Development of Boron Dipyrromethene Based Quenchers as Key Components of Smart Fluorescent Probes for Bioimaging

Project: Research

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Fluorescent probes based on the Forster resonance energy transfer (FRET) mechanismhave been extensively studied for detection of protease activity, nucleic acidhybridization, and real-time polymerase chain reactions. These FRET-based probesgenerally contain a fluorophore and a non-fluorescent quencher which are in closeproximity so that the quencher can effectively quench the fluorescence of thefluorophore via FRET. Upon interaction with a target protease or nucleic acid, these twocomponents are separated thereby restoring the fluorescence. To achieve effectivequenching and subsequent activation, it is desirable that the quencher exhibits a broadabsorption that overlaps with the emission of the fluorophore to facilitate the FRETprocess and an effective non-radiative relaxation pathway to reduce the backgroundsignal. To date, only a few classes of “dark” quenchers have been developed and most ofthem can only pair with blue and green fluorophores (< 500 nm) which limits their usein bioimaging. For in vivo imaging, the fluorophores should absorb and emit in the nearinfraredregion (650-900 nm) to minimize auto-fluorescence and enhance lightpenetration through tissue. To our knowledge, dark quenchers that can work in thisregion are very rare. Even though a few of these quenchers are commercially available,they are very expensive, less versatile in term of the conjugation methods, and unstablein a reducing environment. Therefore, there has been an impetus to develop moreefficient dark quenchers with tunable spectral features and various reactive groups forconjugation. The aim of this project is to develop a series of boron dipyrromethene(BODIPY) based quenchers for FRET-based bioimaging. BODIPYs are highly versatilefunctional dyes which have tunable absorption and photophysical properties, goodsolubility and stability in many solvent systems, and diverse chemical reactivity. In thisproject, we will design and synthesize a series of conjugatable ferrocenyl BODIPYderivatives with different absorption features, in which the ferrocene moiety acts as anelectron donor to effectively quench the fluorescence of the BODIPYs. Emphasis will beplaced on the pi-extended analogues which absorb in the near-infrared region. Toevaluate the usefulness of these dark quenchers, we will conjugate these compoundswith the complementary fluorescent dyes via an enzyme-cleavable linker and study theirfluorescence quenching efficiency and subsequent activation by the enzymes both inaqueous solutions and in vitro. Finally, the efficacy of these probes for in vivo imagingon mouse xenografts will be examined.


Project number9042162
Grant typeGRF
Effective start/end date1/01/1524/06/19