2.4- Dinitrofluorobenzene derivatives with

The DNP derivative, prepared from 2,4-dinitrofluorobenzene is released from the nitrogen with an anionic ion exchange resin." When used for histidine protection, the DNP group has been observed to migrate to nearby [ysine residues during Fmoc cleavage. ... 

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. 

Fluorobenzene-type compounds have been used as functional groups in homobifunctional crosslinking agents (Chapter 4, Section 4). Their reaction with amines involves nucleophilic displacement of the fluorine atom with the amine derivative, creating a substituted aryl amine bond (Reaction 9). Detection reagents incorporating reactive aryl chemistry include 2,4-dinitrofluorobenzene and trinitrobenzenesulfonate (Eisen et al., 1953). These compounds form... 

In contrast, the 0-aryl derivative of Af-methyl-6-(hydroxyamino)-l,3-dimethylpyrimi-dine-2,4(1/7,3i7)-dione 44, prepared by reaction with 2,4-dinitrofluorobenzene (DNFB), yields the corresponding barbiturate 45 by a spontaneous [3,3]-sigmatropic rearrangement (equation 14). 

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. 

Deacylation of deferriferrichrysin occurs in 20 min. in IN HCi at 100°. Trihydrochloride so obtained may be reacylated and, after removal of O-acetyl in methanolic ammonia and re-insertion of iron, ferrichrysin is obtained. The trihydrochloride hexapeptide may be reduced with platinum oxide and H2. Treatment of the latter compound with 2,4-dinitrofluorobenzene followed by hydrolysis gives N8-DNP ornithine. Hydrolysis of ferrichrysin in 57% HI gives serine, glycine, alanine, ornithine and ammonia in the molar ratios 0.24 1.00 1.50 2.91 0.09. Hydrolysis of deferriferrichrysin in 6N HCI, reduction of the hydrolysate with H2 and platinum oxide and addition of excess fluorodinitrobenzene yields crystalline derivatives of glycine, L-serine and L-ornithine. 

Bcekawa et al. [285] successfully separated thirteen amino acids on a 4-m column coated with 1.5% of SE-30 with temperature programming (170—230°C). Only Thr and Ser were not separated on this column they can, however, be separated on 1.5% of XE-61. Prior to the analysis, DNP derivatives of these amino acids were silylated by using HMDS-TMCS in pyridine (2 1 5) at room temperature for 20 min. The method was applied to the analysis of amino acids in serum, as follows. To 0.5 ml of serum, 1.5 ml of ethanol was added in order to deproteinate it. The mixture was centrifuged, the liquid layer drained off and the residue washed with 0.5 ml of water and 1.5 ml of ethanol. Combined extracts were adjusted to pH 3 and washed twice with 5 ml of chloroform. To the aqueous layer, 2 ml of phosphate buffer (pH 8.8), 1 ml of 5% dinitrofluorobenzene in ethanol and 3 ml of ethanol were added and the mixture was shaken at 40°C for 2 h, then extracted three times with 30 ml of diethyl ether. The ethereal layer was washed with the buffer solution and the washings were added to the aqueous layer, which was then acidified with 15 ml of 10% HC1 and extracted with three 20-ml portions of ethyl acetate. 

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. 

Meisenheimer complex. On the addition of a protic acid, the complex may fragment with the elimination of either the ethoxide or methoxide anions. If the ethoxide anion is eliminated this results in an overall substitution of an ethoxy group for methoxy group at the ipso position of the aromatic ring. Another example would involve the use of 2,4-dinitrofluorobenzene, which is used to form the derivative of the amino terminal of polypeptides. 

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 with 2,4-dinitrofluorobenr-ene. the peptide is hydrolyzed, and the N-terminal amino add is identified by separation as its iV-2,4-din ilrophenyl derivative. Propose a mechanism to account for the initial reaction between peptide and dinitrofluorobenzene. 

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