Fluorescein reactive derivatives

Fluorescein and rhodamine are common fluorophores used for the fluorescent labeling of proteins in biological applications. This is due to a number of advantageous properties such as long absorption maxima, insensitivity to solvent polarity, high molar extinction coefficient, and the availability of a wide variety of reactive derivatives. Fluorescein derivatives are characterized by a multi-ring aromatic structure, due to the planar nature of an upper, fused, three-ring system (Fig. 2). Derivatives of the basic structure... 

The following sections describe the most important fluorescein derivatives having reactive groups commonly used to label biomolecules. 

Two general forms of amine-reactive fluorescein probes are available. Both of them react under alkaline conditions with primary amines in proteins and other molecules to form stable, highly fluorescent derivatives. 

The iodoacetamido derivatives of fluorescein possess a sulfhydryl-reactive iodoacetyl group (Chapter 1, Section 4.2 and Chapter 2, Section 2.1) at either the 5- or 6-carbon position on their lower ring. The isomers are commercially available in purified form, since some reactivity and specificity differences between the 5- and 6-derivatives toward various sulfhydryl sites in proteins may be observed. Both iodoacetamido derivatives are among the most intense fluoro-phores available for labeling biomolecules due to high QY. 

SAMSA-fluorescein, 5- [2(and 3)-5-(acetylmercapto)-succinoyl]amino fluorescein, is a fluorescent probe containing a protected sulfhydryl group. In its protected state, the compound is unre-active. The acetyl-protecting group can be removed by treatment with dilute NaOH at pH 10.0 (Figure 9.9). The resulting free sulfhydryl derivative can be used to label thiol-reactive crosslinkers or to couple with sulfhydryl residues on proteins and other molecules. After activating... 

Aldehyde/Ketone and Cytosine-Reactive Fluorescein Derivatives... 

Although FITC and other reactive fluorescein derivatives still are widely used to label (strept)avidin and other proteins, better fluorescence yield and stability will be obtained if one of the newer hydrophilic fluorescein dyes is used. See Chapter 9, Section 1, for additional details on labeling proteins with fluorescein. 

The second label also may be a fluorescent compound, but doesn t necessarily have to be. As long as the second label can absorb the emission of the first label and modulate its signal, binding events can be observed. Thus, the two labeled DNA probes interact with each other to produce fluorescence modulation only after both have bound target DNA and are in enough proximity to initiate energy transfer. Common labels utilized in such assay techniques include the chemiluminescent probe, N-(4-aminobutyl)-N-ethylisoluminol, and reactive fluorescent derivatives of fluorescein, rhodamine, and the cyanine dyes (Chapter 9). For a review of these techniques, see Morrison (1992). 

DNA modified with a diamine compound to contain terminal primary amines may be coupled with amine-reactive fluorescent labels. The most common fluorophores used for oligonucleotide labeling are the cyanine dyes and derivatives of fluorescein and rhodamine (Chapter 9). However, any of the amine-reactive labels discussed throughout Chapter 9 are valid candidates for DNA applications. 

Williams and Robertson [76] have described a simple inexpensive method for determining reactive chlorine in non-saline waters. It involves addition of bromine, which is oxidised by the reactive chlorine in the sample, and which in turn brominates fluorescein to give a pink derivative this can be measured visually or spectophotometrically, or the decrease in fluorescein can be measured fluorimetrically. Potential applications of the method are indicated. 

Extending the utility of fluorescence to various nonfluorophores is achieved via chemical derivatization methods, also termed labeling or tagging methods (Reaction 11.2). Numerous commercial fluorescent tags are available with disparate reactive functional groups. For example, derivatives of fluorescein, fluorescein isothiocyanate (FITC) are reactive toward nucleophiles such as amines and sulfhydryl groups. 

Extrinsic fluorescence is used whenever the natural fluorescence of a macromolecule is inadequate for accurate fluorescence measurement. In this case, one can attach a fluorescent reporter group by using the reactive isocyanate or isothiocyanate derivatives of fluorescein or rhodamine, two intensely fluorescent molecules. One can covalently also label a protein s a- and e-amino groups with dansyl chloride (/.e., A,A-dimethylaminonaphtha-lenesulfonyl chloride). Another useful reagent is 8-ani-lino-l-naphthalenesulfonic acid (abbreviated ANS). This compound is bound noncovalently by hydrophobic interactions in aqueous solutions, ANS is only very fluorescent, but upon binding within an apolar environment, the quantum yield of ANS becomes about 100 times greater. 

Many derivatives of fluorescein containing a reactive group at the C-5 position are commercially available [11], Fluorescein isothiocyanate, for example, is widely used as protein tag [12]. These substances have essentially the same spectroscopic properties as the parent compound with the additional capability of binding covalently to proteins. Because of their high emission quantum yields, fluorescein conjugates are extensively used as tracers for microinjection in living cells to gather information on the structure and function of cells, localization of proteins, and cell-to-cell and intracellular diffusion [13-17]. 

Rose Bengal differs in reactivity from fluorescein and Eosin because of the iodines at C-2, C-4, C-5, and C-7 which differentiate the reactivity of the phenoxide from the carboxylate and make substitution at C-2 possible in lieu of substitution at C-6. Essentially three different kinds of Rose Bengal derivatives have been synthesized. The most common include derivatives with gegen ions other than sodium at both C-2 and C-6. Many esters of C-2 have been synthesized and compounds with a number of different gegen ions at C-6 have been reported. The rarest derivatives of Rose Bengal are the C-6 ethers, of which only three have been reported. 

---