Amino Acids, Peptides and Proteins - Dévényi T., Gergely J. 1976

End-group analysis and stepwise degradation of proteins and peptides
Determination of N-terminal groups by the 2,4-dinitrofluorobenzene method
Preparation of ε-DNP-lysine

The a-NH₂ group is blocked with Сu²⁺ ions, and the ε-NН₂ group is dinitrophenylated in a weakly alkaline medium. After the removal of Сu²⁺ ions, the ε-N-DNP derivative is isolated [14].

Procedure

0.5 g of Lysine is dissolved in 10 ml of distilled Water, the solution is brought to a boil, and СuСO₃ is added to it in small portions. Excess СuСO₃ is removed by filtration, and 1.5 g of NaHCO₃ and 1.5 g of DNFB dissolved in 20 ml of ethanol are added to the solution. The mixture is shaken at room Temperature for 2 h and filtered. The precipitate is washed with distilled water, ethanol, and finally with ether, and then suspended in 5 ml of distilled water, acidified with a few drops of 1 N HCl, and the vessel with the clear solution is placed in crushed ice. Hydrogen sulfide is passed through the cooled solution to remove Сu²⁺ ions. The CuS precipitate is removed by filtration after adsorption with activated charcoal. The clear filtrate is dried in vacuo, and the DNP derivative is recrystallized from 6 N HCl.

NOTES

The δ-N-DNP derivative of Ornithine can be prepared in a similar manner. a-N-DNP-His is formed very rapidly upon The addition of a small amount of DNFB, and dinitrophenylation of the acetyl derivative yields N-DNP-His.