1,2-Dichloroethane → HCID 131721926, compound

Temperature

0 °C

Boc-Gly-Pro-Nleu-OH (0.77 g, 2 mmol), HClNH2CH3 (0.27 g, 4 mmol.) and HOBt (0.35 g, 2.5 mmol) were dissolved in DMF and the solution was chilled to 0° C. on a water-ice bath. Triethylamine (0.7 ml) was added slowly while stirring the solution. After 5 minutes, EDC (0.46 g, 2.4 mmol.) was added to the solution and the stirring continued overnight. The DMF was removed under reduced pressure and the resulting mixture was poured into 150 ml chloroform. The chloroform solution was washed by H2O, saturated NaHCO3, brine, saturated NaHSO4 and brine again until the pH of the brine layer was approximate 7. The organic layer was dried using Na2SO4 and the chloroform was distilled under reduced pressure to obtain the Boc-Gly-Pro-Nleu-NHCH3. TLC Rf=0.44 (developing solvents: CMA, 85:15:3). This product was used directly in next step without further purification and characterization. 30 ml solution of 30% TFA in DCM was used to remove the Boc group. The deprotection was allowed to proceed 30 minutes. Then, the DCM and TFA was removed under reduced pressure and benzene 3×20 ml was added and distilled to remove trace TFA. The TFA salt of Gly-Pro-Nleu-NHCH3 was dissolved in 50 ml and the solution was chilled to 0° C. Triethylamine was added slowly to neutralize the peptide. After a while, acetic anhydride (2 ml) was added and the acetylation was allowed to proceed 40 minutes. The DCM was removed and the resulting mixture was dissolved in 100 ml chloroform. The chloroform solution was washed by H2O, saturated NaHCO3, brine, saturated NaHSO4 and brine again until the pH of the brine layer was approximately 7. The organic layer was dried using Na2SO4 and the chloroform was distilled under reduced pressure to obtain the final product Ac-Gly-Pro-Nleu-NHCH3 (0.6 g, 88%). Analytical HPLC profile, single peak, RT=10.5 min. (10-50% B in 30 min.). FAB-MS, observed (M+H)+=341. 1H-NMR (500 MHz, DMSO-d6, 28° C., assignment by TOCSY) δ8.04 (m, C-terminal NH), 7.93 (m, Gly-NH), 7.71 (m, C-terminal NH), 7.48 (m, C-terminal NH), 4.94 (d, Pro-α), 4.81 (m, Pro-α), 4.68 (m, Pro-α), 4.50 (m, Pro-α), 4.29-3.52 (sets of bands, Nleu-α, Gly-α), 3.52-3.42 (m, Pro-δ), 3.36 (m, Pro-δ), 3.29-2.86 (sets of bands, Nleu-β), 2.59 (m, C-terminal N-CH3), 2.53 (m, C-terminal N-CH3), 2.31-2.03 (sets of bands, Pro-β), 1.96 (m, Pro-β, Pro-γ), 1.92-1.61 (m, acetyl-CH3, Pro-β, Nleu-α, Pro-γ), 0.86 (dd, Nleu-δ), 0.75 (m, Nleu-δ).

1. customthe stirring continued overnight
2. customThe DMF was removed under reduced pressure
3. additionthe resulting mixture was poured into 150 ml chloroform
4. washThe chloroform solution was washed by H2O, saturated NaHCO3, brine, saturated NaHSO4 and brine again until the pH of the brine layer
5. customThe organic layer was dried
6. distillationthe chloroform was distilled under reduced pressure

ORD record

ord-fe03974b19da4913bbd8a2e974fbb9db

Source dataset

uspto-grants-2001_12

DOI

10.6084/m9.figshare.5104873.v1

Patent number

US06329506B1