*2.1.4 Fluorescent coumarin isothiocyanates*

Isothiocyanates form thioureas upon reaction with amines, but some thiourea products are much less stable than the conjugates that are prepared from the corresponding succinimidyl esters. Most part of isothiocyanate-reactive dyes are hydrophobic molecules and should be dissolved either in anhydrous dimethylformamide (DMF) or in dimethylsulfoxide (DMSO), and their reactions may require a pH 9.0–10.0 for optimal conjugations. As in the previous cases, the buffers used shall not contain nucleophilic compounds. The isothiocyanate conjugations are done at room temperature, but either high or low temperature may be required for a particular labelling reaction [19, 102]. The unique fluorescent coumarin isothiocyanate used for labelling biomolecules is shown in **Table 4**, but new isothiocyanate coumarins have been synthesized, with high potential as fluorescent probes [103, 104].

## **2.2 Thiol-reactive fluorescent coumarins**

Cysteine is, in comparison with lysine, a rare amino acid present in biomolecules, and, for this reason, thiol-reactive reagents are used to label selectively a biomolecule at a defined site, probing their function, interaction, and biological structure. A great number of thiol-reactive dyes have been developed to analyze the proteins' topography in biological membranes, to measure the distances within (or between) proteins, and to observe and understand the changes in protein conformation using environmental sensitive probes.

Maleimides and iodoacetamides are the principal types of thiol-reactive coumarin dyes reported in the literature. Despite many similarities in their reactivity and selectivity toward thiol-reactive moieties, maleimides have a great advantage in relation to iodoacetamides, due to their high stability, solubility in simple solvent mixtures, and their high reactivity in the neutral pH range. Air oxidation of thiol compounds (to


**Table 2.**

*Fluorescent coumarin 4-sulfotetrafluorophenyl (STP) ester used for biomolecule labelling.*


#### **Table 3.**

*Fluorescent coumarin sulfonyl chlorides used for biomolecule labelling.*


#### **Table 4.**

*Fluorescent coumarin isothiocyanate used for biomolecule labelling.*

disulfides) is a major competing reaction for the iodoacetamide modifications of thiol compounds [18, 19, 105]. Due to the disinterest on the development of new coumarin iodoacetamides, for the above reasons, only the fluorescent coumarin maleimides will be focused in this section. **Figure 2** represents, in a general schematic diagram, the thiol-labelling reaction with fluorescent coumarin maleimides.

**167**

**Figure 2.**

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

their physicochemical features and biological applications.

**2.3 Tyrosine-reactive fluorescent coumarins**

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

**Coumarin Ex/Em** 

Triethylammonium 7-amino-3-(2-((5-(2,5 dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl) amino)-2-oxoethyl)-4-methylcoumarin-6 sulfonate (Alexa Fluor™ 350 C5 Maleimide)

N-(5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) pentyl)-6,8-difluoro-7-hydroxycoumarin-3 carboxamide (Pacific Blue™ C5-Maleimide)

Maleimides readily react with thiol moieties of biomolecules to form thioether conjugates even under neutral conditions. The thioether bond formed is quite stable and is known to be responsible for the light produced, especially in the solution. Maleimides require conjugation conditions less rigorous than those of iodoacetamides and do not react with histidine and methionine under physiological conditions. Most labelling reactions can be done at room temperature at neutral pH. However, either elevated or reduced pH or temperature may be required for a particular labelling reaction [18, 19, 68]. In **Table 5**, the most important fluorescent coumarin maleimides used for labelling biomolecules are presented, as the corresponding values of maximal excitation (Ex) and emission (Em) wavelengths and

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

**(nm)**

**Physicochemical features and biological applications**

moderate photostability, water soluble and pH insensitive from pH 4 to pH 10, used for stable signal generation in imaging and flow cytometry

thiol-selective modification, quantitation, and analysis and usually requires a higher pH than reaction of maleimides with thiols. Does not react with methionine, histidine, or tyrosine

345/444 Blue-fluorescent dye, with

410/455 Excellent reagent for

**Reference**

[68, 80, 81, 106–108]

[68, 80, 109]

*2.2.1 Fluorescent coumarin maleimides*

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