Réaction #324360

Équation de réaction

fluorescein

fluorescein-5-isothiocyanate

hydrazine

diisopropylethylamine

→

5-Carboxyfluorescein

Solvants

DMF

DMF

Conditions de réaction

Traitement

1. 1
   Autreto remove the ivDde group
2. 2
   LavageThe resin was then washed with DMF (3×10 ml) and twice with CH2Cl2 (10 mL)
3. 3
   Autredried under nitrogen for 1 h
4. 4
   Autrefor 4 h
5. 5
   Filtrationthe solution collected by filtration
6. 6
   AutreThe volatiles were removed under reduced pressure
7. 7
   Autrethe residue was dried under vacuum
8. 8
   AutreThe peptide was precipitated with ether
9. 9
   Autrecollected
10. 10
    Autrethe 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
    Autrethe solution was freeze-dried
14. 14
    workup.DISSOLUTIONThe crude cyclic peptide was dissolved in water
15. 15
    Autrepurified 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
    Autrewere collected
18. 18
    Autrefreeze-dried

Mode opératoire

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).

Source