tert-butyl N-[(2S)-4-chloro-3-oxobutan-2-yl]carbamate

Conditions: temp:22.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A 0.1 M potassium phosphate buffer, 2.0 mM in magnesium chloride, was prepared by combining potassium dihydrogenphosphate (9.86 g, 72.4 mmol), potassium hydrogenphosphate (22.2 g, 127 mmol) and magnesium chloride hexahydrate (0.812 g, 4.0 mmol) in water (2 L); the pH of the resulting solution was 7.05. To this phosphate buffer (1.8 L) was added nicotinamide adenine dinucleotide phosphate, disodium salt trihydrate (1.9 g, 2.4 mmol) and ketoreductase enzyme (Codexis, KRED-P1-E05) (8 g), and the mixture was stirred for 45 minutes at 22° C. to dissolve the ketoreductase. A solution of tert-butyl[(2S)-4-chloro-3-oxobutan-2-yl]carbamate (C43) (50.0 g, 226 mmol) in 2-propanol (200 mL) was added, and the resulting suspension was stirred for 46 hours at 30° C., under a nitrogen flow (10 mL/minute) from a sparge inlet containing 2-propanol and water (1:1, 300 mL). At this point, tert-butyl methyl ether (1 L) was added to the reaction mixture, which was swirled for 20 minutes. The resulting emulsion was filtered through diatomaceous earth (200 g), and the filter cake was broken and washed with tert-butyl methyl ether (3×400 mL). The combined organic layers from the filtrates were dried with sodium sulfate (625 g), filtered, and concentrated in vacuo to afford the crude product as a red oil (50 g). This material was mixed with ethyl acetate (80 mL) and treated with decolorizing carbon (5 g) over 10 minutes with gentle heating. After filtration through Celite, the solution was concentrated in vacuo and mixed with warm hexanes (40 mL) under stirring. After 18 hours, the resulting solid was collected via filtration and rinsed with pentane, providing the product as a white powder (14.14 g). The mother liquor was concentrated under reduced pressure to yield an oil (32 g), which was crystallized in the same way using warm hexanes (30 mL) to obtain additional product as a white powder (13.65 g). Total yield: 27.79 g, 124 mmol, 55%. 1H NMR (400 MHz, CDCl3) δ 4.77 (br s, 1H), 3.77-3.87 (m, 1H), 3.69-3.76 (m, 1H), 3.65 (dd, half of ABX pattern, J=11.1, 3.9 Hz, 1H), 3.53 (dd, half of ABX pattern, J=11.3, 8.0 Hz, 1H), 3.00 (br s, 1H), 1.45 (s, 9H), 1.26 (d, J=6.8 Hz, 3H). to dissolve the ketoreductase the resulting suspension was stirred for 46 hours at 30° C., under a nitrogen flow (10 mL/minute) from a sparge inlet was swirled for 20 minutes The resulting emulsion was filtered through diatomaceous earth (200 g) washed with tert-butyl methyl ether (3×400 mL) The combined organic layers from the filtrates were dried with sodium sulfate (625 g) filtered concentrated in vacuo to afford the crude product as a red oil (50 g) treated with decolorizing carbon (5 g) over 10 minutes with gentle heating After filtration through Celite the solution was concentrated in vacuo mixed with warm hexanes (40 mL) under stirring After 18 hours the resulting solid was collected via filtration rinsed with pentane

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

Procedure: A solution of chloroacetic acid (23.2 g, 246 mmol) in tetrahydrofuran (100 mL) was added over 35 minutes to a −78° C. solution of lithium diisopropylamide in tetrahydrofuran (2.05 M, 240 mL, 492 mmol), at a rate that kept the internal temperature below −65° C. After 30 minutes, the reaction mixture was quickly transferred into a (dry ice)-jacketed addition funnel and added over 5 minutes to a solution of methyl N-(tert-butoxycarbonyl)-L-alaninate (10.0 g, 49.2 mmol) in tetrahydrofuran (120 mL). Mechanical stirring was used for this reaction. The mixture was stirred for 30 minutes, during which time the reaction mixture warmed to 0° C. It was cooled to −78° C. and treated over 10 minutes with a solution of acetic acid (41 mL, 720 mmol) in tetrahydrofuran (41 mL). At this point, the flask was immersed in an ice bath, and stirring was continued for 1.5 hours, while the reaction warmed to 5° C. Water (250 mL) was added, followed by diethyl ether (400 mL); the organic layer was washed with saturated aqueous sodium bicarbonate solution (150 mL) and with saturated aqueous sodium chloride solution (50 mL), dried over magnesium sulfate, filtered, and concentrated in vacuo to afford the product as a waxy, light yellow solid. Yield: 9.47 g, 42.7 mmol, 87%. 1H NMR (400 MHz, CDCl3) δ 5.11 (br d, J=6 Hz, 1H), 4.48-4.58 (m, 1H), 4.28 (AB quartet, JAB=15.9 Hz, ΔνAB=8.3 Hz, 2H), 1.44 (s, 9H), 1.38 (d, J=7.1 Hz, 3H). the internal temperature below −65° C addition funnel was used for this reaction The mixture was stirred for 30 minutes, during which time the reaction mixture It was cooled to −78° C. At this point, the flask was immersed in an ice bath stirring was continued for 1.5 hours, while the reaction warmed to 5° C the organic layer was washed with saturated aqueous sodium bicarbonate solution (150 mL) and with saturated aqueous sodium chloride solution (50 mL) dried over magnesium sulfate filtered concentrated in vacuo to afford the product as a waxy, light yellow solid

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

Procedure: To a stirred solution of L-phenylalanyl-L-leucine, t-butyl ester (2.18 g, 6 mmol) in 10 ml of dimethylformamide (under argon) was added a solution of [(S)-3-chloro-1-methyl-2-oxopropyl]carbamic acid, 1,1-dimethylethyl ester (1.33 g, 6 mmol) in 20 ml of dimethylformamide, followed by sodium iodide (450 mg, 3 mmol) and sodium bicarbonate (504 mg, 6 mmol). The reaction mixture was allowed to stir for 15 hours, then the solvent was evaporated (<25° C., vacuum pump). The yellow residue was taken up into ethyl acetate (250 ml) and washed with water (4×25 ml) and brine (25 ml), then dried (sodium sulfate) and evaporated to a yellow residue (3.44 g). Flash chromatography on silica gel (Merck, 230-400 mesh, 250 g) with hexane-ethyl acetate (1.25:1) yielded 2.29 g of a colorless oil. Rapid recrystallization from ethyl acetate-hexane afforded 1.61 g of a powdery white solid. the solvent was evaporated (<25° C., vacuum pump) washed with water (4×25 ml) and brine (25 ml) dried (sodium sulfate) evaporated to a yellow residue (3.44 g)

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

Procedure: To a stirred solution of L-phenylalanyl-L-leucine, t-butyl ester (2.18 g, 6 mmol) in 10 ml of dimethylformamide (under argon) was added a solution of [(S)-3-chloro-1-methyl-2-oxopropyl]carbamic acid, 1,1-dimethylethyl ester (1.33 g, 6 mmol) in 20 ml of dimethylformamide, followed by sodium iodide (450 mg, 3 mmol) and sodium bicarbonate (504 mg, 6 mmol). The reaction mixture was allowed to stir for 15 hours, then the solvent was evaporated (<25° C., vacuum pump). The yellow residue was taken up into ethyl acetate (250 ml) and washed with water (4×25 ml) and brine (25 ml), then dried (sodium sulfate) and evaporated to a yellow residue (3.44 g). Flash chromatography on silica gel (Merck, 230-400 mesh, 250 g) with hexane-ethyl acetate (1.25:1) yielded 2.29 g of a colorless oil. Rapid recrystallization from ethyl acetate-hexane afforded 1.61 g of a powdery white solid. the solvent was evaporated (<25° C., vacuum pump) washed with water (4×25 ml) and brine (25 ml) dried (sodium sulfate) evaporated to a yellow residue (3.44 g)

Conditions: temp:22.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A 0.1 M potassium phosphate buffer, 2.0 mM in magnesium chloride, was prepared by combining potassium dihydrogenphosphate (9.86 g, 72.4 mmol), potassium hydrogenphosphate (22.2 g, 127 mmol) and magnesium chloride hexahydrate (0.812 g, 4.0 mmol) in water (2 L); the pH of the resulting solution was 7.05. To this phosphate buffer (1.8 L) was added nicotinamide adenine dinucleotide phosphate, disodium salt trihydrate (1.9 g, 2.4 mmol) and ketoreductase enzyme (Codexis, KRED-P1-E05) (8 g), and the mixture was stirred for 45 minutes at 22° C. to dissolve the ketoreductase. A solution of tert-butyl[(2S)-4-chloro-3-oxobutan-2-yl]carbamate (C43) (50.0 g, 226 mmol) in 2-propanol (200 mL) was added, and the resulting suspension was stirred for 46 hours at 30° C., under a nitrogen flow (10 mL/minute) from a sparge inlet containing 2-propanol and water (1:1, 300 mL). At this point, tert-butyl methyl ether (1 L) was added to the reaction mixture, which was swirled for 20 minutes. The resulting emulsion was filtered through diatomaceous earth (200 g), and the filter cake was broken and washed with tert-butyl methyl ether (3×400 mL). The combined organic layers from the filtrates were dried with sodium sulfate (625 g), filtered, and concentrated in vacuo to afford the crude product as a red oil (50 g). This material was mixed with ethyl acetate (80 mL) and treated with decolorizing carbon (5 g) over 10 minutes with gentle heating. After filtration through Celite, the solution was concentrated in vacuo and mixed with warm hexanes (40 mL) under stirring. After 18 hours, the resulting solid was collected via filtration and rinsed with pentane, providing the product as a white powder (14.14 g). The mother liquor was concentrated under reduced pressure to yield an oil (32 g), which was crystallized in the same way using warm hexanes (30 mL) to obtain additional product as a white powder (13.65 g). Total yield: 27.79 g, 124 mmol, 55%. 1H NMR (400 MHz, CDCl3) δ 4.77 (br s, 1H), 3.77-3.87 (m, 1H), 3.69-3.76 (m, 1H), 3.65 (dd, half of ABX pattern, J=11.1, 3.9 Hz, 1H), 3.53 (dd, half of ABX pattern, J=11.3, 8.0 Hz, 1H), 3.00 (br s, 1H), 1.45 (s, 9H), 1.26 (d, J=6.8 Hz, 3H). to dissolve the ketoreductase the resulting suspension was stirred for 46 hours at 30° C., under a nitrogen flow (10 mL/minute) from a sparge inlet was swirled for 20 minutes The resulting emulsion was filtered through diatomaceous earth (200 g) washed with tert-butyl methyl ether (3×400 mL) The combined organic layers from the filtrates were dried with sodium sulfate (625 g) filtered concentrated in vacuo to afford the crude product as a red oil (50 g) treated with decolorizing carbon (5 g) over 10 minutes with gentle heating After filtration through Celite the solution was concentrated in vacuo mixed with warm hexanes (40 mL) under stirring After 18 hours the resulting solid was collected via filtration rinsed with pentane

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

Procedure: A solution of chloroacetic acid (23.2 g, 246 mmol) in tetrahydrofuran (100 mL) was added over 35 minutes to a −78° C. solution of lithium diisopropylamide in tetrahydrofuran (2.05 M, 240 mL, 492 mmol), at a rate that kept the internal temperature below −65° C. After 30 minutes, the reaction mixture was quickly transferred into a (dry ice)-jacketed addition funnel and added over 5 minutes to a solution of methyl N-(tert-butoxycarbonyl)-L-alaninate (10.0 g, 49.2 mmol) in tetrahydrofuran (120 mL). Mechanical stirring was used for this reaction. The mixture was stirred for 30 minutes, during which time the reaction mixture warmed to 0° C. It was cooled to −78° C. and treated over 10 minutes with a solution of acetic acid (41 mL, 720 mmol) in tetrahydrofuran (41 mL). At this point, the flask was immersed in an ice bath, and stirring was continued for 1.5 hours, while the reaction warmed to 5° C. Water (250 mL) was added, followed by diethyl ether (400 mL); the organic layer was washed with saturated aqueous sodium bicarbonate solution (150 mL) and with saturated aqueous sodium chloride solution (50 mL), dried over magnesium sulfate, filtered, and concentrated in vacuo to afford the product as a waxy, light yellow solid. Yield: 9.47 g, 42.7 mmol, 87%. 1H NMR (400 MHz, CDCl3) δ 5.11 (br d, J=6 Hz, 1H), 4.48-4.58 (m, 1H), 4.28 (AB quartet, JAB=15.9 Hz, ΔνAB=8.3 Hz, 2H), 1.44 (s, 9H), 1.38 (d, J=7.1 Hz, 3H). the internal temperature below −65° C addition funnel was used for this reaction The mixture was stirred for 30 minutes, during which time the reaction mixture It was cooled to −78° C. At this point, the flask was immersed in an ice bath stirring was continued for 1.5 hours, while the reaction warmed to 5° C the organic layer was washed with saturated aqueous sodium bicarbonate solution (150 mL) and with saturated aqueous sodium chloride solution (50 mL) dried over magnesium sulfate filtered concentrated in vacuo to afford the product as a waxy, light yellow solid

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

Procedure: To a stirred solution of L-phenylalanyl-L-leucine, t-butyl ester (6 mmol) in 10 ml of dimethylformamide (under argon) was added a solution of [(S)-3-chloro-1-methyl-2-oxopropyl]carbamic acid, 1,1-dimethylethyl ester (1.33 g, 6 mmol) in 20 ml of dimethylformamide, followed by sodium iodide (450 mg, 3 mmol) and sodium bicarbonate (504 mg, 6 mmol). The reaction mixture was allowed to stir for 15 hours, and the solvent was then evaporated (<25° C., vacuum pump). The yellow residue was taken up into ethyl acetate (250 ml) and washed with water (4×25 ml) and brine (25 ml), then dried (sodium sulfate) and evaporated to a yellow residue (3.44 g). This was applied to a column of silica gel (Merck, 230-400 mesh, 250 g) and eluted with hexane-ethyl acetate (1.25:1). Fractions 25-40 (~30 ml each, homogeneous on TLC) were pooled and concentrated yielding 2.29 g of a colorless oil. Rapid recrystallization from ethyl acetate-hexane afforded 1.61 g of the title compound as a powdery white solid. the solvent was then evaporated (<25° C., vacuum pump) washed with water (4×25 ml) and brine (25 ml) dried (sodium sulfate) evaporated to a yellow residue (3.44 g) eluted with hexane-ethyl acetate (1.25:1) concentrated

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

Procedure: To a stirred solution of L-phenylalanyl-L-leucine, t-butyl ester (6 mmol) in 10 ml of dimethylformamide (under argon) was added a solution of [(S)-3-chloro-1-methyl-2-oxopropyl]- carbamic acid, 1,1-dimethylethyl ester (1.33 g, 6 mmol) in 20 ml of dimethylformamide, followed by sodium iodide (450 mg, 3 mmol) and sodium bicarbonate (504 mg,. 6 mmol). The reaction mixture was allowed to stir for 15 hours, and the solvent was then evaporated (<25° C., vacuum pump). The yellow residue was taken up into ethyl acetate (250 ml) and washed with water (4×25 ml) and brine (25 ml), then dried (sodium sulfate) and evaporated to a yellow residue (3.44 g). This was applied to a column of silica gel (Merck, 230-400 mesh, 250 g) and eluted with hexane-ethyl acetate (1.25:1). Fractions 25-40 (~30 ml each, homogeneous on TLC) were pooled and concentrated yielding 2.29 g of a colorless oil. Rapid recrystallization from ethyl acetate-hexane afforded 1.61 g of the title compound as a powdery white solid. the solvent was then evaporated (<25° C., vacuum pump) washed with water (4×25 ml) and brine (25 ml) dried (sodium sulfate) evaporated to a yellow residue (3.44 g) eluted with hexane-ethyl acetate (1.25:1) concentrated

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

Procedure: To a stirred solution of L-phenylalanyl-L-leucine, t-butyl ester (2.18 g, 6 mmol) in 10 ml of dimethylformamide (under argon) was added a solution of [(S)-3-chloro-1-methyl-2-oxopropyl]carbamic acid, 1,1-dimethylethyl ester (1.33 g, 6 mmol) in 20 ml of dimethylformamide, followed by sodium iodide (450 mg, 3 mmol) and sodium bicarbonate (504 mg, 6 mmol). The reaction mixture was allowed to stir for 15 hours, then the solvent was evaporated (<25C° C., vacuum pump). The yellow residue was taken up into ethyl acetate (250 ml) and washed with water (4×25 ml) and brine (25 ml), then dried (sodium sulfate) and evaporated to a yellow residue (3.44 g). Flash chromatography on silica gel (Merck, 230-400 mesh, 250 g) with hexane-ethyl acetate (1.25:1) yielded 2.29 g of a colorless oil. Rapid recrystallization from ethyl acetate-hexane afforded 1.61 g of a powdery white solid. the solvent was evaporated (<25C° C., vacuum pump) washed with water (4×25 ml) and brine (25 ml) dried (sodium sulfate) evaporated to a yellow residue (3.44 g)

Conditions: temp:22.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A 0.1 M potassium phosphate buffer, 2.0 mM in magnesium chloride, was prepared by combining potassium dihydrogenphosphate (9.86 g, 72.4 mmol), potassium hydrogenphosphate (22.2 g, 127 mmol) and magnesium chloride hexahydrate (0.812 g, 4.0 mmol) in water (2 L); the pH of the resulting solution was 7.05. To this phosphate buffer (1.8 L) was added nicotinamide adenine dinucleotide phosphate, disodium salt trihydrate (1.9 g, 2.4 mmol) and ketoreductase enzyme (Codexis, KRED-P1-E05) (8 g), and the mixture was stirred for 45 minutes at 22° C. to dissolve the ketoreductase. A solution of tert-butyl [(2S)-4-chloro-3-oxobutan-2-yl]carbamate (C43) (50.0 g, 226 mmol) in 2-propanol (200 mL) was added, and the resulting suspension was stirred for 46 hours at 30° C., under a nitrogen flow (10 mL/minute) from a sparge inlet containing 2-propanol and water (1:1, 300 mL). At this point, tert-butyl methyl ether (1 L) was added to the reaction mixture, which was swirled for 20 minutes. The resulting emulsion was filtered through diatomaceous earth (200 g), and the filter cake was broken and washed with tert-butyl methyl ether (3×400 mL). The combined organic layers from the filtrates were dried with sodium sulfate (625 g), filtered, and concentrated in vacuo to afford the crude product as a red oil (50 g). This material was mixed with ethyl acetate (80 mL) and treated with decolorizing carbon (5 g) over 10 minutes with gentle heating. After filtration through Celite, the solution was concentrated in vacuo and mixed with warm hexanes (40 mL) under stirring. After 18 hours, the resulting solid was collected via filtration and rinsed with pentane, providing the product as a white powder (14.14 g). The mother liquor was concentrated under reduced pressure to yield an oil (32 g), which was crystallized in the same way using warm hexanes (30 mL) to obtain additional product as a white powder (13.65 g). Total yield: 27.79 g, 124 mmol, 55%. 1H NMR (400 MHz, CDCl3) δ 4.77 (br s, 1H), 3.77-3.87 (m, 1H), 3.69-3.76 (m, 1H), 3.65 (dd, half of ABX pattern, J=11.1, 3.9 Hz, 1H), 3.53 (dd, half of ABX pattern, J=11.3, 8.0 Hz, 1H), 3.00 (br s, 1H), 1.45 (s, 9H), 1.26 (d, J=6.8 Hz, 3H). to dissolve the ketoreductase the resulting suspension was stirred for 46 hours at 30° C., under a nitrogen flow (10 mL/minute) from a sparge inlet was swirled for 20 minutes The resulting emulsion was filtered through diatomaceous earth (200 g) washed with tert-butyl methyl ether (3×400 mL) The combined organic layers from the filtrates were dried with sodium sulfate (625 g) filtered concentrated in vacuo to afford the crude product as a red oil (50 g) treated with decolorizing carbon (5 g) over 10 minutes with gentle heating After filtration through Celite the solution was concentrated in vacuo mixed with warm hexanes (40 mL) under stirring After 18 hours the resulting solid was collected via filtration rinsed with pentane

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

Procedure: A solution of chloroacetic acid (23.2 g, 246 mmol) in tetrahydrofuran (100 mL) was added over 35 minutes to a −78° C. solution of lithium diisopropylamide in tetrahydrofuran (2.05 M, 240 mL, 492 mmol), at a rate that kept the internal temperature below −65° C. After 30 minutes, the reaction mixture was quickly transferred into a (dry ice)-jacketed addition funnel and added over 5 minutes to a solution of methyl N-(tert-butoxycarbonyl)-L-alaninate (10.0 g, 49.2 mmol) in tetrahydrofuran (120 mL). Mechanical stirring was used for this reaction. The mixture was stirred for 30 minutes, during which time the reaction mixture warmed to 0° C. It was cooled to −78° C. and treated over 10 minutes with a solution of acetic acid (41 mL, 720 mmol) in tetrahydrofuran (41 mL). At this point, the flask was immersed in an ice bath, and stirring was continued for 1.5 hours, while the reaction warmed to 5° C. Water (250 mL) was added, followed by diethyl ether (400 mL); the organic layer was washed with saturated aqueous sodium bicarbonate solution (150 mL) and with saturated aqueous sodium chloride solution (50 mL), dried over magnesium sulfate, filtered, and concentrated in vacuo to afford the product as a waxy, light yellow solid. Yield: 9.47 g, 42.7 mmol, 87%. 1H NMR (400 MHz, CDCl3) δ 5.11 (br d, J=6 Hz, 1H), 4.48-4.58 (m, 1H), 4.28 (AB quartet, JAB=15.9 Hz, ΔνAB=8.3 Hz, 2H), 1.44 (s, 9H), 1.38 (d, J=7.1 Hz, 3H). the internal temperature below −65° C addition funnel was used for this reaction The mixture was stirred for 30 minutes, during which time the reaction mixture It was cooled to −78° C. At this point, the flask was immersed in an ice bath stirring was continued for 1.5 hours, while the reaction warmed to 5° C the organic layer was washed with saturated aqueous sodium bicarbonate solution (150 mL) and with saturated aqueous sodium chloride solution (50 mL) dried over magnesium sulfate filtered concentrated in vacuo to afford the product as a waxy, light yellow solid

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

Procedure: To a stirred solution of L-phenylalanyl-L-leucine, t-butyl ester (2.18 g, 6 mmol) in 10 ml of dimethylformamide (under argon) was added a solution of [(S)-3-chloro-1-methyl-2-oxopropyl]carbamic acid, 1,1-dimethylethyl ester (1.33 g, 6 mmol) in 20 ml of dimethylformamide, followed by sodium iodide (450 mg, 3 mmol) and sodium bicarbonate (504 mg, 6 mmol). The reaction mixture was allowed to stir for 15 hours, then the solvent was evaporated (<25° C., vacuum pump). The yellow residue was taken up into ethyl acetate (250 ml) and washed with water (4×25 ml) and brine (25 ml), then dried (sodium sulfate) and evaporated to a yellow residue (3.44 g). Flash chromatography on silica gel (Merck, 230-400 mesh, 250 g) with hexane-ethyl acetate (1.25:1) yielded 2.29 g of a colorless oil. Rapid recrystallization from ethyl acetate-hexane afforded 1.61 g of a powdery white solid. the solvent was evaporated (<25° C., vacuum pump) washed with water (4×25 ml) and brine (25 ml) dried (sodium sulfate) evaporated to a yellow residue (3.44 g)