2.4- Dinitrofluorobenzene , sequencing

Chemical methods for determining the amino acid sequence of a peptide or protein have been developed, and the normal approach is to exploit the properties of the amino group at the Al-terminus. A long-established procedure for identifying the N-terminal amino acid is use of the Sanger reagent 2,4-dinitrofluorobenzene. This reacts with an amine by nucleophilic displacement of the fluorine. 

At this point, you can deduce two possible sequences for Q.) (5) Trypsin hydrolysis of L gives a peptide of composition Ala, Asp, Phe which, with 2,4-dinitrofluorobenzene, gives the 2,4-dinitrophenyl derivative of aspartic acid. (6) Partial acid hydrolysis of eledoisin gives several dipeptides, among them Ser-Lys and Pro-Ser. 

Using procedures such as those outlined in this section more than 100 proteins have been sequenced. This is an impressive accomplishment considering the complexity and size of many of these molecules (see, for example, Table 25-3). It has been little more than two decades since the first amino acid sequence of a protein was reported by F. Sanger, who determined the primary structure of insulin (1953). This work remains a landmark in the history of chemistry because it established for the first time that proteins have definite primary structures in the same way that other organic molecules do. Up until that time, the concept of definite primary structures for proteins was openly questioned. Sanger developed the method of analysis for N-terminal amino acids using 2,4-dinitrofluorobenzene and received a Nobel Prize in 1958 for his success in determining the amino-acid sequence of insulin. 

By the primary structure of a peptide or protein, we mean its amino acid sequence. Complete hydrolysis gives the amino acid content. The N-terminal amino acid can be identified by the Sanger method, using 2,4-dinitrofluorobenzene. The Edman degradation uses phenyl isothiocyanate to clip off one amino acid at a time from the N-terminus. Other reagents selectively cleave peptide chains at certain amino acid links. A... 

Hall and Polis 7 prepared a series of polyarylamines using an aromatic nucleophilic substitution-reduction sequence (Scheme 4.20). Hence, 2,4-dinitrofluorobenzene 66 was treated with />-diaminobenzene to afford tetranitro 67, which was reduced to give the corresponding first generation, diamine 68. Repetition of this sequence afforded the second generation tetradecaamine 69. These starburst polyarylamines were complexed with iodine to form semiconducting materials and were the first dendrimers to be examined by cyclic voltammetry. 

The covalent structure of insulin was established by Frederick Sanger in 1953 after a 10-year effort. This was the first protein sequence determination. Sanger used partial hydrolysis of peptide chains whose amino groups had been labeled by reaction with 2,4-dinitrofluorobenzene to form shorter end-labeled fragments. These were analyzed for their amino acid composition and labeled and hydrolyzed again as necessary. Many peptides had to be analyzed to deduce the sequence of the 21-residue and 30-residue chains that are joined by disulfide linkages in insulin. ... 

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. 

Dinitrophenylamino acids (DNP-amino acids) and phenylthio-hydantoins (PTH-amino acids) are formed when proteins or peptides are treated with dinitrofluorobenzene [151—154] or phenyl isothiocyanate [155], respectively, and the reaction product suitably broken down. Their separation from the reaction mixture and their identification is of considerable practical importance since systematic application enables sequence analysis of peptide structures to be performed. Numerous investigators have worked on this problem... 

An alkylator which has found use in analytical work (sequencing of proteins the determination of the sequence in which the amino acid monomers are arranged in the protein polymer) is 2,4-dinitrofluorobenzene Sanger s reagent). 

---