Design, Syntheses and Biological Applications of Through-bond Energy Transfer Cassettes and Novel Non-covalently Cell Penetrating Peptides



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A xanthene-BODIPY cassette is used as a ratiometric intracellular pH reporter for imaging protein-dye conjugates in living cells. A model was hypothesized to explain the pH-dependent energy transfer efficiencies from the donor to the acceptor based on the electronic chemistry data. Sulfonation conditions were developed for BODIPY dyes to give water-soluble functionalized monosulfonation and disulfonation donors. A water-soluble TBET cassette, which has good photophysical properties, was synthesized using a bissulfonated BODIPY dye as the donor, and their applications for in vitro protein labeling is achieved. Chemoselective cross-coupling reactions were demonstrated for C-S bonds in the BODIPY dye, and similar reactions were applied to make the acceptor of the watersoluble cassette. Chemiluminescent energy transfer cassettes based on fluorescein and Nile Red were synthesized and their spectral properties were studied. Pep-1 (also known as Chariot), R8 (which is not often used as a non-covalent protein carrier), and a new synthesized compound, Azo-R8, was used for the study of non-covalent delivery of four different proteins into mammalian cells. Data from confocal spectroscopy revealed that all three carriers are effective for translocating protein cargos into live cells. At 37 dgrees C, import into endocytic compartments dominates, but at 4 degrees C weak, diffuse fluorescence is observed in the cytosol indicative of a favorable mode of action.