Dinitrophenyl -amino acids

N-(4-nitrophenyl)-amino acids N-(2,4-dinitrophenyl)-amino acids 8) and 3-nitro-2-pyridyl amino acids have been of considerable interest. A generally applicable mechanistic scheme does not exist so far, and except for 3-nitrophenyl acetate in ortho and/or -para position of the phenyl (pyridyl) ring seems to be a prerequisite for efficient decarboxylation. 

The high reactivity of di- and trinitrophenyl fluorides towards nucleophiles has been used for the arylation of various N-nucleophiles. A method was developed for the determination of N-terminal amino acids in peptides. Thus, nucleophilic attack of the amino acid nitrogen at Sanger s reagent (2,4-dinitrophenyl fluoride, 4), hydrolysis and subsequent analysis of the N-(2,4-dinitrophenyl)amino acid allows determination of the amino acid.162,163 Although this method has been replaced by more efficient procedures, it marked a milestone in the elucidation of peptide structures (Nobel Prize 1958). A variety of N-nuclcophilcs (no amino acids) which have been used in the nucleophilic substitution of 2,4-dinitrophcnyl fluoride is listed. 

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 ... 

Fig.4.6. General scheme for the formation of dinitrophenyl-amino acids.

SOLVENTS FOR THE SEPARATION OF DINITROPHENYL-AMINO ACIDS WHICH ARE SOLUBLE IN DIETHYL ETHER... 

The reaction of iV-(2,4-dinitrophenyl)amino acids with base in aqueous dioxane has been shown to give benzimidazole iV-oxides (7). The rate-determining step is likely to be formation of an iV-alkylidene-2-nitrosoaniline intermediate (6), which is followed by rapid cyclization and decarboxylation.19 The loss of carbon dioxide from perbenzoate anions has been investigated by mass spectrometry and electronic structure calculations. The results, including isotopic labelling experiments, support a mechanism involving initial intramolecular nucleophilic attack at either the ortho- or ipso-ring positions. They also indicate that epoxides may be intermediates en route to the phenoxide products.20 There has also been a theoretical study of the formation of trichlorinated dibenzo-/ -dioxins by reaction of 2,4,5-trichlorophenolate ions with 2,4-dichlorophenol.21... 

Very recently, pressurized gradient capillary electrochromatography was used to separate seventeen 2,4-dinitrophenyl amino acid derivatives using a C18 reversed-phase column (3 pm, 130 mm x 75 pm i.d.) using 50 mM sodium acetate, pH 6.4, as... 

Identification of the C-terminal residue is usually done by reaction with hydrazine (100 °C for 12 h). This converts all amino acids other than the C-terminal residue to the H2N-CHR-CONHNH2 derivatives after solvent extraction to remove these hydrazide derivatives, the intact C-terminal amino acid is treated with 2,4-dinitro-phenol and identified chromatographically as the 2,4-dinitrophenyl amino acid, as above. 

In a few cases, alkaline hydrolysis has proved applicable to special problems. Tryptophan is not destroyed in alkali, and analysis of alkaline hydrolyzates forms the basis of one method for quantitative determination of this amino acid (e.g., Dreze, 1960). Despite the fact that tryptophan-containing peptides should be more stable in alkali than acid, partial alkaline hydrolysis has not been employed for identification of this type of peptide. Amino acids often can be regenerated by alkaline hydrolysis from derivatives obtained by the amino-terminal end-group methods. Dinitrophenyl amino acids and phenylthiohydantoin (Fraenkel-Conrat et al., 1955) as well as hydantoin (Stark and Smyth, 1963) derivatives of amino acids can be treated in this manner. 

N-(2,4-Dinitrophenyl)amino acid Unlabeled amino acids (DNP.AA)... 

It has been applied to the separation and enrichment of organic compounds such as indole auxins, bromoacetyl thyroxine and its analogs, dinitrophenyl amino acids. 

Park et al. reported a method to modify zirconia by adsorption with BSA and, subsequently, crosslinked by glutaraldehyde. The BSA-zirconia showed good enantios-electivity for some enantiomers and could be used for RPLC separation in mobile phases of alkaline pH. They also developed a carboxymethyl-p-cyclodextiin-coated zirconia stationary phase for the separation of racemic 2,4-dinitrophenyl amino acids. 

Park, S.Y. Park, J.K. Park, J.H. McNeff, C.V. Carr, P.W. Separation of racemic 2,4-dinitrophenyl amino acids on carboxymethyl-beta-cyclodextrin coated zirconia in RPLC. Microchem. J. 2001, 70, 179-185. 

In the early prototype instrument [1], the columns were fabricated from relatively large-bore PTFE tubing of 4.6 mm inner diameter (i.d.) with PTFE disk inserts having 0.8-mm-diameter holes. These disks were spaced in 3-mm intervals to form 47 locules in each unit. A number of column units were connected in series to provide 5000 locules with a total capacity of 100 mL. The capability of the system was demonstrated with the separation of DNP (dinitrophenyl)-amino acids using a two-phase solvent system composed of chloroform-acetic acid-0.IM HCl at a 2 2 1 volume ratio. In this system, nine DNP-amino acids were resolved within 70 h at about 3000 theoretical plates. 

J. L. Sandlin and Y. Ito, Gram quantity separation of DNP (dinitrophenyl) amino acids with multi-layer coil countercurrent chromatography (CCC), /. Liquid Chromatogr. 7(2) 323-340 (1984). 

B23. Brenner, M., Niederwieser, A., and Pataki, G., Thin layer chromatography of amino acid derivatives using Kieseigel G. N-(2,4-dinitrophenyl)-amino acid and 3-phenyl-2-thiohydantoin). Experientia 17, 145-153 (1961). 

W8. Wang, K. T., and Wang, I. S. Y., Polyamide layer chromatography. Further studies on dinitrophenyl amino acids. J. Chromatogr. 24, 460-463 (1966). 

Li, S and WC Purdy (1991). Liquid chromatographic separation of the enantiomers of dinitrophenyl amino acids using a j8-cyclodextrin-bonded stationary phase. Journal of Chromatography, 543,105-112. 

S. Li and W.C. Purdy, Liquid Chromatographic Separation of the Enantiomers of Dinitrophenyl Amino Acids Using a P-Cyclodextrin-bonded Stationary Phase, /. Chromatogr., 543(1991)105. 

Frey JR, de Week AL, Geleick H, Lergier W (1969) The immunogenicity of dinitrophenyl amino acids. J Exp Med 130 1123-1143... 

Figure 7 Detection of amino acids in plasma as their dinitrophenyl amino acid methyl esters by negative ion chemical ionization mass spectrometry. (Reproduced with permission from the Ph.D. Dissertation of Todd Williams, University of Maine, December 1987 Figure 11, p. 99.)...

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