tert-butyl 8-oxo-6-azaspiro[2.5]octane-6-carboxylate

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

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

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

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

Conditions: temp:35.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: D(+)-glucose monoydrate (300 g) and magnesium chloride hexahydrate (1.0 g) were dissolved in 10 mM MES buffer pH 6.5 (2.4 L; Sigma M3671). After addition of 4-oxo-6-aza-spiro[2.5]octane-6-carboxylic acid tert-butyl ester (300 g; 1.33 mmol) and □-NAD (3.0 g; free acid; Roche Diagnostics Cat. No. 10 004 626) the pH was re-adjusted and the suspension heated to 35° C. The reaction was started by adding ketoreductase KRED-NADH-117 (3.0 g; former Biocatalytics, now Codexis) and glucose dehydrogenase GDH-102 (300 mg; Biocatalytics). The suspension was vigorously stirred at 35° C. keeping the pH constant at 6.5 by the controlled addition (pH-stat) of 1.0 M aq. sodium hydroxide solution. After a consumption of 1.307 L (corresponding to 98% conversion; after 17 h) the reaction mixture was extracted with EtOAc (10 L). The organic phase was dried over sodium sulfate and concentrated in vacuo (200 mbar/45° C.) until evaporation fell off. Upon cooling the oily residue (411 g) started to crystallize and was stirred with heptane (1 L) for 2 h. The crystals were filtered off and the filtrate evaporated to dryness, redissolved in EtOAc (150 ml) and concentrated in vacuo as described above. The crystal suspension formed again upon cooling was stirred with heptane (200 ml; 2 h) and the crystals filtered off. Both crops of crystals were washed with heptane and dried under high vacuum to yield the title compound in 93% yield (250.77 g and 34.60 g white crystals), each having a purity of >98.5% GC and 99.8% ee. [α]D=−44.97° (c=1.00, CHCl3). After a consumption of 1.307 L (corresponding to 98% conversion; after 17 h) the reaction mixture was extracted with EtOAc (10 L) The organic phase was dried over sodium sulfate concentrated in vacuo (200 mbar/45° C.) until evaporation Upon cooling the oily residue (411 g) to crystallize was stirred with heptane (1 L) for 2 h The crystals were filtered off the filtrate evaporated to dryness redissolved in EtOAc (150 ml) concentrated in vacuo The crystal suspension formed again upon cooling the crystals filtered off Both crops of crystals were washed with heptane dried under high vacuum

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

Procedure: To a solution of oxalyl chloride (42.35 ml, 0.480 mol) in CH2Cl2 (910 ml) was added a solution of dimethylsulfoxide (68.24 ml, 0.961 mol) in CH2Cl2 (910 ml) at −70° C. within 45 min. The solution was stirred for 15 min and a solution of 4-hydroxy-6-aza-spiro[2.5]octane-6-carboxylic acid tert-butyl ester (91.00 g, 0.400 mol) in CH2Cl2 (910 ml) was added within 40 min keeping the internal temperature at below −60° C. The mixture was stirred for 35 min and triethylamine (280.4 ml, 2.00 mol) was added at below −60° C. within 10 min. The cooling bath was removed and the yellow suspension was stirred for 1 h then quenched with water (1.4 l). The organic phase was separated, washed with water (3×1 l) and sat. aq. NaCl solution (3 l) and evaporated. The residual orange powder was dissolved in TBME (1.40 l), the turbid solution filtered (Hyflo Speedex) to remove some insoluble material and the clear filtrate evaporated to provide crude 4-oxo-6-aza-spiro[2.5]octane-6-carboxylic acid tert-butyl ester as yellow powder (91.9 g). The material was re-dissolved in TBME (300 ml) and purified by filtration over silica gel (700 g) using a 3:1 heptane/TBME mixture (6.5 l). Evaporation and drying (10 mbar, 40° C.) afforded 4-oxo-6-aza-spiro[2.5]octane-6-carboxylic acid tert-butyl ester as whitish powder (80.58 g, 89%), GC purity 100a %. at below −60° C The mixture was stirred for 35 min The cooling bath was removed the yellow suspension was stirred for 1 h then quenched with water (1.4 l) The organic phase was separated washed with water (3×1 l) and sat. aq. NaCl solution (3 l) evaporated The residual orange powder was dissolved in TBME (1.40 l) the turbid solution filtered (Hyflo Speedex) to remove some insoluble material the clear filtrate evaporated

Conditions: temp:-5.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a solution of 4-hydroxy-6-aza-spiro[2.5]octane-6-carboxylic acid tert-butyl ester (20.0 g, 88.0 mmol) in CH2Cl2 (170 ml) was added sodium bromide (1.092 g, 10.6 mmol), sodium bicarbonate (2.439 g, 29.0 mmol) and 2,2,6,6-tetramethylpiperidine 1-oxyl (237.1 mg, 1.49 mmol). The mixture was cooled to −5° C. and sodium hypochlorite solution (9.5% in water, 55.16 ml) was added within 10 min resulting in a red coloration and a temperature rise to 9° C. The mixture was stirred for 35 min at 0-5° C. and, as conversion was incomplete (2.5% starting material remaining), additional sodium hypochlorite solution (9.5% in water, 7.0 ml) was added within 30 min and the mixture stirred for another 30 min at 0° C. GC analysis indicated complete conversion (<0.1% starting material remaining). Sodium thiosulfate solution (10% in water, 100 ml) was added within 10 min resulting in decoloration. The organic phase was separated, washed with water (100 ml), dried over sodium sulfate (50 g), filtered and evaporated (15 mbar, 40° C.) to afford 4-oxo-6-aza-spiro[2.5]octane-6-carboxylic acid tert-butyl ester as yellowish powder (19.84 g), GC purity 99a %. The powder was dissolved in warm TBME (20 ml), heptane (60 ml) was added to induce crystallization and the white suspension stirred at 0-5° C. for 1.5 h. Filtration, washing with heptane (20 ml) and drying (10 mbar, 45° C.) afforded 4-oxo-6-aza-spiro[2.5]octane-6-carboxylic acid tert-butyl ester (17.25 g, 87%) as white crystalline material, GC purity 100a %. 1H-NMR (CDCl3, 300 MHz): 4.08 (s, CH2 (5)), 3.66 (m, CH2 (7)), 1.88 (m, CH2 (8)), 1.48 (s, tert-Bu), 1.40 (m, 2H), 0.81 (m, 2H). resulting in a red coloration to 9° C was added within 30 min the mixture stirred for another 30 min at 0° C resulting in decoloration The organic phase was separated washed with water (100 ml) dried over sodium sulfate (50 g) filtered evaporated (15 mbar, 40° C.)

Conditions: temp:35.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: D(+)-glucose monoydrate (300 g) and magnesium chloride hexahydrate (1.0 g) were dissolved in 10 mM MES buffer pH 6.5 (2.4 L; Sigma M3671). After addition of 4-oxo-6-aza-spiro[2.5]octane-6-carboxylic acid tert-butyl ester (300 g; 1.33 mmol) and β-NAD (3.0 g; free acid; Roche Diagnostics Cat. No. 10 004 626) the pH was re-adjusted and the suspension heated to 35° C. The reaction was started by adding ketoreductase KRED-NADH-117 (3.0 g; former Biocatalytics, now Codexis) and glucose dehydrogenase GDH-102 (300 mg; Biocatalytics). The suspension was vigorously stirred at 35° C. keeping the pH constant at 6.5 by the controlled addition (pH-stat) of 1.0 M aq. sodium hydroxide solution. After a consumption of 1.307 L (corresponding to 98% conversion; after 17 h) the reaction mixture was extracted with ethyl acetate (10 L). The organic phase was dried over sodium sulfate and concentrated in vacuo (200 mbar/45° C.) until evaporation fell off. Upon cooling the oily residue (411 g) started to crystallize and was stirred with heptane (1 L) for 2 h. The crystals were filtered off and the filtrate evaporated to dryness, redissolved in ethyl acetate (150 ml) and concentrated in vacuo as described above. The crystal suspension formed again upon cooling was stirred with heptane (200 ml; 2 h) and the crystals filtered off. Both crops of crystals were washed with heptane and dried under high vacuum to yield the title compound in 93% yield (250.77 g and 34.60 g white crystals), each having a purity of >98.5% GC and 99.8% ee. [α]D=−44.97° (c=1.00, CHCl3). After a consumption of 1.307 L (corresponding to 98% conversion; after 17 h) the reaction mixture was extracted with ethyl acetate (10 L) The organic phase was dried over sodium sulfate concentrated in vacuo (200 mbar/45° C.) until evaporation Upon cooling the oily residue (411 g) to crystallize was stirred with heptane (1 L) for 2 h The crystals were filtered off the filtrate evaporated to dryness redissolved in ethyl acetate (150 ml) concentrated in vacuo The crystal suspension formed again upon cooling the crystals filtered off Both crops of crystals were washed with heptane dried under high vacuum

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

Procedure: To a solution of oxalyl chloride (42.35 ml, 0.480 mol) in dichloromethane (910 ml) was added a solution of dimethylsulfoxide (68.24 ml, 0.961 mol) in dichloromethane (910 ml) at −70° C. within 45 min. The solution was stirred for 15 min and a solution of 4-hydroxy-6-aza-spiro[2.5]octane-6-carboxylic acid tert-butyl ester (91.00 g, 0.400 mol) in dichloromethane (910 ml) was added within 40 min keeping the internal temperature at below −60°. The mixture was stirred for 35 min and triethylamine (280.4 ml, 2.00 mol) was added at below −60° C. within 10 min. The cooling bath was removed and the yellow suspension was stirred for 1 h then quenched with water (1.4 l). The organic phase was separated, washed with water (3×1 l) and sat. aq. sodium chloride solution (3 l) and evaporated. The residual orange powder was dissolved in tert-butyl methyl ether (1.40 l), the turbid solution filtered (Hyflo Speedex) to remove some insoluble material and the clear filtrate evaporated to provide crude 4-oxo-6-aza-spiro[2.5]octane-6-carboxylic acid tert-butyl ester as yellow powder (91.9 g). The material was re-dissolved in tert-butyl methyl ether (300 ml) and purified by filtration over silica gel (700 g) using a 3:1 heptane/tert-butyl methyl ether mixture (6.5 l). Evaporation and drying (10 mbar, 40° C.) afforded 4-oxo-6-aza-spiro[2.5]octane-6-carboxylic acid tert-butyl ester as whitish powder (80.58 g, 89%), GC purity 100a %. at below −60° The mixture was stirred for 35 min The cooling bath was removed the yellow suspension was stirred for 1 h then quenched with water (1.4 l) The organic phase was separated washed with water (3×1 l) and sat. aq. sodium chloride solution (3 l) evaporated The residual orange powder was dissolved in tert-butyl methyl ether (1.40 l) the turbid solution filtered (Hyflo Speedex) to remove some insoluble material the clear filtrate evaporated to provide crude 4-oxo-6-aza-spiro[2.5]octane-6-carboxylic acid tert-butyl ester as yellow powder (91.9 g) purified by filtration over silica gel (700 g) Evaporation drying (10 mbar, 40° C.)

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

Procedure: To a solution of oxalyl chloride (42.35 ml, 0.480 mol) in dichloromethane (910 ml) was added a solution of dimethylsulfoxide (68.24 ml, 0.961 mol) in dichloromethane (910 ml) at −70° C. within 45 min. The solution was stirred for 15 min and a solution of 4-hydroxy-6-aza-spiro[2.5]octane-6-carboxylic acid tert-butyl ester (91.00 g, 0.400 mol) in dichloromethane (910 ml) was added within 40 min keeping the internal temperature at below −60°. The mixture was stirred for 35 min and triethylamine (280.4 ml, 2.00 mol) was added at below −60° C. within 10 min. The cooling bath was removed and the yellow suspension was stirred for 1 h then quenched with water (1.4 l). The organic phase was separated, washed with water (3×1 l) and sat. aq. sodium chloride solution (3 l) and evaporated. The residual orange powder was dissolved in tert-butyl methyl ether (1.40 l), the turbid solution filtered (Hyflo Speedex) to remove some insoluble material and the clear filtrate evaporated to provide crude 4-oxo-6-aza-spiro[2.5]octane-6-carboxylic acid tert-butyl ester as yellow powder (91.9 g). The material was re-dissolved in tert-butyl methyl ether (300 ml) and purified by filtration over silica gel (700 g) using a 3:1 heptane/tert-butyl methyl ether mixture (6.5 l). Evaporation and drying (10 mbar, 40° C.) afforded 4-oxo-6-aza-spiro[2.5]octane-6-carboxylic acid tert-butyl ester as whitish powder (80.58 g, 89%), GC purity 100a %. at below −60° The mixture was stirred for 35 min The cooling bath was removed the yellow suspension was stirred for 1 h then quenched with water (1.4 l) The organic phase was separated washed with water (3×1 l) and sat. aq. sodium chloride solution (3 l) evaporated The residual orange powder was dissolved in tert-butyl methyl ether (1.40 l) the turbid solution filtered (Hyflo Speedex) to remove some insoluble material the clear filtrate evaporated to provide crude 4-oxo-6-aza-spiro[2.5]octane-6-carboxylic acid tert-butyl ester as yellow powder (91.9 g) purified by filtration over silica gel (700 g) Evaporation drying (10 mbar, 40° C.)

Conditions: temp:-5.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a solution of 4-hydroxy-6-aza-spiro[2.5]octane-6-carboxylic acid tert-butyl ester (20.0 g, 88.0 mmol) in dichloromethane (170 ml) was added sodium bromide (1.092 g, 10.6 mmol), sodium bicarbonate (2.439 g, 29.0 mmol) and 2,2,6,6-tetramethylpiperidine 1-oxyl (237.1 mg, 1.49 mmol). The mixture was cooled to −5° C. and sodium hypochlorite solution (9.5% in water, 55.16 ml) was added within 10 min resulting in a red coloration and a temperature rise to 9° C. The mixture was stirred for 35 min at 0-5° C. and, as conversion was incomplete (2.5% starting material remaining), additional sodium hypochlorite solution (9.5% in water, 7.0 ml) was added within 30 min and the mixture stirred for another 30 min at 0° C. GC analysis indicated complete conversion (<0.1% starting material remaining). Sodium thiosulfate solution (10% in water, 100 ml) was added within 10 min resulting in decoloration. The organic phase was separated, washed with water (100 ml), dried over sodium sulfate (50 g), filtered and evaporated (15 mbar, 40° C.) to afford 4-oxo-6-aza-spiro[2.5]octane-6-carboxylic acid tert-butyl ester as yellowish powder (19.84 g), GC purity 99a %. The powder was dissolved in warm tert-butyl methyl ether (20 ml), heptane (60 ml) was added to induce crystallization and the white suspension stirred at 0-5° C. for 1.5 h. Filtration, washing with heptane (20 ml) and drying (10 mbar, 45° C.) afforded 4-oxo-6-aza-spiro[2.5]octane-6-carboxylic acid tert-butyl ester (17.25 g, 87%) as white crystalline material, GC purity 100a %. 1H-NMR (CDCl3, 300 MHz): 4.08 (s, CH2(5)), 3.66 (m, CH2(7)), 1.88 (m, CH2(8)), 1.48 (s, tert-Bu), 1.40 (m, 2H), 0.81 (m, 2H). resulting in a red coloration to 9° C was added within 30 min the mixture stirred for another 30 min at 0° C resulting in decoloration The organic phase was separated washed with water (100 ml) dried over sodium sulfate (50 g) filtered evaporated (15 mbar, 40° C.)

Conditions: temp:35.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: D(+)-glucose monoydrate (300 g) and magnesium chloride hexahydrate (1.0 g) were dissolved in 10 mM MES buffer pH 6.5 (2.4 L; Sigma M3671). After addition of 4-oxo-6-aza-spiro[2.5]octane-6-carboxylic acid tert-butyl ester (300 g; 1.33 mmol) and β-NAD (3.0 g; free acid; Roche Diagnostics Cat. No. 10 004 626) the pH was re-adjusted and the suspension heated to 35° C. The reaction was started by adding ketoreductase KRED-NADH-117 (3.0 g; former Biocatalytics, now Codexis) and glucose dehydrogenase GDH-102 (300 mg; Biocatalytics). The suspension was vigorously stirred at 35° C. keeping the pH constant at 6.5 by the controlled addition (pH-stat) of 1.0 M aq. sodium hydroxide solution. After a consumption of 1.307 L (corresponding to 98% conversion; after 17 h) the reaction mixture was extracted with ethyl acetate (10 L). The organic phase was dried over sodium sulfate and concentrated in vacuo (200 mbar/45° C.) until evaporation fell off. Upon cooling the oily residue (411 g) started to crystallize and was stirred with heptane (1 L) for 2 h. The crystals were filtered off and the filtrate evaporated to dryness, redissolved in ethyl acetate (150 ml) and concentrated in vacuo as described above. The crystal suspension formed again upon cooling was stirred with heptane (200 ml; 2 h) and the crystals filtered off. Both crops of crystals were washed with heptane and dried under high vacuum to yield the title compound in 93% yield (250.77 g and 34.60 g white crystals), each having a purity of >98.5% GC and 99.8% ee. [α]D=−44.97° (C=1.00, CHCl3). After a consumption of 1.307 L (corresponding to 98% conversion; after 17 h) the reaction mixture was extracted with ethyl acetate (10 L) The organic phase was dried over sodium sulfate concentrated in vacuo (200 mbar/45° C.) until evaporation Upon cooling the oily residue (411 g) to crystallize was stirred with heptane (1 L) for 2 h The crystals were filtered off the filtrate evaporated to dryness redissolved in ethyl acetate (150 ml) concentrated in vacuo The crystal suspension formed again upon cooling the crystals filtered off Both crops of crystals were washed with heptane dried under high vacuum