反应详情

6.26 g (10 mM) of 2HBr.H-D-Leu-Gly-Arg-pNA (prepared according to Example 7f, see Table 3) were dissolved in 30 ml of distilled DMF. The solution was cooled to -15°, and first 2.80 ml (20 mM) of Et3N and immediately afterwards 1.91 g (10 mM) of tosyl chloride were added, while stirring, in the absence of humidity. After a reaction time of about 2 to 3 hours at -15°, the reaction mixture was allowed to further react overnight at room temperature. The reaction mixture was again cooled to -15°. The precipitated mixture of Et3N.HBr and Et3N.HCl was removed by filtration and washed with a small amount of cold DMF. The filtrate and the washing solution were combined and concentrated to dryness in vacuo at 50°. The residue was dissolved in 75 ml of 50% AcOH. The solution was purified on a column of "Sephadex" G-15 equilibrated with 50% AcOH. The main fraction of the AcOH eluate which was split by treatment with trypsin with release of p-nitroaniline was concentrated to dryness in vacuo at 40°. The residue was dissolved in 100 ml of MeOH, and the solution was again concentrated to dryness. The residue was dried in a vacuum desiccator at 60° over P2O5 to obtain 6.65 g (95.0% of the theory) of amorphous compound Tos-D-Leu-Gly-Arg-pNA.HBr which was homogeneous in the SS as shown by TLC. Elementary analysis and calculation from the empirical formula C27H39N8SO7Br gave the following values: C=46.85% (46.35%), H=5.66% (5.62%), N=16.28% (16.02%), S=4.43% (4.58%) and Br=11.22% (11.42%).

1. temperatureThe solution was cooled to -15°
2. customAfter a reaction time of about 2 to 3 hours
3. customat -15°
4. customto further react overnight at room temperature
5. temperatureThe reaction mixture was again cooled to -15°
6. customwas removed by filtration
7. washwashed with a small amount of cold DMF
8. concentrationconcentrated to dryness in vacuo at 50°
9. dissolutionThe residue was dissolved in 75 ml of 50% AcOH
10. customThe solution was purified on a column of "Sephadex" G-15
11. customThe main fraction of the AcOH eluate which was split by treatment with trypsin with release of p-nitroaniline
12. concentrationwas concentrated to dryness in vacuo at 40°
13. dissolutionThe residue was dissolved in 100 ml of MeOH
14. concentrationthe solution was again concentrated to dryness
15. dry with materialThe residue was dried in a vacuum desiccator at 60° over P2O5