1. Field of Invention
This application relates to the design and synthesis of long wavelength fluorescent ion indicators.
2. Prior Art
The most popular fluorescent calcium indicators that are ratiometric include Fura-2 (excitation shifting) and Indo-1 (emission shifting)i. Ratiometry has the unique advantage of eliminating the variable effects in calcium measurement such as indicator concentration, degree of cell loading, photobleaching, detector sensitivity, cell thickness, and optical path. It is essentially an ideal property that allows for calibration and quantitative measurements. However, these popular ratiometric dyes operate in ultraviolet light. UV optics are expensive and hence have limited the general use of these dyes. The high energy UV light can also be harmful to the cell. The intrinsic fluorescence due to nucleotides and some amino acids when exciting with UV light also causes interference during measurement, although ratiometry does attenuate this problem.
The popular visible wavelength fluorescent ion indicators, Fluo dyesii, do not exhibit ratiometry. They simply markedly enhance emission intensity upon binding calcium.
The baseline is not easily delineated, but using manganese or nickel has allowed a useful assessment of the baseline or background in experiments.
There are two ratiometric long wavelength indicators. The first, BTCiii, has a very high dissociation constant Kd that does not allow for measurement of small changes in cytosolic concentrations (nanomolar) of calcium. The second, Fura-Rediv, has a low quantum yield in water due to its hydrophobicity and therefore has not found much use in cellular studies.
The present invention is a new family of calcium indicators which emit in the red or near infrared of the visible spectrum and shift emission wavelengths on binding calcium, depending on the wavelength of excitation. Exciting at their maximal absorption wavelengths produces an enormous increase in fluorescence, without shift in wavelength, on binding calcium. This new family of calcium indicators is represented by four distinct compounds and their variants.
The first of these compounds comprises a BAPTA backbone conjugated via carbon-carbon bond to a fluorophore, either a putative seminaphthofuorescein or seminaphthorhodamine. The emission maxima of these dyes are in the red at 650 nm.
The pKa of the seminaphthofuorescein and seminaphthorhodamine is reduced by halogenation of the naphthol moiety with fluorines and chlorines. Thus, the pKa of the nonhalogenated seminaphthofluorescein is reduced from 7.8 to 5.5 via di-orthohalogenation, making the dies unresponsive to small pH changes in the cell.
Compound I:
The second of these compounds comprises a BAPTA backbone conjugated via carbon-carbon bond to a putative naphthofluorescein fluorophore. The pKa of the dyes is reduced by a substitution of fluorines and chlorines on the napthol moiety. Thus the normal pKa of the naphthofluorosceins is reduced from above 8 to 5.6. The emission of these dyes is extended by the extra aromatic ring to near infrared 690 nm.
Compound II:
Both compounds I and II have dissociation constants between 300 nM and 400 nM and pKa's of about 5.5. Compound I has and excitation maximum of 540 nm and an emission maximum of 650 nm (
The third of these compounds has a unique chelating backbone. The BAPTA has an extra carboxylic function ortho to the fluorophore. The dye conjugated to this BAPTA most closely resembles 6-aminoseminaphthorhodamine for an enhanced quantum yield. The naphthols have chlorine or fluorine substitution to reduce the pKa from greater than 8 to 5.5. The emission maximum is in the red at 650 nm.
Compound III:
The fourth compound has the same unique chelating backbone as compound III, with an carboxylic function ortho to the fluorophore, closely resembling 6-aminoseminaphthofluorescein. The naphthols have chlorine or fluorine substitution to reduce the pKa from greater than 8 to 5.5. The emmission is maximum is 650 nm.
Compound IV:
These four compounds have been modified at the R2 and R3 positions to provide variations which give them special functions in the cell as needed.
a mitochondrial version results, allowing calcium measurement in mitochondria rather than in the cytosol.
a near-membrane version results, allowing calcium measurement near the membrane without affecting fluorescent properties.
These variations extend the utility of the new inventions for calcium studies in the cell.
These and other objects and advantages of the present invention are set forth below and further made clear by reference to the drawings therein:
In the present specifications and claims, reference will be made to phrases and terms of art which are expressly defined for use herein as follows:
As used herein [Ca2+] means intracellular free calcium.
As used herein EGTA means ethylene glycol bis-(-beta-aminoethylether-N,N,N′,N′ tetraacetic acid.
As used herein “BAPTA-like” means substituted derivatives of BAPTA which retain the essential characteristics of the two bis(carboxyethyl)amino-substituted phenyl rings, said rings being linked at the positions ortho to the amines through a four atom bridge wherein the atom adjacent to each phenyl ring is O and the two center atoms are each carbon. The 6-position on one of the phenyl rings linked to the fluorophore may be a carboxylic function or H.
As used herein AM ester means acetoxymethyl ester, or any ester form to facilitate cell loading.
As used herein “μM” means 10−6 moles/liter and “nM” means 10−9 moles/liter.
As used herein MOPS means 3-(N-morpholino)propanesulfonic acid.
The present invention introduces a new family of calcium indicators with emissions in the red and near infrared regions of the electromagnetic spectrum. The new indicators are derived from a BAPTA or BAPTA-like backbone for calcium chelation, and the red or infrared fluorescence is achieved by linking the chelating portion with a chlorinated and fluorinated Benzo[c]xanthenone. When the xanthenone is derived from a combination of resorcinol and naphthalene diol, the omission is 650 nm, and a derivation from two napthalene diols gives an emission of 690 nm. As calcium indicators they respond to intracellular calcium. The longer wavelength causes a slight reduction of quantum yield.
6-hydroxynapthoic acid was esterified and the resulting ester was geminally difluorinated with Selectfluor to give (2), immediately followed by reduction with zinc and acetic acid to give the monofluoro compound (3); the fluorinated compound was chlorinated to give (4) and the phenol was then protected with benzyl bromide to give (5). The final compound was obtained by first reducing the methyl ester of (5) with lithium aluminum hydride to alcohol (6) and oxidizing the alcohol to aldehyde (7) with pyridinium chlorochromate. The aldehyde was converted to napthol (8) by Baeyer-Villiger reaction and (9) was obtained by deprotection of the benzyl protection with BBr3.
Commercially available 3-hydroxy-4-nitrobenzoic acid was esterified and then coupled with 2-bromoethoxy-4-methyl-nitrobenzene to form the BAPTA backbone. After hydrolyzing the ester to the free acid, the mixed anhydride was formed and reacted with 4-haloresorcinol to give the benzophenone. The phenol groups were protected as benzyl ethers prior to reducing the nitro groups with stannous chloride and alkylating the resulting anilines. Debenzylating with palladium and hydrogen prepared the ketone to form dye by coupling with 5,7-dihalo-1,6-dihydroxynaphthalene in the presence of methanesulfonic acid. The dye was subsequently hydrolyzed to the calcium-chelating potassium salt. Acidification gave the free acid, which was then alkylated with bromomethyl acetate to give the membrane permeable acetoxymethyl (AM) ester.
The aldehyde precursor from Grynkiewicz et al was coupled to 4-haloresorcinol and 5,7-dihalo-1,6-dihydroxynaphthalene in the presence of methanesulfonic acid. The ester was then hydrolyzed to the calcium-chelating potassium salt. Acidification gave the free acid which was alkylated to give the acetoxymethyl (AM) form
The aldehyde precursor from Grynkewicz et al1 was coupled to 5,7-dihalo-1,6-dihydroxynaphthalene in the presence of methanesulfonic acid. The ester was then hydrolyzed to the calcium-chelating potassium salt. Acidification gave the free acid which was alkylated to give the acetoxymethyl (AM) form.
Commercially available 4-hydroxyphthalic acid was esterified to the dimethyl phthalate prior to nitration at the 5 position. Reaction with 2-bromoethoxy-4-methyl-nitrobenzene yielded the BAPTA backbone. After hydrolyzing the methyl esters, the phthalic acid in acetic anhydride formed the cyclic anhydride. Friedel-crafts acylation with 4-halo resorcinol gave the isomeric benzophenones, which were separated by column chromatography before proceeding further. The phenols and free acid were benzylated prior to reducing the nitro groups with stannous chloride, followed by alkylation of the resulting anilines. Debenzylation with palladium and hydrogen allowed dye formation when coupled to 5,7-dihalo-1,6-dihydroxynaphthalene in the presence of methanesulfonic acid. The dye was hydrolyzed to the calcium-chelating potassium salt. Acidification yielded the free acid, which was alkylated to give the membrane-permeable AM form.
Syntheses of Compounds I and II
One aspect of the invention is the ability to lower the pKa of the seminaphthofluorescein, dinaphthofluorescein, and seminaphthorhodamine-like fluorophores. The precursor that enabled such a change is 5-fluoro-7-chloro-1,6-naphthalene diol (
6-hydroxynaphthoic acid (25 g) was dissolved in methanol (250 ml) and adding sulfuric acid (25 ml). The mixture was heated at 80° C. overnight. The reaction mixture was allowed to cool and diluted with ethyl acetate and washed with brine, then sodium bicarbonate, and dried over sodium sulfate. Evaporation of the solvent gave the ester as a creamy solid (24 g). The ester (10 g) was dissolved in acetonitrile (500 ml) and two equivalents of Selectfluor were added and stirred at room temperature for 3 hours at which time the starting material was consumed. The reaction mixture was diluted with ethyl acetate, washed with brine, and dried over sodium sulfate to give geminally difluorinated compound (2) as cream-colored solid (7 g). This solid was reduced to the monofluoro compound with 2 g zinc powder in 200 mL acetic acid. The solids were filleted and the solvents removed under reduced pressure. Column chromatography (6:1 hexanes/ethyl acetate) gave 6 g of compound 3.
Compound (3) 5 g was dissolved and toluene (50 ml) and sec-butylamine (500 μl) was added and the reaction mixture kept at 66° C. on an oil bath and sulfuryl chloride (1.2 equivalents) was added over 3 hours. The mixture was diluted with ethyl acetate and washed with brine, the 5% sodium thiosulfate dried over sodium sulfate and evaporated to give (4). The residue was dissolved in acetonitrile (10 ml), and potassium carbonate (4 g), followed by benzyl bromide (3 ml), were added. The mixture was heated under reflux for two hours when TLC showed all starting material was consumed. The reaction mixture was diluted with ethyl acetate and washed with brine, dried and evaporated to get oily residue which was purified by column chromatography in 9:1 hexane to ethyl acetate to give (5) as white solid (3 g).
Compound (5) (5.8 g) was dissolved in diethylether (100 ml) and added dropwise to lithium aluminum hydride (2 equivalents) in 75 mL of dry ether. When addition was complete, stirring was continued for another 30 minutes, and the reaction mixture was diluted with ethyl acetate and stirred with dilute sulfuric acid. The organic extract was dried and evaporated to give a creamy residue. The residue was dissolved and dry methylene chloride (75 ml) and added to pyridinium chlorochromate (1.5 equivalent) in CH2Cl2 (75 ml) and stirred for 2 hours at room temperature. Ethyl acetate was added to the reaction mixture, which was then filtered through a 300 cm3 of silica. Evaporation of the organic filtrate gave (6) as 4 g of a light brown solid.
Compound 6 (3 g) was dissolved in benzene (30 ml), and m-chloroperbenzoic acid (2 equivalents) was added, and the mixture was heated at 70° C. for 24 hours. 10 mls of 1M KOH were added as the mixture stirred to hydrolyze the formate ester. The reaction mixture was acidified with 1M HCl and extracted with ethyl acetate, washed with brine, dried over sodium sulfate and evaporated to give a gummy residue, purified by column chromatography in 4:1 hexane to ethyl acetate to give (8) as creamy solid 2 g.
Compound 8 (2 g) was dissolved in methylene chloride (20 mls), boron tribromide (1M in CH2Cl2, three equivalents) was added, and the solution was stirred for three hours. Addition of ice water to the reaction and extraction with methylene chloride, followed by ethyl acetate gave (9) after washing and evaporation as light brown solid (1 g). It was ready for use directly for synthesis of the dyes.
Synthesis of Compound I Experimental:
Compound I, Ethyl Ester:
BAPTA aldehyde from Grynkewicz et al1 (1 mmol), 4-fluororesorcinol (1 mmol), and 5-fluoro-7-chloro-1,6-naphthalene diol (1 mmol) were dissolved in 10 mL methanesulfonic acid at room temperature for one hour. The reaction was poured into 200 mL 3M sodium acetate in ice, and the violet precipitate was filtered. After drying under vacuum over phosphorous pentoxide, the solid was dissolved in 20 mL 1:1 chloroform/methanol and p-chloranil (2 equivalents) was added. The solution was refluxed overnight. The reaction mixture was evaporated to dryness, and then the violet solid was redissolved in methanol. Insoluble p-chloranil was filtered off. The filtrate was evaporated and purified by column chromatography using a 5-15% methanol/chloroform gradient to give Compound I, Ethyl ester.
Compound I, Pentapotassium Salt:
Pure Compound I, Methyl ester (0.5 mmol) was hydrolyzed with 2 M aqueous KOH (4.0 mmol) in 4 mL methanol at room temperature for thirty minutes. 2 mL water were added, and the methanol was removed under reduced pressure. The fully aqueous reaction was stirred at room temperature for one hour, at which point C18 TLC (3:2 methanol/brine) showed the hydrolysis was complete. Ice was added to the reaction mixture, and 2M HCl was added until the pH was 7.5 to 8.5. The mildly basic solution was loaded onto 30 g of LH-20 column packed in water and eluted with water. Pure product fractions were stripped of solvent at 45° C. under vacuum to give Compound I, Pentapotassium Salt.
Compound I, Acetoxymethyl (AM) Ester:
Compound I, Pentapotassium Salt (0.1 mmol) was dissolved in 1 mL ice water. 2M HCl was added to bring the pH to 1.5, and the precipitated free acid was filtered. The violet solid was dried under vacuum over phosphorous pentoxide. It was then dissolved in 1 mL dry dimethylformamide, and 1.5 mmol diisopropylethylamine were added. After stirring for five minutes at room temperature, 1 mmol bromomethyl acetate was added, and the reaction was stirred at room temperature for two hours. The mixture was diluted with 50 mL ethyl acetate and washed twice with 50 mL 0.1M citric acid and once with 50 mL brine. The organics were dried over sodium sulfate, filtered, and stripped free of solvents under reduced pressure. Column chromatography (1:1 hexanes/ethyl acetate) gave pure Compound I, AM ester.
Compound II, Ethyl Ester:
BAPTA aldehyde from Grynkewicz et al1 (1 mmol) and 5-fluoro-7-chloro-1,6-naphthalene diol (2.2 mmol) were dissolved in 10 mL methanesulfonic acid at room temperature for one hour. The reaction was poured into 200 mL 3M sodium acetate in ice, and the blue-violet precipitate was filtered. After drying under vacuum over phosphorous pentoxide, the solid was dissolved in 20 mL 1:1 chloroform/methanol and p-chloranil (2 equivalents) was added. The solution was refluxed overnight. The reaction mixture was evaporated to dryness, and then the blue-violet solid was redissolved in methanol. Insoluble p-chloranil was filtered off. The filtrate was evaporated and purified by column chromatography using a 5-10% methanol/chloroform gradient to give Compound II, Ethyl ester.
Compound II, Pentapotassium Salt:
Pure Compound II, Ethyl ester (0.5 mmol) was hydrolyzed with 2 M aqueous KOH (4.0 mmol) in 4 mL methanol at room temperature for thirty minutes. 2 mL water were added, and the methanol was removed under reduced pressure. The fully aqueous reaction was stirred at room temperature for one hour, at which point C18 TLC (3:2 methanol/brine) showed the hydrolysis was complete. Ice was added to the reaction mixture, and 2M HCl was added until the pH was 7.5 to 8.5. The mildly basic solution was loaded onto 30 g of LH-20 column packed in water and eluted with water. Pure product fractions were stripped of solvent at 45° C. under vacuum to give Compound II, Pentapotassium Salt.
Compound II, Acetoxymethyl (AM) Ester:
Compound II, Pentapotassium Salt (0.1 mmol) was dissolved in 1 mL ice water. 2M HCl was added to bring the pH to 1.5, and the precipitated free acid was filtered. The blue-violet solid was dried under vacuum over phosphorous pentoxide. It was then dissolved in 1 mL dry dimethylformamide, and 1.5 mmol diisopropylethylamine were added. After stirring for five minutes at room temperature, 1 mmol bromomethyl acetate was added, and the reaction was stirred at room temperature for two hours, going colorless over time. The mixture was diluted with 50 mL ethyl acetate and washed twice with 50 mL 0.1M citric acid and once with 50 mL brine. The organics were dried over sodium sulfate, filtered, and stripped free of solvents under reduced pressure. Column chromatography (2:1 hexanes/ethyl acetate) gave pure Compound II, AM ester.
This application is related to U.S. Provisional Patent Application No. 61/252,654 filed Oct. 17, 2010 by applicants, and claims the priority date of that application.
Number | Date | Country | |
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61252654 | Oct 2009 | US |