Amino acids fluorescent derivative

The replacement of the dimethylamino group of DNS-C1 by the di-N-butylamino group has led to some improvements in the fluorescence labeling of amino acids. The derivatives are formed in the same manner as the DNS-amino acids (see above), but are less polar and more easily extracted from the reaction mixture [82,83]. The fluorescence characteristics of these derivatives do not differ greatly from the DNS derivatives, but the fluorescence quantum yields are ca. 15% larger in ethyl acetate solution. Bansyl (BNS) derivatives are also suitable for evaluation by MS (see Section 4.4). 

The quantum yields of amine and amino acid Dns derivatives are of the same order of magnitude (Table 2). Phenol and imidazole derivatives, however, have much lower fluorescence efficiencies. In Table 3, the spectral characteristics of the Dns derivatives of an amine, an aminophenol and a phenol are compared. Interestingly, the aminophenol derivative (0,N-bis-Dns-p-tyramine) shows much lower fluorescence efficiency than the analogous amine derivative although it bears two apparently independent fluorophores, and possesses... 

In light of the improved host properties we found for modified CD, we investigated the potential effects of HP- -CD on a wide array of amino acid OPA-derived isoindoles, to see if significant fluorescence enhancements could be observed, and in addition, whether a stabilization of the isoindoles could be obtained (this was not observed in the above-mentioned previous studies) [99]. Our measurements showed modest fluorescence enhancements for some of the derivatives, including lysine and glycine, with values between 2 and 3. However,... 

The mixture of free amino acids is reacted with OPA (Fig. 7-8) and a thiol compound. When an achiral thiol compound is used, a racemic isoindole derivative results. These derivatives from different amino acids can be used to enhance the sensitivity of fluorescence detection. Figure 7-9 shows the separation of 15 amino acids after derivatization with OPA and mercaptothiol the racemic amino acids may be separated on a reversed-phase column. If the thiol compound is unichiral, the amino acid enantiomers may be separated as the resultant diastereomeric isoindole compound in the same system. Figure 7-10 shows the separation of the same set of amino acids after derivatization with the unichiral thiol compound Wisobutyryl-L-cysteine (IBLC). 

The advantages of this method are a short reaction time and the nonfluorescence of the OPA reagent. Therefore, excess reagent must not be removed before the chromatography stage. Using this method, it is possible to measure tryptophan, but not secondary amino acids such as proline or hydroxyproline. Cysteine and cystine can be measured, but because of the low fluorescence of their derivatives, they must be detected using an UV system, or alternatively oxidized to cysteic acid before reaction. 

Reagents which form a derivative that strongly absorbs UV/visible radiation are called chromatags an example is the reagent ninhydrin, commonly used to obtain derivatives of amino acids which show absorption at about 570 nm. Derivatisation for fluorescence detectors is based on the reaction of non-fluorescent reagent molecules (fluorotags) with solutes to form fluorescent... 

Excess of the reagent hydrolyses to a non-fluorescent residue and the reagent itself does not fluoresce. The optimum wavelength of the excitation light is 390 nm and that of the emitted light 475 nm. This regent is, however, less sensitive than Fluoropa and the derivative is unstable consequently, it must be injected onto the column immediately after formation if used in pre-column derivatization. It has been used successfully in the separation and analysis of polyamines (32), catecholamines (33) and amino acids (34). 

Applications of the oxalate-hydrogen peroxide chemiluminescence-based and fluorescence-based assays with NDA/CN derivatives to the analysis of amino acids and peptides are included. The sensitivity of the chemiluminescence and fluorescence methods is compared for several analytes. In general, peroxyoxalate chemiluminescence-based methods are 10 to 100 times more sensitive than their fluorescence-based counterparts. The chief limitation of chemiluminescence is that chemical excitation of the fluorophore apparently depends on its structure and oxidation potential. 

A number of drawbacks in the application of the 0PA/2-ME reagent system include the instability of the fluorescent isoindole derivative (5-7) the use of the noisome reagent 2-mercaptoethanol the low and solvent-dependent fluorescence efficiencies (8,9) of the isoindole and—perhaps the most limiting—the effective restriction of the OPA assay to primary aliphatic amines and to amino acids. 

The stability of the CBI derivative is sufficient for its isolation and complete characterization (11), an accomplishment that is not realized with most OPA adducts. Thus, the CBI derivatives of a number of representative amino acids and amines have been isolated and their fluorescent properties determined as a function of the media and other relevant parameters encountered in reverse-phase HPLC (RP-HPLC). 

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. 

Steroids, e.g. cholesterol, triolein, androsterone sugars, e.g. fructose, glucose, ribose amino acids, pyrimidines, purines, alkaloids 110-150°C, 2-12 h Conversion to fluorescent derivatives by heating. [5]... 

In the presence of mercaptoethanol [3, 12] o-phthalaldehyde reacts with primary amines and amino acids to yield fluorescent isoindole derivatives. 

In the first step tin(Il) chloride in acetic acid solution reduces the aromatic nitro groups to amino groups. The aromatic amines produced then react with fluorescamine in weakly basic medium to yield fluorescent derivatives (cf. reagent monograph Fluorescamine Reagent , Volume la). 

Chio, K.S. and Tappel, A.L. (1969). Synthesis and characterisation of the fluorescent products derived from malonylalde-hyde and amino acids. Biochemistry 8, 2827-2832. 

Wu, S. and Dovichi, N. J., Capillary zone electrophoresis separation and laser-induced fluorescence detection of zeptomole quantities of fluorescein thiohy-dantoin derivatives of amino acids, Talanta, 39, 173, 1992. 

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