2.4- Dinitrofluorobenzene, Sanger reagent

Dinitrofluorobenzene (Sanger s reagent) [70-34-8] M 186.1, m 25-27 , b 133 /2mm, 140-141 /5mm, d 1.483. Crystd from ether or EtOH. Vacuum distd through a Todd Column (see p. 174). If it is to be purified by distn in vacuo, the distn unit must be allowed to cool before air is allowed into the apparatus otherwise the residue carbonises spontaneously and an EXPLOSION may occur. The material is a skin irritant and may cause serious dermatitis. 

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. 

Fluoride ion is usually a poor leaving group because it is not very polarizable. Fluoride serves as the leaving group in the Sanger reagent (2,4-dinitrofluorobenzene), used in the determination of peptide structures (Chapter 24). Explain why fluoride works as a leaving... 

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. 

A method for determining the N-terminal amino acid of a peptide. The peptide is treated with 2,4-dinitrofluorobenzene (Sanger s reagent), then completely hydrolyzed. The derivatized amino acid is easily identified, but the rest of the peptide is destroyed in the hydrolysis, (p. 1180)... 

Determination of N-terminal amino acid residues by use of 2,4-dinitrofluorobenzene (Sanger s reagent). 

Solution 5-dimethylaminonaphthalenesulfonyl chloride (dansyl chloride) reacts with an amino acid in the same manner as does 2,4 dinitrofluorobenzene (Sanger s reagent) it bonds with the unprotected a-amino group. 

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. 

Dinitrofluorobenzene (CAS 70-34-8), 10% aqua (Sanger reagent) (Garcia-Perez 1978)... 

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. 

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). 

Acid fluondes such as oxalyl fluoride, tnchloroacetyl fluonde, propionyl fluonde, as well as Sanger s reagent, 2,4-dinitrofluorobenzene, easily cleave N,N-acetals (aminals) to give high yields of fluoromethyldialkylamines [82] (equation 20) (Table 5)... 

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. 

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. 

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... 

Hitherto we have concentrated on electrophilic aromatic substitution. However, certain n-deficient aromatic rings are deactivated towards electrophilic attack but are susceptible to nucleophilic addition and a subsequent elimination. A particular example is 2,4-dinitrochloroben-zene. The electron-withdrawing nitro groups facilitate a Michael-type addition of a nucleophile to give a so-called Meisenheimer intermediate (Scheme 4.8). Collapse of the Meisenheimer intermediate and reversion to the aromatic system may lead to expulsion of the halide ion, as exemplified by the preparation of 2,4-dinitrophenylhydrazine. 2,4-Dinitrofluorobenzene is known as Sanger s reagent and is used for the detection of the N-terminal amino acids in peptides. 

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