反应 #554639

反应方程式

fluorescein-5-isothiocyanate

fluorescein

hydrazine

lysine

diisopropylethylamine

→

5-Carboxyfluorescein

溶剂

DMF

DMF

反应条件

后处理

1. 1
   其他to remove the ivDde group
2. 2
   洗涤The resin was then washed with DMF (3×10 mL) and twice with CH2Cl2 (10 mL)
3. 3
   其他dried under nitrogen for 1 h
4. 4
   其他for 4 h
5. 5
   过滤the solution collected by filtration
6. 6
   其他The volatiles were removed under reduced pressure
7. 7
   其他the residue was dried under vacuum
8. 8
   其他The peptide was precipitated with ether
9. 9
   其他collected
10. 10
    其他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
    其他the solution was freeze-dried
14. 14
    workup.DISSOLUTIONThe crude cyclic peptide was dissolved in water
15. 15
    其他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
    其他were collected
18. 18
    其他freeze-dried

实验过程

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% TFA).

来源