Aurore Loudet

A Ratiometric pH Reporter For Imaging Protein-dye Conjugates In Living Cells

A molecule that transfers energy through bonds from a donor to an acceptor was prepared with a pH sensitive donor function (fluorescein). At pH values above 6.5, minimal energy transfer occurred, and the probe emitted green fluorescence (ca. 520 nm) when excited at the donor (488 nm). Below pH 6.0 however, energy transfer is efficient; hence excitation at the donor causes emission at the acceptor part (600 nm). This probe was used to image a conjugate of the probe with bovine serum albumin that was imported into endosomes or in the cytosol using the noncovalently bound carrier, Pep-1, at 37 and 4 degrees C, respectively. The more acidic environment of the endosomes was conspicuous from the red fluorescence of the probe.

B,O-chelated azadipyrromethenes as near-IR probes.

Complementary synthetic routes to a new class of near-IR fluorophores are described. These allow facile access (four synthetic steps) to the core fluorophore and substituted derivatives with emissions between 740 and 780 nm in good quantum yields.

Syntheses and spectroscopic properties of energy transfer systems based on squaraines

The purpose of this project was to prepare fluorescent dyes that could absorb energy at relatively short wavelengths, and fluoresce in the near-IR region. To achieve this, copper- and palladium-mediated C–N couplings were used to prepare the ‘cassettes’, i.e the carbazole derivative 3b and the carbazole-, phenothiazine-, and phenoazine-squaraines 4b–d. These compounds have carbazole, phenothiazine, and phenoazine donor-components that absorb around about 300–320 nm, and squaraine acceptor-parts that fluoresce in the range 650–700 nm. The efficiencies of energy transfer from the donor to the acceptor, and the overall quantum yields of the cassettes were determined.

Intracellular imaging of organelles with new water-soluble benzophenoxazine dyes

Five new water-soluble derivatives of Nile Red 1-5 were prepared. These benzophenoxazine dyes fluoresce between 640 and 667 nm with quantum yields of 0.17-0.33 in pH 7.4 phosphate buffer, and at slightly shorter wavelengths and higher quantum yields in EtOH. Two dyes, 3 and 4 permeated into Clone 9 cells and selectively stained mitochondria and golgi, respectively.

An Asymmetric Hydrogenation Route To (−)-Spongidepsin

(-)-Spongidepsin 1, a cytotoxic marine natural product, was prepared via two iridium-catalyzed hydrogenation reactions; both were highly stereoselective, giving convenient access to pivotal intermediates. This synthesis was modified to give several spongidepsin analogues, and their cytotoxicities were compared with those of the natural product.

Energy transfer cassettes in silica nanoparticles target intracellular organelles

Lipophilic energy transfer cassettes like 1 and 2 are more conveniently synthesized than the corresponding hydrophilic compounds, but they are not easily used in aqueous media. To overcome the latter issue, cassettes 1 and 2 were separately encapsulated in silica nanoparticles (ca. 22 nm) which freely disperse in aqueous media. Photophysical properties of the encapsulated dyes 1-SiO(2) and 2-SiO(2) were recorded. The nanoparticles 1-SiO(2) permeated into Clone 9 rat liver cells and targeted only the ER. A high degree of energy transfer was observed in this organelle such that most of the light fluoresced from the acceptor part, i.e. the particles appeared red. Silica nanoparticles 2-SiO(2) also permeated into Clone 9 rat liver cells and they targeted mitochondria but were also observed in endocytic vesicles (lysosomes or endosomes). In these organelles they fluoresced red and red/green respectively. Thus the cargo inside the nanoparticles influences where they localize in cells, and the environment of the nanoparticles in the cells changes the fluorescent properties of the encapsulated dyes. Neither of these findings were anticipated given that silica nanoparticles of this type are generally considered to be non-porous.

Femtosecond-Domain Dynamical Studies of Energy-Transfer Cassettes

Energy-transfer labeling cassettes, having two different chromophores in the visible region, are widely used in the fluorescence-aided sequencing of DNA. After chemically selective binding of the labeling cassettes to different fragments of DNA followed by gel separation, the mixture is irradiated at a single laser wavelength. The resulting fluorescence in four different wavelength bands provides a means to color-code the fragments, which in turn can identify the terminal bases and provide a method for sequencing. Commercial labeling agents employ a fluorescein derivative as an energy absorber near 500 nm and four different chemically attached rhodamine derivatives to provide fluorescent signals in four resolvable bands. The dominant energy-transfer mechanism in such cassettes, where the chromophores are linked by partially saturated chains, is usually modeled according to a Forster energy transfer mechanism. New applications of fluorescent labels generate the need for alternative probe molecules, having greater chemical stability and rigid structure.