**2.1 Amine reactive fluorescent coumarins**

Presently, amine reactive fluorescent coumarins are widely used to label biomolecules, as peptides, proteins, oligonucleotides, and nucleic acids, among others. The fluorescent bioconjugates obtained are very useful in fluorescence in situ hybridization (FISH), receptor labelling immunochemistry, cell tracing, and fluorescent analog cytochemistry studies. Almost all of the techniques used in these tests implicate a robust fluorescent conjugate able to support rigorous incubation, hybridization, and washing steps, which is provided by the stability of the covalent bond between the amine reactive dye and biomolecule. Chemically, the amine labelling reaction proceeds usually through acylation pathway producing stable amide (or thiourea) bonds. The "ideal" reactions are those which require the same conditions as proteins, like functional group tolerance, compatibility, selectivity, water as solvent (or pH ~ 7), room temperature, high reaction rates, low reactant concentration, and nontoxic reagents.

A number of fluorescent amino-reactive coumarins have been developed to label various biomolecules, and the resultant conjugates are widely used in biological applications. Four major classes of amine-reactive fluorescent reagents are currently used to label biomolecules: succinimidyl esters (SE), 4-sulfotetrafluorophenyl (STP) esters, sulfonyl chlorides, and isothiocyanates [68]. **Figure 1** represents, in a general schematic diagram, the referred labelling reactions, between an amine group of a biomolecule and a fluorescent amino-reactive coumarin.
