2.4- Dinitrophenylation 2.4- Dinitrofluorobenzene

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-... 

Treatment of 228 with 2,4-dinitrofluorobenzene (227) provided 230. The assumed intermediate A -benzoyl-A -(2,4-dinitrophenyl)-A -phenylhydrazine (229) underwent the denitrocyclization reaction in its enol form (80JOC3677). Similar reaction is probably involved also in the thermal cyclization of antraquinone 231 leading to 232 (Scheme 35), which took place even during attempts to crystallize the compound (60T107). 

Harada et al. started from preparing inclusion complexes by adding an aqueous solution of PEG bisamine (PEG-BA) to a saturated aqueous solution of a-CD at room temperature and then allowing the complexes formed to react with an excess of 2,4-dinitrofluorobenzene. They examined the product by column chromatography on Sephadex G-50, with DMSO as the solvent, and obtained the elution diagram shown in Fig. 46. They identified the first, second, and third fraction, respectively, as the desired product, i.e., a polyrotaxane, dinitrophenyl derivatives of PEG, and uncomplexed a-CD, by measurement of both optical rotation and UV absorbance at 360 nm for the first, UV absorbance at 360 nm for the second, and optical rotation for the third. 

Dinitrofluorobenzene (DNFB) reacts with phenols and, which is not of interest here, with amino groups. Hydrogen fluoride is eliminated. DNFB does not react w ith carboxylic acids. Alcohols, if they react at all, form dinitrophenyl ethers very slowly. Very weakly dissociated phenolic hydroxyl groups, e.g., in salicylic acid (pK = 13.4), are inert towards DNFB. 

Determination of the iV-terminal acid in the peptide can be made by treatment of the peptide with 2,4-dinitrofluorobenzene, a substance very reactive in nucleophilic displacements with amines but not amides (see Section 14-6B). The product is an N-2,4-dinitrophenyl derivative of the peptide which, after hydrolysis of the amide linkages, produces an iV-2,4-dinitrophenyl-amino acid ... 

Treatment of the peptide (P) with carboxypeptidase released alanine, and with 2,4-dinitrofluorobenzene followed by hydrolysis gave the 2,4-dinitrophenyl derivative of valine. These results establish the TV-terminus as valine and the C-terminus as alanine. The known structural elements now are... 

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. 

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. 

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 Sanger end-group determination is sometimes used as an alternative to the Edman degradation. In the Sanger method, a peptide is allowed to react witi> 2.4-dinitrolluorobenr ae. the peptide is hydrolyzed, and the N-terminal amino add is identified by separation as its iV-2,4-dinitrophenyl derivative. Propose a mechanism to account for the initial reaction between peptide and dinitrofluorobenzene. 

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... 

Numerous methods were proposed for this purpose, but only few withstood the test of time. A reliable procedure is hydrazinolysis (Akabori et al. 1952) which involves the heating of a solution of the peptide in ca. 97 % hydrazine in a sealed tube at 100 °C for 12 hours. The peptide bonds are cleaved by hydrazine and the amino acid constituents thus converted to amino acid hydrazides except the C-terminal residue which is merely hberated in the process. Its separation is faciUtated by dinitrophenylation of the mixture with 2,4-dinitrofluorobenzene. The DNP-amino acid hydrazides as neutral substances are extracted from the aqueous, bicarbonate containing mixture with an organic solvent while the sodium salt of the DNP-derivative of the C-terminal amino acid remains dissolved ... 

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. 

Dinitrofluorobenzene will react with any free amino group in a polypeptide, including the e-amino group of lysine, and this fact complicates Sanger analyses. Only the N-terminal amino acid residue of a peptide will bear the 2,4-dinitrophenyl group at its a-amino group, however. Nevertheless, the Edman method of N-terminal analysis is much more widely used. ... 

Treatment of the heptapeptide with 2,4-dinitrofluorobenzene followed by incomplete hydrolysis gave, among other products valine labeled at the a-amino group, lysine labeled at the s-amino group, and a dipeptide, DNP—VL (DNP = 2,4-dinitrophenyl-). 

It has to be pointed out that, with a few exceptions, the acceleration by polyelectrolytes was associated with decreases and Table IV gives the thermodynamic parameters for the aquation reactions of Co(NH3)5Br induced by Ag". Similar decreases in and AS were found for various reactions the Hg -induced aquation of Co(NH3)5Br , the SpjAr reaction of dinitrochlorobenzoic acid with OH [51], the hydrolysis of 2,4-dinitrophenyl phosphates [reaction (E)] [33], the outer-sphere electron-transfers between Co-complexes [Co(NH3)5N3, Co(NH3)5Br , Co(en)2Cl2 ] and Ru(NH3)6 or [8, 20] [en ethylenediamine], the polyvinyl-imidazole-accelerated solvolysis of p-nitrophenylacetate [52], the coupling reactions of dinitrofluorobenzene with aminoacids [53], dipeptides [53] and aniline [54], the lignin sulfonic acid-accelerated hydrolysis of methyl acetate [55], and the hydrolysis of nitrophenyl esters [37]. The opposite tendency (acceleration caused by increases... 

Dinitrophenyl-thioethers are cleaved under very mild conditions ( thiolysis of the thioether), with mercaptoethanol at pH 8. These derivatives are obtained by reacting the thiol with 2,4-dinitrofluorobenzene in presence of base. 

Dinitrophenylation according to Lockhard and Abraham , 60—160 pg of the peptide are dissolved in 0.1 ml of a 1.6% aqueous solution of trimethyl-ammonium carbonate (pH 9.3) and 0.2 ini of a 6% alcoholic solution of dinitrofluorobenzene is added. The mixture is left in the dark for 2V2 hours. The ethanol is then evaporated off in vacuo, 0.24 ml more of trimethylammonium carbonate... 

Binitrophenylatton Levy and Li [163] dissolve at least 0.2 pmole of the material in 3 ml of 0.05N aqueous KCl at 40° C and adjust the pH to 8 with 0.05 potassium hydroxide using an auto-titrator. About 0.1 ml of dinitrofluorobenzene is added and the solution energetically stirred in the dark at constant pH and temperature. The end of the reaction is indicated when alkali consumption ceases. The solution is then extracted three times with ether and the aqueous phase acidified in order to precipitate the dinitrophenyl derivative. The precipitate is centrifuged off, washed with water, acetone and ether and dried in a desiccator over P2O5. 

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. 

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