Dinitrophenyl-derivatized amino acids

Benzyloxycarbonyl, N-(3,5-dinitrobenzyloxy carbonyl), 9-fluorenylmethoxycarbonyl, benzoyl, acetyl and N-(2,4-dinitrophenyl) derivatized amino acids and profens WAX (weak anion-exchange) type CSP tert. -butylcarbamoylquinine as chiral selector on Hypersil silica gel), 3 pm Acetonitrile-methanol (80 20)+400 mM acetic acid+4 mM triethylamine 335 mm x 100 pm i.d. 250 mm effective length, chiral separation... 

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. 

Wang and Porter [92] resolved the enantiomers of oxazepam, lorazepam, and temazepam using /1-cyclodextrin as the CMPA by CEC. The authors varied separation parameters such as voltage and mobile phase. Wei et al. [93] resolved the enantiomers of phenylephrine and synephrine by varying the concentration of /1-cyclodextrin (CMPA), pH, electrolyte concentration, and temperature. Lelievre et al. [99] separated the enantiomers of chlorthalidone using hydroxypropyl /1-cyclodextrin as the CMPA. Lammerhofer and Lindner [90] resolved the enantiomers of N-derivatized amino acids (e.g., 3,5-dinitrobenzoyl, 3,5-dinitro-benzyloxycarbonyl, 2,4-dinitrophenyl, and 9-fluorenylmethoxycarbonyl amino... 

The common methods for determining N-terminal residue employ l-fluoro-2,4-dinitrobenzene (Sanger s reagent) and 1-dimethylamino phthalene-5-sulfonyl (dansyl) chloride. The derivatized peptide is hydrolyzed, and the labeled N-terminal residue is identified by its yellow color as dinitrophenyl (DNP) amino acid or by fluorescence as dansyl... 

When FDAA was replaced by l-FDLA (l-fluoro-2,4-dinitrophenyl-5-L-leucine amide), an enhanced sensitivity, hydrophobicity, and thermal stability (in comparison to l-FDAA) were observed. Electrospray ionization and frit-fast atom bombardment (Frit-EAB) were applied as the interface [25,26]. It also required changing the original mobile phase of acetonitrile-phosphate buffer to acetonitrile-TEA as the former was not volatile. Derivatized amino acids with FDLA showed almost the same retention behavior as that with FDAA. 

Bruckner, H. and Keller-Hoehl, C., HPLC separation of DL-amino acids derivatized with Af -(5-fluoro-2,4-dinitrophenyl)-L-amino acid amides, Chroma-tographia, 30, 621, 1990. 

A simple and rapid method of separating optical isomers of amino acids on a reversed-phase plate, without using impregnated plates or a chiral mobile phase, was described by Nagata et al. [27]. Amino acids were derivatized with /-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA or Marfey s reagent). Each FDAA amino acid can be separated from the others by two-dimensional elution. Separation of L- and D-serine was achieved with 30% of acetonitrile solvent. The enantiomers of threonine, proline, and alanine were separated with 35% of acetonitrile solvent and those of methionine, valine, phenylalanine, and leucine with 40% of acetonitrile solvent. The spots were scraped off the plate after the... 

Amim acids. Some aromatic amino acids such as kynurenin can be resolved on the BSA CSP without derivatization (92). However, most amino acids require precolumn derivatization of the amine moiety. The N-deriva-tives that have been used include acetyl, benzenesulphonyl, phthalimido, DANSYL, 2,4-dinitrophenyl, and 2,4,6-trinitrophenyl (85,92,107,108). 

The identification of N-terminal amino acids in peptides and proteins is of considerable practical importance because it constitutes an essential step in the process of sequential analysis of peptide structures. Many N-amino acid derivatives have been proposed for this purpose and the ones most commonly studied by TLC are 2,4-dinitrophenyl (DNP)-and 5-dimethylaminonaphthalene-l-sulfonyl (dansyl, Dns)-amino acids, and 3-phenyl-2-thiohydantoins (PTH-amino acids). Recently, 4-(dimethylamino)azobenzaie-4/-isothio-cyanate (DABITC) and phenyl-isothiocyanate (PITC) have also been investigated as derivatizing agents of amino acids. 

As shown in Table 1.5, betaines are widespread in both the animal and plant kingdoms. Derivatization of amino acids by reaction with l-fluoro-2,4-dinitrobenzene (FDNB) yields N-2,4-dinitrophenyl amino acids (DNP-amino acids), which are yellow compounds and crystallize readily. The reaction is important for labeling N-terminal amino acid residues and free 8-amino groups present in peptides and proteins the DNP-amino acids are stable under conditions of acidic hydrolysis (cf. Reaction 1.33). 

Derivatization of an amino acid with FDAA produces a diastereomer referred to as 2,4-dinitrophenyl-5-L-alanine amide amino acid or simply DNPA-amino acid. Aqueous solutions (50 vaM) of amino acids (D- and L-isomers) are used as starting materials for synthesis. Solution of each of the amino acid (50 lA, 2.5 /tmol) is placed in separate 2 ml plastic tubes. To each is added 100 ii of 1% acetone solution of FDAA (1 mg, 3.6 /zmol), the molar ratio of FDAA to amino acid is 1.4 1, followed by NaHCOs (1 M, 20 ii, 20 umol). The contents are mixed and heated over a hot plate at 30-40°C for 1 h with frequent mixing. After cooling to room temperature, HCl (2 Af, 10 /u-1, 20 /umol) is added to each reaction mixture. After mixing, the contents are dried in a vacuum desiccator over NaOH pallets. Each residue is then dissolved in DMSO (0.5 ml). A1 1 dilution of these is made (2.5 mM) and 5 /ul sample of each is pooled and analyzed, for example, injected for HPLC. 

Racemic and nonracemic amino acids were derivatized with chiral reagent l-fluoro-2,4-dinitrophenyl-5-L-phenylalanine amide (FDPA) according to Marfey s method [11]. Twelve proteinaceous a-amino acids were derivatized with FDPA, a chiral variant of MR. The resulting diastereomers were separated... 

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