반응 #324360

반응 방정식

fluorescein

fluorescein-5-isothiocyanate

hydrazine

diisopropylethylamine

→

5-Carboxyfluorescein

용매

DMF

DMF

반응 조건

후처리

1. 1
   기타to remove the ivDde group
2. 2
   세척The resin was then washed with DMF (3×10 ml) and twice with CH2Cl2 (10 mL)
3. 3
   기타dried under nitrogen for 1 h
4. 4
   기타for 4 h
5. 5
   여과the solution collected by filtration
6. 6
   기타The volatiles were removed under reduced pressure
7. 7
   기타the residue was dried under vacuum
8. 8
   기타The peptide was precipitated with ether
9. 9
   기타collected
10. 10
    기타the 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
    기타the solution was freeze-dried
14. 14
    workup.DISSOLUTIONThe crude cyclic peptide was dissolved in water
15. 15
    기타purified 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
    기타were collected
18. 18
    기타freeze-dried

실험 절차

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).

출처