Amino acids reactions with dinitrofluorobenzene

Following reaction with 2,4-dinitrofluorobenzene, all amide bonds of the pol) eptide chain are hydrolyzed and the amino acid labeled with a 2,4-dinitrophenyl group is separated by either paper or column chromatography and identified. 

The dinitrophenyl group has been used to protect the imidazole — NH group in histidines (45% yield)" by reaction with 2,4-dinitrofluorobenzene and potassium carbonate. Imidazole —NH groups, but not a-amino acid groups, are quantitatively regenerated by reaction with 2-mercaptoethanol (22°, pH 8, 1 h)." The 2,4-... 

Nucleophilic aromatic substitution is much less common than electrophilic substitution but nevertheless does have certain uses. One such use is the reaction of proteins with 2,4-dinitrofluorobenzene, known as Sanger s reagent, to attach a "label" to the terminal NH2 group of the amino acid at one end of the protein chain. 

A related reaction is that of 2,4-dinitrofluorobenzene with the amino groups of peptides and proteins, and this reaction provides a means for analysis of the N-terminal amino acids in polypeptide chains. (See Section 25-7B.)... 

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. 

Write the equation for the reaction of an amino acid with 2,4-dinitrofluorobenzene. 

The Sanger method for N-terminus determination is a less common alternative to the Edman degradation. In the Sanger method, the peptide is treated with the Sanger reagent, 2,4-dinitrofluorobenzene, and then hydrolyzed by reaction with 6 M aqueous HC1. The N-terminal amino acid is recovered as its 2,4-dinitrophenyl derivative and identified. 

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. ... 

One of the most historically significant examples of aromatic nucleophilic substitution is the reaction of amines with 2,4-dinitrofluorobenzene. This reaction was used by Sanger " to develop a method for identification of the N-terminal amino acid in proteins, and the process opened the way for structural characterization of proteins and other biopolymers. 

N-2,4-Dinitrophenyl-a-amino acids (32), obtained from the reaction of 2,4-dinitrofluorobenzene with a-amino acids (Sanger, 1945), show an intense... 

The determination of AT-terminal amino acids of polypeptides or proteins by means of reaction with 2,4-dinitrofluorobenzene is used as a chemical method for the estimation of molecular weight. This operation is rather complex because of the need for hydrolysis, separation of DNP-amino acids, and colorimetric comparison with a standard curve. Schiedt and Restle (1954) have estimated the degree of polymerization of oligopeptides using dinitrophenylation and infrared spectroscopic... 

In Section 17-12A, we saw that the Sanger reagent, 2,4-dinitrofluorobenzene, is a good substrate for nucleophilic aromatic substitution. Show the product, and propose a mechanism for the reaction of the Sanger reagent with the amino acid phenylalanine. 

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. 

The TLC method was developed for amino acids therefore, in principle, it is equally applicable to peptides. Peptides, such as amino acids, are generally hydrophilic. There are, however, limits to this analogy. Dinitrophenylamino acid derivatives (DNP amino acids) and phenylthiohydantoin derivatives (PTH amino acids) are obtained when reaction of peptides or proteins with dinitrofluorobenzene or phenyl mustards are properly degraded. Their separation from reaction mixtures and their identification are considerable practical importance because they constitute essential steps... 

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... 

Free amino acids instead of proteins or peptides can of course be reacted with dinitrofluorobenzene (DNFB). This can arise when an amino acid mixture cannot be chromatographed because of contaminants the DNP-amino acids can often be more easily converted into a form capable of being chromatographed, than can the free amino acids. The fact that these derivatives are coloured facilitates quantitative analysis since all the difficulties associated with subsequent colour reactions are obviated. 

FIGURE 23.32 The amino add at the amino terminus can be identified by reaction with 2,4-dinitrofluorobenzene, followed by hydrolysis. Only the amino acid at the amino terminus of the chain will be labeled. 

Lessen rearrangement and identification of the amino acid involved in intermediate formation. The dinitrophenylation method and Lessen rearrangement procedure followed the description of Gross and Morrell (22). The solution of hydroxamate modified was adjusted to pH 8 by the use of sodium bicarbonate and incubated with an equal amount of 1% 2,4-dinitrofluorobenzene in ethanol for 30 min at room temperature. After the reaction, the excess of 2,4-dinitrofluorobenzene was extracted with ether several times, until no yellow color was observed. The protein was dissolved in 0.1 N NaOH and the resultant solution was immersed in boiling water for 10 min. After acidification, the product was lyophilized, then hydrolyzed in a sealed tube containing 6 N HCl for 24 hr at 110 (23). The amino acid analysis was performed on a 2 x 53-cm column of PA-15 in a Beckman amino acid analyzer Model 121, eluting with 0.35 N sodium citrate, pH 3.8, at 30o. The peak of 2,3-diaminopropionic acid appeared at 231 min, and 2,4-diaminobutyric acid was eluted at 240 min. 

Derivatives for the identification of amino acids are prepared by substituting the amino group. In addition to the acetylation (85, 86), benzoylation (87), and 3,5-dinitrobenzoylation (88), the reaction with 2,4-dinitrofluorobenzene (89) is also suitable for the preparation of derivatives. This reagent reacts with amino acids at room temperature. 

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