH (left) under irradiation with a 365 nm UV lamp. FIG. 3 shows the fluorescence response of Glutathione Green (10 μΜ) incubated with different thiol compounds (5 mM) after 30 minutes in HEPES buffer (20 mM, pH=7.4) under excitation at 470 run.

FIG. 4 displays the fluorescence responses of Glutathione Green (10 μΜ) toward different analytes at 522 nm (black bar: 5mM analyte concentration; white bar: 1 mg/mL analyte concentration).

FIG. 5 shows time dependent fluorescent response of Glutathione Green (10 μΜ) to GSH (2 mM) in HEPES buffer (20 mM, pH=7.4) with different percentages of DMSO ( 1 %, 5%, 10%, 25% and 50% from top to bottom).

FIG. 6 shows HPLC-MS spectral characterization of Glutathione Green

(upper panel), GSH incubated with Glutathione Green in 50% DMSO for 10 min (middle panel), and GSH incubated with Glutathione Green in 50% DMSO for 2h (lower panel). The resulting new peaks have a mass of 840.0 [M+H] (mono- substitution) (middle panel), 1 147.2 [M+H] (di-substitution) and 574.2 [(M+2H)/2] (di-substitution; not shown here) (lower panel), which correspond, respectively, to structures 1 and 2 in Scheme 1.

FIG. 7 contains bar graphs demonstrating the cytotoxic effect of Glutathione Green in 3T3, HeLa, Chang and HepG2 cell lines.

FIG. 8a shows GSH concentration in cell extract calculated by using a commercial GSH fluorimetric kit. Similarly, FIG. 8b shows GSH concentration in cell extract calculated by using Glutathione Green. FIG. 8c shows the average image intensity ratio (F_green/F_red) collected from two emission windows, FITC (green) and Texas Red (red) channel obtained in cell imaging. FIG. 8d displays a fluorescent microscopic image of live 3T3 cells stained with 2μΜ Glutathione Green upon pre-incubate of NMM (1 mM) for 30 minutes (bottom), LPA (250 μΜ) for 48 hours (middle) and the control (top). In the FITC channel of FIG. 8d, glutathione concentration clearly increases with treatment of LPA, as measured by image intensity. Conversely, upon treatment with NMM, the image intensity of glutathione is vastly depleted.

FIG. 9 displays rat liver tissue GSH imaging in normal liver tissue and tumor-containing liver tissue. Specifically, normal (upper panels) and cancerous (lower panels) liver tissues were stained by Glutathione Green and Hoechst 33342. Higher GSH levels in the cancerous tissue, which is clearly distinguishable by abnormal morphology of nucleus (DAPI filter, blue) is shown by lower red (Tx Red)/green (FITC) signal ratio (R/G) compared to that of normal tissue. Scale bar, 100 μΜ.

FIG. 10a shows a plot of of k' versus concentration of GSH. FIG. 1 Ob shows the time dependent fluorescent response of glutathione green (10 μΜ) to GSH (2 mM) in HEPES buffer (20 raM, pH=7.4). FIG. 10c shows a kinetics measurement by fluorescence of Glutathione Green (10 μΜ) incubated with GSH (5 mM). The pseudo-ivcst order reaction constant to Glutathione Green is 2.5 s^"1.

FIG. 11 is a bar graph showing GSH concentration in cell extract, as calculated by a commercial GSH fluoremetric kit (dark gray) and as calculated with glutathione green (light gray).

DETAILED DESCRIPTION OF THE INVENTION

A description of example embodiments of the invention follows.

Glossary

All definitions of substituents set forth below are further applicable to the use of the term in conjunction with another substituent.

"Alkyl" as used alone or as part of a larger moiety as in "arylalkyl" or "aryloxyalkyl" means a saturated aliphatic branched or straight-chain monovalent hydrocarbon radical. Optionally, the alkyl group has a specified number of carbon atoms. Thus, "(Ci-C₆) alkyl" means a radical having from 1- 6 carbon atoms in a linear or branched arrangement. "(C₁-C₆)alkyl" includes methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, and hexyl.

The term "(C6-Ci₀)aryl" used alone or as part of a larger moiety as in

"arylalkyl", "arylalkoxy", or "aryloxyalkyl", means carbocyclic aromatic rings. The term "carbocyclic aromatic group" may be used interchangeably with the terms "aryl", "aryl ring" "carbocyclic aromatic ring", "aryl group" and "carbocyclic aromatic group". An aryl group typically has 6-10 ring atoms.

The term "alkoxy" means -O-alkyl, where alkyl is defined as above; (Ci-

C₆)alkoxy refers to an alkoxy group having from 1 to 6 carbons; "arylalkoxy" means an alkoxy group substituted at any carbon by an aryl group. Further, "hydroxy(C!- C₆)alkyl" means an alkyl group having from 1 to 6 carbons, substituted at any carbon with hydroxy; "halo(Ci-C₆)alkyl means an alkyl group having from 1 to 6 carbons, substituted at any carbon with a halogen. A "halogen" as defined herein, is a fluorine, a chlorine, a bromine or an iodine atom. Typically, an alkyl group, an alkoxy group, a hydroxyalkyl group and a haloalkyl group have between 1 and 6 carbon atoms.

The term "Alkenyl" means a straight or branched hydrocarbon radical having carbon atoms and including at least one double bond. An alkenyl group generally has between 2 and 6 carbon atoms.

The term "heteroaryl", "heteroaromatic", "heteroaryl ring", "heteroaryl group" and "heteroaromatic group", used alone or as part of a larger moiety as in "heteroarylalkyl" or "heteroarylalkoxy", refers to aromatic ring groups having five to ten ring atoms selected from carbon and at least one (typically 1 - 4, more typically 1 or 2) heteroatoms (e.g., oxygen, nitrogen or sulfur). They include monocyclic rings and polycyclic rings in which a monocyclic heteroaromatic ring is fused to one or more other carbocyclic aromatic or heteroaromatic rings. The term "5-10 membered heteroaryl" as used herein means a monocyclic, bicyclic or tricyclic ring system containing one or two aromatic rings and from 5 to 10 atoms of which, unless otherwise specified, one, two, three, four or five are heteroatoms independently selected from N, NH, N(Ci-C₆)alkyl, O and S.

Pharmaceutically acceptable salts of the compounds of the present invention are also included. For example, an acid salt of a compound of the present invention containing an amine or other basic group can be obtained by reacting the compound with a suitable organic or inorganic acid, resulting in pharmaceutically acceptable anionic salt forms. Examples of anionic salts include the acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, glyceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate,

hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, methylsulfate, mucate, napsylate, nitrate, pamoate, pantothenate, phosphate/diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, teoclate, tosylate, and triethiodide salts.

A "ratiometric probe" is a voltage-based sensor measuring fluorescence, wherein the voltage output from the sensor is proportional to the voltage input.

A "biological analyte" as used herein, is a molecular species for which a diagnostic test is performed. A biological analyte may be a biopolymer, or small molecule bioactive material, and may also be, without limitation, a protein, peptide, carbohydrate, polysaccharide, glycoprotein, hormone, receptor, antigen, antibody, virus, substrate, metabolite, cofactor, inhibitor, drug, dye, nutrient, or growth factor.

A "cellular extract" is lysed cells from which insoluble matter has been removed via centrifugation.

A "tissue section" is a portion of tissue suitable for analysis. A tissue section can refer to a single tissue section or a plurality of tissue sections.

A "live cell" is a living cell culture for in vitro analysis. A live cell can refer to a single cell or a plurality of cells.

"Pro-fluorescent" as used herein, refers to an analytical probe, wherein the fluorescence of the probe is altered when bound to or associated with an analyte. "Altering" the fluorescence means, for example, turning on the fluorescence, changing the intensity of the fluorescence, or changing the wavelength or fluorescence maxima k_f\u) of the fluorescence signal.

The terms "selectivity" or "selective", as used herein, refer to an analytical probe, for example a fluorescent dye, that produces a response for a target analyte that is distinguishable from responses of all other analytes. Selectivity can also refer to the analytical probe preferentially binding to a target analyte over all other analytes.

The terms "specificity" or "specific", as used herein, refer to an analytical probe, for example a fluorescent dye, that produces a response for only one single analyte. Specificity can also refer to the analytical probe exclusively binding with a target analyte.

The term "ratiometric" refers to a system having two wavelengths of signal that change directly proportional to input. As used herein, "spectroscopy" encompasses any method by which matter reacts with radiated energy. This includes, but is in no way limited to, microscopy, fluorescence microscopy, UV Vis spectrometry, and flow cytometry.

A "change in fluorescence signal" as used herein, can be used to indicate a change in the fluorescence intensity of a sample after exposure to an analyte, as compared to a baseline exposure. For example, a fluorophore, such as a BODIPY- based fluorophore having the structure of Formula (I), exhibits a change in fluorescence intensity after exposure to an analyte such as glutathione. In some embodiments of the invention, the change in fluorescence intensity is an increase in fluorescence intensity. Alternately, a change in fluorescence can be a change in the color of the fluorescence. A change in the color of the fluorescence can be a change in the color hue of the fluorescence (e.g. a green hue versus a red hue), or can be a change in the tint or saturation of the fluorescence (e.g. a light red versus a dark red).

As used herein, the term "incubation" or alternately, "incubating" a sample means mixing a sample. Alternately, incubating means mixing and heating a sample. "Mixing" can comprise mixing by diffusion, or alternately by agitation of a sample.

In some embodiments of the invention, "detecting a fluorescence signal" means analysis utilizing a fluorescence reader, fluorescence spectroscopy, fluorescence meter or another method that can quantify fluorescence. In alternate embodiments of the invention, "detecting a fluorescence signal" means a visual analysis carried out by the human eye. In some embodiments of the invention, detecting fluorescence by visual analysis is carried out under visible light. In other embodiments of the invention, detecting fluorescence by visual analysis is carried out under certain wavelengths of light, e.g. about 365 run (ultra-violet light), about 532 nm (green laser light).

Description of Embodiments of the Invention

Glutathione (GSH) is an important antioxidant, preventing damage of cellular components caused by reactive oxygen species such as peroxides and free radicals. The importance of monitoring the level of glutathione in biosystems is especially crucial in aging and in diseases such as cancer, AIDS, cystic fibrosis, and neurodegenerative diseases. Furthermore, glutathione in cancer cells is known to confer resistance to chemotherapeutic drugs. Therefore, detection and analysis of glutathione levels in cancer cells and tissues has potential applications in selecting or altering a therapy for the treatment of a subject suffering from cancer. The invention herein is directed to a method for detection and measurement of glutathione in cellular extracts and tissues by utilization of a new ratiometric glutathione probe having a BODIPY-based structure.

The present invention relates to the fluorescence-based detection of biothiols in a cellular extract or in a li rmula (I):

wherein the furyl ring is independently substituted by R at the 4-position, the 5- position or the 4- and 5-positions and R is selected from (CrC₆)alkyl, (C -C₁₀)aryl, (C₄-Ci₀)heteroaryl, or (C₂-C₆)alkenyl, wherein each R is optionally and

independently substituted with one or more substituents selected from a halogen, hydroxyl, nitro, cyano, amino, -B(OH)₂, (CrC^alkyl, halo(C_!-C₆)alkyl,

hydroxy(C(-C₆)alkyl, (C[-C6)alkoxy, or (C₆-C|₀)aryl; or a pharmaceutically acceptable salt thereof.

In certain embodiments of the invention, the fluorescent dye has the structure of Formula (I), wherein the furyl ring is substituted at the 5-position by R, wherein R is selected from (Ci-C₆)alkyl or (C₆-C₁₀)aryl, optionally and independently substituted by a halogen, a hydroxyl, or a (Ci-C₆)alkoxy. In certain other

embodiments, the fluorescent dye has the structure of any one of formulas BDD-22, BDD-195, BDD-238, BDD-284, BDD-319, BDD-371, BDD-393, or BDD-428. The fluorescent response of each of these dyes to glutathione is shown in FIG. 1.

or a pharmaceutically acceptable salt thereof. Compounds of the invention that are useful for glutathione detection fall within the scope of Formula (I), and thus include a furanyl ring. Without being bound to theory, it is believed that the furan ring is a structural feature that enables glutathione binding to the fluorescent dye.⁶

The structure of Formula (II) is known as Glutathione Green⁷ (BDD-135, FIG. 1), and exhibits absorption maxima ( _abs) at 565 nm (ε=51,000 M^"'cm^"1) and fluorescence maxima ( _f|_U) at 585 nm with quantum yield of 0.47 in DMSO. When bound to GSH, the fluorescence and absorbance spectra of Glutathione Green exhibit a profound ratiometric change, undergoing a significant hypsochromic shift, with the fluorescence maxima shifting to 522 nm with an apparent isosbestic point at 562 nm, and the absorbance maxima shifting to 512 nm with an apparent isosbestic point at 522 nm in 20% DMSO/HEPES buffer solution (FIG. 2). In certain embodiments of the invention, the fluorescent dyes of the present invention show a ratiometric response to biothiol analytes containing thiol groups, such as cysteine, dithiothreitol (DTT), 2-mercaptoethanol (2-ME), and glutathione (GSH). In alternate embodiments, the fluorescent dyes have a response to oxidized glutathione (GSSG). The fluorescence response of Glutathione Green to a number of target analytes is shown in FIG. 3. In particular embodiments, Glutathione Green is highly selective for GSH over other classes of analytes with respect to the response at 522 nm. For example, Glutathione Green shows good selectivity for GSH against actual concentrations of biothiol compounds in biosystems such as cysteine (FIG. 4). Without being bound to theory, it is believed that other thiols may not significantly interfere in the GSH response to the fluorescent probe, because they are present in relatively low concentrations in biological systems.

In certain embodiments of the invention, the reaction between a fluorescent dye having the structure of Formula (I) and a biological analyte occurs during incubation. Incubation can occur in liquid media, for example in water-based buffers. In certain embodiments, the water-based buffer may contain up to 50% dimethylsulfoxide (DMSO), up to 50% methanol, or up to 50% acetonitrile. Higher concentrations of organic solvents, however, slow the reaction, as demonstrated in Example 3 and FIG. 5. In embodiments of the invention where the reaction takes place in a water-based buffer, and in the absence of organic solvent, a change in fluorescence, for example, a change in solution color, is immediately observable. In alternate embodiments of the invention, wherein up to 50% DMSO, methanol, or acetonitrile is present in the reaction, the color change occurs relatively slowly, such that a step-wise change in fluorescence is observable. For example, at 50% DMSO, the reaction of Glutathione Green with glutathione changes the color of the solution from red to orange, and then from orange to green (Example 3, FIG. 6). Based on analytical data, the orange intermediate is a product of mono-addition of glutathione to Glutathione Green, while the green final product results from di-addition of glutathione to Glutathione Green.

In certain embodiments of the invention, a change in fluorescence signal change occurs via incomplete reaction of the fluorescence probe with the biological analyte. For example, as shown in Scheme 1 , Glutathione Green can react once with Glutathione to form the mono-addition product 1, which produces a fluorescence signal that is distinct from the fluorescence signal of Glutathione Green. Under certain conditions, intermediate 1 will undergo Michael Addition with a second glutathione molecule to form the di-addition product 2, which produces a fluorescence signal that is distinct from the fluorescence signal of 1 and Glutathione Green. Both 1 and 2 are indicative of the presence of the target biological analyte.

The good sensitivity, selectivity, fast reaction speed and ratiometric response of fluorescent dyes of Formula (I) allow both quantitative measurement of biothiol analytes in cell extracts and direct visualization of biothiol analytes in live cells and tissue sections. Glutathione Green (Formula (II)) is particularly useful in the detection of glutathione and biothiols due to the fact that it is minimally toxic to cells over a 24 hour incubation period (Example 7, FIG. 7). In certain embodiments of the invention, the fluorescent dyes of Formula (I) are used to detect a biological analyte in a live cell. In alternate embodiments, the fluorescent dyes of Formula (I) are used to detect a biological analyte in a tissue section. In particular embodiments, the dye is Glutathione Green and the analyte is glutathione. As demonstrated in Example 8 and FIG. 8d, Glutathione Green can detect and visualize GSH in live cells. This detection can occur in real time, and be used in live-imaging the cells, even as environmental or chemical factors alter the levels of GSH present in the cells.

In certain embodiments, the fluorescent dyes of Formula (I) are used in quantitative measurement of a biological analyte. Glutathione Green, for example, can be used in the quantitative measurement of glutathione concentration in a cell extract or live cell. Specifically, the cell extract or live cell is incubated with glutathione green under conditions sufficient to enable a change in a fluorescence signal, and then the fluorescence signal is measured by a quantitative method such as fluorescence meter or fluorescence spectroscopy. Glutathione Green is capable of measuring glutathione concentration with close precision as compared to the measurements of a commercially available glutathione fluoremetric kit (FIG. 1 1).

Increase of GSH in rapidly proliferating hepatocyte and hepatocellular carcinoma is known.¹⁰ In certain embodiments of the invention, the fluorescent dyes of Formula (I) are used to assess the change in biothiol concentration in

carcinogensis in tissues. In particular embodiments, the fluorescent dye is

Glutathione Green and the biothiol is glutathione.

While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

EXAMPLES

The following examples are provided to illustrate one or more preferred embodiments of the invention, but are not limited embodiments thereof. Numerous variations can be made to the following examples that lie within the scope of the invention. All the reagents and solvents were purchased from Aldrich, Alfa, Acros Organics, or another commercial source and used without further purification.

Column chromatography was performed on Merck 60 silica gel (230-400 mesh). Analytical characterization was performed on a HPLC-MS (Agilent- 1200 series) with a DAD detector and a single quadrupole mass spectrometer (6130 series) with an ESI probe. 1H-NMR and ^l3C-NMR spectra were recorded on a Bruker Avance 300 NMR and 500 NMR spectrometers. Chemical shifts are reported as δ in units of parts per million (ppm) and coupling constants are reported as a J value in Hertz (Hz). High Resolution mass spectrometry (HRMS) data was recorded on a

Micromass VG 7035 (Mass Spectrometry Laboratory at National University of Singapore (NUS)). Spectroscopic and quantum yield data were measured on spectroscopic measurements, performed on a fluorometer and UV/VIS instrument, Synergy 4 of Bioteck Company. The slit width was 1 nm for both excitation and emission and the data analysis was performed using GraphPrism 5.0. Alternately compound mass was determined by LC-MS of Agilent Technologies with an electrospray ionization source. Fluorescence assays were performed with a Gemini XS fluorescence plate reader.

Example 1: Synthesis of Glutathione Green

Following the synthesis published by Zhai, et al. , Glutathione Green was prepared and characterized. HRMS m/z (C22H₂oBCl₃F₂N₂03) calculated: 514.0601 found: 495.0534 (M-F). Ή NMR (300 MHz, CDC1₃): 7.40 (d, J=16.2 Hz, IH), , 7.04 (d, J=16.2 Hz, IH), 7.01 (s, IH), 6.84 (d, J=3.9 Hz, IH), 6.64 (s, IH), 6.51 (d, J=3.3 Hz, IH), 6.29 (d, J=3.9 Hz, IH), 6.10 (d, J=3.3 Hz, IH), 4.78 (s, 2H), 3.40 (t, J=7.5 Hz, 2H), 2.96 (t, J=7.5 Hz, 2H), 2.40 (s, 3H), 2.27 (s, 3H). ^l3C NMR (75.5 MHz, CDC1₃): 1 1.3, 22.7, 23.8, 31.9, 74.1, 93.9, 109.2, 1 10.4, 1 14.9, 1 15.5, 1 16.2, 116.7, 121.2, 124.2, 125.6, 126.5, 129.5, 137.2, 144.6, 151.1, 155.9, 171.1.

Example 2: Reaction of Glutathione Green and GSH

1 xL of Glutathione Green DMSO solution (1 mM) was directly mixed with 99 of GSH buffer solution (5 mM). The reaction between Glutathione Green and GSH followed pseudo-first order kinetics with a large excess of GSH (5 mM), and the pseudo-first order reaction constant k' = 2.5 s^"1 (FIG. 10). The plot of k' versus concentration of GSH was a straight line through the origin, indicating that the reaction is second-order overall, first order with respect to Glutathione Green and first order with respect to GSH, with k = 0.28 M^"'s^"'.

Example 3: Reaction of Glutathione Green and GSH in DMSO solution

GSH was incubated with Glutathione Green in 50% DMSO. The incubation media was characterized by HPLC-MS at 10 minutes and 2 hours. As compared to the HPLC-MS for Glutathione Green, the HPLC-MS at 10 minutes indicated a new peak at 840.0 [M+H] for the monosubstituted product, an orange solution, and a new peak at 1147.2 [M+H] for the disubstituted product, a green solution (FIG. 6)

Example 4: Cell Culture

3T3 fibroblast cells were cultured on a cell culture dish in Dulbecco's

Modified Eagle Medium (Sigma) with 10% newborn calf serum and 5 mM L- glutamine and 5 mg/mL gentamicin. Cell cultures were maintained in an incubator at 37 °C with 5% C0₂. Cells were cultured in glass-bottom, 96-well black plates for imaging experiments, 24-36 hours prior to conducting experiments.

Example 5: Tissue Preparation

Frozen tissue sections (10 μπν) were prepared on a cryostat and mounted on lysine coated slide glasses. After drying for 30 minutes at room temperature, the tissues were incubated with 2 μΜ of Glutathione Green diluted in PBS for 1 hr and briefly washed with PBS. Then the tissues were mounted with PBS containing 1 g ml Hoechst 33342 and cover slipped.

Example 6: Image acquisition

Glutathione Green stock solution in DMSO (5 mM) was added directly to the cell culture wells to reach the desired concentration. After 1 hour incubation at 37 °C, cells were subjected for imaging with an automated fluorescence microscope ImageXpress^Micr0 (Molecular Devices). FITC Long Pass (ex 450-490nm, em 515 nm) and Texas Red filters were used for fluorescence image acquisition. For tissues, microscope images were acquired by an inverted fluorescence microscope Ti (Nikon) using DAPI, FITC and Texas Red filters. Green and red images were merged and red/green ratio was viewed using NIS Element software.

Example 7: Cytotoxic Effect of Glutathione Green in 3T3, HeLa, Chang and HepG2 Cell lines. The cytotoxic effect of Glutathione Green was tested by the MTS (3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium, inner salt) assay using the CellTiter 96 nonradioactive cell proliferation colorimetric assay kit (Promega) on mESC cells. First, the cells (1 X 10⁴ cells in 100 μΐ of media) were seeded onto 96 well plate and then different concentrations (0 μΜ, 2 μΜ, 5 μΜ, 10 μΜ and 50 μΜ) of Glutathione Green was added to the cells on the following day and incubated them at 37°C for 24h. After 4 h, 20 μΐ of MTS solution was added to each well and incubated for 1 hour before the absorbance was measured at 490 nm. The same experiments were done for 10 h and 24 h. The control cells were 100% alive without compound condition. At least 70% of the Glutathione Green treated cells were alive after 24 h of incubation at 10 μΜ concentration. These results indicated that Glutathione Green is nontoxic to different cell lines.

Example 8: Visualization of glutathione in live cells a-lipoic acid (LP A) is known to induce the synthesis of GSH, and NMM

(N-methylmaleimide)⁹ is known to protect the thiol group inside a variety of cells. 3T3 cells were pretreated with α-lipoic acid for 48 h, then after fluorescence analysis, treated with NMM for 30 min. The cell extracts were analyzed by

Glutathione Green, as well as a commercial GSH fluorimetric kit. The results from both the methods correlated very well with each other, indicating that Glutathione Green is able to be used for GSH quantification in vitro (FIGs. 4a and 4b).

Glutathione Green was also tested to monitor intracellular GSH concentration using 3T3 cells. The image intensity of the 3T3 cells labeled with Glutathione Green acquired from two emission windows (green: FITC; red: texas red) gave an average ratio of F_green F_red⁼l .61. Glutathione Green also responses to the change of GSH concentration in cellular environment: the F_green Fred increased to 2.59 when the cells were pre-incubated with α-lipoic acid for 48 h, and decreased to 0.14 upon treatment to the cells with NMM for 30 min (FIGs. 4c and 4d).

Example 9: Visualization of glutathione in tissue during carcinogenesis

To assess whether the change of GSH concentration in liver carcinogenesis can be detected by Glutathione Green, tumors generated by injecting hepatoma cell line MH39248 into the rat liver and normal rat liver were flash frozen in dry ice, cryosectioned and incubated with Glutathione Green followed by Hoechst 33342 on a slide glass. Abnormal nucleus morphology of tumor visualized by Hoechst 33342 was clearly distinguishable from that of normal tissue. Green and red staining patterns in tumor tissue were uneven, while the patterns are even in normal rat liver tissue. Within the tumor section, the green signal was even higher in certain groups of cells than in the other cancer cells. Overall green signal intensity was stronger and red was weaker in tumor compared to normal tissue, which was more clearly visualized by red/green ratio view (FIG. 9). This result suggests that GSH level changes in tumorous region of liver tissue can be simply and easily detected by Glutathione Green.

References:

1. Z. A. Wood, et al., Trends Biochem. Sci. 2003, 28, 32-40.

2. R. Kizek, et al. , Bioelectrochemistry 2004, 63, 19-24.

3. D. M. Townsend, et al., Biomed. Pharmacother. 2003, 57, 145-155.

4. Balendiran, G. K. et al., Cell Biochemistry and Function 2004, 22, 343-352.

5. U.S. Patent Application Publication No. US 2011/0130306 Al (Published June 2, 201 1) (C. J. Chang, applicant).

6. D. Zhai, et al, Chem. Commun. 2013, 49, 7207.

7. D. Zhai, et al, ACS Comb. Sci, 2012, 14, 81-84.

8. B. Hultberg, et al , Toxicology 2002, 175, 103- 110.

9. C. R. Yellaturu, et al. , J. Biol. Chem. 2002, 277, 40148-40155.

10. Z. Z. Huang, et al , FASEB J. 2001, 15, 19-21. The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

CLAIMS is claimed is:

A method for detection of a biological analyte in a cellular extract or in a live cell, comprising:

a) contacting a cellular extract or a live cell with a compound of formula

(i);

wherein the furyl ring is independently substituted by R at the 4- position, the 5-position or the 4- and 5-positions and R is independently selected from (C)-C₆)alkyl, (C₆-Ci₀)aryl, (C₄- Ci₀)heteroaryl, or (C₂-C6)alkenyl, wherein each R is optionally and independently substituted with one or more substituents selected from a halogen, hydroxyl, nitro, cyano, amino, -B(OH)₂, (C_rC₆)alkyl, halo(Ci-C₆)alkyl, hydroxy(C C₆)alkyl, (C_rC₆)alkoxy, or (C₆-C₁₀)aryl;

or a pharmaceutically acceptable salt thereof;

to form an incubation media;

incubating the incubation media of step (a) under conditions sufficient to enable detection of a biological analyte by fluorescence, if present in the incubation media; and

detecting a fluorescence signal in the incubated media, wherein a change in the fluorescence signal as compared to a fluorescence signal of the compound of Formula (I) not in the presence of the cellular extract or the live cell is indicative of the presence of the biological analyte in the cellular extract or live cell.

2. The method of Claim 1 , wherein: R is selected from (Ci-C₆)alkyl or (C₆-Cio)aryl, wherein each R is optionally and independently substituted with one or more substituents selected from a halogen, hydroxyl or (Ci-C₆)alkoxy; or a pharmaceutically acceptable salt thereof.

The method of Claim 1 , wherein the compound of formula (I) has the structure of form

.

or a pharmaceutically acceptable salt thereof.

The method of Claim 1, wherein the biological analyte is glutathione.

The method of Claim 2, wherein the biological analyte is glutathione.

The method of Claim 3, wherein the biological analyte is glutathione.

The method of Claim 1 , wherein the detection of a biological analyte is the quantitative measurement of a biological analyte.

The method of Claim 4, wherein the detection of a biological analyte is the quantitative measurement of a biological analyte.

A method for visualizing a biological analyte in a tissue or a live cell, comprising:

a) contacting a tissue section or a live cell with a compound of formula (I),

wherein the furyl ring is independently substituted by R at the 4-position, the 5-position or the 4- and 5-positions and R is selected from (Ci-C₆)alkyl, (C₆-C₁₀)aryl, (C₄-C₁₀)heteroaryl, or (C₂-C₆)alkenyl, wherein each R is optionally and independently substituted with one or more substituents selected from a halogen, hydroxyl, nitro, cyano, amino, - B(OH)₂, (Ci-C₆)alkyl, halo(d-C₆)alkyl, hydroxy(C₁-C₆)alkyl, (Ci-C₆)alkoxy, or (C₆-C₁₀)aryl;

or a pharmaceutically acceptable salt thereof;

to form an incubation media;

b) incubating the incubation media of step (a) for a period of time under conditions sufficient to form an incubated mixture; and c) imaging the incubated mixture of step (b) to visualize a biological analyte in the tissue section or the live cell using fluorescence spectroscopy.

The method of Claim 9, wherein:

R is selected from (C C₆)alkyl or (C₆-Ci₀)aryl, wherein each R is optionally and independently substituted with one or more

substituents selected from a halogen, hydroxyl, or (C₁-C₆)alkoxy; or a pharmaceutically acceptable salt thereof.

11. The method of Claim 9, wherein the compound of formula (I) has the

structure of formula (II):

or a pharmaceutically acceptable salt thereof.

12. The method of Claim 9, wherein the biological analyte is glutathione.

13. The method of Claim 10, wherein the biological analyte is glutathione. 14. The method of Claim 1 1, wherein the biological analyte is glutathione.