Trimeric and Tetrameric Cationic Styryl Dyes as Novel Fluorescence and CD Probes for ds-DNA and ds-RNA.

The wide use of mono- or bis-styryl fluorophores in biomedical applications prompted the presented design and study of a series of trimeric and tetrameric homo-analogues, styryl moieties arranged around a central aromatic core. The interactions with the most common biorelevant targets, ds-DNA and ds-RNA, were studied by a set of spectrophotometric methods (UV-VIS, fluorescence, circular dichroism, thermal denaturation). All studied dyes showed strong light absorption in the 350-420 nm range and strongly Stokes-shifted (+100-160 nm) emission with quantum yields (Φf) up to 0.57, whereby the mentioned properties were finely tuned by the type of the terminal cationic substituent and number of styryl components (tetramers being red-shifted in respect to trimers). All studied dyes strongly interacted with ds-DNA and ds-RNA with 1-10 nM-1 affinity, with dye emission being strongly quenched. The tetrameric analogues did not show any particular selectivity between ds-DNA or ds-RNA due to large size and consequent partial, non-selective insertion into DNA/RNA grooves. However, smaller trimeric styryl series showed size-dependent selective stabilization of ds-DNA vs. ds-RNA against thermal denaturation and highly selective or even specific recognition of several particular ds-DNA or ds-RNA structures by induced circular dichroism (ICD) bands. The chiral (ICD) selectivity was controlled by the size of a terminal cationic substituent. All dyes entered efficiently live human cells with negligible cytotoxic activity. Further prospects in the transfer of ICD-based selectivity into fluorescence-chiral methods (FDCD and CPL) is proposed, along with the development of new analogues with red-shifted absorbance properties.

Oligo(styryl)benzenes liposomal AIE-dots for bioimaging and phototherapy in an in vitro model of prostate cancer.

Whilst the development of advanced organic dots with aggregation-induced emission characteristics (AIE-dots) is being intensively studied, their clinical translation in efficient biotherapeutic devices has yet to be tackled. This study explores the synergistic interplay of oligo(styryl)benzenes (OSBs), potent fluorogens with an increased emission in the aggregate state, and Indocyanine green (ICG) as dual Near Infrared (NIR)-visible fluorescent nanovesicles with efficient reactive oxygen species (ROS) generation capacity for cancer treatment using photodynamic therapy (PDT). The co-loading of OSBs and ICG in different nanovesicles has been thoroughly investigated. The nanovesicles' physicochemical properties were manipulated via molecular engineering by modifying the structural properties of the lipid bilayer and the number of oligo(ethyleneoxide) chains in the OSB structure. Diffusion Ordered Spectroscopy (DOSY) NMR and spectrofluorometric studies revealed key differences in the structure of the vesicles and the arrangement of the OSB and ICG in the bilayer. The in vitro assessment of these OSB-ICG nanovesicles revealed that the formulations can increase the temperature and generate ROS after photoirradiation, showing for the first time their potential as dual photothermal/photodynamic (PTT/PDT) agents in the treatment of prostate cancer. Our study provides an exciting opportunity to extend the range of applications of OSB derivates to potentiate the toxicity of phototherapy in prostate and other types of cancer.

Fluorescent and Magnetic Radical Dendrimers as Potential Bimodal Imaging Probes.

Dual or multimodal imaging probes have emerged as powerful tools that improve detection sensitivity and accuracy in disease diagnosis by imaging techniques. Two imaging techniques that are complementary and do not use ionizing radiation are magnetic resonance imaging (MRI) and optical fluorescence imaging (OFI). Herein, we prepared metal-free organic species based on dendrimers with magnetic and fluorescent properties as proof-of-concept of bimodal probes for potential MRI and OFI applications. We used oligo(styryl)benzene (OSB) dendrimers core that are fluorescent on their own, and TEMPO organic radicals anchored on their surfaces, as the magnetic component. In this way, we synthesized six radical dendrimers and characterized them by FT-IR, 1H NMR, UV-Vis, MALDI-TOF, SEC, EPR, fluorimetry, and in vitro MRI. Importantly, it was demonstrated that the new dendrimers present two properties: on one hand, they are paramagnetic and show the ability to generate contrast by MRI in vitro, and, on the other hand, they also show fluoresce emission. This is a remarkable result since it is one of the very few cases of macromolecules with bimodal magnetic and fluorescent properties using organic radicals as the magnetic probe.

Synergic activity of oligostyrylbenzenes with amphotericin B against Candida tropicalis biofilms.

Antimicrobial drug resistance is a serious challenge in clinical settings worldwide, with biofilm formation having been associated with this problem. In the present study, the synergism of oligostyrylbenzene (OSB) compounds in combination with amphotericin B (AmB) against Candida tropicalis biofilms was investigated. In addition, the toxicity in human blood cells was determined. Synergistic combinations of OSBs and AmB were evaluated to consider future effects of OSBs in vivo. The checkerboard microdilution method was used to study the interactions of one anionic (1) and two cationic (2 and 3) OSBs with AmB. We investigated the effects of OSBs on reactive oxygen species (ROS) and the levels of the reactive nitrogen intermediates (RNIs). The cellular stress affected biofilm growth through an accumulation of ROS and RNI, at synergistic concentrations of OSBs and AmB. Furthermore, significant surface topography differences were noted upon treatment with the OSB 2/AmB combination, using confocal laser scanning microscopy in conjunction with the image analysis software COMSTAT. The results revealed a low toxicity to leukocytes and red blood cells at synergistic combinations of cationic OSBs with AmB. These findings demonstrated the antibiofilm effects of OSBs and the synergism of AmB with cationic OSBs against biofilms of C. tropicalis for the first time.

Novel antifungal activity of oligostyrylbenzenes compounds on Candida tropicalis biofilms.

As sessile cells of fungal biofilms are at least 500-fold more resistant to antifungal drugs than their planktonic counterparts, there is a requirement for new antifungal agents. Olygostyrylbenzenes (OSBs) are the first generation of poly(phenylene)vinylene dendrimers with a gram-positive antibacterial activity. Thus, this study aimed to investigate the antifungal activity of four OSBs (1, 2, 3, and 4) on planktonic cells and biofilms of Candida tropicalis. The minimum inhibitory concentration (MIC) for the planktonic population and the sessile minimum inhibitory concentrations (SMIC) were determined. Biofilm eradication was studied by crystal violet stain and light microscopy (LM), and confocal laser scanning microscopy (CLSM) was also utilized in conjunction with the image analysis software COMSTAT. Although all the OSBs studied had antifungal activity, the cationic OSBs were more effective than the anionic ones. A significant reduction of biofilms was observed at MIC and supraMIC50 (50 times higher than MIC) for compound 2, and at supraMIC50 with compound 3. Alterations in surface topography and the three-dimensional architecture of the biofilms were evident with LM and CLSM. The LM analysis revealed that the C. tropicalis strain produced a striking biofilm with oval blastospores, pseudohyphae, and true hyphae. CLSM images showed that a decrease occurred in the thickness of the mature biofilms treated with the OSBs at the most effective concentration for each one. The results obtained by microscopy were supported by those of the COMSTAT program. Our results revealed an antibiofilm activity, with compound 2 being a potential candidate for the treatment of C. tropicalis infections. LAY SUMMARY: This study aimed to investigate the antifungal activity of four OSBs (1, 2, 3, and 4) on planktonic cells and biofilms of Candida tropicalis. Our results revealed an antibiofilm activity, with compound 2 being a potential candidate for the treatment of C. tropicalis infections.

Understanding the Driving Mechanisms of Enhanced Luminescence Emission of Oligo(styryl)benzenes and Tri(styryl)-s-triazine.

This work is focused on unraveling the mechanisms responsible for the aggregation-induced enhanced emission and solid-state luminescence enhancement effects observed in star-shaped molecules based on 1,3,5-tris(styryl)benzene and tri(styryl)-s-triazine cores. To achieve this, the photophysical properties of this set of molecules were analyzed in three states: free molecules, molecular aggregates in solution, and the solid state. Different spectroscopy and microscopy experiments and DFT calculations were conducted to scrutinize the causative mechanisms of the luminescence enhancement phenomenon observed in some experimental conditions. Enhanced luminescence emission was interpreted in the context of short- and long-range excitonic coupling mechanisms and the restriction of intramolecular vibrations. Additionally, we found that the formation of π-stacking aggregates could block E/Z photoisomerization through torsional motions between phenylene rings in the excited state, and hence, enhancing the luminescence of the system.

Shape-persistent and adaptive multivalency: rigid transgeden (TGD) and flexible PAMAM dendrimers for heparin binding.

This study investigates transgeden (TGD) dendrimers (polyamidoamine (PAMAM)-type dendrimers modified with rigid polyphenylenevinylene (PPV) cores) and compares their heparin-binding ability with commercially available PAMAM dendrimers. Although the peripheral ligands are near-identical between the two dendrimer families, their heparin binding is very different. At low generation (G1), TGD outperforms PAMAM, but at higher generation (G2 and G3), the PAMAMs are better. Heparin binding also depends strongly on the dendrimer/heparin ratio. We explain these effects using multiscale modelling. TGD dendrimers exhibit "shape-persistent multivalency"; the rigidity means that small clusters of surface amines are locally well optimised for target binding, but it prevents the overall nanoscale structure from rearranging to maximise its contacts with a single heparin chain. Conversely, PAMAM dendrimers exhibit "adaptive multivalency"; the flexibility means individual surface ligands are not so well optimised locally to bind heparin chains, but the nanostructure can adapt more easily and maximise its binding contacts. As such, this study exemplifies important new paradigms in multivalent biomolecular recognition.

Modeling and analysis of the San Francisco City Clinic Cohort (SFCCC) HIV-epidemic including treatment.

We investigate two HIV/AIDS epidemic models. The first model represents the early San Francisco men having sex with men (MSM) epidemic. We use data from the San Francisco City Clinic Cohort Study (SFCCC), documenting the onset of HIV in San Francisco (1978-1984). The second model is a 'what-if' scenario model including testing and treatment in the SFCCC epidemic. We use compartmental, population-level models, described by systems of ordinary differential equations. We find the basic reproductive number R0 for each system, and we prove that if R0<1, the system has only the disease-free equilibrium (DFE) which is locally and globally stable, whereas if R0>1, the DFE is unstable. In addition, when R0>1, both systems have a unique endemic equilibrium (EE). We show that treatment alone would not have stopped the San Francisco MSM epidemic, but would have significantly reduced its impact.

Imine formation as a simple reaction to construct copper-reactive cruciform fluorophores.

We developed a series of new conjugated cruciform fluorophores (XF) featuring imine groups. The condensation of an XF containing aldehyde functionalities and selected primary amines leads to several XF-imine derivatives. Upon addition of Cu(2+) or Zn(2+) ions to solutions of the imine XFs in different solvents, a red-shifted emission is detected, resulting in an altered emission color. The imine acts as a simple modular metallo-reactive fluorophore.

Water-soluble distyrylbenzenes: one core with two sensory responses--turn-on and ratiometric.

The synthesis of four water-soluble distyrylbenzenes (compounds 1-4) is reported. Their acidochromicity in aqueous media was investigated. Blue shifts and increases in the quantum yields were observed as a general response. The pH-dependent photophysics of 1b-3b in water reveal unexpected protonation sequences upon titration: compound 1b is green-yellow fluorescent at high pH (10) but becomes very weakly fluorescent between pH 5 and pH 3, whereas below pH 2 strong blue fluorescence is observed. This behavior can be explained in terms of the interplay in the protonation of aniline and of the carboxylate groups. In compound 4, a higher basicity of the amino group is observed and ratiometric fluorescence change takes place upon protonation or on reaction with zinc salts in water. Compound 4 can therefore act as a weak ratiometric zinc ligand in water, even though it has only a dimethylamino unit as a binding motif.

Unsymmetrical cruciforms.

Five new, unsymmetrical 1,4-distyryl-2,5-bisphenylethynylbenzenes (cruciforms, XF) have been prepared by a sequential Horner reaction of the bisphosphonate of 2,5-diiodo-1,4-xylene with two different aromatic aldehydes. The obtained diiodide was coupled to phenylacetylene under Sonogashira conditions with (Ph(3)P)(2)PdCl(2) as catalyst. The resulting XFs carry dibutylamino, pyridyl, cyano, and diphenylamino residues on their styryl arms to give rise to donor-, acceptor-, and donor-acceptor-substituted XFs. The optical properties of these XFs were investigated. Titration studies using trifluoroacetic acid tracked changes in the electronic structure of the XFs upon protonation. Donor XFs display a blue shift in absorption and emission upon protonation, while the pyridyl-substituted XF displays red shift in absorption and emission upon protonation. In the case of the donor-acceptor XF carrying a pyridyl and an aminostyryl arm, the first protonation occurs either on the pyridine or on the dibutylamino arm; a red shift is seen in absorption (for the former) and a blue shift is observed in emission (for the latter). The titration studies indicate that the protonated XFs do not display kinetic photoacidity when operating either in dichloromethane or acetonitrile solutions. The trends observed for protonation were mirrored when the XFs bind to metal cations. While the binding constants of the metal cations to the XFs were lower than for that for protons, as in some cases full metalation of the XF could not be obtained, the results were qualitatively the same. We did not find dynamic excited-state decomplexation events in the XFs that we have investigated. The XFs, stilbene derivatives, are different from other reported, similarly structured fluorophores as they show significant ratiometric changes in emission upon metal complexation; thus, distyrylbenzene-derived fluorophores may be, in the end, viable choices as platforms for metal ion detection.

Bis(4'-dibutylaminostyryl)benzene: spectroscopic behavior upon protonation or methylation.

We have investigated the UV/Vis absorption and emission of 1,4-bis(4'-dibutylaminostyryl)benzene (1) upon protonation with trifluoroacetic acid in dichloromethane and in acetonitrile. We find that 1 does not display significantly dynamic acidity in the excited state, that is, it is not a photoacid. Three protonation states of 1 were investigated, all of which, neutral, singly protonated, and bis-protonated, are fluorescent. As an isolable model for the mono-protonated species, a methylated derivative of 1 was prepared by reaction with methyl triflate. This species displays redshifted emission but similar absorption as the non-quaternized parent, 1. The strong observed emission of all three protonated states of 1 suggests that the kinetic photoacidity of 1* is low or absent. We assume that the lifetime of the excited state is too short to allow solvent reorganization and therefore we cannot make a statement on the thermodynamic photoacidity of the protonated forms of 1.

Hyperbranched: a universal conjugated polymer platform.

Out on a limb: Sonogashira coupling of a suitable AB(2) monomer containing two iodine and one alkyne group forms a hyperbranched conjugated polymer that is studded with iodine end groups (see picture: I purple). These iodine groups are a perfect handle for convenient, efficient, and high-yielding post-functionalization to access hyperbranched, fluorescent poly(phenyleneethynylene)s.

Water soluble cruciforms: effect of surfactants on fluorescence.

Brighten up! Adding surfactants to aqueous solutions of three different water-soluble cruciforms (XF) improves their fluorescence quantum yields. Additionally, changes are observed in the emission wavelength of the XF around the critical micelle concentration (cmc) of the surfactant. Three 1,4-bis-(aminostyryl)-2,5-bis(phenylethynyl) benzenes carrying four, six, or eight acetic acid units were investigated for their surfactochromicity. Anionic, cationic, and nonionic surfactants as well as a surfactant-like protein (bovine serum albumin, BSA) were added to buffered solutions of the XFs in water, causing-in most cases-the fluorescence quantum yield to increase significantly and a blue- or red-shifted emission to be observed. The addition of cationic (cetyltrimethylammonium bromide, CTAB) and neutral (Brij 35, TWEEN 20 and Triton X-100) surfactants to XFs causes a red shift in their emission at low or very low surfactant concentrations that we attribute to surfactant-induced excimer formation. The fluorescence quantum yield is in most cases a monotonous function of surfactant concentration. For the investigated ionic surfactants, the fluorescence quantum yield of the XFs does not change much after the critical micelle concentration (CMC) of the surface is reached. However, in the case of non-ionic surfactants, the fluorescence quantum yields of the XFs starts to increase after the CMC has been reached, suggesting that different effects are involved.

Water-soluble cruciforms: response to protons and selected metal ions.

The syntheses of three water-soluble cruciform fluorophores (XF) carrying aniline- N, N-bisacetic acid, 2-hydroxyaniline- N, N, O-trisacetic acid, and 1,2-phenylenediamine- N, N, N', N'-tetrakisacetic acid are reported. The XF skeleton was synthesized by a Horner reaction to assemble the distyrylbenzene unit followed by a Sonogashira coupling to attach the phenyleneethynylene modules. The photophysics of both the sodium salts and the ethyl esters of the three carboxylated 1,4-bis(aminostyryl)-2,5-bis(phenylethynyl)benzenes were investigated in chloroform and in aqueous buffered solution at a pH of 7.0 and compared to that of 1,4-bis(dibutylaminostyryl)-2,5-bis(phenylethynyl)benzene (BDB). The attachment of the carboxylate units to the aniline nitrogens influenced the photophysics and the sensory responses of the XFs, as the combined effect of steric bulk and charge repulsion led to a blue-shifted absorption when compared to that of BDB. While the fluorescence of the water-soluble XFs is sensitive toward metal cations, the mode of sensing action is different from that of BDB, where direct complexation to the aniline nitrogen lowers the energy of the HOMO (but not of the LUMO), leading to a blue-shifted emission. In the case of the 2-hydroxyaniline- N, N, O-trisacetic acid and 1,2-phenylenediamine- N, N, N', N'-tetrakisacetic acid-functionalized XFs, interaction with metal cations in aqueous buffered solution is guided by a breakup of excimers that form in water at XF concentrations as low as 50 micromol x L (-1).

Control of surface functionality in poly(phenylenevinylene) dendritic architectures.

The efficient synthesis of new asymmetric poly(phenylenevinylene) dendritic macromolecules using a stepwise convergent-growth approach is described. By an iterative methodology that made use of the Horner-Wadsworth-Emmons (HWE) reaction, dendrons and dendrimers up to the third generation, with eight different functional groups located at the periphery, were prepared in good yields. Both the number and placement of functionalities can be accurately controlled to afford a large variety of dendritic architectures.