tert-butyl (2S)-2-amino-2-phenylacetate

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

Procedure: To a solution of Fmoc-Cys(Acm)-OH (855 mg, 2 mmol) in 4 ml dry DCM at 0° C., HOBt (270 mg, 2 mmol) was added. Then an ice-cold solution of H-Phg-OtBu (415 mg, 2 mmol) in 4 ml of dry DCM was added. Subsequently, DIC (319 μl, 2 mmol) was introduced dropwise. After 1 h at 0° C. and 4 h at 4° C., the solution was evaporated under vacuum. The residue was dissolved in 50 ml EtOAc and washed with sat. aq. NaHCO3, 0.5 M HCl and water. After drying and evaporation of the organic phase, the residue was chromatographed on silica gel to give the protected dipeptide 1 as a foam. Yield 1.14 g (95%), Rf 0.35 (DCM/MeOH, 95:5). 1H NMR (CDCL3): δ1.45 (9H, s, tBu), 1.92 (3H, s, CH3 Acm), 2.71-3.1 (2H, m, CβH2 Cys), 3.8 (1H, m, CoH Cys) 4.2-4.5 (5H,m, CH2 Acm. CH—CH2 Fmoc), 4.56-4.85 (3H, m, CoH Phg, Fmoc), 5.4 (1H, d, J=7.3 Hz, NH) 6.47 (1H, d, J=8.4 Hz, NH), 7.2-8.05 (14H, Fmoc+Phg aromatic, NH Acm). 13C NMR (CDCl3): δ23.7 (CH2, Acm), 27.7 (3×CH3, tBu), 34.9 (Cβ, Cys), 41.1 (CH2, Acm), 41.9 (CH2, Fmoc), 57.4 (CαPhg), 83.7 (Cq tBu), 119.7-127.5 (aromatic, Fmoc, Phg), 141.0 and 143.6 (4C, Fmoc), 156.6 (CO, Fmoc), 170.2 (CO, Cys), 173.5 (2×CO, Acm, tBu ester). 4 h at 4° C., the solution was evaporated under vacuum The residue was dissolved in 50 ml EtOAc washed with sat. aq. NaHCO3, 0.5 M HCl and water After drying evaporation of the organic phase the residue was chromatographed on silica gel

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

Procedure: (2R)-({[4-((2R,3R)-1-(4-Fluorophenyl)-3-{[2-(4-fluorophenyl)-2-oxoethyl]thio}-4-oxoazetidin-2-yl)phenoxy]acetyl}amino)(phenyl)acetic acid (0.040 g, 0.065 mmol) was dissolved in CH2Cl2 (5 ml). tert-Butyl (2S)-amino(phenyl)acetate (0.016 g, 0.078 mmol) and N-methyl-morphiline (0.020 g, 0.195 mmol) were added. After 5 minutes, TBTU (0.027 g, 0.084 mmol) was added. The reaction mixture was stirred at room temperature for 3 h. The resulting tert-butyl ester was purified on silica gel and eluted with EtOAc/CH2Cl2 (25/75). The pure fractions were concentrated. The residue was dissolved in CH2Cl2 (3 ml) and TFA (0.5 ml). The reaction mixture was stirred overnight at room temperature. The resulting acid was concentrated and the remaining trace of TFA was removed by co-evaporation with toluene (3 ml). The acid was dissolved in MeOH (3 ml) and NaBH (0.010 g, 0.260 mmol) was added. After 5 minutes, full conversion to the corresponding alcohol was obtained. The reaction was quenched by the addition of 1 ml of 0.1M NH4OAc buffer. Concentration followed by purification on preparative HPLC using a gradient of 20-50% CH3CN in 0.1M NH4OAc buffer as mobile phase and freeze-drying of the pure fractions afforded the title compound (0.037 g, 76%) as a colourless solid. M/z: 750.6 (M−1). 1H NMR [(CD3)2SO), 400 MHz] δ 2.87-2.94 (m, 2H), 4.25-4.28 (m, 1H), 4.58-4.78 (m, 3H), 5.01-5.07 (m, 2H), 5.65-5.74 (m, 1H), 6.94-7.39 (m, 22H), 8.53-8.72 (m, 2H). The resulting tert-butyl ester was purified on silica gel eluted with EtOAc/CH2Cl2 (25/75) The pure fractions were concentrated The residue was dissolved in CH2Cl2 (3 ml) The reaction mixture was stirred overnight at room temperature The resulting acid was concentrated the remaining trace of TFA was removed by co-evaporation with toluene (3 ml) The acid was dissolved in MeOH (3 ml) After 5 minutes

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

Procedure: (2R)-({[4-((2R,3R)-1-(4-Fluorophenyl)-3-{[2-(4-fluorophenyl)-2-oxoethyl]thio}-4-oxoazetidin-2-yl)phenoxy]acetyl}amino)(phenyl)acetic acid (0.040 g, 0.065 mmol) was dissolved in CH2Cl2 (5 ml). tert-Butyl (2S)-amino(phenyl)acetate (0.016 g, 0.078 mmol) and N-methyl-morphiline (0.020 g, 0.195 mmol) were added. After 5 minutes, TBTU (0.027 g, 0.084 mmol) was added. The reaction mixture was stirred at room temperature for 3 h. The resulting tert-butyl ester was purified on silica gel and eluted with EtOAc/CH2Cl2 (25/75). The pure fractions were concentrated. The residue was dissolved in CH2Cl2 (3 ml) and TFA (0.5 ml). The reaction mixture was stirred overnight at room temperature. The resulting acid was concentrated and the remaining trace of TFA was removed by co-evaporation with toluene (3 ml). The acid was dissolved in MeOH (3 ml) and NaBH (0.010 g, 0.260 mmol) was added. After 5 minutes, full conversion to the corresponding alcohol was obtained. The reaction was quenched by the addition of 1 ml of 0.1M NH4OAc buffer. Concentration followed by purification on preparative HPLC using a gradient of 20-50% CH3CN in 0.1M NH4OAc buffer as mobile phase and freeze-drying of the pure fractions afforded the title compound (0.037 g, 76%) as a colourless solid. M/z: 750.6 (M−1). 1H NMR [(CD3)2SO), 400 MHz] δ 2.87-2.94 (m, 2H), 4.25-4.28 (m, 1H), 4.58-4.78 (m, 3H), 5.01-5.07 (m, 2H), 5.65-5.74 (m, 1H), 6.94-7.39 (m, 22H), 8.53-8.72 (m, 2H). The resulting tert-butyl ester was purified on silica gel eluted with EtOAc/CH2Cl2 (25/75) The pure fractions were concentrated The residue was dissolved in CH2Cl2 (3 ml) The reaction mixture was stirred overnight at room temperature The resulting acid was concentrated the remaining trace of TFA was removed by co-evaporation with toluene (3 ml) The acid was dissolved in MeOH (3 ml) After 5 minutes

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

Procedure: To a solution of Fmoc-Cys(Acm)-OH (855 mg, 2 mmol) in 4 ml dry DCM at 0° C., HOBt (270 mg, 2 mmol) was added. Then an ice-cold solution of H-Phg-OtBu (415 mg, 2 mmol) in 4 ml of dry DCM was added. Subsequently, DIC (319 μl, 2 mmol) was introduced dropwise. After 1 h at 0° C. and 4 h at 4° C., the solution was evaporated under vacuum. The residue was dissolved in 50 ml EtOAc and washed with sat. aq. NaHCO3, 0.5 M HCl and water. After drying and evaporation of the organic phase, the residue was chromatographed on silica gel to give the protected dipeptide 1 as a foam. Yield 1.14 g (95%), Rf 0.35 (DCM/MeOH, 95:5). 1H NMR (CDCL3): δ1.45 (9H, s, tBu), 1.92 (3H, s, CH3 Acm), 2.71-3.1 (2H, m, CβH2 Cys), 3.8 (1H, m, CoH Cys) 4.2-4.5 (5H,m, CH2 Acm. CH—CH2 Fmoc), 4.56-4.85 (3H, m, CoH Phg, Fmoc), 5.4 (1H, d, J=7.3 Hz, NH) 6.47 (1H, d, J=8.4 Hz, NH), 7.2-8.05 (14H, Fmoc+Phg aromatic, NH Acm). 13C NMR (CDCl3): δ23.7 (CH2, Acm), 27.7 (3×CH3, tBu), 34.9 (Cβ, Cys), 41.1 (CH2, Acm), 41.9 (CH2, Fmoc), 57.4 (CαPhg), 83.7 (Cq tBu), 119.7-127.5 (aromatic, Fmoc, Phg), 141.0 and 143.6 (4C, Fmoc), 156.6 (CO, Fmoc), 170.2 (CO, Cys), 173.5 (2×CO, Acm, tBu ester). 4 h at 4° C., the solution was evaporated under vacuum The residue was dissolved in 50 ml EtOAc washed with sat. aq. NaHCO3, 0.5 M HCl and water After drying evaporation of the organic phase the residue was chromatographed on silica gel

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

Procedure: To a solution of Fmoc-Cys(Acm)-OH (855 mg, 2 mmol) in 4 ml dry DCM at 0° C., HOBt (270 mg, 2 mmol) was added. Then an ice-cold solution of H-Phg-OtBu (415 mg, 2 mmol) in 4 ml of dry DCM was added. Subsequently, DIC (319 μl, 2 mmol) was introduced dropwise. After 1 h at 0° C. and 4 h at 4° C., the solution was evaporated under vacuum. The residue was dissolved in 50 ml EtOAc and washed with sat. aq. NaHCO3, 0.5 M HCl and water. After drying and evaporation of the organic phase, the residue was chromatographed on silica gel to give the protected dipeptide 1 as a foam. Yield 1.14 g (95%), Rf 0.35 (DCM/MeOH, 95:5). 1H NMR (CDCL3): δ1.45 (9H, s, tBu), 1.92 (3H, s, CH3 Acm), 2.71-3.1 (2H, m, CβH2 Cys), 3.8 (1H, m, CoH Cys) 4.2-4.5 (5H,m, CH2 Acm. CH—CH2 Fmoc), 4.56-4.85 (3H, m, CoH Phg, Fmoc), 5.4 (1H, d, J=7.3 Hz, NH) 6.47 (1H, d, J=8.4 Hz, NH), 7.2-8.05 (14H, Fmoc+Phg aromatic, NH Acm). 13C NMR (CDCl3): δ23.7 (CH2, Acm), 27.7 (3×CH3, tBu), 34.9 (Cβ, Cys), 41.1 (CH2, Acm), 41.9 (CH2, Fmoc), 57.4 (CαPhg), 83.7 (Cq tBu), 119.7-127.5 (aromatic, Fmoc, Phg), 141.0 and 143.6 (4C, Fmoc), 156.6 (CO, Fmoc), 170.2 (CO, Cys), 173.5 (2×CO, Acm, tBu ester). 4 h at 4° C., the solution was evaporated under vacuum The residue was dissolved in 50 ml EtOAc washed with sat. aq. NaHCO3, 0.5 M HCl and water After drying evaporation of the organic phase the residue was chromatographed on silica gel

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

Procedure: (2R)-({[4-((2R,3R)-1-(4-Fluorophenyl)-3-{[2-(4-fluorophenyl)-2-oxoethyl]thio}-4-oxoazetidin-2-yl)phenoxy]acetyl}amino)(phenyl)acetic acid (0.040 g, 0.065 mmol) was dissolved in CH2Cl2 (5 ml). tert-Butyl (2S)-amino(phenyl)acetate (0.016 g, 0.078 mmol) and N-methyl-morphiline (0.020 g, 0.195 mmol) were added. After 5 minutes, TBTU (0.027 g, 0.084 mmol) was added. The reaction mixture was stirred at room temperature for 3 h. The resulting tert-butyl ester was purified on silica gel and eluted with EtOAc/CH2Cl2 (25/75). The pure fractions were concentrated. The residue was dissolved in CH2Cl2 (3 ml) and TFA (0.5 ml). The reaction mixture was stirred overnight at room temperature. The resulting acid was concentrated and the remaining trace of TFA was removed by co-evaporation with toluene (3 ml). The acid was dissolved in MeOH (3 ml) and NaBH (0.010 g, 0.260 mmol) was added. After 5 minutes, full conversion to the corresponding alcohol was obtained. The reaction was quenched by the addition of 1 ml of 0.1M NH4OAc buffer. Concentration followed by purification on preparative HPLC using a gradient of 20-50% CH3CN in 0.1M NH4OAc buffer as mobile phase and freeze-drying of the pure fractions afforded the title compound (0.037 g, 76%) as a colourless solid. M/z: 750.6 (M−1). 1H NMR [(CD3)2SO), 400 MHz] δ 2.87-2.94 (m, 2H), 4.25-4.28 (m, 1H), 4.58-4.78 (m, 3H), 5.01-5.07 (m, 2H), 5.65-5.74 (m, 1H), 6.94-7.39 (m, 22H), 8.53-8.72 (m, 2H). The resulting tert-butyl ester was purified on silica gel eluted with EtOAc/CH2Cl2 (25/75) The pure fractions were concentrated The residue was dissolved in CH2Cl2 (3 ml) The reaction mixture was stirred overnight at room temperature The resulting acid was concentrated the remaining trace of TFA was removed by co-evaporation with toluene (3 ml) The acid was dissolved in MeOH (3 ml) After 5 minutes

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

Procedure: To a solution of Fmoc-Cys(Acm)-OH (855 mg, 2 mmol) in 4 ml dry DCM at 0° C., HOBt (270 mg, 2 mmol) was added. Then an ice-cold solution of H-Phg-OtBu (415 mg, 2 mmol) in 4 ml of dry DCM was added. Subsequently, DIC (319 μl, 2 mmol) was introduced dropwise. After 1 h at 0° C. and 4 h at 4° C., the solution was evaporated under vacuum. The residue was dissolved in 50 ml EtOAc and washed with sat. aq. NaHCO3, 0.5 M HCl and water. After drying and evaporation of the organic phase, the residue was chromatographed on silica gel to give the protected dipeptide 1 as a foam. Yield 1.14 g (95%), Rf 0.35 (DCM/MeOH, 95:5). 1H NMR (CDCL3): δ1.45 (9H, s, tBu), 1.92 (3H, s, CH3 Acm), 2.71-3.1 (2H, m, CβH2 Cys), 3.8 (1H, m, CoH Cys) 4.2-4.5 (5H,m, CH2 Acm. CH—CH2 Fmoc), 4.56-4.85 (3H, m, CoH Phg, Fmoc), 5.4 (1H, d, J=7.3 Hz, NH) 6.47 (1H, d, J=8.4 Hz, NH), 7.2-8.05 (14H, Fmoc+Phg aromatic, NH Acm). 13C NMR (CDCl3): δ23.7 (CH2, Acm), 27.7 (3×CH3, tBu), 34.9 (Cβ, Cys), 41.1 (CH2, Acm), 41.9 (CH2, Fmoc), 57.4 (CαPhg), 83.7 (Cq tBu), 119.7-127.5 (aromatic, Fmoc, Phg), 141.0 and 143.6 (4C, Fmoc), 156.6 (CO, Fmoc), 170.2 (CO, Cys), 173.5 (2×CO, Acm, tBu ester). 4 h at 4° C., the solution was evaporated under vacuum The residue was dissolved in 50 ml EtOAc washed with sat. aq. NaHCO3, 0.5 M HCl and water After drying evaporation of the organic phase the residue was chromatographed on silica gel

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

Procedure: (2R)-({[4-((2R,3R)-1-(4-Fluorophenyl)-3-{[2-(4-fluorophenyl)-2-oxoethyl]thio}-4-oxoazetidin-2-yl)phenoxy]acetyl}amino)(phenyl)acetic acid (0.040 g, 0.065 mmol) was dissolved in CH2Cl2 (5 ml). tert-Butyl (2S)-amino(phenyl)acetate (0.016 g, 0.078 mmol) and N-methyl-morphiline (0.020 g, 0.195 mmol) were added. After 5 minutes, TBTU (0.027 g, 0.084 mmol) was added. The reaction mixture was stirred at room temperature for 3 h. The resulting tert-butyl ester was purified on silica gel and eluted with EtOAc/CH2Cl2 (25/75). The pure fractions were concentrated. The residue was dissolved in CH2Cl2 (3 ml) and TFA (0.5 ml). The reaction mixture was stirred overnight at room temperature. The resulting acid was concentrated and the remaining trace of TFA was removed by co-evaporation with toluene (3 ml). The acid was dissolved in MeOH (3 ml) and NaBH (0.010 g, 0.260 mmol) was added. After 5 minutes, full conversion to the corresponding alcohol was obtained. The reaction was quenched by the addition of 1 ml of 0.1M NH4OAc buffer. Concentration followed by purification on preparative HPLC using a gradient of 20-50% CH3CN in 0.1M NH4OAc buffer as mobile phase and freeze-drying of the pure fractions afforded the title compound (0.037 g, 76%) as a colourless solid. M/z: 750.6 (M−1). 1H NMR [(CD3)2SO), 400 MHz] δ 2.87-2.94 (m, 2H), 4.25-4.28 (m, 1H), 4.58-4.78 (m, 3H), 5.01-5.07 (m, 2H), 5.65-5.74 (m, 1H), 6.94-7.39 (m, 22H), 8.53-8.72 (m, 2H). The resulting tert-butyl ester was purified on silica gel eluted with EtOAc/CH2Cl2 (25/75) The pure fractions were concentrated The residue was dissolved in CH2Cl2 (3 ml) The reaction mixture was stirred overnight at room temperature The resulting acid was concentrated the remaining trace of TFA was removed by co-evaporation with toluene (3 ml) The acid was dissolved in MeOH (3 ml) After 5 minutes