sodium;2-oxopropanoate

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

Procedure: D-Erythrose 4-phosphate (0.45 mL, 12 mM, pH 7.0), sodium pyruvate (0.054 mL, 100 mM, pH 7.0), CoCl2 (0.027 mL, 10 mM), NAD (0.054 mL, 1 mM), DHQ synthase (2 units) and DHQ dehydratase (2 units) are incubated with KDPGal aldolase (100 units) from E. coli at room temperature for 2 h. Protein is subsequently removed by ultrafiltration using Millipore PM-10 membrane. 3-Dehydroshikimate was formed in 90% yield, as determined by 1H NMR analysis. Protein is subsequently removed by ultrafiltration

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

Procedure: The process according to claim 1, wherein 3-isobutyl-cyclohexanone and sodium pyruvate are heated in a HCl and acetic acid solution to give a mixture of condensation products and said condensation products are heated for 4 to 5 hours with pyridine hydrochloride to give p-isobutylhydratropic acid. to give a mixture of condensation products condensation products

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

Procedure: The process according to claim 1, wherein 3-isobutyl-cyclohexanone and sodium pyruvate are heated in a HCl and acetic acid solution to give a mixture of condensation products and said condensation products are heated for 4 to 5 hours with pyridine hydrochloride to give p-isobutylhydratropic acid. to give a mixture of condensation products condensation products

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

Procedure: After 10.0 g (49.2 mmol) of indole-3-pyruvic acid was added to and dissolved in 98 ml of aqueous saturated sodium carbonate solution, the resulting solution was adjusted to pH 12.4 using aqueous 25% sodium hydroxide solution. After 16.3 g (147.6 mmol) of sodium pyruvate was added for reaction at ambient temperature for 2 hours, a reaction solution containing 4-hydroxy-4-(3-indolylmethyl)-2-ketoglutaric acid was obtained. 13.7 g (197 mmol) of hydroxylamine hydrochloride salt was added to the reaction solution while the reaction solution was adjusted to a pH value around neutrality using aqueous 25% sodium hydroxide solution, and agitated at ambient temperature for 4 hours. Using conc. hydrochloric acid, the reaction solution was adjusted to an acidic pH value, to extract the organic matter in ethyl acetate. The organic layer was rinsed with aqueous saturated sodium chloride, dried over anhydrous magnesium sulfate and subsequently concentrated, to obtain the residue. The residue was recrystallized in aqueous 28% ammonia and ethanol, to obtain 5.51 g of 4-hydroxy-4-(3-indolylmethyl)-2-hydroxyiminoglutaric acid (16.2 mmol; a yield of 32% vs. indole-3-pyruvic acid) in crystal form. was obtained to extract the organic matter in ethyl acetate The organic layer was rinsed with aqueous saturated sodium chloride dried over anhydrous magnesium sulfate subsequently concentrated to obtain the residue The residue was recrystallized in aqueous 28% ammonia and ethanol

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

Procedure: After 10.0 g (49.2 mmol) of indole-3-pyruvic acid was added to and dissolved in 98 ml of aqueous saturated sodium carbonate solution, the resulting solution was adjusted to pH 12.4 using aqueous 25% sodium hydroxide solution. After 16.3 g (147.6 mmol) of sodium pyruvate was added for reaction at ambient temperature for 2 hours, a reaction solution containing 4-hydroxy-4-(3-indolylmethyl)-2-ketoglutaric acid was obtained. 13.7 g (197 mmol) of hydroxylamine hydrochloride salt was added to the reaction solution while the reaction solution was adjusted to a pH value around neutrality using aqueous 25% sodium hydroxide solution, and agitated at ambient temperature for 4 hours. Using conc. hydrochloric acid, the reaction solution was adjusted to an acidic pH value, to extract the organic matter in ethyl acetate. The organic layer was rinsed with aqueous saturated sodium chloride, dried over anhydrous magnesium sulfate and subsequently concentrated, to obtain the residue. The residue was recrystallized in aqueous 28% ammonia and ethanol, to obtain 5.51 g of 4-hydroxy-4-(3-indolylmethyl)-2-hydroxyiminoglutaric acid (16.2 mmol; a yield of 32% vs. indole-3-pyruvic acid) in crystal form. was obtained to extract the organic matter in ethyl acetate The organic layer was rinsed with aqueous saturated sodium chloride dried over anhydrous magnesium sulfate subsequently concentrated to obtain the residue The residue was recrystallized in aqueous 28% ammonia and ethanol

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

Procedure: The activity of the HEXaspC mutant proteins for the production of S,S-monatin was measured using the following reaction conditions: Each 1 mL reaction contained 50 mM TAPS, pH 8.2, 4 mM MgCl2, 3 mM sodium phosphate, pH 8.0, 200 mM sodium pyruvate (pH adjusted to 8), 5 mM α-ketoglutarate (pH adjusted to 8), 50 mM tryptophan, 0.05 mM pyridoxal 3-phosphate, 50 μg/mL ProA aldolase (added as a cell free extract) and varying concentrations (approximately 50 and 500 μg/mL) of aminotransferase (added as a cell free extract). Deaerated water was used to prepare the stock solutions and to adjust the volume of the reaction mixtures to 1.0 mL. The pyridoxal phosphate was added just prior to the addition of the enzymes. The reaction tubes were incubated at 30° C. with gentle shaking for 4 h. Samples (0.01 mL) were withdrawn at 1, 2, and 4 h after the addition of the enzymes, filtered, and analyzed by LC/MS/MS, as described in Example 1. Monatin production was normalized based on the amount of aminotransferase present in the reactions. Under the conditions of these assays, the HEXaspC and the HEXaspCT156A produced the most total monatin per mg of aminotransferase while the P9T/R122G protein produced the least, followed by HEXaspCW130F. The HEXaspCW130F and P9T/R122G enzymes showed the greatest stereoselectivity for S-MP (greater than 98% S,S-monatin), even when high enzyme concentrations were used (greater than 300 μg/mL). The percentage of S,S-monatin product decreased to less than 90% in the enzymatic reactions containing the P9T/T156A enzyme at high concentration. The other mutants showed a product stereoselectivity very similar to the original HEXaspC mutant (approximately 95% S,S-monatin). Analysis of the product of the reaction containing the HEXaspC enzyme using the FDAA derivitazation reagent described in Example 1 showed that the second stereoisomer formed is R,S-monatin. the following reaction conditions Each 1 mL reaction added as a cell free extract) and concentrations (approximately 50 and 500 μg/mL) of aminotransferase ( added as a cell free extract) to prepare the stock solutions to the addition of the enzymes The reaction tubes were incubated at 30° C. Samples (0.01 mL) were withdrawn at 1, 2, and 4 h after the addition of the enzymes filtered

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

Procedure: The activity of the HEXaspC mutant proteins for the production of S,S-monatin was measured using the following reaction conditions: Each 1 mL reaction contained 50 mM TAPS, pH 8.2, 4 mM MgCl2, 3 mM sodium phosphate, pH 8.0, 200 mM sodium pyruvate (pH adjusted to 8), 5 mM α-ketoglutarate (pH adjusted to 8), 50 mM tryptophan, 0.05 mM pyridoxal 3-phosphate, 50 μg/mL ProA aldolase (added as a cell free extract) and varying concentrations (approximately 50 and 500 μg/mL) of aminotransferase (added as a cell free extract). Deaerated water was used to prepare the stock solutions and to adjust the volume of the reaction mixtures to 1.0 mL. The pyridoxal phosphate was added just prior to the addition of the enzymes. The reaction tubes were incubated at 30° C. with gentle shaking for 4 h. Samples (0.01 mL) were withdrawn at 1, 2, and 4 h after the addition of the enzymes, filtered, and analyzed by LC/MS/MS, as described in Example 1. Monatin production was normalized based on the amount of aminotransferase present in the reactions. Under the conditions of these assays, the HEXaspC and the HEXaspCT156A produced the most total monatin per mg of aminotransferase while the P9T/R122G protein produced the least, followed by HEXaspCW130F. The HEXaspCW130F and P9T/R122G enzymes showed the greatest stereoselectivity for S-MP (greater than 98% S,S-monatin), even when high enzyme concentrations were used (greater than 300 μg/mL). The percentage of S,S-monatin product decreased to less than 90% in the enzymatic reactions containing the P9T/T156A enzyme at high concentration. The other mutants showed a product stereoselectivity very similar to the original HEXaspC mutant (approximately 95% S,S-monatin). Analysis of the product of the reaction containing the HEXaspC enzyme using the FDAA derivitazation reagent described in Example 1 showed that the second stereoisomer formed is R,S-monatin. the following reaction conditions Each 1 mL reaction added as a cell free extract) and concentrations (approximately 50 and 500 μg/mL) of aminotransferase ( added as a cell free extract) to prepare the stock solutions to the addition of the enzymes The reaction tubes were incubated at 30° C. Samples (0.01 mL) were withdrawn at 1, 2, and 4 h after the addition of the enzymes filtered

Conditions: temp:0.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Sodium pyruvate (24.2 g, 0.22 mol) was dissolved in 200 ml 1N aqueous sodium hydroxide at 20° C. To this solution there was added anisaldehyde (27.4 g, 0.20 mol) at 0.2 ml/min. The solution was aged twenty-four hours at 20° C., cooled at 0° C. and acidified with 6N NCl to pH 1.0 while maintaining a temperature of 0° C. The 4-(p-methoxyphenyl)-2-oxo-3-butenoic acid product was then extracted with 500 ml ethyl acetate and isolated as a yellow solid by concentration under vacuum. The yield by HPLC analysis was 88%. The solution was aged twenty-four hours at 20° C. while maintaining a temperature of 0° C The 4-(p-methoxyphenyl)-2-oxo-3-butenoic acid product was then extracted with 500 ml ethyl acetate isolated as a yellow solid by concentration under vacuum The yield by HPLC analysis

Conditions: temp:20.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Sodium pyruvate (24.2 g, 0.22 moL) was added to a cold (0° C.) solution of 1N sodium hydroxide (200 mL) and ethanol (10 mL). p-Chlorobenzaldehyde (28.11 g, 0.20 moL) was then added, maintaining the temperature below 5° C. The reaction mixture was aged six hours at 0°-5° C. and then allowed to warm to 20° C. where it was aged an additional six hours to effect complete reaction. The mixture was cooled to -10° C. and the pH adjusted to 1.0 with cold 6N HCl. The p-chlorobenzalpyruvic acid product was then extracted with 800 mL ethyl acetate in 94% yield. maintaining the temperature below 5° C was aged an additional six hours reaction The mixture was cooled to -10° C. The p-chlorobenzalpyruvic acid product was then extracted with 800 mL ethyl acetate in 94% yield

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

Procedure: Sodium pyruvate (60.5 g, 0.55 mol) was first added to a cold (0° C.) 1N aqueous solution of sodium hydroxide (550 ml). Benzaldehyde (53 g, 0.55 mol) was then added at such a rate that the temperature was maintained below 5° C. The reaction mixture was then aged four hours at 0° C. with good agitation. Following the age period, the mixture was acidified to pH 1.0 with 6N HCl maintaining a temperature of 0°-5° C. After acidification, the benzalpyruvic acid product was extracted with 500 ml ethyl acetate. Yield: 75-77% by HPLC analysis. Anal. Calcd. for C10H7O3K: C, 56.08; H, 3.30; K, 18.21. Found: C, 56.11; H, 3.31; K, 18.20. 1H NMR (acetone-d6) δ: 7.2 (d, 1H), 7.8 (d, 1H), 7.5 (s, 5H). IR (KBr) cm-1 : 3500sb, 3050-2900s, 1740s, 1695s, 1230s, 1020s. was maintained below 5° C a temperature of 0°-5° C After acidification, the benzalpyruvic acid product was extracted with 500 ml ethyl acetate

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

Procedure: 293 cells (human transformed embryonic kidney cell line; ATCC cat#: CRL-1573) were cultivated in DMEM containing 2 mM L-glutamine, 0.1 mM non-essential amino acids, 1.5 g/l sodium bicarbonate, 1 mM sodium pyruvate, 10% FBS, penicillin 100 U/mL and streptomycin 100 microg/mL. A549 (Human lung carcinoma cell lines; ATCC) were cultivated in Ham's F12 media containing 2 mM L-glutamine; 1.5 g/L sodium bicarbonate; 10% FBS, Penicillin 100 U/mL, Streptomycin 100 microg/mL. Human peripheral blood mononuclear cells (PBMC) (peripheral blood mononuclear cells) were obtained from healthy volunteers. PBMC were isolated by Ficoll-Hypaque density gradient centrifugation of EDTA K3 blood from normal donors. PBMC were plated at 2×106 cells/mL/well in 12 well plates in AIM-V culture media supplemented with Penicillin 100 U/mL, Streptomycin 100 microg/mL. CYNOM-K1 cells were cultivated in EMEM containing 2 mM L-glutamine, 1% non-essential amino acids, 10% FBS, penicillin 100 U/mL and streptomycin 100 microg/mL. NHBE cells were cultivated in BEBM (500 mL supplemented with BPE, 2 ml; Hydrocortisone, 0.5 ml; hEGF, 0.5 ml; Epinephrine, 0.5 ml; Transferrin, 0.5 ml; Insulin, 0.5 ml; Retinoic Acid, 0.5 ml; Triiodothyronine, 0.05 ml). Human peripheral blood mononuclear cells (PBMC) (peripheral blood mononuclear cells) were obtained from healthy volunteers

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

Procedure: 293 cells (human transformed embryonic kidney cell line; ATCC cat#: CRL-1573) were cultivated in DMEM containing 2 mM L-glutamine, 0.1 mM non-essential amino acids, 1.5 g/l sodium bicarbonate, 1 mM sodium pyruvate, 10% FBS, penicillin 100 U/mL and streptomycin 100 microg/mL. A549 (Human lung carcinoma cell lines; ATCC) were cultivated in Ham's F12 media containing 2 mM L-glutamine; 1.5 g/L sodium bicarbonate; 10% FBS, Penicillin 100 U/mL, Streptomycin 100 microg/mL. Human peripheral blood mononuclear cells (PBMC) (peripheral blood mononuclear cells) were obtained from healthy volunteers. PBMC were isolated by Ficoll-Hypaque density gradient centrifugation of EDTA K3 blood from normal donors. PBMC were plated at 2×106 cells/mL/well in 12 well plates in AIM-V culture media supplemented with Penicillin 100 U/mL, Streptomycin 100 microg/mL. CYNOM-K1 cells were cultivated in EMEM containing 2 mM L-glutamine, 1% non-essential amino acids, 10% FBS, penicillin 100 U/mL and streptomycin 100 microg/mL. NHBE cells were cultivated in BEBM (500 mL supplemented with BPE, 2 ml; Hydrocortisone, 0.5 ml; hEGF, 0.5 ml; Epinephrine, 0.5 ml; Transferrin, 0.5 ml; Insulin, 0.5 ml; Retinoic Acid, 0.5 ml; Triiodothyronine, 0.05 ml). Human peripheral blood mononuclear cells (PBMC) (peripheral blood mononuclear cells) were obtained from healthy volunteers