Amino acids dansyl derivatives

Carboxylic acids, dansyl derivates of amino acids Aminopropyl derivate of the ergot alkaloid (+ )-terguride Methanol—acetate, 205 pH range 3.8—5.5... 

Dansylation is often used for the determination of free and N-terminal amino acids. Dansyl chloride (5-dimethylaminonaphthalene-l-sulfonyl chloride, DNS-C1) reacts with the amino substituent of amino acids to form highly fluorescent derivatives [75,76]. The method is particularly useful for the analysis of trace components due to the high sensitivity of the products. The derivatives are usually separated by TLC on various types of layers. Separations of DNS-amino acids by flat-bed techniques have been reviewed [77]. Separations by column chromatography have been examined on polyamide [78] and Amberlite IRC-SO [79]. Although many variations of the dansylation reaction with amino acids have been reported, the one described below [77] appears to be the most common. 

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... 

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]. 

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. 

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... 

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. 

Dansyl chloride (dimethylaminonaphthalene-5-sulphonyl chloride) will react with free amino groups in alkaline solution (pH 9.5-10.5) to form strongly fluorescent derivatives (Figure 10.14). This method can also be used in combination with chromatographic procedures for amino acid identification in a similar manner to the FDNB reagent but shows an approximately 100-fold increase in sensitivity. This makes it applicable to less than 1 nmol of material and more amenable for use with very small amounts of amino acids liberated after hydrolysis of peptides. The dansyl amino acids are also very resistant to hydrolysis and they can be located easily after chromatographic separation by viewing under an ultraviolet lamp see Procedure 10.1. 

Figure 10.14 The reaction of dansyl chloride with compounds containing a free amino group. At an alkaline pH, the reaction results in the formation of fluorescent derivatives of free amino acids and the N-tenninal amino acid residue of peptides.

Procedure 10.1 Preparation of dansyl derivatives of amino acids for sub e quest separation by thin layer chromatography... 

The formation of DNP or dansyl amino acid derivatives followed by chromatography or electrophoresis is a useful technique in certain circumstances. The preparation of DNP derivatives may be indicated when the sample for analysis contains a variety of other substances, removal of which would be complicated, leading possibly to considerable analytical errors. However, the derivative formation and extraction is time consuming and itself can introduce inaccuracies into the analysis and should be used only when it offers an advantage over the separation of untreated amino acids. 

The use of dansyl derivatives is not recommended for routine analysis of free amino acids but is very suitable in the identification of an unknown amino acid that has been selectively extracted from the original sample and is present in small quantities. Both kinds of derivative can be easily separated by chromatography or electrophesis and no locating reagent is required for either because the DNP derivatives are themselves yellow in colour and the dansyl derivatives are fluorescent. 

A comparative study was made of the RP-HPLC analysis of free amino acids in physiological concentrations in biological fluids, with pre-column derivatization by one of the four major reagents o-phthalaldehyde (73) in the presence of 2-mercaptoethanol, 9-fluorenylmethyl chloroformate (90), dansyl chloride (92) and phenyl isothiocyanate (97, R = Ph) (these reagents are discussed separately below). Duration of the analysis was 13-40 min. Sensitivity with the latter reagent was inferior to the other three however, its use is convenient in clinical analysis, where sample availability is rarely a problem. The derivatives of 73 were unstable and required automatized derivatization lines. Only 92 allowed reliable quantation of cystine. All four HPLC methods compared favorably with the conventional ion-exchange amino acid analysis188. 

Suitably protected amino acids (112) (cysteine, serine, and lysine) have been added via the side-chain heteroatom (S, O, and N, respectively) to conjugated alkynones, alkynoic ester and alkynoic amide (113). The expected heterosubstituted vinyl product (114) was formed in each case, mainly as the ii-isomer. In an accompanying paper, this type of addition was applied to the derivatives of fluorescein, 7-hydroxycoumarin, Sudan 1, and dansyl chloride with linker arms containing a conjugated terminal alkyne. 

These techniques have been used successfully in the micro-Zdman degradation of the enzyme mouse sarcoma dihydrofolate reductase to obtain the amino acid sequence of the first 25 amino acids 455). Similarly, RPC has been used in coqjunction with the automated Edman technique for sequencing 32 residues of myoglobin 456). Methionine and its oxidation products, methionine sulfoxide and methionine sulfone, in methionine fortified foods have been analyzed as their dansyl derivatives 457). Lysine has been determined as its dansyl derivative in a study in which the stability of lysine in fortified wheat flour was evaluated (458). 

However, the CMPA concept for CLEC. introduced by Lindner and co-workers139 for the resolution of dansyl amino acids using a chiral triamine and Zn(II) as transient metal ions parallel to the work of Gil-Av146, allowed the further development and successful use of amino acids and their derivatives together with Cu(II) as CMPAs in reverse-phase systems for the direct resolution of, for example, a-amino acids146. 

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. 

Another useful reaction of amino side chains is that with dansyl chloride (Eq. 3-29). Many lysine derivatives can be determined quantitatively by amino acid analysis.280... 

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

Even more versatile than the dansyl method is the Edman method (Figure E2.4). The NH2-terminal amino acid is removed as its phenylthiohydan-toin (PTH) derivative under anhydrous acid conditions, while all other amide bonds in the peptide remain intact. The derivatized amino acid is then extracted from the reaction mixture and identified by paper, thin-layer, gas, or high-performance liquid chromatography. The intact peptide (minus the original NH2-terminal amino acid) may be isolated and recycled by reaction with phenylisothiocyanate. Since this method is nondestructive to the remaining peptide (aqueous acid hydrolysis is not required) and results in good yield, it can be used for stepwise sequential analysis of peptides. The method is now automated. 

Period 2 Part B—Work up the dansyl hydrolysate and spot on the TLC plate with standard dansyl amino acids. Part A. 1—Work up peptide hydrolysate and prepare FMOC derivatives of amino acids for analysis by HPLC or CE. Part A.2-If applicable, develop paper chromatogram in solvent system. 

Draw a picture in your notebook of the polyamide thin-layer plate exposed under UV light after each of the two or three solvent developments. These pictures should look similar to Figure E2.7. Three fluorescent areas should be evident after solvent 2 however, better separation is achieved by solvent 3. A blue fluorescent area at the bottom of the plate is dansic acid (DNS-OH), which is a hydrolysis product of dansyl chloride. A blue-green fluorescent spot about one-third to one-half up the plate is dansyl amide (DNS-NH2), which is produced by reaction of dansyl chloride with ammonia. A third spot, which usually fluoresces green, is the dansyl derivative of the NH2-terminus amino acid. Note the positions of the standard dansyl amino acids and compare with the unknown. What is the identity of the NH2-terminal amino acid Are any other fluorescent spots evident on the plate Using polarity or nonpolarity, try to explain the position of each molecule on the thin-layer plate. 

Dansyl chloride and phenylisothiocyanate (PITC) are the derivatizating agents most used in UV detection. Dansyl chloride reacts with the primary and secondary amino groups of peptides in a basic medium (pH 9.5), forming dansylated derivatives that are very stable to hydrolysis but are photosensitive. The derivatives are detectable in UV at 254 nm and by fluorescence. Dansyl sulfonic acid is formed as a by-product of the reaction, and excess reagent reacts with the dansyl derivatives to form dansyl amide the conditions of derivatization must therefore be optimized in order to avoid the formation of such by-products to the extent possible. The conditions of the reaction with dansyl chloride and of the separation of the derivatives thus formed have been thoroughly studied (83,84). Martin et al. (85) carried out derivatization using an excess concentration of dansyl chloride of 5 -10-fold in a basic medium (lithium carbonate, pH 9.5) in darkness for 1 h. 

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