Blue Derivatives

The following sections discuss the most important Cascade Blue derivatives that are available for covalent modification purposes. 

Br-BODIPY 493/503 is insoluble in aqueous reaction mixtures, but may be dissolved in DMF or DMSO as a concentrated stock solution prior to addition of a small amount to a buffered solution. Coupling to sulfhydryl-containing molecules is rapid, leading to the formation of a thioether linkage. The reaction may be done in 50mM sodium borate, 5mM EDTA, pH 8.3. An important consideration is to protect the iodoacetyl derivative from light which may generate iodine and reduce the reactivity of the probe. 

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The blue derivatives formed with the reagent by alkaloids remain stable for at least one day and usually much longer (cover the chromatogram with a glass plate) [2, 6, 7]. The shade of color produced can be affected by fluorescence indicators incorporated in the silica gel layer [7]. Tertiary amine alkaloids do not react at room temperature with the acetaldehyde-containing reagent [2]. 

Table 7. Hydrophilic and lipophilic Nile Blue derivatives.

The compound 251 decarbonylates on photolysis to bis(4-hydroxyaryl) acetylene 253, which is easily oxidized to the quinonoid cumulene 254. This is also obtained by thermal decarbonylation of the product of oxidation of cyclopropenone 251, the diquinocyclopropanone 252. Likewise, the blue derivative of 3-radialene 256 (a phenylogue of triketo cyclopropane) is formed from tris-(4-hydroxyaryl) cyclopropenium cation 255 by oxidation34. ... 

One Cascade Blue derivative is available for creating linkages with amine-containing molecules. The acetyl azide functionality of this reagent reacts with primary amines at ambient temperatures or below to create amide bond derivatives (Lanier and Recktenwald, 1991 Oparka et al.,... 

Figure 9.38 The acetyl azide group of this Cascade Blue derivative has dual functions. It can react with amine groups to form amide bonds, or it can be converted to an isocyanate at high temperatures to couple with hydroxyl functional groups, creating a carbamate linkage.

Cascade Blue cadaverine and Cascade Blue ethylenediamine both contain a carboxamide-linked diamine spacer off the 8-methoxy group of the pyrene trisulfonic acid backbone. The cadaverine version contains a 5-carbon spacer, while the ethylenediamine compound has only a 2-carbon arm. Both can be coupled to carboxylic acid-containing molecules using a carbodiimide reaction (Chapter 3, Section 1). Since Cascade Blue derivatives are water-soluble, the carbodiimide EDC can be used to couple these fluorophores to proteins and other carboxylate-containing molecules in aqueous solutions at a pH range of 4.5-7.5. The reaction forms amide bond linkages (Figure 9.39). 

These fluorophores have excitation maxima at 377-378 nm and at 398-399nm and emission maxima at 422-423 nm. The extinction coefficients of the molecules in water are about 27,000 M 1cm 1. The Cascade Blue derivatives can be used along with Lucifer Yellow... 

Figure 9.39 The side-chain primary amine group of this Cascade Blue derivative can be coupled to carboxylate-containing molecules using a carbodiimide reaction.

Whitaker, J.E., fdaugland, R.P., Moore, P.L., Hewitt, P.C., Reese, M., and Haugland, R.P. (1991) Cascade blue derivatives Water soluble, reactive, blue emission dyes evaluated as fluorescent labels and tracers. 

Many of the premetallised direct dyes are symmetrical structures in the form of bis-1 1 complexes with two copper(II) ions per disazo dye molecule. Scheme 5.12 illustrates conversion of the important unmetallised royal blue Cl Direct Blue 15 (5.43), derived from tetrazotised dianisidine coupled with two moles of H acid, to its much greener copper-complex Blue 218 (5.44) with demethylation of the methoxy groups as described above. Important symmetrical red disazo structures of high light fastness, such as Cl Direct Red 83 (5.45), contain two J acid residues linked via their imino groups. Unsymmetrical disazo blues derived from dianisidine often contain a J acid residue as one ligand and a different coupler as the other, such as Oxy Koch acid in Cl Direct Blue 77 (5.46), for example. 

Alternatively, the use of ionophores may enhance the selectivity of the matrix. Several ionophores have been used for the detection of metals. Simon reported a neutral ionophore with a Nile Blue derivative and an azo compound for the detection ofpotassium.(86)Table 7.4 shows the selectivity coefficients of several ionophores. 

Surfactants Methylene blue derivatives of sulfate esters and 652... 

Anthraquinoneazoles. In contrast to the older yellow Algol dyes, which contain two thiazole rings (e g., 2,2-bisanthra [2,1 d thiazole-6,11-quinonyl), the red to blue oxazoles and thiazoles derived from l-aminoanthraquinone-2-carboxylic acid and 3-amino-2-hydroxy- or -mercaptoanthraquinones exhibit good lightfastness. The good fastness to atmospheric conditions and chlorine of the blue deriv-... 

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