Photoinduced Chromophore Hydration in the Fluorescent Protein Dreiklang Is Triggered by Ultrafast Excited-State Proton Transfer Coupled to a Low-Frequency Vibration

Because of growing applications in advanced fluorescence imaging, the mechanisms and dynamics of photoinduced reactions in reversibly photoswitchable fluorescent proteins are currently attracting much interest. We report the first time-resolved study of the photoswitching of Dreiklang, so far the only fluorescent protein to undergo reversible photoinduced chromophore hydration. Using broadband femtosecond transient absorption spectroscopy, we show that the reaction is triggered by an ultrafast deprotonation of the chromophore phenol group in the excited state in 100 fs. This primary step is accompanied by coherent oscillations that we assign to its coupling with a low-frequency mode, possibly a deformation of the chromophore hydrogen bond network. A ground-state intermediate is formed in the picosecond–nanosecond regime that we tentatively assign to the deprotonated water adduct. We suggest that proton ejection from the phenol group leads to a charge transfer from the phenol to the imidazolinone ring, which triggers imidazolinone protonation by nearby Glu222 and catalyzes the addition of the water molecule

Circularly Permuted Fluorogenic Proteins for the Design of Modular Biosensors

Fluorescent reporters are essential components for the design of optical biosensors that are able to image intracellular analytes in living cells. Herein, we describe the development of circularly permuted variants of Fluorescence-Activating and absorption-Shifting Tag (FAST) and demonstrate their potential as reporting module in biosensors. Circularly permutated FAST (cpFAST) variants allow one to condition the binding and activation of a fluorogenic ligand (and thus fluorescence) to analyte recognition by coupling them with analyte-binding domains. We demonstrated their use for biosensor design by generating multicolor plug-and-play fluorogenic biosensors for imaging the intracellular levels of Ca2+ in living mammalian cells in real time

Circularly Permuted Fluorogenic Proteins for the Design of Modular Biosensors

Fluorescent reporters are essential components for the design of optical biosensors that are able to image intracellular analytes in living cells. Herein, we describe the development of circularly permuted variants of Fluorescence-Activating and absorption-Shifting Tag (FAST) and demonstrate their potential as reporting module in biosensors. Circularly permutated FAST (cpFAST) variants allow one to condition the binding and activation of a fluorogenic ligand (and thus fluorescence) to analyte recognition by coupling them with analyte-binding domains. We demonstrated their use for biosensor design by generating multicolor plug-and-play fluorogenic biosensors for imaging the intracellular levels of Ca2+ in living mammalian cells in real time

Light-Activated Proteolysis for the Spatiotemporal Control of Proteins

The regulation of proteolysis is an efficient way to control protein function in cells. Here, we present a general strategy enabling to increase the spatiotemporal resolution of conditional proteolysis by using light activation as trigger. Our approach relies on the auxin-inducible degradation system obtained by transposing components of the plant auxin-dependent degradation pathway in mammalian cells. We developed a photoactivatable auxin that acts as a photoactivatable inducer of degradation. Upon local and short light illumination, auxin is released in cells and triggers the degradation of a protein of interest with spatiotemporal control

Circularly Permuted Fluorogenic Proteins for the Design of Modular Biosensors

Fluorescent reporters are essential components for the design of optical biosensors that are able to image intracellular analytes in living cells. Herein, we describe the development of circularly permuted variants of Fluorescence-Activating and absorption-Shifting Tag (FAST) and demonstrate their potential as reporting module in biosensors. Circularly permutated FAST (cpFAST) variants allow one to condition the binding and activation of a fluorogenic ligand (and thus fluorescence) to analyte recognition by coupling them with analyte-binding domains. We demonstrated their use for biosensor design by generating multicolor plug-and-play fluorogenic biosensors for imaging the intracellular levels of Ca2+ in living mammalian cells in real time

Synthesis and Structure–Activity Relationships of the First Ferrocenyl-Aryl-Hydantoin Derivatives of the Nonsteroidal Antiandrogen Nilutamide

We present here the first synthesis of organometallic complexes derived from the nonsteroidal antiandrogen nilutamide, bearing a ferrocenyl substituent at position N(1) or at C(5) of the hydantoin ring; for comparison, we also describe the C(5) p-anisyl organic analogue. All of these complexes retain a modest affinity for the androgen receptor. The N-substituted complexes show a weak or moderate antiproliferative effect (IC50 around 68 µM) on hormone-dependent and -independent prostate cancer cells, while the C(5)-substituted compounds exhibit toxicity levels 10 times higher (IC50 around 5.4 µM). This strong antiproliferative effect is probably due to a structural effect linked to the aromatic character of the ferrocene rather than to its organometallic feature. In addition, it seems connected to a cytotoxic effect rather than an antihormonal one. These results open the way toward a new family of molecules that are active against both hormone-dependent and hormone-independent prostate cancer cells

A [3]Ferrocenophane Polyphenol Showing a Remarkable Antiproliferative Activity on Breast and Prostate Cancer Cell Lines

We have previously shown that modification of polyphenols with a ferrocenyl group can dramatically enhance their cytotoxicity. We now present two new [3]ferrocenophane compounds, one of which has an antiproliferative effect seven times stronger than the corresponding noncyclic species, with IC50 values of 90 and 94 nM on hormone-independent MDA-MB-231 breast and PC-3 prostate cancer cell lines, respectively. Solubility studies in water using methylated β-cyclodextrin and electron transfer studies are also presented

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A simple imaging protocol for autofluorescence elimination and optical sectioning in fluorescence endomicroscopy: supplementary materia

Synthesis and Structure Activity Relationship of Organometallic Steroidal Androgen Derivatives

We present here the synthesis and the structure activity relationship of a series of organometallic complexes of the steroidal androgens testosterone and dihydrotestosterone (DHT) substituted at the C-17 position of the steroid skeleton with an ethynyl substituent grafted with various sandwich or semisandwich organometallic units [ferrocenyl, (η5-C5H4)-Re(CO)3, (η5-C5H4)-Mn(CO)3, (η6-C6H5)-Cr(CO)3] and of 3β-androstanediol substituted at C-16 and C-17 respectively by a ferrocenyl vinyl and a ferrocenyl ethynyl unit. In contrast to the estradiol series, there are currently very few examples of organometallic steroidal androgens in the literature. The ethynyltestosterone derivatives were obtained via a Stille coupling reaction between the appropriate iodo-organometallics and 17β-ethynyltestosterone stannyl derivatives. The ethynyl-DHT derivatives were synthesized by addition of the corresponding acetylide to the C-17 carbonyl of the steroid. The crystal structures of two ferrocenyl and one rhenium complexes were determined by X-ray diffraction and had confirmed that the organometallic moiety points toward the α face of the steroid skeleton. All the complexes retain a modest affinity for the androgen receptor. The ferrocenyl derivatives of ethynyl testosterone, 8 and 12, show a strong antiproliferative effect on the hormone-independent prostate cancer cells PC-3 with IC50 values of respectively 4.7 and 8.3 μM. These values are very similar, for 12, or slightly better, for 8, than those found recently for the most active ferrocenyl derivative of the nonsteroidal antiandrogen nilutamide (IC50 value of 5.4 μM). The ferrocenyl complexes described here are the first examples of organometallic steroidal androgens possessing a strong antiproliferative activity on prostate cancer cells