Reaction #1435696

Reaction equation

fluorescein-5-isothiocyanate

fluorescein

hydrazine

lysine

diisopropylethylamine

→

5-Carboxyfluorescein

Solvents

DMF

DMF

Conditions

Workup

1. 1
   Otherto remove the ivDde group
2. 2
   WashThe resin was then washed with DMF (3×10 mL) and twice with CH2Cl2 (10 mL)
3. 3
   Otherdried under nitrogen for 1 h
4. 4
   Otherfor 4 h
5. 5
   Filtrationthe solution collected by filtration
6. 6
   OtherThe volatiles were removed under reduced pressure
7. 7
   Otherthe residue was dried under vacuum
8. 8
   OtherThe peptide was precipitated with ether
9. 9
   Othercollected
10. 10
    Otherthe 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
    Otherthe solution was freeze-dried
14. 14
    workup.DISSOLUTIONThe crude cyclic peptide was dissolved in water
15. 15
    Otherpurified by RP-HPLC on a C18 column with linear gradient of acetonitrile into water (both phases
16. 16
    workup.ADDITIONFractions containing the pure product
17. 17
    Otherwere collected
18. 18
    Otherfreeze dried

Procedure

Peptide-resin obtained via from 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 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% TFA).

Source