反応 #324360

反応方程式

fluorescein

fluorescein-5-isothiocyanate

hydrazine

diisopropylethylamine

→

5-Carboxyfluorescein

溶媒

DMF

DMF

反応条件

後処理

1. 1
   その他to remove the ivDde group
2. 2
   洗浄The resin was then washed with DMF (3×10 ml) and twice with CH2Cl2 (10 mL)
3. 3
   その他dried under nitrogen for 1 h
4. 4
   その他for 4 h
5. 5
   ろ過the solution collected by filtration
6. 6
   その他The volatiles were removed under reduced pressure
7. 7
   その他the residue was dried under vacuum
8. 8
   その他The peptide was precipitated with ether
9. 9
   その他collected
10. 10
    その他the precipitate was dried under a stream of nitrogen
11. 11
    workup.ADDITIONThe precipitate was added to water (1 mg/mL)
12. 12
    workup.WAITCyclization of the peptide was carried out for 48 h
13. 13
    その他the solution was freeze-dried
14. 14
    workup.DISSOLUTIONThe crude cyclic peptide was dissolved in water
15. 15
    その他purified by RP-HPLC on a C18 column with a linear gradient of acetonitrile into water (both phases
16. 16
    workup.ADDITIONFractions containing the pure product
17. 17
    その他were collected
18. 18
    その他freeze-dried

実験手順

Peptide-resin obtained via Method 5 containing an ivDde protecting group on the epsilon nitrogen of lysine, was mixed with a solution of hydrazine in DMF (10% hydrazine/DMF, 2×10 mL, 10 min) to remove the ivDde group. The epsilon nitrogen of the lysine was labeled with fluorescein-5-isothiocyanate (0.12 mmol) and diisopropylethylamine (0.12 mmol) in DMF . The mixture was agitated for 12 h (fluorescein-containing compounds were protected from light). The resin was then washed with DMF (3×10 ml) and twice with CH2Cl2 (10 mL) and dried under nitrogen for 1 h. The peptide was cleaved from the resin using reagent B for 4 h and the solution collected by filtration. The volatiles were removed under reduced pressure, and the residue was dried under vacuum. The peptide was precipitated with ether, collected and the precipitate was dried under a stream of nitrogen. The precipitate was added to water (1 mg/mL) and the pH of the mixture was adjusted to 8 with 10% aqueous meglumine. Cyclization of the peptide was carried out for 48 h and the solution was freeze-dried. The crude cyclic peptide was dissolved in water and purified by RP-HPLC on a C18 column with a linear gradient of acetonitrile into water (both phases contained 0.1% TFA). Fractions containing the pure product were collected and freeze-dried. The peptides were characterized by ES-MS and the purity was determined by RP-HPLC (linear gradient of acetonitrile into water/0.1% TEA).

出典