BODIPY fluorophores

Fluorophores containing 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene as a core skeleton are commonly designated as BODIPY fluorophores. Due to their useful photophysical properties including high fluorescence quantum yields, high molar absorption coefficient, narrow absorption and emission band width, and their high photostability [50], BODIPY dyes are proven to be extremely versatile and useful in many biological applications Fig. 11 [68]. 

The first BODIPY fluorophore was synthesized by Treibs and Kreuzer in 1968 [69]. As described in recent review article [70], BODIPY dyes can be categorized into... 

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. 

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... 

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.

Figure 9.32 Reaction of this BODIPY fluorophore with aldehyde groups creates hydrazone linkages.

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

BODIPY 530/550 C3 is 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (Molecular Probes). This derivative of the basic BODIPY structure contains two phenyl rings off the No. 5 and 7 carbon atoms and a propionic acid group on the No. 3 carbon atom. The carboxylate group may be used to attach the fluo-rophore to amine-containing molecules via a carbodiimide reaction to create an amide bond. The substituents on this BODIPY fluorophore result in alterations to its spectral properties, pushing its excitation and emission maximums up to higher wavelengths. 

Several BODIPY fluorophores with different excitation and emission maxima are available, and each can have alternative linkage chemistries. It is important to note that the linkage chemistry affects the fluorescence intensity. One cannot therefore quantitatively compare peptides labelled using different linkage chemistries. Once having selected a fluorophore/ linkage chemistry it is better to stick with it. 

Benzo[i7]thiophen-3-yl)pyrrole (37) was reacted sequentially with mesityl aldehyde, DDQ and Bp3-OEt2 to afford 4,4-difluoro-3,5-di(benzo [ 7]thiophen-3-yl)-8-mesityl-4-bora-3a,4a-diaza-s-indacene (156) (Equation (46)), a representative of the novel BODIPY fluorophore family in 34% overall yield (09CEJ5823). 

The BODIPY fluorophore displayed an intense red-shifted fluorescence emission in CH2Q2 (625 nm, 0.84 fluorescence quantum yield), fully preserved in the solid state. 

---