**1. Introduction**

Important areas such as environmental sciences, medicine, medicinal chemistry, and cellular biology are dependent on very sensitive analytical techniques to detect and track biomolecules (amino acids, peptides, proteins, antibodies, oligonucleotides, nucleic acids, carbohydrates, and other biological molecules). Many of these techniques often require labelling with reporters or sensors, such as isotope labels [1], radioactive tracers [2], colorimetric biosensors [3], photoswitchable biomaterials [4], photochromic compounds [5, 6], electrochemical sensors [7], or fluorescent labels [8, 9]. The fluorescent labelling presents numerous advantages, when compared to the other techniques, due to the high sensitivity of the fluorescence technique and also due to its non-destructive nature that allows the use of small sample quantities and their fluorescent labels. The fluorescence process occurs in certain molecules called fluorophores or fluorescent dyes, and a fluorescent probe is nothing more than a fluorophore enabled to detect particular components of complex biomolecular assemblies, including live cells, with complex sensitivity and selectivity [10]. The organic fluorophores may form covalent

or non-covalent linkages with the sample to be analyzed, producing the respective bioconjugates (or complexes) that can show fluorescence, from short to very long wavelengths, depending on the label used. The bioconjugation technique depends on two interrelated chemistries: the reactive functionality present on the fluorescent label and the functional groups present on the target biomolecules to be labeled. The knowledge of the basic mechanisms by which the reactive groups couple to target functionalities provides the means to intelligently design the bioconjugation strategy. Choosing the correct fluorescent label that can react with the chemical groups available on target biomolecules forms the basis for successful labelling [11].

In general, the fluorescent label should be small in size and chemically stable, with minimal interference on the structure and biological functions of the unlabeled biomolecules, producing high fluorescence quantum yield bioconjugates.

On the other hand, the labelling reaction should be extremely efficient with high yields, preferably establishing a stable covalent linkage between the fluorescent label and a specific residue in the target biomolecule. The efficiency and selectivity of several fluorescent-labeled biomolecules have been used to study and understand their dynamics, kinetics, and photophysical properties [12–18].

The amine reactive fluorescent labels are the most frequently used to prepare stable bioconjugates to a great number of biological applications since amino groups are either abundant or easily introduced into biomolecules. In contrast, to study some particular protein structures and functions, thiol-reactive reagents are chosen due to the smaller presence of thiol groups, when compared with lysine, in biomolecules [19]. In this context, cysteine is generally the amino acid chosen to label when it is desired to label selectively a protein in vitro, due to its relatively low abundance and high nucleophilicity compared to other amino acid side chains. Specific and noninterfering dual fluorescent labelling in a peptide or protein molecule allows conformational investigations in terms of intramolecular distances [20].

The expeditious development of the fluorescent-labelling techniques allowed to explore and discover several cellular functions. To study, and understand, the activity of signal transduction by visualizing protein binding or folding, the fluorescence correlation spectroscopy (FCS) and the fluorescence resonance energy transfer (FRET) are widely used [21]. Molecular tags that specifically bind to particular membranepermeable dyes [22] allow to study protein dynamics and trafficking by fluorescence recovery after photobleaching (FRAP) as well the protein turnover [23, 24].

The great development of fluorescent-labelling techniques combined with the enormous technological advances in the field of fluorescence microscopy allowed to study, in vivo and in vitro systems, the protein distribution as well as their translocation and their interactions [25]. With specific and efficient fluorescent labelling, the proteins can be visualized in real time for the elucidation of their functions in a complex biological network, which also allows the detection of the protein-protein interactions, fundamental to understand intra- and intercellular communications [26].

Coumarins (benzopyranones or 2H-chromen-2-ones), whether natural products or synthetic ones, have also aroused a growing interest of the scientific community in the last decades due to their very significant pharmacological activity [27–37]. The nature and substitution pattern in the coumarins grant them diversified and exceptional optical properties with high fluorescence quantum yields [38]. Coumarins constitute the major class of fluorescent dyes [39–63], used as fluorescent labels and probes for physiological measurement [43–47], fluorescent whiteners [48], optical brighteners [49, 50], nonlinear optical chromophores [51–53], emission layers in organic light-emitting diodes (OLED) [54–57], and more recently, in caging [58–61], and labelling [62, 63]. Due to strong blue fluorescence of coumarin, it is easy to distinguish its light from green, yellow, and red,

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*Coumarins as Fluorescent Labels of Biomolecules DOI: http://dx.doi.org/10.5772/intechopen.85973*

**2. Chemical labelling**

and the coumarins may play a leading role in this field.

**2.1 Amine reactive fluorescent coumarins**

concentration, and nontoxic reagents.

*2.1.1 Fluorescent coumarin succinimidyl esters*

an enormous advantage in multicolored fluorescence investigation. Developments from the last decade show that the introduction of appropriated substituents into the coumarin ring contributes to structures with improved photophysical and spectroscopic properties [64–66]. The synthesis of new fluorophores, with absorption and emission at long wavelengths, is of extreme importance for biological purposes,

Of all different fluorescent-labelling techniques, the chemical labelling is actually one of the most used as it allows novel types of experiments in biomolecules using a wider range of reactive fluorescent chromophores available. The covalent attachment of the chemical probes with specific amino acid has the advantage of being an irreversible process when compared to the non-covalent binding [67]. The chemical labelling methodology produces very stable bioconjugates, easy to manipulate with high efficiency, in a great number of available fluorophores that can be coupled covalently to the target biomolecule. Chemical labelling methods produce better results in in vitro studies rather than in vivo [18]. The most used methods in chemical labelling, in the biomolecules' native functional groups, under mild aqueous conditions, and using fluorescent coumarins, are discussed below.

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

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

Succinimidyl esters (SE) are proven to be very good reagents for amine modifications. These kinds of reagents are generally stable and show good reactivity and selectivity with aliphatic amines, such as the amine group of lysine side chain. Some of these kinds of reactive dyes are hydrophobic molecules and should be previously dissolved in anhydrous dimethylformamide (DMF) or dimethylsulfoxide (DMSO), but the sulfo-succinimidyl esters are water soluble. The amine labelling

group of a biomolecule and a fluorescent amino-reactive coumarin.

an enormous advantage in multicolored fluorescence investigation. Developments from the last decade show that the introduction of appropriated substituents into the coumarin ring contributes to structures with improved photophysical and spectroscopic properties [64–66]. The synthesis of new fluorophores, with absorption and emission at long wavelengths, is of extreme importance for biological purposes, and the coumarins may play a leading role in this field.
