Amino dansylation

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... 

Tnethanolaimne dansyl amino acids, enhancement dansyl amides... 

Triton X-100 dansyl amides and amino acids 30 to 110-fold spray solution, 20% in chloroform [265, 290]... 

Sodiiun dodecylsulfate, cetyltrimethylam-monium chloride, sodium cholate, -cyclodextrin dansylated amino acids and polycyclic aromatic hydrocarbons > 45-fold 1% in water the greatest enhancement of fluorescence is that of sodium cholate on pyrene [263]... 

These workers used binary solvent systems over a range of mole fractions to determine, for each solute, the constants a and b of equation (8.2). For methyl and phenacyl esters, TLC was used, while overpressured layer chromatography (OPLC) was used for dansyl amino acids. Nurok and co-workers (11) also evaluated how the quality of a simulated separation varies with changing solvent strength by using the inverse distance function (IDF) or planar response function (PRF), as follows ... 

A CSP with a smaller (i-cyclodextrin moiety (seven glucose units) immobilized on silica gel (ChiraDex ) is able to separate the dansyl-derivatives [5-(dimethy-lamino)-naphthalin-l-sulfonylchloride] of amino acids [26]. 

Righetti and co-workers [11] were one of the first to demonstrate the utility of classical isoelectric focusing for the chiral separation of small molecules in a slab gel configuration. In their system, dansylated amino acids were resolved enan-tiomerically through complexation with (i-cyclodextrin. Preferential complexation between the cyclodextrin and the derivatized amino acid induced as much as a 0.1 pH unit difference in the pK s of the dansyl group. 

Perhaps most encouraging in these discoveries was the observation that NDA/CN worked equally well for derivatization of dipeptides and higher homologues of the primary amino acid series. Again, a stable, fluorescent, isolatable derivative was obtained. One of the most important initial findings was the high fluorescence efficiency of the CBI adduct (12). Tables 1 and 2 list the efficiencies for a representative group of mono-, di-, and tripeptides and a limited comparison of the CBI efficiencies with the more traditional OPA (8) and dansyl (9) derivatives, respectively. 

Table VIII. Quenching Effects by Oxalates and Product Phenols on CBI- and Dansyl-Labeled Amino Acids...

Reports on the use of fluorescent derivatives abound (5). Some reagents have become widely used. The dansyl group is probably the most thoroughly studied. Dansyl chloride has been widely used as a fluorescent derivatizing reagent for HPLC (6,7). It reacts readily with primary and secondary amino groups (7) and with phenols (8), but forms derivatives of alcohols very slowly (9). The lower detection limit for dansyl derivatives of aliphatic amines is in the range of 300 femtomoles per injection. 

Dansylamides 104,106 — 108 Dansylamino acids 73,107,108 Dansylation 72 ff amines (prim, and sec.) 73 amino acids 73 -, phenols 73 prechromatographic 72 ff with dansyl semicadaveride 73, 53... 

Woods, R. and Wang, Kung-Tsung "Separation of Dansyl Amino Acids by Polyamide Layer Chromatography". Blochlm. Blophys. Acta., (1967), 133, 369. 

Mixing the additive in the eluent used as a mobile phase can also modify the chromatographic system (dynamic modification), but the use of modified adsorbents has led to an improvement of resolution. Example works include that by Armstrong and Zhou [11], who used a macrocyclic antibiotic as the chiral selector for enantiomeric separations of acids, racemic drugs, and dansyl amino acid on biphenyl-bonded silica. 

Cyclodextrins as chemically banded layers [102] or mobile phase additives [103-105] have been used successfully to resolve a wide variety of alkaloids, steroids and dansyl- and naphthylamide-amino acid derivatives. The low solubility in aqueous solution and f high cost of cyclodextrins restricted the use of these additives > initially. These limitations were overcome by the availability of ... 

Dansyl chloride is the most widely used of the derivatizing reagents. It forms derivatives with primary and secondary amines readily, less rapidly with phenols and imidazoles, and very slowly with alcohols. The reaction medium is usually an aqueous-organic sixture (e.g., 1 1 acetone-water) adjusted to a pH of 9.5-10. Dansyl chloride has two major application areas. It is used to determine small amounts of amines, amino acids and phenols, as... 

Figure 14 Chiral separation of dansyl amino acids by chiral polyacrylamide gel electrophoresis. Peak identification 1 = dansyl-L-glutamic acid, 2 = dansyl-D-glutamic acid, 3 = dansyl-L-serine, 4 = dansyl-D-serine, 5 = dansyl-L-leucine, 6 = dansyl-D-leu-cine. (From Guttman, A., Paulus, A., Cohen, A. S., Grinberg, N. and Karger, B. L., /. Chromatogr., 448, 41, 1988. With permission.)...

For the sulfonation step, amino acids were added as their sodium salts. The reactions were carried out in cold aqueous solution, in which the sulfonamides were immediately precipitated. By the same method dansylations of amino acids could be accomplished with N-( 1 -dimethylaminonaphthalene-5-sulfonyl)-Af -methylimidazolium chloride ... 

The Af-dansylated amino acid (e.g., glycine, leucine, proline) exhibits a yellow fluorescence. The sensitivity of detection for amino acids by this method is about 10 9 mol of amino acid. The advantage of this prodecure in comparison with that using dansyl chloride is the fact that it can be carried out in homogenous aqueous solution without addition of a cosolvent. 191... 

Capillary zone electrophoresis, an up-to-date high resolution separation method useful for proteins and peptides, has been shown to be a useful method for determining electrophoretic mobilities and diffusion coefficients of proteins [3], Diffusion coefficients can be measured from peak widths of analyte bands. The validity of the method was demonstrated by measuring the diffusion coefficients for dansylated amino acids and myoglobin. 

The amine-reactive 5-(dimethylamino)naphthalene-l-sulfonyl (dansyl) chloride 28 [80] and related fluorophores [81, 82], as well as the 5-((2 aminoethyl)amino) naphthalene-1-sulfonic acid (EDANS) 29, are included in the naphthalene fluorophore family. Derivatives of the latter, such as compound 30, exhibit a Lm.ix/ Lem 336/520 nm, molar absorptivity (e) of 6.1 x 103 M-1 cm-1, and a fluorescent quantum yield of 0.27 in water [83], The use of EDANS is particularly interesting in FRET experiments [84, 85]. Furthermore, 4-amino-3,6-disulfonylnaphthalimides (e.g., Lucifer yellow 31), associated to a longer absorption (Lmax 428 nm) [86] are suitable polar tracers [87]. 

Amino acid analysers based on ion exchange resins are available commercially. These achieve good separations of amino acid mixtures. Fluorescent derivatives of separated amino acids constitute a very sensitive means of detecting these compounds in seawater [256,258]. Fluorescent derivatives that have been studied include o-phthalaldehyde [259], dansyl [260], fluo-rescamine [261], and ninhydrin [261]. 

The amino acid analyser using fluorescamine as the detecting reagent has been used to measure 250 picomoles of individual amino acids routinely [262], and dansyl derivatives have been detected fluorometrically at the 10 15 M level [260]. Where the amounts of amino acid are high enough, the fluorescamine method, with no concentration step, can be recommended for its simplicity. At lower concentrations, the dansyl method, with an extraction of the fluorescent derivatives into a non-polar solvent, should be more sensitive and less subject to interferences. For proteins and peptides, the fluorescamine method seems to be the most sensitive available method. 

Dansylated amino acids Synthetic 0.1 jtmol L"1 (glutamine)... 

Enhancements in the sensitivity with which amino acids containing a primary amine group can be determined have been achieved by derivatization. Chen and Sato [37] reported derivatization with divinyl-sulfone-reduced limits of detection by several orders of magnitude, while Lee and Nieman [38] reported derivatization with dansyl-chloride-reduced limits of detection by a factor of three. 

Amino acids TCP0-H202-Dansyl chloride Borate buffer (pH 8.9)... 

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