*2.1.1 Fluorescent coumarin succinimidyl esters*

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

**Figure 1.**

*Schematic diagram of amine labelling techniques using succinimidyl esters (A), 4-sulfotetrafluorophenyl esters (B), sulfonyl chlorides (C), and isothiocyanates (D).*

reaction with succinimidyl esters has a handicap, due to its great pH dependence. Succinimidyl esters react with non-protonated aliphatic amine groups, and the amine acylation reaction must be carried out at pH > 7.5. In the specific case of protein labelling by succinimidyl esters, the reactions require a pH between 7.5 and 8.5. Buffers used in labelling reactions shall not contain nucleophilic compounds because they may react with the labelling reagent to form unstable intermediates that could destroy the reactive dye. Most conjugations are done at room temperature, but either high or low temperature may be required for a particular labelling reaction. Some of the fluorescent coumarin succinimidyl esters contain a seven-atom aminohexanoyl spacer between the fluorophore and the reactive group, providing better solubility and spatial separation between the fluorophore and the target molecule being labeled. This separation potentially reduces the quenching that typically occurs upon conjugation and makes the dye more available for recognition by secondary detection reagents [68]. The most important fluorescent coumarin succinimidyl esters used for labelling biomolecules are shown in **Table 1**, as the corresponding values of maximal excitation (Ex) and emission (Em) wavelengths and their physicochemical features and biological applications [19, 68].
