1-Fluoro-2,4-dinitrobenzene


1-Fluoro-2,4-dinitrobenzene is a chemical that reacts with the N-terminal amino acid of polypeptides. This can be helpful for sequencing proteins.

Preparation

In 1936, Gottlieb presented a synthesis in which 1-chloro-2,4-dinitrobenzene reacted with potassium fluoride in nitrobenzene:

Uses

In 1945, Frederick Sanger described its use for determining the N-terminal amino acid in polypeptide chains, in particular insulin. Sanger's initial results suggested that insulin was a smaller molecule than previously estimated, and that it consisted of four chains, with the chains cross-linked by disulfide bonds. Sanger continued work on insulin, using dinitrofluorobenzene in combination with other techniques, eventually resulted in the complete sequence of insulin.
Following Sanger's initial report of the reagent, the dinitrofluorobenzene method was widely adopted for studying proteins, until it was superseded by other reagents for terminal analysis and other general methods for sequence determination.
Dinitrofluorobenzene reacts with the amine group in amino acids to produce dinitrophenyl-amino acids. These DNP-amino acids are moderately stable under acid hydrolysis conditions that break peptide bonds. The DNP-amino acids can then be recovered, and the identity of those amino acids can be discovered through chromatography. More recently, Sanger's reagent has also been used for the rather difficult analysis of distinguishing between the reduced and oxidized forms of glutathione and cysteine in biological systems in conjunction with HPLC. This method is so rugged that it can be performed in such complex matrices as blood or cell lysate.

Literature

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