Amino acids dinitrophenyl derivatives

Lepri et al. investigated the chromatographic behavior of racemic dinitropyridyl, dinitrophenyl, dinitrobenzoyl, 9-fluorenylmethoxycarbonyl amino acids, tryptophanamides, lactic acid derivatives, and unusual enantiomers such as binaphthols on reversed phase TLC plates developed with aqueous-organic mobile phase containing bovine serum albumin (BSA) as chiral agent. More than 75 racemates has been separated in these experiments with planar chromatography using BSA in mobile phase. BSA showed enantioselectivity towards racemates with structures completely different from amino acids, their derivatives, and similar compounds such as hydroxy acids. 

Appllca.tlons. Various A/-derivatives of amino acids (qv) are resolvable on BSA columns. These /V-amino acid derivatives include ben2enesulfonyl-, phthalimido-, S-dimethylarnino-l-naphthalenesulfonyl- (DANSYL-), 2,4-dinitrophenyl- (DNP-), and 2,3,6-trinitrophenyl- (TNP-) derivatives (30). Amines such as Prilocain, ( )-2-(prop5lamino)-(9-propiono-toluidide, a local anesthetic (Astra Pharm. Co.), are also resolved on BSA. The aromatic amino acids DL-tryptophan, 5-hydroxy-DL-tryptophan, DL-kynurenine [343-65-7] C qH 2N 2 3 3-hydroxy-DT.-kynurenine [484-78-6] and dmgs... 

Sugars and Amino Acids, Using the AT-(2 4-dinitrophenyl) Derivatives. Science [Washington] 113, 354 (1951). 

Shaltiel, S. (1967) Thiolysis of some dinitrophenyl derivatives of amino acids. Biochem. Biophys. Res. Comm. 29, 178. 

In earlier studies the in vitro transition metal-catalyzed oxidation of proteins and the interaction of proteins with free radicals have been studied. In 1983, Levine [1] showed that the oxidative inactivation of enzymes and the oxidative modification of proteins resulted in the formation of protein carbonyl derivatives. These derivatives easily react with dinitrophenyl-hydrazine (DNPH) to form protein hydrazones, which were used for the detection of protein carbonyl content. Using this method and spin-trapping with PBN, it has been demonstrated [2,3] that protein oxidation and inactivation of glutamine synthetase (a key enzyme in the regulation of amino acid metabolism and the brain L-glutamate and y-aminobutyric acid levels) were sharply enhanced during ischemia- and reperfusion-induced injury in gerbil brain. 

Figure 10.13 The reaction of FDNB with compounds containing a free amino group. The reaction at pH 9.5 between FDNB and amino acids or peptides results in the formation of yellow-coloured dinitrophenyl derivatives.

Various procedures are used to analyze protein primary structure. Several protocols are available to label and identify the amino-terminal amino acid residue (Fig. 3-25a). Sanger developed the reagent l-fluoro-2,4-dinitrobenzene (FDNB) for this purpose other reagents used to label the amino-terminal residue, dansyl chloride and dabsyl chloride, yield derivatives that are more easily detectable than the dinitrophenyl derivatives. After the amino-terminal residue is labeled with one of these reagents, the polypeptide is hydrolyzed to its constituent amino acids and the labeled amino acid is identified. Because the hydrolysis stage destroys the polypeptide, this procedure cannot be used to sequence a polypeptide beyond its amino-terminal residue. However, it can help determine the number of chemically distinct polypeptides in a protein, provided each has a different amino-terminal residue. For example, two residues—Phe and Gly—would be labeled if insulin (Fig. 3-24) were subjected to this procedure. 

Dinitrophenyl derivatives of the ct-amino groups of other amino acids could not be found. 

High-performance liquid chromatographic techniques have been applied with success to the analysis of phenylthiohydantoin, 2,4-dinitrophenyl, and dansyl amino acid derivatives. 

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 ... 

These amino-acid derivatives can be separated from the ordinary amino acids resulting from hydrolysis of the peptide because the low basicity of the 2,4-dinitrophenyl-substituted nitrogen (Section 23-7C) greatly reduces the solubility of the compound in acid solution and alters its chromatographic behavior. The main disadvantage to the method is that the entire peptide must be destroyed in order to identify the one A-terminal acid. 

Abbreviations DNP, dinitrophenyl SNP, sulfonyloxynitrophenyl TFA, tri-fluoroacetyl and others as explained in Table V footnote. In the polyamino acid derivatives the subscript is the total number of residues added to the protein molecule. These residues are distributed among the number of amino groups listed in column 3. In column 3, analytical number of modified residues is given except where an amino acid prefix occurs the number then refers to the position in the amino acid sequence. 

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 first ionically bonded phase was presented by Pirkle it contained (/ )-3,5-dinitrobenzoyl phenylglycine [13]. The most commonly used 7t-acid moiety is the 3,5-dinitrophenyl group introduced by the reaction of 3,5-dinitrobenzoyl chloride (DNB-C1) on chiral selectors such as amino acids, amino alcohols, and amines. In addition, pentafluorobenzoyl derivatives have also been reported [14,15]. The 7r-basic phases are complimentary to the re-acidic phases. These CSPs include the presence of phenyl- or alkyl-substituted phenyl groups. Macaudiere et al. [9] designed a CSP containing both re-acidic and re-basic... 

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... 

A reaction worth noting in the context of nitro-group interactions with <-amino functions involves the rapid photolytic breakdown of 2,4-dinitrophenyl derivatives of a-amino acids. The lability of such derivatives in sunlight has long been known to protein chemists who... 

Treatment with FDNB. FDNB (l-fluoro-2,4-dinitrobenzene) reacted with free amino (but not amido or guanidino) groups in proteins to produce dinitrophenyl (DNP) derivatives of amino acids ... 

Dinitrophenyl (DNP) derivatives of amino acids are used to identify terminal amino acids in proteins. They are determined by GC after the conversion of free carboxyl groups into methyl esters [283—285] with the aid of either diazomethane or BF3—methanol. 

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