(S)-3-氨基-2-氮杂环庚烷酮

反应条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

实验步骤: According to the procedure described in Example 8(a), 2(RS)-(3-biphenyl4-yl-1H-imidazol-1-yl)-succinic acid 4-benzyl ester (1.20 g, 2.82 mmol) and L-α-amino-ε-caprolactam (469 mg, 3.67 mmol) was coupled in DMF with pyBOP to furnish 1.02 g (67%) of 3(RS)-(3-biphenyl-4-yl-1H-imidazol-1-yl)-N-(hexahydroazepin-2-on-3(S)-yl)succinamic acid benzyl ester.

反应条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

实验步骤: To a solution of 2,5-dioxopyrrolidin-1-yl-5-(2,4-difluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylate (Example 16; 300 mg, 0.677 mmol) in dichloromethane (3 mL) was added (S)-3-aminoazepan-2-one (prepared according to Example 4; 173 mg, 1.35 mmol) and the reaction was allowed to stir overnight at ambient temperature. The reaction mixture was diluted with ethyl acetate and washed with 1M hydrochloric acid, 1M sodium hydroxide, saturated sodium bicarbonate, and saturated sodium chloride. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography on a silica gel column, eluting with 80% ethyl acetate/hexanes to yield (S)-5-(2,4-difluorophenoxy)-1-isobutyl-N-(2-oxoazepan-3-yl)-1H-indazole-6-carboxamide (Ic) (290 mg, 94%). Mass spec: 457 (M+1). 1H NMR (CDCl3) δ 9.11 (br, 1H), 8.33 (s, 1H), 7.86 (s, 1H), 7.16 (m, 1H), 7.04 (s, 1H), 7.00 (m, 1H), 6.89 (m, 1H), 6.14 (br, 1H), 4.78 (ddd, 1H), 4.22 (d, 2H), 3.34 (m, 1H), 3.24 (m, 1H), 2.35 (m, 1H), 2.26 (m, 1H), 2.07 (m, 1H), 1.89 (m, 2H), 1.57 (m, 1H), 1.42 (m, 1H), 0.93 (d, 6H). washed with 1M hydrochloric acid, 1M sodium hydroxide, saturated sodium bicarbonate, and saturated sodium chloride The organic layer was dried over anhydrous sodium sulfate filtered concentrated The residue was purified by column chromatography on a silica gel column eluting with 80% ethyl acetate/hexanes

反应条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

实验步骤: To a solution of 2,5-dioxopyrrolidin-1-yl-5-(2,4-difluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylate (Example 16; 300 mg, 0.677 mmol) in dichloromethane (3 mL) was added (S)-3-aminoazepan-2-one (prepared according to Example 4; 173 mg, 1.35 mmol) and the reaction was allowed to stir overnight at ambient temperature. The reaction mixture was diluted with ethyl acetate and washed with 1M hydrochloric acid, 1M sodium hydroxide, saturated sodium bicarbonate, and saturated sodium chloride. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography on a silica gel column, eluting with 80% ethyl acetate/hexanes to yield (S)-5-(2,4-difluorophenoxy)-1-isobutyl-N-(2-oxoazepan-3-yl)-1H-indazole-6-carboxamide (Ic) (290 mg, 94%). Mass spec: 457 (M+1). 1H NMR (CDCl3) δ 9.11 (br, 1H), 8.33 (s, 1H), 7.86 (s, 1H), 7.16 (m, 1H), 7.04 (s, 1H), 7.00 (m, 1H), 6.89 (m, 1H), 6.14 (br, 1H), 4.78 (ddd, 1H), 4.22 (d, 2H), 3.34 (m, 1H), 3.24 (m, 1H), 2.35 (m, 1H), 2.26 (m, 1H), 2.07 (m, 1H), 1.89 (m, 2H), 1.57 (m, 1H), 1.42 (m, 1H), 0.93 (d, 6H). washed with 1M hydrochloric acid, 1M sodium hydroxide, saturated sodium bicarbonate, and saturated sodium chloride The organic layer was dried over anhydrous sodium sulfate filtered concentrated The residue was purified by column chromatography on a silica gel column eluting with 80% ethyl acetate/hexanes

反应条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

实验步骤: According to the procedure described in Example 8(a), 2(RS)-(3-biphenyl-4-yl-1H-imidazol-1-yl)-succinic acid 4-benzyl ester (1.20 g, 2.82 mmol) and L-α-amino-ε-caprolactam (469 mg, 3.67 mmol) was coupled in DMF with pyBOP to furnish 1.02 g (67%) of 3(RS)-(3-biphenyl-4-yl-1H-imidazol-1-yl)-N-(hexahydroazepin-2-on-3(S)-yl)succinamic acid benzyl ester. FABMS: 537.5 (C32H33N4O4 ; M+H+).

反应条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

实验步骤: According to the procedure described in Example 8(a), 2(RS)-(3-biphenyl-4-yl-1H-imidazol-1-yl)-succinic acid 4-benzyl ester (1.20 g, 2.82 mmol) and L-α-amino-ε-caprolactam (469 mg, 3.67 mmol) was coupled in DMF with pyBOP to furnish 1.02 g (67%) of 3(RS)-(3-biphenyl-4-yl-1H-imidazol-1-yl)-N-(hexahydroazepin-2-on-3(S)-yl)succinamic acid benzyl ester. FABMS: 537.5 (C32H33N4O4 ; M+H+).

反应条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

实验步骤: According to the procedure described in Example 8(a), 2(RS)-(3-biphenyl-4-yl-1H-imidazol-1-yl)-succinic acid 4-benzyl ester (1.20 g, 2.82 mmol) and L-α-amino-ε-caprolactam (469 mg, 3.67 mmol) was coupled in DMF with pyBOP to furnish 1.02 g (67%) of 3(RS)-(3-biphenyl-4-yl-1H-imidazol-1-yl)-N-(hexahydroazepin-2-on-3(S)-yl)succinamic acid benzyl ester. FABMS: 537.5 (C32H33N4O4; M+H+).

反应条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

实验步骤: According to the procedure described in Example 8(a), 2(RS)-(3-biphenyl4-yl-1H-imidazol-1-yl)-succinic acid 4-benzyl ester (1.20 g, 2.82 mmol) and L-α-amino-ε-caprolactam (469 mg, 3.67 mmol) was coupled in DMF with pyBOP to furnish 1.02 g (67%) of 3(RS)-(3-biphenyl-4-yl-1H-imidazol-1-yl)-N-(hexahydroazepin-2-on-3(S)-yl)succinamic acid benzyl ester.

反应条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

实验步骤: According to the procedure described in Example 8(a), 2(RS)-(3-biphenyl-4-yl-1H-imidazol-1-yl)-succinic acid 4-benzyl ester (1.20 g, 2.82 mmol) and L-α-amino-ε-caprolactam (469 mg, 3.67 mmol) was coupled in DMF with pyBOP to furnish 1.02 g (67%) of 3(RS)-(3-biphenyl-4-yl-1H-imidazol-1-yl)-N-(hexahydroazepin-2-on-3(S)-yl)succinamic acid benzyl ester. FABMS: 537.5 (C32H33N4O4; M+H+).

反应条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

实验步骤: According to the procedure described in Example 8(a), 2(RS)-(3-biphenyl-4-yl-1H-imidazol-1-yl)-succinic acid 4-benzyl ester (1.20 g, 2.82 mmol) and L-α-amino-ε-caprolactam (469 mg, 3.67 mmol) was coupled in DMF with pyBOP to furnish 1.02 g (67%) of 3(RS)-(3-biphenyl-4-yl-1H-imidazol-1-yl)-N-(hexahydroazepin-2-on-3(S)-yl)succinamic acid benzyl ester. FABMS: 537.5 (C32H33N4O4; M+H+).

反应条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

实验步骤: Then D-α-amino-ε-caprolactam and pyridoxal-5-phosphate were added to the disintegrated cell suspensions of each monoculture to concentrations of 5 g/l and 0.01 mM respectively. After 1.5 h of incubation at 20° C. and 20 rpm, 2 ml samples were transferred into 1 ml of 1 M H3PO4. After removal of all particulates by centrifugation followed by filtration through a 0.22 μM filter, concentrations D- and L-α-amino-ε-caprolactam in the samples were determined by HPLC using the protocol as described in Example 1. Finally, monocultures that formed L-α-amino-ε-caprolactam from the D-α-amino-ε-caprolactam substrate were sent to the NCIMB for strain determination. After removal of all particulates by centrifugation followed by filtration through a 0.22 μM filter concentrations D- and L-α-amino-ε-caprolactam in the samples

反应条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

实验步骤: Then 100 mg of these lyophilized cells from each monoculture were added to a reaction mixture consisting of 10 mM HEPES-NaOH buffer (pH 7.7), toluene (2,5 v/v %), pyridoxal-5-phosphate (20 μM), and L-α-amino-ε-caprolactam (2.5 m %). Chemical blanks (with the assay mixture without the lyophilized cells) were used as a negative control. All reaction mixtures (including the blanks) were incubated for approximately 72 hours at 40° C. before the reaction was stopped by the addition of 9 volumes of 1 M H3PO4. After removal of the cells by filtration through a 0.45 μM filter, these reaction mixtures were analyzed by the HPLC method described above to determine the concentration of D- and L-α-amino-ε-caprolactam. Finally, monocultures that formed D-α-amino-ε-caprolactam from the L-α-amino-ε-caprolactam substrate were sent to the NCIMB for strain determination. Nb. no D-α-amino-ε-caprolactam could be determined in the chemical blanks. Then 100 mg of these lyophilized cells from each monoculture were added to a reaction mixture All reaction mixtures ( After removal of the cells by filtration through a 0.45 μM filter these reaction mixtures

反应条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

实验步骤: Then 100 mg of these lyophilized cells from each monoculture were added to a reaction mixture consisting of 10 mM HEPES-NaOH buffer (pH 7.7), toluene (2,5 v/v %), pyridoxal-5-phosphate (20 μM), and L-α-amino-ε-caprolactam (2.5 m %). Chemical blanks (with the assay mixture without the lyophilized cells) were used as a negative control. All reaction mixtures (including the blanks) were incubated for approximately 72 hours at 40° C. before the reaction was stopped by the addition of 9 volumes of 1 M H3PO4. After removal of the cells by filtration through a 0.45 μM filter, these reaction mixtures were analyzed by the HPLC method described above to determine the concentration of D- and L-α-amino-ε-caprolactam. Finally, monocultures that formed D-α-amino-ε-caprolactam from the L-α-amino-ε-caprolactam substrate were sent to the NCIMB for strain determination. Nb. no D-α-amino-ε-caprolactam could be determined in the chemical blanks. Then 100 mg of these lyophilized cells from each monoculture were added to a reaction mixture All reaction mixtures ( After removal of the cells by filtration through a 0.45 μM filter these reaction mixtures