2-oxopropanoate

Conditions: temp:0.00; pressure:0.00; stir:

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

Procedure: The yields were increased by adding auxiliary enzymes to reduce pyruvate to lactate. Lactate dehydrogenase requires NADH as a cofactor. The NADH was regenerated using formate dehydrogenase. A solution containing 5,5,5-trifluoro-2-oxopentanoic acid (100 mg, 0.588 mmoles), D,L-alanine (200 mg, 2.244 mmoles), 0.02 mM pyridoxal phosphate, sodium formate (68 mg, 1 mmole), NAD (3.31 mg, 5 μmoles) L-lactate dehydrogenase cloned from rabbit muscle (Biocatalytics LDH-103, 0.107 mg, 15 units), and formate dehydrogenase (0.5 mL, 15 units cloned from Pichia pastoris and expressed in Escherichia coli) in 0.1 M potassium phosphate buffer, pH 7.5, was incubated with R-transaminase AT-103 from Biocatalytics (5 mg, 44 units) or cloned R-transaminase from Bacillus thuringiensis SC 16569 (0.5 mL, 10 units) at 30° C. in a total volume of 5 mL in 15 mL tubes. Reaction yields of (R)-5,5,5-trifluoro-2-aminopentanoic acid of 94% and 91% were obtained with AT-103 and BMS transaminases, respectively. Ee was 100% in both cases. The yields were increased by adding auxiliary enzymes Reaction

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

Procedure: In 10.0 mM 2-morpholinoethanesulfonic acid (MES) buffer, 17.6 mM MgCl2, 17.6 mM KCl, 175.7 mM phosphoenolpyruvic acid (PEP), 17.6 mM ATP, 123 U/mL pyruvate kinase (PK), 97 U/mL hexokinase, and 20 U/mL ascorbic acid oxidase were dissolved in this composition, and the pH was adjusted to 7.0 to obtain a glucose converting reagent.

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

Procedure: The KHG aldolases from B. subtilis, E. coli, and S. meliloti were also used with the E. coli L-aspartate aminotransferase to produce monatin enzymatically. The following reaction conditions were used: 50 mM NH4—OAc pH 8.3, 2 mM MgCl2, 200 mM pyruvate, 5 mM glutamate, 0.05 mM pyridoxal phosphate, deaerated water to achieve a final volume of 0.5 mL after the addition of the enzymes, 3 mM potassium phosphate, 20 μg/mL of recombinant B. subtilis KHG aldolase (purified), ca. 400 μg/mL of E. coli L-aspartate aminotransferase (AspC) unpurified from cell extract, and 12 mM indole-3-pyruvate. The reactions were incubated at 30° C. for 30 minutes with shaking. The amount of monatin produced using the B. subtilis enzyme was 80 ng/mL, and increased with increasing amounts of aldolase. If indole-3-pyruvate and glutamate were replaced by saturating amounts of tryptophan and 5 mM alpha-ketoglutarate, the production of monatin was increased to 360 ng/mL. Reactions were repeated with 30 μg/mL of each of the three KHG enzymes in 50 mM Tris pH 8.3, with saturating amounts of tryptophan, and were allowed to proceed for an hour in order to increase detection. The Bacillus enzyme had the highest activity as in Example 4, producing approximately 4000 ng/mL monatin. The E. coli KHG produced 3000 ng/mL monatin, and the S. meliloti enzyme produced 2300 ng/mL. The following reaction conditions aldolase (purified), ca. 400 μg/mL of E coli L-aspartate aminotransferase (AspC) unpurified from cell extract

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

Procedure: In a 10 mL of 0.1M potassium phosphate buffer (pH 7.5) containing 10 mM DTT and 0.5M pyruvate were dissolved 50 mg of 6-azido-6-deoxy-N-acetylmannosamine and 1.5 mg of NeuAc aldolase. The starting material was consumed in 14 hours. The solution was lyophilized and the purification was carried out with Bio Gel P-2 gel filtration (3×90 cm) chromatography with a flow rate of 6 mL/40 minutes, at 4° C. The fractions containing the product were pooled and freezed dry to give Compound 203 in 84 percent yield. The starting material was consumed in 14 hours the purification was carried out with Bio Gel P-2 gel filtration (3×90 cm) chromatography with a flow rate of 6 mL/40 minutes, at 4° C The fractions containing the product freezed dry

Conditions: temp:37.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a 10 mL potassium phosphate buffer (0.1M, pH 7.5) were added 10 mM DTT, 0.5M pyruvate, 0.1M D-mannose and 1.5 mg NeuAc aldolase. The reaction mixture was shaken at 37° C. for three days. The reaction mixture was chromatographed with a Dowex-1 (HCO3-) resin, eluted with zero to 1M ammonium bicarbonate gradient. The fractions containing KDN were pooled and lyophilized three times repeatedly to remove the volatile salt to give a 78 percent yield of KDN. The physical data of the product were are consistent with the reported. The reaction mixture was chromatographed with a Dowex-1 (HCO3-) resin eluted with zero to 1M ammonium bicarbonate gradient The fractions containing KDN to remove the volatile salt to give a 78 percent yield of KDN

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

Procedure: AT-103 transaminase was part of a transaminase library purchased from BioCatalytics (Pasadena, Calif.) and the enzyme was tested for production of monatin in coupled reactions using the ProA aldolase from C. testosteroni. The aldolase was prepared as described in WO 03/091396 A2. AT-103 is a broad specificity D-transaminase (E.C. 2.6.1.21) from a Bacillus species that requires a D-amino acid (such as D-glutamate, D-aspartate, or D-alanine) as the amino acid donor. Enzymes and additional components/substrates were added directly to the reaction buffer provided in the kit, which contained 100 mM potassium phosphate buffer pH 7.5, 100 mM amino donor, and 0.1 mM PLP. To one mL of reaction buffer were added: 4 mg indole-3-pyruvate, 20 mg pyruvate, approximately 50 μg ProA provided in a cellular extract, 1 μL 2 M MgCl2, and 2 mg of aminotransferase enzyme. Reactions were performed in duplicate. The reactions were incubated overnight at 30° C. with gentle shaking (100 rpm). The samples were filtered and submitted for reversed-phase LC/MS/MS analysis as described in Example 1. The results indicated that approximately 370 μg/mL monatin were produced using AT-103 enzyme. The results were further analyzed to determine ratios of S,R/R,S versus R,R/S,S monatin, on the basis of the peak areas of the two stereoisomer pools that resolve during the chromatographic separation. Of the total monatin produced by AT-103, 69% was R,R/S,S monatin in comparison to the mixed isomers. This enzyme is homologous to the Bacillus subtilis DAT enzyme described in WO 03/091396 A2, which is known to have a broad specificity for D-amino acids. Chiral analysis was performed using the FDAA methodology described in Example 1, which verified that the D-aminotransferase was making predominantly R,R monatin, and some S,R monatin as expected. Further transamination experiments with S,S monatin or R,R monatin and α-ketoglutarate as substrates verified that the BioCatalytics enzyme was highly selective for the D-configuration at carbon 4, as expected. In these experiments, no glutamate was detected in the reaction with S,S monatin and α-ketoglutarate as substrates. The aldolase was prepared Enzymes and additional components/substrates were added directly to the reaction buffer provided in the kit, which To one mL of reaction buffer were added

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

Procedure: Human cervix adenocarcinoma HeLa cells are maintained as exponentially growing monolayers in Dulbecco modified Eagle's minimum essential medium with 4.5 g/l glucose, 0.1 g/l pyruvate, 105 Ul/l penicilline, 0.1 g/l streptomycine, 0.86 g/l Glutamax I and 10% v/v foetal calf serum (37° C., 5% CO2), without or with 5 μM DL-threo-1-phenyl-2-hexadecanoyl-amino-3-morpholino-1-propanol (PPMP) to inhibit Gb3 expression. When present, PPMP is introduced into pre-cultures for at least 6 days prior to cytotoxicity assays and is present for up to drug wash. Human cervix adenocarcinoma HeLa cells are maintained as exponentially growing monolayers in Dulbecco wash

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

Procedure: In a 10 mL of 0.1M potassium phosphate buffer (pH 7.5) containing 10 mM DTT and 0.5M pyruvate were dissolved 50 mg of 6-azido-6-deoxy-N-acetylmannosamine and 1.5 mg of NeuAc aldolase. The starting material was consumed in 14 hours. The solution was lyophilized and the purification was carried out with Bio Gel P-2 gel filtration (3×90 cm) chromatography with a flow rate of 6 mL/40 minutes, at 4° C. The fractions containing the product were pooled and freezed dry to give Compound 203 in 84 percent yield. The starting material was consumed in 14 hours the purification was carried out with Bio Gel P-2 gel filtration (3×90 cm) chromatography with a flow rate of 6 mL/40 minutes, at 4° C The fractions containing the product freezed dry

Conditions: temp:37.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a 10 mL potassium phosphate buffer (0.1M, pH 7.5) were added 10 mM DTT, 0.5M pyruvate, 0.1M D-mannose and 1.5 mg NeuAc aldolase. The reaction mixture was shaken at 37° C. for three days. The reaction mixture was chromatographed with a Dowex-1 (HCO3-) resin, eluted with zero to 1M ammonium bicarbonate gradient. The fractions containing KDN were pooled and lyophilized three times repeatedly to remove the volatile salt to give a 78 percent yield of KDN. The physical data of the product were are consistent with the reported. The reaction mixture was chromatographed with a Dowex-1 (HCO3-) resin eluted with zero to 1M ammonium bicarbonate gradient The fractions containing KDN to remove the volatile salt to give a 78 percent yield of KDN

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

Procedure: AT-103 transaminase was part of a transaminase library purchased from BioCatalytics (Pasadena, Calif.) and the enzyme was tested for production of monatin in coupled reactions using the ProA aldolase from C. testosteroni. The aldolase was prepared as described in WO 03/091396 A2. AT-103 is a broad specificity D-transaminase (E.C. 2.6.1.21) from a Bacillus species that requires a D-amino acid (such as D-glutamate, D-aspartate, or D-alanine) as the amino acid donor. Enzymes and additional components/substrates were added directly to the reaction buffer provided in the kit, which contained 100 mM potassium phosphate buffer pH 7.5, 100 mM amino donor, and 0.1 mM PLP. To one mL of reaction buffer were added: 4 mg indole-3-pyruvate, 20 mg pyruvate, approximately 50 μg ProA provided in a cellular extract, 1 μL 2 M MgCl2, and 2 mg of aminotransferase enzyme. Reactions were performed in duplicate. The reactions were incubated overnight at 30° C. with gentle shaking (100 rpm). The samples were filtered and submitted for reversed-phase LC/MS/MS analysis as described in Example 1. The results indicated that approximately 370 μg/mL monatin were produced using AT-103 enzyme. The results were further analyzed to determine ratios of S,R/R,S versus R,R/S,S monatin, on the basis of the peak areas of the two stereoisomer pools that resolve during the chromatographic separation. Of the total monatin produced by AT-103, 69% was R,R/S,S monatin in comparison to the mixed isomers. This enzyme is homologous to the Bacillus subtilis DAT enzyme described in WO 03/091396 A2, which is known to have a broad specificity for D-amino acids. Chiral analysis was performed using the FDAA methodology described in Example 1, which verified that the D-aminotransferase was making predominantly R,R monatin, and some S,R monatin as expected. Further transamination experiments with S,S monatin or R,R monatin and α-ketoglutarate as substrates verified that the BioCatalytics enzyme was highly selective for the D-configuration at carbon 4, as expected. In these experiments, no glutamate was detected in the reaction with S,S monatin and α-ketoglutarate as substrates. The aldolase was prepared Enzymes and additional components/substrates were added directly to the reaction buffer provided in the kit, which To one mL of reaction buffer were added

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

Procedure: AT-103 transaminase was part of a transaminase library purchased from BioCatalytics (Pasadena, Calif.) and the enzyme was tested for production of monatin in coupled reactions using the ProA aldolase from C. testosteroni. The aldolase was prepared as described in WO 03/091396 A2. AT-103 is a broad specificity D-transaminase (EC 2.6.1.21) from a Bacillus species that requires a D-amino acid (such as D-glutamate, D-aspartate, or D-alanine) as the amino acid donor. Enzymes and additional components/substrates were added directly to the reaction buffer provided in the kit, which contained 100 mM potassium phosphate buffer pH 7.5, 100 mM amino donor, and 0.1 mM pyridoxal-5′-phosphate (“PLP”). To one mL of reaction buffer were added: 4 mg indole-3-pyruvate, 20 mg pyruvate, approximately 50 μg ProA provided in a cellular extract, 1 μL. 2 M MgCl2, and 2 mg of the aminotransferase enzyme (AT-103). Reactions were performed in duplicate. The reactions were incubated overnight at 30° C. with gentle shaking (100 rpm). The samples were filtered and submitted for reversed-phase LC/MS/MS analysis as described in Example 1. The results indicated that approximately 370 μg/mL monatin were produced using AT-103 enzyme. The results were further analyzed to determine ratios of S,R/R,S versus R,R/S,S monatin, on the basis of the peak areas of the two stereoisomer pools that resolve during the chromatographic separation. Of the total monatin produced by AT-103, 69% was R,R/S,S monatin in comparison to the mixed isomers. This enzyme (AT-103) is homologous to the Bacillus subtilis DAT enzyme described in WO 03/091396 A2, which is known to have a broad specificity for D-amino acids. Chiral analysis was performed using the FDAA methodology described in Example 1, which verified that the D-aminotransferase was making predominantly R,R monatin, and some S,R monatin as expected. Further transamination experiments with S,S monatin or R,R monatin and α-ketoglutarate as substrates verified that the BioCatalytics enzyme was highly selective for the D-configuration at carbon 4, as expected. In these experiments, no glutamate was detected in the reaction with S,S monatin and α-ketoglutarate as substrates. The aldolase was prepared Enzymes and additional components/substrates were added directly to the reaction buffer provided in the kit, which To one mL of reaction buffer were added