BODIPY derivatives

BODIPY fluorophores are a relatively new class of probes based on the fused, multiring structure 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (Fig. 225) (note BODIPY is a registered trademark of Molecular Probes 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 diferent charaaeristics. The modifications cause spectral shifts in its excitation and emission wavelengths and can provide sites for chemical coupling to label other molecules. 

The BODIPY derivatives typically have high extinction coefficients and excellent quantum yields, often greater than 0.8. Their spectral characteristics are relatively insensitive to changes in pH. Luminescent changes with shifts in pH usually are due to 

The BODIPY Structure 4,4-Difluoro4-bora-3a,4a-diaza-s-indacene 

The emission spectra of BODIPY derivatives normally display narrow bandwidths, providing intensely fluorescent labels for biomolecules. Unfortunately, they also have very small Stokes shifts, typically on the order of only 10—20 nm. Excitation at the optimal wavelength may cause some interference in measurements at the emission wavelength due to light scattering or cross-over from the wide bandwidth of the excitation source. The dyes usually require excitation at suboptimal wavelengths to prevent this problem. 

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

Luminescent changes with shifts in pH usually are due to reconfiguration of a fluorophore s tt-electron cloud if an atom on the ring system becomes protonated or unprotonated. Since the BODIPY structure lacks an ionizable group, alterations in pH have no effect on its spectral attributes. 

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Fig. 13 Structure-photophysical property relationship at the C-l and C-7 position of BODIPY 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. 

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

Hydrazide groups react with aldehyde and ketone groups to form hydrazone linkages (Chapter 2, Section 5.1). Three BODIPY derivatives are available that contain a hydrazine group modification of carboxylate side chains. Biomolecules such as proteins that don t normally possess aldehyde residues can be modified to contain them by a number of chemical means (Chapter 1, Section 4.4). 

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

Figure 9.34 The long side chain of this BODIPY derivative contains a sulfhydryl-reactive iodoacetamide group that can couple to a thiol group to form a thioether bond.

BODIPY 593/503 C3 hydrazide is 4,4-difluoro-l,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-propionyl hydrazide (Molecular Probes). Unlike BODIPY 530/550 C3 hydrazide, this BODIPY derivative contains substituents that shift to lower wavelengths the spectral characteristics of its fluorescent properties. The molecule is highly reactive toward aldehyde-containing compounds, including glycoproteins that have been oxidized with sodium periodate to create the requisite groups (Fig. 230). 

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