Fluorescent deriv Chapter

The majority of compounds can be chromatographed by TLC without derivatization [1-5]. On the other hand, it is much easier to devise and follow separations if the compounds are already coloured, and here Chapter 8 will be helpful. Coloured derivatives are particularly useful for compounds that do not themselves chromatograph well, such as amines. The advantage of TLC is that, when all else fails, separated organic compounds can be revealed by charring. Fluorescent derivatives (Chapter 9) may also be useful. For optically active compounds special TLC plates can also effect resolution (Chapter 10). 

Electromagnetic radiation energy can be used to stimulate substances to fluorescence after separation by thin-layer chromatography. Its action makes it possible to convert some nonfluorescent substances into fluorescent derivatives. The active sorbents often act as catalysts in such processes (cf. Chapter 1.1). 

The supply of thermal energy by a heater or IR lamp is a second method of converting the separated substances into fluorescent derivatives. Here too, at about their decomposition temperatures, many substances react to form fluorescent derivatives, generally with the catalytic participation of the active sorbent . These fluorescent derivative often provide speciflc evidence concerning the nature of the substances being detected (cf. Chapter 1.2). 

Three main forms of amine-reactive AMCA probes are commonly available. One of them is simply the free acid form of AMCA, which can be used to couple to amine-containing molecules using the carbodiimide reaction (Chapter 3, Section 1.1). The other two are active-ester derivatives of AMCA—the water-insoluble NHS ester and the water-soluble sulfo-NHS ester forms—both of which spontaneously react with amines to create stable amide linkages. All of them react under mild conditions with primary amines in proteins and other molecules to form highly fluorescent derivatives. 

A number of BODIPY derivatives that contain reactive groups able to couple with amine-containing molecules are commonly available. The derivatives either contain a carboxy-late group, which can be reacted with an amine in the presence of a carbodiimide to create an amide bond, or an NHS ester derivative of the carboxylate, which can react directly with amines to form amide linkages. The three discussed in this section are representative of this amine-reactive BODIPY family. The two NHS ester derivatives react under alkaline conditions with primary amines in molecular targets to form stable, highly fluorescent derivatives. The carboxylate derivative can be coupled to an amine using the EDC/sulfo-NHS reaction discussed in Chapter 3, Section 1.2. 

BODIPY 530/550 C3 is insoluble in aqueous solution, but it may be dissolved in DMF or DMSO as a concentrated stock solution prior to addition of a small aliquot to a reaction. Coupling to amine-containing molecules may be done using the EDC/sulfo-NHS reaction as discussed in Chapter 3, Section 1.2 (Figure 9.29). However, modification of proteins with this fluorophore probably won t yield satisfactory results, since BODIPY fluorophores are easily quenched if substitutions on a molecule exceed a 1 1 stoichiometry. For labeling molecules which contain only one amine group, such as DNA probes modified at the 5 end to contain an amine (Chapter 27, Section 2.1), BODIPY 530/550 C3 will give intensely fluorescent derivatives. 

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). 

Flow injection analysis is discussed in more detail in Chapter 3. Some simple chemical reactions which result in the formation of fluorescent derivatives are shown in Table 7.2. All of these reactions could be adapted to enable analysis by FIA. 

Fluorescein-5-thiosemicarbazide is a hydrazide derivative of fluorescein that can spontaneously react with aldehyde- or ketone-containing molecules to form a covalent, hydrazone linkage (Fig. 208) (Pierce). It also can be used to label cytosine residues in DNA or RNA by use of the bisulfite activation procedure (Chapter 17, Section 2.1). The resulting fluorescent derivative exhibits an excitation maximum at a wavelength of 492 nm and a maximal emission wavelength of 519 nm when dissolved in buffer at pH 8.6. In the same buffered environment, the compound has an extinction coefficient of approximately 78,000 M-1cm 1 at 492 nm. 

Other avidin-enzyme conjugates that convert solid substrates to colored precipitates can be used. Likewise, fluorescent derivatives of avidin can be used, and the sample can be viewed with a fluorescent microscope two different fluorophores can be employed in double-labeling experiments (ieethis vol., Chapters 11 and 42). Preformed complexes containing avidin and biotinylated enzyme can also be used in place of the covalently coupled conjugate. 

If a compound is nonfluorescent, it may be converted to a fluorescent derivative. For example, nonfluorescent steroids may be converted to fluorescent compounds by dehydration with concentrated sulfuric acid. These cyclic alcohols are converted to phenols. Similarly, dibasic acids, such as malic acid, may be reacted with j8-naphthol in concentrated sulfuric acid to form a fluorescing derivative. White and Argauer have developed fluorometric methods for many metals by forming chelates with organic compounds (see Ref. 23). Antibodies may be made to fluoresce by condensing them with fluorescein isocyanate, which reacts with the free amino groups of the proteins. NADH, the reduced form of nicotinamide adenine dmucleotide, fluoresces. It is a product or reactant (cofactor) in many enzyme reactions (see Chapter 24), and its fluorescence serves as the basis of the sensitive assay of enzymes and their substrates. Most amino acids do not fluoresce, but fluorescent derivatives are formed by reaction with dansyl chloride. 

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