We have employed diheteroarylethenes as acceptors for photochromic FRET (pcFRET), a technique introduced for the quantitative determination of fluorescence resonance energy transfer (FRET). In pcFRET, the fluorescent emission of the donor is modulated by cyclical transformations of a photochromic acceptor. Light induces a reversible change in the structure and, concomitantly, in the absorption properties of the acceptor. Only the closed forms of the selected diheteroarylethenes 2a and 2b have an absorption band overlapping the emission band of the donor, 1. The corresponding variation in the overlap integral (and thus critical transfer distance R(o)) between the two states provides the means for reversibly switching the process of FRET on and off, allowing direct and repeated evaluation of the relative changes in the donor fluorescence quantum yield. The diheteroarylethenes demonstrate excellent stability in aqueous media, an absence of thermal back reactions, and negligible fatigue. The equilibration of these systems after exposure to near-UV or visible light follows simple monoexponential kinetics. We developed a general conceptual scheme for such coupled photochromic-FRET reactions, allowing quantitative interpretations of the photostationary and kinetic data, from which the quantum yields for the cyclization and cycloreversion reactions of the photochromic acceptor were calculated.

A novel surface architecture was developed to generate biocompatible and stable photoswitchable quantum dots (psQDs). Photochromic diheteroarylethenes, which undergo thermally stable photoconversions between two forms with different spectral properties in organic solvents, were covalently linked to an amphiphilic polymer that self-assembles with the lipophilic chains surrounding commercial hydrophobic core-shell CdSe/ZnS QDs. This strategy creates a small (∼7 nm diameter) nanoparticle (NP) that is soluble in aqueous medium. The NP retains and even enhances the desirable properties of the original QD (broad excitation, narrow emission, photostability), but the brightness of its emission can be tailored by light. The modulation of emission monitored by steady-state and time-resolved fluorescence was 35-40%. The psQDs exhibit unprecedented photostability and fatigue resistance over at least 16 cycles of photoconversion.

Photoswitchable semiconductor nanoparticles, quantum dots (QDs), couple the advantages of conventional QDs with the ability to reversibly modulate the QD emission, thereby improving signal detection by rejection of background signals. Using a simple coating methodology with polymers incorporating a diheteroarylethene photochromic FRET acceptor as well as a spectrally distinct organic fluorophore, photoswitchable QDs were prepared that are small, biocompatible, and feature ratiometric dual emission. With programmed irradiation, the fluorescence intensity ratio can be modified by up to ∼100%.

Introduction of the dialkylaminophenyl group in position 7 of 3-hydroxychromone changes the orientation of the excited-state dipole moment and leads to superior solvatochromic properties (>170 nm emission shift in aprotic media). The excited-state intramolecular proton-transfer (ESIPT) reaction of 7-aryl-3-hydroxychromones is almost completely inhibited in most solvents. Methylation of the 3-OH abolishes ESIPT completely and also leads to improved photostability. The probes exhibit a ∼100-fold increase in fluorescence intensity and large Stokes shifts upon binding to membranes, reflecting differences in membrane phase and charge by a >40 nm spread in the emission band position.

Cystic fibrosis is an autosomal recessive genetic disease, produced by a mutation in the CFTR gene that impairs its function as a chloride channel. In this work, we have examined the effects of interleukin-1beta (IL-1beta) on the expression of CFTR in human colonic T84 cells. Treatment of T84 cells with IL-1beta (0.25 ng/ml) for 4 h resulted in an increased CFTR expression (mRNA and protein). However, higher doses of IL-1beta (1 ng/ml and over) produced inhibition of CFTR mRNA and protein expression. The protein kinase C (PKC) inhibitors H7 (50 microM) and GF109203X (1 microM) inhibited the stimulatory effect of IL-1beta. Similar effects were seen in the presence of the protein tyrosine kinase (PTK) inhibitors genistein (60 microM) and herbymicin A (2 microM). These results suggest that some PKC isoform(s) and at least a PTK might be involved in the CFTR up-regulation induced by IL-1beta. The repression of CFTR up-regulation by cycloheximide (35.5 microM) suggests the participation of a de novo synthesized protein. Results obtained by using the RNA polymerase II inhibitor DRB (78 microM), suggest that the increased mRNA levels seen after IL-1beta treatment are not due to an increased stability of the message. We conclude that the CFTR mRNA and protein levels are modulated by IL-1beta, this cytokine being the first extracellular protein known to up-regulate CFTR gene expression.

Photoswitchable fluorescent diheteroarylethenes are promising candidates for applications in superresolution molecular localization fluorescence microscopy thanks to their high quantum yields and fatigue-resistant photoswitching characteristics. We have studied the effect of varying substituents on the photophysical properties of six sulfone derivatives of diheteroarylethenes, which display fluorescence in one (closed form) of two thermally stable photochromic states. Electron-donating substituents displace the absorption and emission spectra towards the red without substantially affecting the fluorescence quantum yields. Furthermore, ethoxybromo, a very electron-donating substituent, stabilizes the excited state of the closed isomer to the extent of almost entirely inhibiting its cycloreversion. Multi-parameter Hammett correlations indicate a relationship between the emission maxima and electron-donating character, providing a useful tool in the design of future photochromic molecules. Most of the synthesized compounds exhibit small bathochromic shifts and shorter fluorescence lifetimes with an increase in solvent polarity. However, the ethoxybromo-substituted fluorescent photochrome is unique in its strong solvatochromic behaviour, constituting a photoactivatable ( photochromic), fluorescent and highly solvatochromic small organic compound. The Catalán formalism identified solvent dipolarity as the principal basis of the solvatochromism, reflecting the highly polarized nature of this molecule.

A photomodulatable amphiphilic polymer has been synthesized with a backbone of poly[isobutylene-alt-maleic anhydride] and pendant dodecyl alkyl chains, Lucifer Yellow (LY) fluorescent probes, and diheteroarylethenes photochromic (PC) groups. The latter serve as reversible UV-activated FRET acceptors for the LY donors. We characterized the spectral and switching properties of the polymer in an organic solvent (CHCl(3)). In an aqueous medium the polymer forms polymersomes, constituting fluorescence probes ~75 nm in diameter. Self-assembly of the polymer on the surface of a quantum dot (QD) serving as a template creates a dual-color photoswitchable nanoparticle (psNP) with improved properties due to the increase in polymer density and efficiency of PC photoconversion. The psNP exhibits a second QD red emission band that functions as an internal standard requiring only a single excitation wavelength, and is much reduced in size (<20 nm diameter) compared to the polymersomes. The QD template also greatly increases the depth of modulation by photochromic FRET of the LY emission monitored by both steady-state and time-resolved (lifetime) fluorescence (from 20%→70%, and from 12%→55%, respectively).

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