2.4- dinitrofluorobenzene DNFB Sanger

The reaction of 2,4-dinitrofluorobenzene (DNFB) (Sanger s reagent [10]) with amino acids is another useful technique which is often employed for the analysis of N-terminal amino acids by TLC and column chromatography after derivatization. The reaction involved in product formation is shown in Fig.4.6. The separated derivatives are determined by measuring the quenching of fluorescence on TLC plates or by UV analysis after column chromatography. The generalized absorption curves of dinitrophenyl (DNP)-amino acids in acidic and alkaline solutions are shown in Fig. 4.7. 

Another method for sequence analysis is the Sanger N-terminal analysis, based on the use of 2,4-dinitrofluorobenzene (DNFB). When a polypeptide is treated with DNFB in mildly basic solution, a nucleophilic aromatic substitution reaction (SnAt, Section 21.1 lA) takes place involving the free amino group of the N-terminal residue. Subsequent hydrolysis of the polypeptide gives a mixture of amino acids in which the N-terminal amino acid is labeled with a 2,4-dinitrophenyl group. After separating this amino acid from the mixture, it can be identified by comparison with known standards. 

Scheme 12.68. A representation of the Sanger method of degradation of a peptide at the N-terminus. The example chosen shows the reaction between the N-terminus amino acid alanine (Ala, A) and 2,4-dinitrofluorobenzene (DNFB). A phenylalanine (Phe, F) is shown adjacent to the terminal alanine (Ala, A) as the second from the N-terminus. The remainder of the peptide is not shown.The products of the reaction are shown as the A-(2,4-dinitrophenyl) alanine and the peptide chain where the phenylalanine (Phe, F) is now the new terminal amino acid.

Dinitrofluorobenzene (DNFB) or Sangers Reagent has also been used for similar applications such as the analysis of amino acids and amines (32). Tt reacts with both primary and secondary amines and phenols but not with aliphatic alcohols consequently it is useful for determining phenols in the presence of aliphatic hydroxy compounds. The sensitivity realized from this reagent is similar to that obtained from the dinitrobenzoyl derivatives. 

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