The interaction of amino acids with macrocyclic pH probes of pseudopeptidic nature.

The fluorescence quenching, by a series of amino acids, of pseudopeptidic compounds acting as probes for cellular acidity has been investigated. It has been found that amino acids containing electron-rich aromatic side chains like Trp or Tyr, as well as Met quench the emission of the probes mainly via a collisional mechanism, with Stern-Volmer constants in the 7-43 M-1 range, while other amino acids such as His, Val or Phe did not cause deactivation of the fluorescence. Only a minor contribution of a static quenching due to the formation of ground-state complexes has been found for Trp and Tyr, with association constants in the 9-24 M-1 range. For these ground-state complexes, a comparison between the macrocyclic probes and an open chain analogue reveals the existence of a moderate macrocyclic effect due to the preorganization of the probes in the more rigid structure.

Recognition of free tryptophan in water by synthetic pseudopeptides: fluorescence and thermodynamic studies.

Pseudopeptidic receptors containing an acridine unit have been prepared and their fluorescence response to a series of amino acids was measured in water. Free amino acids, not protected either at the C or the N terminus, were used for this purpose. The prepared receptors display a selective response to tryptophan (Trp) versus the other assayed amino acids under acidic conditions. The macrocyclic nature of the receptor is important as the fluorescence quenching is higher for the macrocyclic compound than for the related open-chain receptor. Notably, under the experimental acidic conditions used, both the receptor and guest are fully protonated and positively charged; thus, the experimental results suggest the formation of supramolecular species that contain two positively charged organic molecular components in proximity stabilized through aromatic-aromatic interactions and a complex set of cation-anion-cation interactions. The selectivity towards Trp seems to be based on the existence of a strong association between the indole ring of the monocharged amino acid and the acridinium fragment of the triprotonated form of the receptor, which is established to be assisted by the interaction of the cationic moieties with hydrogen sulfate anions.

Fluorescent macrocyclic probes with pendant functional groups as markers of acidic organelles within live cells.

A new family of acidity sensitive fluorescent macrocycles has been synthesized and fully characterized. Their photophysical properties including emission quantum yield and fluorescence lifetime have been determined. The acid-base properties of the new molecules can be tuned by the incorporation of pendant functional groups. The nature of such functional groups (carboxylic acid or ester) influences dramatically the pKa of the probes, two compounds of which exhibit low values. Preliminary intracellular studies using confocal microscopy together with emission spectra of the probes from the cellular environment have shown that the synthesized fluorescent macrocycles mark the acidic organelles of RAW 264.7 macrophage cells.

Photoluminescence enhancement of CdSe quantum dots: a case of organogel-nanoparticle symbiosis.

Highly fluorescent organogels (QD-organogel), prepared by combining a pseudopeptidic macrocycle and different types of CdSe quantum dots (QDs), have been characterized using a battery of optical and microscopic techniques. The results indicate that the presence of the QDs not only does not disrupt the supramolecular organization of the internal fibrillar network of the organogel to a significant extent, but it also decreases the critical concentration of gelator needed to form stable and thermoreversible organogels. Regarding the photophysical properties of the QDs, different trends were observed depending on the presence of a ZnS inorganic shell around the CdSe core. Thus, while the core-shell QDs preserve their photophysical properties in the organogel medium, a high to moderate increase of the fluorescence intensity (up to 528%) and the average lifetime (up to 1.7), respectively, was observed for the core QDs embedded in the organogel. The results are relevant for the development of luminescent organogels based on quantum dots, which have potential applications as advanced hybrid materials in different fields.

Fluorescent acridine-based receptors for H2PO4(-).

Two new pseudopeptidic molecules (one macrocyclic and one open chain) containing an acridine unit have been prepared. The fluorescence response of these receptors to a series of acids was measured in CHCl(3). Receptors are selective to H(2)PO(4)(-) versus HSO(4)(-), and an even higher selectivity is found over other anions such as Cl(-), Br(-), CH(3)COO(-), and CF(3)COO(-). We show that the macrocyclic receptor is more selective for H(2)PO(4)(-) than the related open chain receptor. The supramolecular interactions of triprotonated receptors with different anions have been modeled in silico and have been studied by different experimental techniques. Optimized geometries obtained by computational calculations agree well with experimental data, in particular fluorescence experiments, suggesting that the selective supramolecular interaction takes places through coordination of the anions to the triprotonated form of the receptor.

Dual use of porphyrazines as sensitizers and viscosity markers in photodynamic therapy.

Porphyrazines have recently emerged as a useful class of tetrapyrroles suitable for photodynamic therapy of cancer (PDT) with excellent uptake and retention properties in vivo. Here we demonstrate that the photophysical properties of cyano-aryl porphyrazine pz1 are strongly viscosity dependent, i.e. the fluorescence lifetime and the quantum yield of pz1 increase as a function of solution viscosity. We have calibrated pz1 as a red-emitting fluorescent 'molecular rotor' in a large range of viscosities from 80 to ca. 5500 cP, in solutions of various solvent compositions and temperatures. On the other hand, pz1 works as an efficient PDT sensitiser, i.e. it induces apoptosis and necrosis in cells upon irradiation with red light through formation of singlet oxygen. We demonstrate that PDT in cells using pz1 is accompanied by a significant viscosity increase by monitoring the fluorescence lifetime of the rotor. We suggest that this increase could be used as a completely new type of diagnostic and dosimetry tool in a PDT treatment.

The interactions between a small molecule and G-quadruplexes are visualized by fluorescence lifetime imaging microscopy.

Guanine-rich oligonucleotides can fold into quadruple-stranded helical structures known as G-quadruplexes. Mounting experimental evidence has gathered suggesting that these non-canonical nucleic acid structures form in vivo and play essential biological roles. However, to date, there are no small-molecule optical probes to image G-quadruplexes in live cells. Herein, we report the design and development of a small fluorescent molecule, which can be used as an optical probe for G-quadruplexes. We demonstrate that the fluorescence lifetime of this new probe changes considerably upon interaction with different nucleic acid topologies. Specifically, longer fluorescence lifetimes are observed in vitro for G-quadruplexes than for double- and single-stranded nucleic acids. Cellular studies confirm that this molecule is cell permeable, has low cytotoxicity and localizes primarily in the cell nucleus. Furthermore, using fluorescence lifetime imaging microscopy, live-cell imaging suggests that the probe can be used to study the interaction of small molecules with G-quadruplexes in vivo.

NADPH-diaphorase activity increases during estrous phase in the bed nucleus of the accessory olfactory tract in the female rat.

We investigated the presence of nitric oxide in the bed nucleus of the accessory olfactory tract (BAOT) in males, diestrous females and estrous females using NADPH-diaphorase. Our results demonstrate a significant increase in the density of the medium-stained cells in the estrous female rats suggesting that during estrous a specific subpopulation of nitrinergic cells are activated in the BAOT. This might be related to the physiological and behavioral changes that occurs in estrous.

Chemical and transient spectroscopic evidence for C2-C3 cleavage of 2,3-diaryloxetane radical cations.

Electron transfer cycloreversion of the methoxy substituted oxetane 1b results in the production of trans-anethole and benzaldehyde through C2-C3 bond cleavage. trans-Anethole radical cation has been detected as transient intermediate by laser flash photolysis.

A molecular rotor for measuring viscosity in plasma membranes of live cells.

Molecular rotors have emerged as versatile probes for microscopic viscosity in live cells, however, the exclusive localisation of rotors in the plasma membrane has remained elusive. We report the synthesis, spectroscopic characterisation and live cell imaging of a new BODIPY-based molecular rotor suitable for mapping viscosity in the cell plasma membrane.

Photoluminescence of CdSe/ZnS core-shell quantum dots stabilized in water with a pseudopeptidic gemini surfactant.

The use of pseudopeptidic gemini surfactants as stabilizers of hydrophobic quantum dots in water is discussed. Compound 1a acts as an intercalator with hydrophobic ligands of QDs transferring them from toluene to pure water yielding a fluorescent nanoparticle resistant to quenching by chloride anion (up to 0.1 M).

Use of fluorescence spectroscopy to study polymeric materials with porous structure based on imprinting by self-assembled fibrillar networks.

Different polymeric materials have been prepared from the organogels formed by a polymerizable methacrylic mixture (methyl methacrylate/ethylene glycol dimethacrylate, 1:1, w/w) and the macrocyclic pseudopeptide 1. The use of (2,4,6-trimethylbenzoyl)diphenylphosphine oxide as a very efficient radical initiator allows polymeric materials in which the structure of the fibrils formed by self-assembly of the organogelator 1 is truly preserved to be obtained. Removal of the pseudopeptidic molecule provides materials with a porous structure reflecting that of the original self-assembled fibrils. The use of fluorescent probes such as rhodamine B and pyrene greatly facilitate the study of the porous structures formed and, accordingly, that of the morphology of the original fibrils. Those studies reveal the presence of a permanent porosity and the organization of the substructures as a porous network. This confirms the existence of a nucleation and growth mechanism for the generation of the fibrils, giving rise to the formation of spherulitic structures. Those spherulites are additionally linked by connections of variable size. A series of diffusion experiments allowed establishment of a direct dependence of the inner porosity of the materials on the amount of self-organizing template used for their preparation.

Theoretical calculations on the cycloreversion of oxetane radical cations.

The molecular mechanism for the cycloreversion of oxetane radical cations has been studied at the UB3LYP/6-31G* level. Calculations support that the cycloreversion takes place via a concerted but asynchronous process, where C-C bond breaking at the transition state is more advanced than O-C breaking. This allows a favorable rearrangement of the spin electron density from the oxetane radical cation (with the spin density located mainly on the oxygen atom) to the alkene radical cation which is one of the final products. Inclusion of solvent effects does not modify the gas-phase results.

Stepwise cycloreversion of oxetane radical cations with initial C-O bond cleavage.

2,4,6-Triaryl(thia)pyrylium salts have been used as electron-transfer photosensitizers for the cycloreversion of the oxetane ring system. The radical cation of 2,3-diphenyl-4-hydroxymethyloxetane (1) undergoes stepwise splitting via initial O-C2 cleavage. Spin and charge in the resulting intermediate are located in the oxygen and carbon atoms, respectively. Subsequent intramolecular nucleophilic attack produces 2,3-diphenyl-4-hydroxytetrahydrofuran (4a). Formation of this product occurs in the submicrosecond time scale, competing with C3-C4 cleavage to the detectable (lambdamax = 470 nm) trans-stilbene radical cation.

Photosensitiser-controlled regioselectivity in the electron-transfer cycloreversion of 2,3-diphenyloxetanes.

The regioselectivity of the oxidative electron-transfer cycloreversion of trans,trans-2,3-diphenyl-4-methyloxetane is photosensitiser dependent. With chloranil the reaction proceeds through the trans-beta-methylstyrene radical cation; however, in the case of pyrylium salts, the trans-stilbene radical cation is formed.

Involvement of triplet excited states and olefin radical cations in electron-transfer cycloreversion of four-membered ring compounds photosensitized by (thia)pyrylium salts.

Cycloreversion of 1,2,3,4-tetraphenylcyclobutanes 1a,b and oxetane 2 is achieved using (thia)pyrylium salts as electron-transfer photosensitizers. Radical cation intermediates involved in the electron-transfer process have been detected using laser flash photolysis. The experimental results are consistent with the reaction taking place from the triplet excited state of the sensitizer.

Reductive PET cycloreversion of oxetanes: singlet multiplicity, regioselectivity, and detection of olefin radical anion.

Cycloreversion of 2-(p-cyanophenyl)-4-methyl-3-phenyloxetane (1) is achieved using 1-methoxynaphthalene (2) as electron-transfer photosensitizer. The experimental results are consistent with the reaction taking place from the singlet excited state of the sensitizer. Ring splitting of the radical anion 1*- occurs with cleavage of O-C2 and C3-C4 bonds, leading to products (acetaldehyde and p-cyanostilbene) different from the reagents used in the Paterno-Büchi synthesis of 1. The olefin radical anion involved in the electron-transfer process has been detected by means of laser flash photolysis.

The role of aromatic radical cations and benzylic cations in the 2,4,6-triphenylpyrylium tetrafluoroborate photosensitized oxidation of ring-methoxylated benzyl alcohols in CH2Cl2 solution.

A steady-state and laser flash photolysis (LFP) study of the TPPBF(4)-photosensitized oxidation of ring-methoxylated benzyl alcohols has been carried out. Direct evidence on the involvement of intermediate benzyl alcohol radical cations and benzylic cations in these reactions has been provided through LFP experiments. The reactions lead to the formation of products (benzaldehydes, dibenzyl ethers, and diphenylmethanes) whose amounts and distributions are influenced by the number and relative position of the methoxy substituents. This behavior has been rationalized in terms of the interplay between the stabilities of benzyl alcohol radical cations and benzyl cations involved in these processes. A general mechanism for the TPPBF(4)-photosensitized reactions of ring-methoxylated benzyl alcohols has been proposed, where the alpha-OH group of the parent substrate acts as the deprotonating base promoting alpha-C-H deprotonation of the benzyl alcohol radical cation (formed after electron transfer from the benzyl alcohol to TPP) to give a benzyl radical and a protonated benzyl alcohol, precursor of the benzylic cation. This hypothesis is in contrast with previous studies, where formation of the benzyl cation was suggested to occur from the neutral benzyl alcohol through the Lewis acid action of excited TPP(+) (TPP).