sodium;butanoate

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: The same medium and parameters as the inoculum were used. A 500 ml flask containing 100 ml broth was sterilized for 20 min. at 121° C. A 2.5 ml aliquot of sterilized 20% sodium butyrate solution (pH 7.0) and 1 ml. of the 24 hours grown culture were added. The flask was incubated at 30° C. and 150 rpm for 24 hours. Sterile 10% sodium cinnamate (pH 7.0) was added to each flask as follows: 5 g/l at 24 hours, 4 g/l at 72 hours, and 6 g/l at 80 hours. Periodically, 2 ml. samples of broth were acidified to pH 4.0 using 50% sulfuric acid, extracted with an equal volume of ethyl acetate, and analyzed by GC. The acetophenone concentration was estimated by external standard. At the conclusion of the fermentation, the culture broth was acidified to pH 4 as above and extracted twice with ½ volume of ethyl acetate each time. The extracts were combined and solvent was removed under vacuum using a rotary evaporator. The crude product was distilled in a micro-distillation oven and the distillate analyzed by GC. The yield of 1.8 g/L having a purity of 94.3% was obtained. A 500 ml flask containing 100 ml of the 24 hours were added was incubated at 30° C. 150 rpm for 24 hours 5 g/l at 24 hours 4 g/l at 72 hours 6 g/l at 80 hours extracted with an equal volume of ethyl acetate The acetophenone concentration extracted twice with ½ volume of ethyl acetate each time solvent was removed under vacuum a rotary evaporator The crude product was distilled in a micro-distillation oven was obtained

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A 12 L round-bottom flask was equipped with a mechanical stirrer, thermometer and reflux condenser. 385 g sodium butanoate (3.5 mol) and 2 L THF were added to form a slurry. 2.625 L lithium diisopropylamide, 2M in heptane/ THF/ethyl-benzene (5.25 mol, 50% excess) were added at ambient temperature, and then stirred for 24 hours. Then 705 g 2-chlorobenzyl chloride (4.375 mol, 25% excess) was added, and the reaction stirred for another 24 hours. Once completed, the reaction was quenched by adding 1500 mL H2O, and the solution allowed to separate. The aqueous layer, pH=13, was separated and neutralized by addition of 12N HCl to obtain pH=7.0, at which point a phase separation occurs. 2-(2-chlorobenzyl) butanoic acid was concentrated in the organic layer. Synthesis of 2-ethyl-7-chloroindene was completed by the same sequence of reactions and method as described in Example 1, beginning with the addition of SOCl2. See FIGS. 1 and 3. The product, 2-ethyl-7-chloroindene, was obtained by distillation at 110-114° C. under 1-3 mm Hg. Yield was 205 g (33% overall) of a clear, colorless oil. A 12 L round-bottom flask was equipped with a mechanical stirrer thermometer and reflux condenser to form a slurry the reaction stirred for another 24 hours the reaction was quenched by adding 1500 mL H2O to separate The aqueous layer, pH=13, was separated neutralized by addition of 12N HCl to obtain pH=7.0 at which point a phase separation 2-(2-chlorobenzyl) butanoic acid was concentrated in the organic layer Synthesis of 2-ethyl-7-chloroindene

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A 12 L round-bottom flask was equipped with a mechanical stirrer, thermometer and reflux condenser. 385 g sodium butanoate (3.5 mol) and 2 L THF were added to form a slurry. 2.625 L lithium diisopropylamide, 2M in heptane/THF/ethylbenzene (5.25 mol, 50% excess) were added at ambient temperature, and then stirred for 24 hours. Then 705 g 2-chlorobenzyl chloride (4.375 mol, 25% excess) was added, and the reaction stirred for another 24 hours. Once completed, the reaction was quenched by adding 1500 mL H2O, and the solution allowed to separate. The aqueous layer, pH=13, was separated and neutralized by addition of 12N HCl to obtain pH=7.0, at which point a phase separation occurs. 2-(2-chlorobenzyl) butanoic acid was concentrated in the organic layer. Synthesis of 2-ethyl-7-chloroindene was completed by the same sequence of reactions and method as described in Example 1, beginning with the addition of SOCl2. See FIGS. 1 and 3. The product, 2-ethyl-7-chloroindene, was obtained by distillation at 110°-114° C. under 1-3 mm Hg. Yield was 205 g (33% overall) of a clear, colorless oil. A 12 L round-bottom flask was equipped with a mechanical stirrer thermometer and reflux condenser to form a slurry the reaction stirred for another 24 hours the reaction was quenched by adding 1500 mL H2O to separate The aqueous layer, pH=13, was separated neutralized by addition of 12N HCl to obtain pH=7.0 at which point a phase separation 2-(2-chlorobenzyl) butanoic acid was concentrated in the organic layer Synthesis of 2-ethyl-7-chloroindene

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: 9.86 Grams of methyl acetoacetate were dissolved in 15 ml of water, and 12.67 g of a 30% aqueous sodium hydroxide solution were added thereto by drops while cooling the mixture to 25° C. or less. After having been stirred at 30°-35° C. for 3 hours, the mixture was adjusted to pH 7.0 with a concentrated aqueous hydrochloric acid solution. Thereafter, 1.10 g of sodium butyrate and 2.42 g of tetrabutylammonium bromide were added thereto and then an additional concentrated aqueous hydrochloric acid solution was added to the mixture so that the mixture was adjusted to pH 6.8. Further, 29.45 g of a toluene solution containing 31.2% of 3-(4-bromophenylthio)propanal were added thereto and was stirred at 45°-50° C. for 9 hours. The organic layer separated from the mixture was washed with a 5% aqueous acetic acid solution and then with a saturated aqueous sodium chloride solution. Then the layer was dried over anhydrous magnesium sulfate. Removing the solvent from the layer under reduced pressure gave 14.54 g of crude 6-(4-bromophenylthio)-4-hydroxy-2-hexanone (purity: 81.8%, yield: 99.5%). A purified product was obtained by recrystallization from diisopropyl ether. The purified product had a melting point of 53.0°-54.0° C. by drops while cooling the mixture to 25° C. or less were added was stirred at 45°-50° C. for 9 hours The organic layer separated from the mixture was washed with a 5% aqueous acetic acid solution Then the layer was dried over anhydrous magnesium sulfate Removing the solvent from the layer under reduced pressure

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Crude 5b (23.6 g, 96 mmol) is added during 30 min to a stirred suspension of sodium butanoate (10.5 g, 96 mmol) in dry DMF (125 mL) at -20° C. followed by stirring overnight at ambient temperature. After filtration and washing with DMF (5 mL) and Et2O (10 mL),Et2O (250 mL) and ice-cold H2O (100 mL) are added to the combined filtrates. The aqueous phase is separated and extracted with Et2O (100 mL). The combined organic phases are washed (all solutions ice-cold) with 5% aqueous NaHCO3 (50 mL), H2O (50 mL), 0.01N HCl (100 mL), and H2O (2×50 mL). After drying (MgSO4) and evaporation of solvents the residue is distilled to yield 15.0 g (76%) of 6db, bp 75°-80° C./0.5 mbar. After filtration washing with DMF (5 mL) and Et2O (10 mL),Et2O (250 mL) and ice-cold H2O (100 mL) are added to the combined filtrates The aqueous phase is separated extracted with Et2O (100 mL) The combined organic phases are washed (all solutions ice-cold) with 5% aqueous NaHCO3 (50 mL), H2O (50 mL), 0.01N HCl (100 mL), and H2O (2×50 mL) After drying (MgSO4) and evaporation of solvents the residue is distilled

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: The same medium and parameters as the inoculum were used. A 500 ml flask containing 100 ml broth was sterilized for 20 min. at 121° C. A 2.5 ml aliquot of sterilized 20% sodium butyrate solution (pH 7.0) and 1 ml. of the 24 hours grown culture were added. The flask was incubated at 30° C. and 150 rpm for 24 hours. Sterile 10% sodium cinnamate (pH 7.0) was added to each flask as follows: 5 g/l at 24 hours, 4 g/l at 72 hours, and 6 g/l at 80 hours. Periodically, 2 ml. samples of broth were acidified to pH 4.0 using 50% sulfuric acid, extracted with an equal volume of ethyl acetate, and analyzed by GC. The acetophenone concentration was estimated by external standard. At the conclusion of the fermentation, the culture broth was acidified to pH 4 as above and extracted twice with ½ volume of ethyl acetate each time. The extracts were combined and solvent was removed under vacuum using a rotary evaporator. The crude product was distilled in a micro-distillation oven and the distillate analyzed by GC. The yield of 1.8 g/L having a purity of 94.3% was obtained. A 500 ml flask containing 100 ml of the 24 hours were added was incubated at 30° C. 150 rpm for 24 hours 5 g/l at 24 hours 4 g/l at 72 hours 6 g/l at 80 hours extracted with an equal volume of ethyl acetate The acetophenone concentration extracted twice with ½ volume of ethyl acetate each time solvent was removed under vacuum a rotary evaporator The crude product was distilled in a micro-distillation oven was obtained

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: 9.86 Grams of methyl acetoacetate were dissolved in 15 ml of water, and 12.67 g of a 30% aqueous sodium hydroxide solution were added thereto by drops while cooling the mixture to 25° C. or less. After having been stirred at 30°-35° C. for 3 hours, the mixture was adjusted to pH 7.0 with a concentrated aqueous hydrochloric acid solution. Thereafter, 1.10 g of sodium butyrate and 2.42 g of tetrabutylammonium bromide were added thereto and then an additional concentrated aqueous hydrochloric acid solution was added to the mixture so that the mixture was adjusted to pH 6.8. Further, 29.45 g of a toluene solution containing 31.2% of 3-(4-bromophenylthio)propanal were added thereto and was stirred at 45°-50° C. for 9 hours. The organic layer separated from the mixture was washed with a 5% aqueous acetic acid solution and then with a saturated aqueous sodium chloride solution. Then the layer was dried over anhydrous magnesium sulfate. Removing the solvent from the layer under reduced pressure gave 14.54 g of crude 6-(4-bromophenylthio)-4-hydroxy-2-hexanone (purity: 81.8%, yield: 99.5%). A purified product was obtained by recrystallization from diisopropyl ether. The purified product had a melting point of 53.0-54.0° C. by drops while cooling the mixture to 25° C. or less were added was stirred at 45°-50° C. for 9 hours The organic layer separated from the mixture was washed with a 5% aqueous acetic acid solution Then the layer was dried over anhydrous magnesium sulfate Removing the solvent from the layer under reduced pressure

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: 9.86 Grams of methyl acetoacetate were dissolved in 15 ml of water, and 12.67 g of a 30% aqueous sodium hydroxide solution were added thereto by drops while cooling the mixture to 25° C. or less. After having been stirred at 30°-35° C. for 3 hours, the mixture was adjusted to pH 7.0 with a concentrated aqueous hydrochloric acid solution. Thereafter, 1.10 g of sodium butyrate and 2.42 g of tetrabutylammonium bromide were added thereto and then an additional concentrated aqueous hydrochloric acid solution was added to the mixture so that the mixture was adjusted to pH 6.8. Further, 29.45 g of a toluene solution containing 31.2% of 3-(4-bromophenylthio)propanal were added thereto and was stirred at 45°-50° C. for 9 hours. The organic layer separated from the mixture was washed with a 5% aqueous acetic acid solution and then with a saturated aqueous sodium chloride solution. Then the layer was dried over anhydrous magnesium sulfate. Removing the solvent from the layer under reduced pressure gave 14.54 g of crude 6-(4-bromophenylthio)-4-hydroxy-2-hexanone (purity: 81.8%, yield: 99.5%). A purified product was obtained by recrystallization from diisopropyl ether. The purified product had a melting point of 53.0-54.0° C. by drops while cooling the mixture to 25° C. or less were added was stirred at 45°-50° C. for 9 hours The organic layer separated from the mixture was washed with a 5% aqueous acetic acid solution Then the layer was dried over anhydrous magnesium sulfate Removing the solvent from the layer under reduced pressure

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A 12L round-bottom flask was equipped with a mechanical stirrer, thermometer and reflux condenser. 385 g sodium butanoate (3.5 mol) and 2L THF were added to form a slurry. 2.625L lithium diisopropylamide, 2M in heptane/THF/ethyl-benzene (5.25 mol, 50% excess) were added at ambient temperature, and then stirred for 24 hours. Then 705 g 2-chlorobenzyl chloride (4.375 mol, 25% excess) was added, and the reaction stirred for another 24 hours. Once completed, the reaction was quenched by adding 1500 mL H2O, and the solution allowed to separate. The aqueous layer, pH=13, was separated and neutralized by addition of 12N HCl to obtain pH=7.0, at which point a phase separation occurs. 2-(2-chlorobenzyl) butanoic acid was concentrated in the organic layer. Synthesis of 2-ethyl-7-chloroindene was completed by the same sequence of reactions and method as described in Example 1, beginning with the addition of SOCl2. See FIGS. 1 and 3. The product, 2-ethyl-7-chloroindene, was obtained by distillation at 110-114° C. under 1-3 mm Hg. Yield was 205 g (33% overall) of a clear, colorless oil. thermometer and reflux condenser to form a slurry the reaction stirred for another 24 hours the reaction was quenched by adding 1500 mL H2O to separate The aqueous layer, pH=13, was separated neutralized by addition of 12N HCl to obtain pH=7.0 at which point a phase separation 2-(2-chlorobenzyl) butanoic acid was concentrated in the organic layer Synthesis of 2-ethyl-7-chloroindene

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: It is not necessary that the copper(II) carboxylate be added directly to the reaction mixture. For example, when copper(II) chloride and the sodium salt of a carboxylic acid, e.g., sodium acetate or sodium butyrate, are charged to the reaction vessel, the corresponding copper(II) carboxylate, e.g., copper(II) acetate or copper(II) butyrate, are formed in situ in the reaction mixture. Preferably, the copper(II) salt is dried before use to remove any water of crystallization and obtain substantially anhydrous salt. be added directly to the reaction mixture

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: The slides produced with cells grown in presence of either Cd+Cr+H2O2 or 5-azacytidine, or sodium butyrate are incubated overnight at 4° C. in the presence of 30 μl of an anti-5 methylcytosine sheep antibody (Abcam, Ab1884) diluted 1/100 in PBS. The slides are washed once in PBS and then incubated for 15 seconds in the presence of 30 μl of NaOH 7×10−5 N. After washing with PBS, the slides are finally incubated 30 minutes in the presence of a donkey anti-sheep IgG antibody (southern Biotechnology Associates, Inc) diluted at a concentration of 0.1 μg 1 1×106 cells in PBS. The slides produced with cells diluted 1/100 in PBS The slides are washed once in PBS After washing with PBS the slides are finally incubated 30 minutes in the presence of a donkey anti-sheep IgG antibody (southern Biotechnology Associates, Inc) diluted at a concentration of 0.1 μg 1 1×106 cells in PBS

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: The H2O2-UV irradiated slides or slides produced with cells grown in presence of either Cd+Cr+H2O2 or 5-azacytidine, or sodium butyrate are incubated overnight at 4° C. in the presence of 30 μl of an anti-8-oxo-dG mouse monoclonal antibody (Gentaur) diluted 1/100 in 10 mM Tris-HCl, pH 7.5, 10% Foetal Bovine Serum. The slides are washed once in PBS and then incubated for 40 minutes in the presence of a goat anti-mouse IgG1 antibody (southern Biotechnology Associates, Inc) diluted at a concentration of 0.1 μg/1×106 cells in PBS. produced with cells The slides are washed once in PBS diluted at a concentration of 0.1 μg/1×106 cells in PBS