BODIPY molecules

BODIPY FL C3-SE is 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, succinimidyl ester (Invitrogen). The derivatization to the base BODIPY molecule... 

BODIPY fluorophores are a class of probes based on the fused, multi-ring structure, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (Figure 9.27) (Invitrogen) (U.S. patent 4,774,339). This fundamental molecule can be modified, particularly at its 1, 3, 5, 7, and 8 carbon positions, to produce new fluorophores with different characteristics. The modifications cause spectral shifts in its excitation and emission wavelengths, and can provide sites for chemical coupling to label biomolecules. 

The following sections discuss the major BODIPY derivatives that are reactive toward particular functional groups in proteins and other molecules. 

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. 

Since BODIPY fluorophores are easily quenched if substitutions on a molecule exceed a 1 1 stoichiometry, modification of proteins with this fluorophore probably won t yield satisfactory... 

Figure 9.28 The side-chain NHS ester of this BODIPY derivative can be used to modify amine-containing molecules, forming amide bond linkages.

The excitation maximum for the molecule occurs at 535 nm and its emission at 552 nm. Its Stoke s shift is slightly greater than some of the other BODIPY fluorophores, producing a 17nm separation between excitation and emission peaks. BODIPY 530/550 C3 has an extinction coefficient in methanol of about 62,000 M 1 cm-1 at 535 nm. 

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. 

Figure 9.29 This BODIPY fluorophore contains a carboxylate group that can be attached to amine-containing molecules using a carbodiimide reaction.

The excitation maximum for BODIPY 493/503 C3 hydrazide occurs at 498 nm and its emission at 506 nm. Since this is an extremely small Stoke s shift, it may be difficult to avoid completely problems of excitation-light scattering interference in critical emission measurements unless sub-optimal excitation wavelengths are used. The molecule has an extinction coefficient in methanol of about 92,000M-1cm 1 at 493 nm. 

Figure 9.33 Modification of aldehyde-containing molecules can be done through this BODIPY derivative s hydrazide group.

Figure 9.35 The iodoacetamide group of this BODIPY fluorophore can react with sulfhydryl-containing molecules to form thioether linkages.

BODIPY 530/550 IA is insoluble in aqueous solution, but may be dissolved in DMF or DMSO as a concentrated stock solution prior to addition of a small aliquot to a reaction mixture. Coupling to sulfhydryl-containing molecules occurs rapidly with the formation of a thioether linkage. The reaction may be done in 50 mM sodium borate, 5mM EDTA, pH 8.3. 

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