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

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

Shaking a mixture of DNFB and Amino Acids in an alkaline alcoholic solution yields DNP-Amino Acid Derivatives.

Procedure

10 mmol of The amino acid is dissolved or suspended in 40 mL of Water, and 2 g of anhydrous Nа2СO3 is added. The solution is heated to 40° C and 10 mmol of DNFB is added. The reaction is complete within 30— 50 min, as indicated by the complete dissolution of the reaction mixture components. The mixture is acidified by adding a few drops of concentrated НСl. The DNP-amino acids are recrystallized from the appropriate Solvents (Table 11).

Class="center">Table 11 Solvents for recrystallization of DNP-amino acids

DNP-amino acid

Solvent

Ala, Cys, (Cys)2, Phe, Glu, Met, Leu, norVal, Hyp, Pro, Thr, Val

Ether — petroleum ether

Asn, Gly, Glu, Ser

Methanol — water

Arg, His

Acetone — water

Lys, Tyr, Trp

Acetone — ether

ß-Ala

Ethanol — water

NOTES

Upon acidification, Some amino acids separate as an oily material, which is precipitated with acetone. The resulting suspension is diluted with acetone, dried over anhydrous Na2SO4, filtered, and most of the acetone is evaporated; the DNP-amino acid is then precipitated with benzene and petroleum ether. If the material remains oily, the entire procedure is repeated. The precipitate of DNP-amino acids is recrystallized from the appropriate solvent.



Last update: 19/08/2026

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