Fluorescamine. properties

Fluorescamine (VI) is more advantageous because of its specificity. The reaction can be performed with any compound that has a primary amine group or that can be chemically treated to yield a primary amine this is the case with a pesticide such as fenitrothion, that has a nitro group which can be reduced to an amino group. This property of fenitrothion has been used to advantage and a very reliable quantitative method has been developed (36). 

A high degree of sensitivity and selectivity can be obtained with certain biomolecules by the chemical attachment of fluorophores. The most common fluorescent derivatization reagents include fluorescamine, dansyl chloride, pyridoxal, pyridoxal 5-phosphate, dansyl hydrazine, and pyr-idoxamine. Such derivatization procedures can be used to enhance the fluorescence of compounds with low quantum yields as well as impart fluorescent properties to compounds that do not fluoresce naturally. 

Fluorescence detection is often used where no other property of the solute (e.g. UV of RI detection) is convenient and can be either an intrinsic property of the solute itself or a derivatised form of the solute. Solution studies have indicated that the sensitivity of detection can be increased by up to three orders of magnitude over UV. This has increased the popularity of post-column fluorescence detection methods for may compounds, including physiological fluids, catecholamines, and other polyamines. A popular use of fluorescence detection is in peptide chemistry where no convenient intrinsic chro-mophore is present. Derivatising agents such as orthophthalaldehyde and fluorescamine are used extensively in both pre- and post-column systems allowing detection of low picomole quantities (Chapter 11). In addition, detection can be performed using the intrinsic fluorescence of many compounds such as steroids, vitamins, and nucleotides. 

Therefore, it is advisable to measure the first (at around 380 nm) and the second (at around 336-308 nm) Cotton effects and, when possible, also the third (at 285-264 nm). The importance of the second Cotton effect was clearly recognized and demonstrated during an investigation of the chiroptical properties of fluorescamine condensation compounds with a-amino acid esters in situ (Toome and Wegrzynski, 1978). It was found that the... 

On the basis of these results, it is apparent that the derivation of the absolute configuration of a COOH- or NH2-terminal amino acid in a dipeptide from its chiroptical properties is hazardous, and a comparison of spectral data with those of proper model peptides is needed. Thus, a more reliable technique is to react the NH2-terminal (or COOH-terminal) amino acid of a small peptide with a proper chromophoric reagent, as discussed above (via Edman degradation or other techniques). Cyclic peptides have to undergo at least partial acid hydrolysis before they can be reacted with methyl isothiocyanate, fluorescamine, etc. 

Methoxy-2-diphenyl-3(2//)-furanone, which is structurally related to fluorescamine, has the same unique properties. The reaction of MDPF with primary amines proceeds rapidly only above pH 9 (Weigele et al, 1973). Much higher reagent concentrations are often required, e.g., 10 mg/ml, in order to obtain nearly quantitative fluorophor formation (Wideman et al, 1978). This requirement is due to the more rapid hydrolysis of MDPF. In contrast with fluorescamine, MDPF dissolved in methanol is as stable and as reactive as when it is dissolved in acetone. The MDPF fluorophors also have excitation and emission maxima at 390 and 475 nm, respectively. Various peptide fluorophors have been found to be stable between pH 2 and 11, and it has been possible to lyophilize the fluorophors without any decomposition. The fluorescence intensity also varies with solvent composition, but not with pH. 

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