**2.3 Tyrosine-reactive fluorescent coumarins**

The hydroxyl groups of the amino acids can be labeled with the same reagents used for the lysine residues, but the labelling reaction is carried out in organic solvent, like anhydrous dimethylformamide (DMF) or dimethylsulfoxide (DMSO), which absorbs the formed water molecule avoiding possible hydrolysis reactions. The amino acid

#### **Figure 2.**

*Schematic diagram of thiol-labelling technique using maleimides.*



**Table 5.**

*Fluorescent coumarin maleimides used for biomolecule labelling.*

hydroxyl groups do not allow highly specific labelling reactions due to the existence of several hydroxyl groups in biomolecules (serine, threonine, and tyrosine) [113].

One of the well-known labelling methods is the reaction with diazonium salts resulting in the formation of azo compounds, as 4-trifluoromethylcoumarin-7-diazonium chloride [114]. Although these aryl diazonium ions are promising for the desired application, their storage and delivery are challenging, and they often require in situ generation. The pH range should be between 8 and 10 for the formation of a phenolate anion [115].
