(2S)-3-amino-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoic acid

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

Procedure: Commercially available (S)-3-amino-2-tert-butoxycarbonylamino-propionic acid (BOC-Dap-OH) (18.09 g, 88.56 mmol) was suspended in 100 ml DCM with N-hydroxy-succinimide (16.99 g, 147.61 mmol) and N,N′-diisopropylcarbodiimide (11.43 ml, 73.8 mmol), then 5-Chloro-thiophene-2-carboxylic acid (12 g, 73.8 mmol) and DIPEA (25.51 ml, 147.61 mmol) in 60 ml DCM were slowly added. The mixture was stirred at RT for 2 h, then cooled to 0° C. and TMS-diazomethane was slowly added. The reaction was stirred for 2 h at RT, HCl solution (0.5 M) was added and the organic layer was separated, washed with H2O, dried with Na2SO4, filtered and evaporated to dryness. The resulting solid was triturated 16 h with diisopropyl ether, filtered and washed with diisopropyl ether and dried under vacuum at 60° C. 24.4 g of crude product were obtained and used without further purification. cooled to 0° C. The reaction was stirred for 2 h at RT the organic layer was separated washed with H2O dried with Na2SO4 filtered evaporated to dryness The resulting solid was triturated 16 h with diisopropyl ether filtered washed with diisopropyl ether dried under vacuum at 60° C

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

Procedure: Commercially available (S)-3-Amino-2-tert-butoxycarbonylamino-propionic acid (BOO-Dap-OH) (408 mg, 2 mmol) was suspended in 4 ml DCM and DIPEA (1.75 ml, 10 mmol). 5-Chloro-thiophene-2-carbonyl chloride (362 mg, 2 mmol) (described in WO 2005068456) in 4 ml DCM was added slowly and the mixture was stirred at RT for 2 h. The reaction was filtered and the solvent evaporated. The resulting solid (895 mg) was used without further purification in the next step. The reaction was filtered the solvent evaporated The resulting solid (895 mg) was used without further purification in the next step

Conditions: temp:80.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: (2S)-2-tert-Butoxycarbonylamino-3-(2-nitrophenyl-amino)-propionic acid. (2S)-2-tert-Butoxycarbonylamino-3-aminopropionic acid (10 g, 49 mmol), 2-fluoronitrobenzene (5.7 ml, 54 mmol), and NaHCO3 (8.25 g, 98 mmol) was taken into 130 ml of DMF and heated at 80° C. for 18 h. The reaction was evaporated in vacuo to give a viscous orange residue that was dissolved in 300 ml of H2O and extracted with Et2O (3×150 ml). The aq. solution was acidified to pH 5 with 10% NaHSO4 and extracted with EtOAc (3×250 ml). The combined extracts were dried over anhydrous Na2SO4, filtered, and evaporated to give 12.64 g (83%) of the title compound as an orange amorphous solid: 1H NMR (CD3OD) δ 8.15-8.10 (1H,d), 7.54-7.48 (1H,t), 7.13-7.08 (1H, d), 6.73-6.65 (1H, t), 4.45-4.35 (1H, m), 3.9-3.8 (1H, dd), 3.65-3.55 (1H, dd), 1.45 (9H, s). The reaction was evaporated in vacuo to give a viscous orange residue that extracted with Et2O (3×150 ml) extracted with EtOAc (3×250 ml) The combined extracts were dried over anhydrous Na2SO4 filtered evaporated

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

Procedure: A stirring mixture of the N-Boc-Dap-OH 1 (10.00 g, 49.02 mmol), THF (90 mL), water (30 mL), K2CO3 (13.53 g, 98.04 mmol) and KOH (2.75 g, 49.02 mmol) was cooled to −5° C. Cbz-Cl solution (50 wt % in toluene, 25 mL, 73.53 mmol) was then added dropwise over a period of an hour. The mixture was allowed to stir for 8 h before being concentrated under reduced pressure to remove the THF. The aqueous residue was extracted with diethyl ether (50 mL), acidified to pH 4 by solid citric acid and the resulting precipitate was extracted by CH2Cl2 (2 50 mL). Combined organic layers were dried (Na2SO4) and evaporated to furnish the free acid 2 (15.57 g, 94% yield) as a gummy white solid which was used without further purification. 1H NMR (300 MHz, CDCl3) δ10.76 (1H), 7.25 (5H), 6.24, 5.67 (2H), 5.85 (1H), 5.08, 5.02 (2H), 4.36-4.23 (1H), 3.63-3.35 (2H), 1.38 (9H); 13C NMR (75 MHz, CDCl3)δ 173.5, 157.5, 156.2, 136.4, 128.6, 128.2, 80.6, 67.3, 54.4, 42.9, 28.6. before being concentrated under reduced pressure to remove the THF The aqueous residue was extracted with diethyl ether (50 mL) the resulting precipitate was extracted by CH2Cl2 (2 50 mL) Combined organic layers were dried (Na2SO4) evaporated

Conditions: temp:16.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A slurry of N-Boc-L-asparagine (5.00 g, 21.5 mmol), EtOAc (24 mL), acetonitrile (24 mL), water (12 mL), and PIDA (8.32 g, 25.8 mmol) was cooled and stirred at 16° C. for 30 min. The temperature was then allowed to reach 20° C., and the reaction was stirred until completion (ca. 4 h). The mixture was cooled to 0° C. and filtered. The filter cake was washed with EtOAc (10 mL) and dried in vacuo to give 1 (2.73 g, 62% yield). 1H NMR (CD3OD and a drop of TFA) δ4.05 (1H), 3.20-3.08 (2H), 1.45 (9H). to reach 20° C. the reaction was stirred until completion (ca. 4 h) The mixture was cooled to 0° C. filtered The filter cake was washed with EtOAc (10 mL) dried in vacuo

Conditions: temp:-10.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: (S)-2-tert-Butoxycarbonylamino-3-amino-propionic acid (14.9 g, 73.2 mmol) was dissolved in a mixture of THF (45 mL) and 3 N aq. NaOH (45 mL). The reaction mixture was cooled to −10° C. and nosyl chloride (17.9 g, 80.5 mmol) was added as a THF solution (75 mL) drop wise over 30 min. The reaction mixture was stirred at −10° C. for 45 min followed by stirring at ambient temperature for 30 min. The reaction mixture was diluted with water (150 mL), acidified with 2% aqueous H2SO4 (to pH ˜2) and diluted with additional water (450 mL). The product was extracted with EtOAc (600 mL total) and washed with water (3×400 mL) and brine (100 mL). The organic layer was separated, dried over Na2SO4, filtered and condensed in vacuo to afford compound A (20.0 g, 70% yield) as a cream solid. LC-MS [M+H-Boc] 290.3 (C14H19N3O8S+H, calc: 390.4). Purity >95% (UV/254 nm). Compound A was used without further purification. by stirring at ambient temperature for 30 min The product was extracted with EtOAc (600 mL total) washed with water (3×400 mL) and brine (100 mL) The organic layer was separated dried over Na2SO4 filtered condensed in vacuo

Conditions: temp:-10.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: (S)-2-tert-Butoxycarbonylamino-3-amino-propionic acid (14.9 g, 73.2 mmol) was dissolved in a mixture of THF (45 mL) and 3 N aq. NaOH (45 mL). The reaction mixture was cooled to −10° C. and nosyl chloride (17.9 g, 80.5 mmol) was added as a THF solution (75 mL) drop wise over 30 min. The reaction mixture was stirred at −10° C. for 45 min followed by stirring at ambient temperature for 30 min. The reaction mixture was diluted with water (150 mL), acidified with 2% aqueous H2SO4 (to pH ˜2) and diluted with additional water (450 mL). The product was extracted with EtOAc (600 mL total) and washed with water (3×400 mL) and brine (100 mL). The organic layer was separated, dried over Na2SO4, filtered and condensed in vacuo to afford compound A (20.0 g, 70% yield) as a cream solid. LC-MS [M+H-Boc] 290.3 (C14H19N3O8S+H, calc: 390.4). Purity >95% (UV/254 nm). Compound A was used without further purification. by stirring at ambient temperature for 30 min The product was extracted with EtOAc (600 mL total) washed with water (3×400 mL) and brine (100 mL) The organic layer was separated dried over Na2SO4 filtered condensed in vacuo

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

Procedure: Cbz-Phe-Tetrazole (2.25 g, 6.25 mmole) was then dissolved in ethanol (100 ml) and treated with Pd/C 10% (500 mg, 0.3125 mmole) and stirred under hydrogen for 2 hr. The reaction mixture was filtered through a celite pad and concentrated to dryness. This material was reconstituted in dichloromethane (80 ml) and treated with Boc-Dap (1.88 g, 6.54 mmole), DEPC (1.42 ml, 9.38 mmole) and DIEA (3.8 ml, 21.88 mmole). After 16 hr volatiles were removed to afford crude Boc-Dap-Phe-Tetrazole which was purified by preparative HPLC to give 704 mg (20%) of 1001 as a white powder. Reverse-phase HPLC analysis: 99% at 6.3 min; LCMS (ESI): m/z 458.905 (MH)+, 480.874 (M+Na)+, eluted at 12.95 min. The reaction mixture was filtered through a celite pad concentrated to dryness After 16 hr volatiles were removed to afford crude Boc-Dap-Phe-Tetrazole which was purified by preparative HPLC

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

Procedure: Commercially available (S)-3-amino-2-tert-butoxycarbonylamino-propionic acid (BOC-Dap-OH) (18.09 g, 88.56 mmol) was suspended in 100 ml DCM with N-hydroxy-succinimide (16.99 g, 147.61 mmol) and N,N′-diisopropylcarbodiimide (11.43 ml, 73.8 mmol), then 5-Chloro-thiophene-2-carboxylic acid (12 g, 73.8 mmol) and DIPEA (25.51 ml, 147.61 mmol) in 60 ml DCM were slowly added. The mixture was stirred at RT for 2 h, then cooled to 0° C. and TMS-diazomethane was slowly added. The reaction was stirred for 2 h at RT, HCl solution (0.5 M) was added and the organic layer was separated, washed with H2O, dried with Na2SO4, filtered and evaporated to dryness. The resulting solid was triturated 16 h with diisopropyl ether, filtered and washed with diisopropyl ether and dried under vacuum at 60° C. 24.4 g of crude product were obtained and used without further purification. cooled to 0° C. The reaction was stirred for 2 h at RT the organic layer was separated washed with H2O dried with Na2SO4 filtered evaporated to dryness The resulting solid was triturated 16 h with diisopropyl ether filtered washed with diisopropyl ether dried under vacuum at 60° C

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

Procedure: Commercially available (S)-3-Amino-2-tert-butoxycarbonylamino-propionic acid (BOO-Dap-OH) (408 mg, 2 mmol) was suspended in 4 ml DCM and DIPEA (1.75 ml, 10 mmol). 5-Chloro-thiophene-2-carbonyl chloride (362 mg, 2 mmol) (described in WO 2005068456) in 4 ml DCM was added slowly and the mixture was stirred at RT for 2 h. The reaction was filtered and the solvent evaporated. The resulting solid (895 mg) was used without further purification in the next step. The reaction was filtered the solvent evaporated The resulting solid (895 mg) was used without further purification in the next step

Conditions: temp:80.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: (2S)-2-tert-Butoxycarbonylamino-3-(2-nitrophenyl-amino)-propionic acid. (2S)-2-tert-Butoxycarbonylamino-3-aminopropionic acid (10 g, 49 mmol), 2-fluoronitrobenzene (5.7 ml, 54 mmol), and NaHCO3 (8.25 g, 98 mmol) was taken into 130 ml of DMF and heated at 80° C. for 18 h. The reaction was evaporated in vacuo to give a viscous orange residue that was dissolved in 300 ml of H2O and extracted with Et2O (3×150 ml). The aq. solution was acidified to pH 5 with 10% NaHSO4 and extracted with EtOAc (3×250 ml). The combined extracts were dried over anhydrous Na2SO4, filtered, and evaporated to give 12.64 g (83%) of the title compound as an orange amorphous solid: 1H NMR (CD3OD) δ 8.15-8.10 (1H,d), 7.54-7.48 (1H,t), 7.13-7.08 (1H, d), 6.73-6.65 (1H, t), 4.45-4.35 (1H, m), 3.9-3.8 (1H, dd), 3.65-3.55 (1H, dd), 1.45 (9H, s). The reaction was evaporated in vacuo to give a viscous orange residue that extracted with Et2O (3×150 ml) extracted with EtOAc (3×250 ml) The combined extracts were dried over anhydrous Na2SO4 filtered evaporated

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

Procedure: 4-Chlorobutanoyl chloride (12.1 g, 86 mmol) was added to a solution of Boc-L-Dap (16.0 g, 78 mmol) in dioxane (160 mL) and 10% aqueous Na2CO3 (180 mL) at 0° C. dropwise. The reaction mixture was stirred at 0° C. for 1 h and then allowed to warm to ambient temperature and stirred overnight. The mixture was acidified with 1N aqueous hydrochloric acid to pH=3 and extracted with EtOAc (300 mL×3). The combined organic phases were washed with 1N aqueous hydrochloric acid (300 mL×3) and brine (300 mL×1), dried over anhydrous sodium sulfate, and concentrated to afford (S)-2-(tert-butoxycarbonylamino)-3-(4-chlorobutanamido)propanoic acid (15.5 g, 64% yield), which was used directly without further purification. to warm to ambient temperature stirred overnight extracted with EtOAc (300 mL×3) The combined organic phases were washed with 1N aqueous hydrochloric acid (300 mL×3) and brine (300 mL×1) dried over anhydrous sodium sulfate concentrated