*2.1.2 Fluorescent coumarin 4-sulfotetrafluorophenyl (STP) esters*

Some succinimidyl esters may not be compatible with a specific application due to their insolubility in aqueous solution. To overcome these limitations, the 4-sulfotetrafluorophenyl (STP) ester can be used. These sulfonated esters have higher water solubility than simple succinimidyl esters and sometimes eliminate the need for organic solvents in the conjugation reaction, which is a great advantage to maintain the native characteristics of biomolecules. They are, however, more polar than succinimidyl esters, which makes them less likely to react with buried amines in proteins or to penetrate cell membranes [68, 94]. **Table 2** presents the single fluorescent coumarin 4-sulfotetrafluorophenyl (STP) ester used for labelling biomolecules, as the corresponding values of maximal excitation (Ex) and emission (Em) wavelengths and their physicochemical features and biological applications [95, 96].

#### *2.1.3 Fluorescent coumarin sulfonyl chlorides*

Sulfonyl chlorides (SC) are highly reactive and are unstable in water, especially at high pH required for reaction with aliphatic amines. The labelling reactions with sulfonyl chlorides must be performed, carefully, at very low temperature in a place with local exhaust ventilation. Sulfonyl chlorides present a major reactive handicap as they can also easily react with other reactive groups present in biomolecules as phenols, thiols, aliphatic alcohols, imidazoles, and many others. Fortunately, this

**163**

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

**Coumarin Ex/Em** 

this solvent.

2,5-dioxopyrrolidin-1-yl-7-

2,5-dioxopyrrolidin-1-yl 7-hydroxycoumarin-3-carboxylate

methylcoumarin) acetate

2,5-dioxopyrrolidin-1-yl 7-methoxycoumarin-3-carboxylate

2,5-dioxopyrrolidin-1-yl 2-(7-hydroxy-4-

2,5-dioxopyrrolidin-1-yl 2-(7-amino-4 methylcoumarin-3-yl)acetate

SE)

diethylaminocoumarin-3-carboxylate (DEAC

kind of reactions rarely occurs in proteins or in aqueous solution, allowing the use of this type of chromophores to label proteins. Sulfonyl chloride dyes are generally hydrophobic molecules and should be dissolved in anhydrous dimethylformamide (DMF), but never in dimethylsulfoxide (DMSO) due to their highly instability in

The labelling reactions of amines with SC reagents are strongly pH dependent, and the sulfonylation-based conjugations may require a pH 9.0–10.0 for optimal conjugations, which potentiates the sulfonyl chlorides' degradation by hydrolysis reactions. In general, sulfonylation-based conjugations have much lower yields than the succinimidyl ester-based conjugations. As in the case of succinimidyl esters, the buffers used in sulfonyl chloride reactions shall not contain nucleophilic compounds, because they may react with the labelling reagent to form unstable

**(nm)**

**Physicochemical features and biological applications**

bioconjugates. Quite hydrophobic fluorescent dye, used for labelling live cells

blue-fluorescent dyes for labelling proteins and nucleic acids and increasingly used to label peptides, nucleotides, and carbohydrates

preparing bioconjugates of blue fluorescence but pH-dependent and environment-sensitive fluorescence

nucleotides with strong blue fluorescence and also used to label cell membranes although its fluorescence is quite short

examination of human kidney glomeruli. Reacts under mild conditions

432/472 Strong blue-fluorescent

363/447 One of the most popular

364/458 Widely used for

358/410 Used to label peptides and

350/450 Used for fluorohistochemical

**Ref.**

[19, 69–72]

[19, 73]

[19, 74, 75]

[19, 72, 76, 77]

[78, 79]

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

*Phytochemicals in Human Health*

*(B), sulfonyl chlorides (C), and isothiocyanates (D).*

**Figure 1.**

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].

*Schematic diagram of amine labelling techniques using succinimidyl esters (A), 4-sulfotetrafluorophenyl esters* 

*2.1.2 Fluorescent coumarin 4-sulfotetrafluorophenyl (STP) esters*

and their physicochemical features and biological applications [95, 96].

Sulfonyl chlorides (SC) are highly reactive and are unstable in water, especially at high pH required for reaction with aliphatic amines. The labelling reactions with sulfonyl chlorides must be performed, carefully, at very low temperature in a place with local exhaust ventilation. Sulfonyl chlorides present a major reactive handicap as they can also easily react with other reactive groups present in biomolecules as phenols, thiols, aliphatic alcohols, imidazoles, and many others. Fortunately, this

*2.1.3 Fluorescent coumarin sulfonyl chlorides*

Some succinimidyl esters may not be compatible with a specific application due to their insolubility in aqueous solution. To overcome these limitations, the 4-sulfotetrafluorophenyl (STP) ester can be used. These sulfonated esters have higher water solubility than simple succinimidyl esters and sometimes eliminate the need for organic solvents in the conjugation reaction, which is a great advantage to maintain the native characteristics of biomolecules. They are, however, more polar than succinimidyl esters, which makes them less likely to react with buried amines in proteins or to penetrate cell membranes [68, 94]. **Table 2** presents the single fluorescent coumarin 4-sulfotetrafluorophenyl (STP) ester used for labelling biomolecules, as the corresponding values of maximal excitation (Ex) and emission (Em) wavelengths

**162**

kind of reactions rarely occurs in proteins or in aqueous solution, allowing the use of this type of chromophores to label proteins. Sulfonyl chloride dyes are generally hydrophobic molecules and should be dissolved in anhydrous dimethylformamide (DMF), but never in dimethylsulfoxide (DMSO) due to their highly instability in this solvent.

The labelling reactions of amines with SC reagents are strongly pH dependent, and the sulfonylation-based conjugations may require a pH 9.0–10.0 for optimal conjugations, which potentiates the sulfonyl chlorides' degradation by hydrolysis reactions. In general, sulfonylation-based conjugations have much lower yields than the succinimidyl ester-based conjugations. As in the case of succinimidyl esters, the buffers used in sulfonyl chloride reactions shall not contain nucleophilic compounds, because they may react with the labelling reagent to form unstable



**165**

[103, 104].

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

2,5-dioxopyrrolidin-1-yl 6-(11-oxo-2,3,5,6,7,11 hexahydro-1H-pyrano[2,3-f]pyrido[3,2,1-ij] quinoline-10-carboxamido)hexanoate

(Coumarin 343 X SE)

**Table 1.**

**Coumarin Ex/Em** 

*Fluorescent coumarin succinimidyl esters used for biomolecule labelling.*

intermediates that could destroy the reactive dye [19, 97–99]. **Table 3** shows fluorescent coumarin sulfonyl chlorides used for labelling biomolecules, as the corresponding values of maximal excitation (Ex) and emission (Em) wavelengths and their physicochemical features and biological applications. In addition to the coumarins presented in **Table 3**, new sulfonyl chloride coumarins have been

**(nm)**

**Physicochemical features and biological applications**

be used to design fluorescence resonance energy transfer (FRET)-based assays with fluorescein amidite (FAM) as acceptor and to construct systems which harvest blue light energy

437/477 Blue-emitting coumarin can

**Ref.**

[92, 93]

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

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 confor-

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

developed, with high potential as fluorescent probes [100, 101].

*2.1.4 Fluorescent coumarin isothiocyanates*

**2.2 Thiol-reactive fluorescent coumarins**

mation using environmental sensitive probes.

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


**Table 1.**

*Phytochemicals in Human Health*

7-amino-3-(2-((2,5-dioxo-pyrrolidin-1-yl) oxy)-2-oxoethyl)-4-methylcoumarin-6-sulfonic

Triethylammonium (9-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-8,8-dimethyl-2-oxo-4- (trifluoromethyl)-8,9-dihydro-2H-pyrano[3,2-g] quinolin-6-yl)methanesulfonate (Alexa Fluor™

2,5-dioxopyrrolidin-1-yl 6,8-difluoro-7 hydroxycoumarin-3-carboxylate (Pacific Blue™

2,5-dioxopyrrolidin-1-yl 2-(6,8-difluoro-7 hydroxy-4-methylcoumarin-3-yl)acetate

2,5-dioxopyrrolidin-1-yl 6-(2-(7-amino-4 methylcoumarin-3-yl)acetamido)hexanoate

(Marina Blue™ SE)

(AMCA-X SE)

acid (Alexa Fluor™ 350 SE)

430 SE)

SE)

**Coumarin Ex/Em** 

**(nm)**

**Physicochemical features and biological applications**

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

dye, water soluble and pH insensitive from pH 4 to pH 10. Used for stable signal generation in imaging and flow cytometry

346/442 Blue-fluorescent dye, water

430/545 Bright green-fluorescent

410/455 Conjugates of this dye are

365/460 Conjugates that are strongly

353/442 Conjugates yield blue

fluorescent, even at neutral pH. Optimally detected using optical filters configured for 4′,6-diamidino-2 phenylindole (DAPI)

fluorescence that can be used as a contrasting color in multicolor applications. Because its fluorescence may not be as bright as that of other dyes or may be obscured by autofluorescence, it is only recommended for use with highly abundant targets

strongly fluorescent even at neutral pH. Ideally suited for 405 nm violet diode laser excitation on the Applied Biosystems® Attune™ Acoustic Focusing cytometer and similarly equipped fluorescence microscopes

**Ref.**

[68, 80–83]

[68, 80, 81, 84, 85]

[68, 86–88]

[68]

[68, 89–91]

**164**

*Fluorescent coumarin succinimidyl esters used for biomolecule labelling.*

intermediates that could destroy the reactive dye [19, 97–99]. **Table 3** shows fluorescent coumarin sulfonyl chlorides used for labelling biomolecules, as the corresponding values of maximal excitation (Ex) and emission (Em) wavelengths and their physicochemical features and biological applications. In addition to the coumarins presented in **Table 3**, new sulfonyl chloride coumarins have been developed, with high potential as fluorescent probes [100, 101].
