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Phenothiazine compounds are known as effective inhibitors of a multidrug resistance (MDR) of tumor cells to chemotherapeutic agents. This group consists of many important substances used in human medicine such as antipsychotic drugs in the case of fluphenazine (FPh) or chlorpromazine (CPZ). Fluphenazine was on the World Health Organization (WHO) list of Essential Medicines of 2009, and its new pyrimidine analog (FPh-prm) presented in this work has been documented to have a high anti-MDR activity. In order to discover the character of alterations of the lipid bilayer structure caused by the presence of FPh-prm inside the lipid membrane, which is responsible for the essential increase of an anti-MDR activity of FPh-prm, microcalorimetric (differential scanning calorimetry), Laurdan fluorescence, (31)P nuclear magnetic resonance spectroscopy (NMR), and attenuated total reflectance Fourier transfer infrared spectroscopy (FTIR-ATR) were used for dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes mixed with a different concentration of amine analogue. It was stated that the formation of domains with different content of FPh-prm/DPPC can be a reason for the membrane-related mechanism of chemoprevention associated with the inhibition of the outward transport of anticancer drugs by the glycoprotein P (Pgp) in cancer cells by the pyrimidine analog of FPh. To our best knowledge, this report is the first to show the bilayer structure of domains formed by incomplete miscibility of fluphenazine-related compounds and phospholipid molecules. Our results provide a sound basis for the design of future modifications of anti-MDR drugs by providing very effective inhibitors of the pump activity of Pgp.

Biomarkers are essential part of daily medical practice. Currently, biomarkers are being used both for diagnostic and prognostic purposes. There are many approaches e.g. ELISA by which biomarker levels are detected from patient samples. However, all these approaches are laborious, time consuming and expensive. There is therefore a general need for exploring new technique which can overcome these drawbacks. Here, we present a preliminary study for detection of serum biomarkers by fluorescence correlation spectroscopy (FCS) based diagnostic technique. FCS is a technique basically used for spatial and temporal analysis of molecular interactions of extremely low-concentration biomolecules in solution. FCS is able to measure diffusion time of the fluorescent molecules passing through the open detection volume and it can also measure the average number of fluorescent molecules passing through the detection volume. Because diffusion speed is correlated with shape and molecular mass of the fluorescent molecule, this property makes it possible to study the complex formation between a small fluorescently labelled and a large unlabelled molecule. In this preliminary study, we utilize this FCS property for detection of serum biomarker. Further studies on various pathological serum samples are warranted to explore further aspects of this technique.

Fluorescence correlation spectroscopy (FCS) is an important biophysical technique. FCS is currently being used in many areas of biology to solve several scientific problems. Its properties such as detection at the single molecular level, higher sensitivity, and use of lower sample volume make FCS a promising molecular diagnostic tool. The promising applications of FCS extend from DNA kinetics/dynamics studies to the comprehensive understanding of receptor–ligand interactions. In this article, we review various promising biological applications of FCS.

In a comprehensive picture of inclusion com- plex formation of the highly fluorescent dye coumarin-6 (C6) and betacyclodextrin (beta-CD), which was obtained using various fluorescence spectroscopic methods, it was demonstrated that up to three beta-CD rings can thread on the rod like dye molecule. Interaction of coumarins and modified coumarins with cellular organelles or proteins has been reported in several publications. Especially 7-amino- coumarins are characterized by unique properties like high fluorescence quantum yield and are thus already used successfully in different areas, like staining of fluorescent nanoparticles. We could show that Coumarin-6 made sol- uble by complexation with beta-cyclodextrin is able to stain eukaryotic cells specifically dependent on their origin and cellular behaviour. The staining reaction is indepen- dent from pH, is photo stable, and shows no cross talk with proteins in the cytoplasm and other staining procedures or erythrocytes. Staining with coumarin 6/cyclodextrin com- plexes can thus be used for fast discrimination of different cell types. Importantly, it could be shown that the ideal staining reaction is dependent on the stoichiometry of the complex-formation.

Cancer is one of the big killers of world population. The majority of cancers are diagnosed at a late stage, making a cure almost impossible. Fluorescence spectroscopy is an emerging diagnostic tool for various medical diseases including premalignant and malignant lesions. Fluorescence spectroscopy is a noninvasive technique and has been applied successfully for the diagnosis of multisystem cancers with high sensitivity and specificity. Fluorescence spectroscopy minimizes the need for repetitive biopsy, which is routine practice for cancer patient follow-up. But there are many aspects of this new diagnostic technique that should be discussed in future research to overcome limitations and challenges faced by this technique for diagnosis of cancers.

G protein-coupled receptors have been proposed to exist in signalosomes subject to agonist-driven shifts in the assembly disassembly equilibrium, affected by stabilizing membrane lipids and/or cortical actin restricting mobility. We investigated the highly homologous corticotropin-releasing factor receptors (CRFRs), CRFR1 and -2, which are different within their hydrophobic core. Agonist stimulation of CRFR1 and CRFR2 gave rise to similar concentration-response curves for cAMP accumulation, but CRFR2 underwent restricted collision coupling. Both CRFR1 and CRFR2 formed constitutive oligomers at the cell surface and recruited beta-arrestin upon agonist activation (as assessed by fluorescence resonance energy transfer microscopy in living cells). However, CRFR2, but not CRFR1, failed to undergo agonist-induced internalization. Likewise, agonist binding accelerated the diffusion rate of CRFR2 only (detected by fluorescence recovery after photobleaching and fluorescence correlation spectroscopy) but reduced the mobile fraction, which is indicative of local confinement. Fluorescence intensity distribution analysis demonstrated that the size of CRFR complexes was not changed. Disruption of the actin cytoskeleton abolished the agonist-dependent increase in CRFR2 mobility, shifted the agonist concentration curve for CRFR2 to the left, and promoted agonist-induced internalization of CRFR2. Our observations are incompatible with an agonist-induced change in monomer-oligomer equilibrium, but they suggest an agonist-induced redistribution of CRFR2 into a membrane microdomain that affords rapid diffusion but restricted mobility and that is stabilized by the actin cytoskeleton. Our data show that membrane anisotropy can determine the shape and duration of receptor-generated signals in a subtype-specific manner.

Die LTCC (Low Temperature Co-fired Ceramic)-Technologie, die ursprunglich fur den Aufbau von hoch zuverlassigen Mehrlagenschaltungen entwickelt wurde, eroffnet die Moglichkeit, auch komplexe dreidimensionale Strukturen in einfacher Weise aufzubauen. Am Beispiel eines Monitoring-Moduls zur Erfassung von biologischen Reaktionen wird die Herstellung einer komplexen Mikrofluidik-Applikation vorgestellt. Anhand eines definierten Mischprozesses sowie eines Reaktionsprozesses, welche sich auf numerische Simulationen abstutzen, wird die Funktionalitat des Systems aufgezeigt.

3-Pentadecylphenol (PDP) belongs to a natural group of amphiphilic phenols and plays a lot of different interesting functions. Effects on the phase behavior of bilayer mixtures of DPPC (dipalmitoyl phosphatidylcholine) or MPPC (l-myristoyl-2-palmitoyl-phosphatidylcholine) and PDP has been investigated using electron microscopy, differential scanning calorimetry (DSC) and fluorescence spectroscopy. Addition of 3-pentadecylphenol increases the phase transition temperature T m . Above 20 mole % PDP in DPPC the phase behavior becomes increasingly complex. Coexistence of various phases within the bilayer with different composition and structural properties is suggested and is responsible for the changes of the physico-chemical properties of the lipid bilayer. Excellent analogy between fluorescence and calorimetric data demonstrate that phase effects are primarily caused by the abundance of the large phenolic headgroup. DPPC and MPPC bilayers are similarly affected but MPPC is more sensitive to added PDP.

The photophysical properties of indoline (I) and three of its derivatives, namely, N-methylindoline (MI), 5-cyanoindoline (CI), and 5-cyano-N-methylindoline (CMI), are studied in H-donating solvents of varying polarity. Based on measurements of fluorescence yield and lifetime, and of triplet yield and hydrated-electron formation, two distinct mechanisms of solvent-induced fluorescence quenching are evidenced. The first mechanism involves the cyano substituent and leads to an increase in the rate constant of internal conversion of one order of magnitude in ethanolic solution and of more than two orders of magnitude in water, as compared to solutions in n-hexane or acetonitrile. A similar trend had previously been observed in the case of 4-N,N-dimethylaminobenzonitrile (DMABN). The second mechanism reduces the fluorescence lifetimes of the non-cyanated derivatives in aqueous solution by one order of magnitude and is related to the formation of hydrated electrons. Neither of these mechanisms is influenced by methylation at the ring nitrogen. Quantum chemical calculations are performed on the ground and excited states of the hydrogen-bonded complexes between protic solvents and MI as well as CMI. Stable hydrogen-bonded configurations involving the CN substituent and a solvent OH group are found; these configurations are stable both in the ground and the first excited singlet states, whereas the corresponding complex at the ring amino nitrogen is stable in the ground state only. The CN--HO configuration is therefore a prime candidate for a mechanistic explanation of the observed quenching by the first mechanism. These findings may have useful applications for the design of fluorescence probes for water in biological systems.

Low temperature co-fired ceramic (LTCC) technology was originally developed for the realization of multilayer circuits of high reliability. It was recognized that LTCC-technology is a valuable development in thick film technology which launches new application areas as it becomes evident that complex three-dimensional structures can be easily realized. Aspects of the realization of a sophisticated biological monitoring module comprising a three-dimensional network of channels and cavities is demonstrated.

Human rhinoviruses (HRVs) are composed of 60 identical subunits, each comprising one copy of the viral capsid proteins VP1, 2, 3, and 4. Consequently, 60 symmetry-related epitopes are available for binding of antibodies or receptors. The minor receptor group of HRVs uses members of the low-density lipoprotein receptor family for cell entry. The ligand binding domains of these receptors are composed of various numbers of ligand binding repeats, and several of these modules within a single molecule are believed to attach simultaneously to the star-shaped dome at the 5-fold symmetry axis of the virus. Using fluorescence correlation spectroscopy (FCS), we have now determined the equilibrium binding constants and the mode of attachment of recombinant concatemers of ligand binding module 3 of the human very-low-density lipoprotein receptor to HRV2. We demonstrate that the avidity of the interaction drastically increases with the number of concatenated modules. For the trimer, the binding isotherm was biphasic, indicating that attachment of two and of three modules within the same molecule was resolved. The receptor consisting of seven repeats was found to bind most strongly, but a complete binding isotherm could not be established due to cross-linking of virions. The values of the dissociation constants were about 1 order of magnitude higher than those previously determined by using surface plasmon resonance techniques reflecting the different presentation of the binding partners. As compared to the concatemers, the natural receptors are composed of similar but not identical repeats; thus, cooperativity and different specificity of the ligand-binding modules allow for recognition of many ligands and viral serotypes. Due to the low concentrations and amounts of sample required, FCS is ideally suited for the determination of receptor binding parameters of viruses difficult to produce in high quantities and/or concentrations.

The appearance of malignant cells in body fluids like urine, blood or body-cavity fluids are a clear indication for the existence of a tumor and urine or body-cavity fluid cytology are routine diagnostics today. Cytologic examination of the cellular features of fluids is a valuable adjunct to patient diagnosis and the staging and management of tumors. The German-language literature contains the earliest references to the cytology of malignant cells in fluid specimens. Preparation of the specimen has evolved from unstained wet smears to protocols that generally include centrifugation and the generation of stained smears and a cell block. The smears may be alcohol-fixed direct smears, cytospins, or a liquid-based preparation, and they are usually stained with the Papanicolaou method. Additional techniques, such as immunocytochemistry and flow cytometry, provide significant help in this differential diagnosis. Evaluation of microscopic images after Papanicolaou staining eluded digital pathology, an image-based information environment enabled by computer technology that allows for extracting information from a digital slide. With the advent of full-slide scanning digital methods are regarded as promising way to achieve better, faster and cheaper diagnosis, prognosis and prediction of cancer and other important diseases. One important feature are combinations with immunostaining, FISH technology etc., to elude additional information from the specimen. Circulating tumor cells (CTCs) can be found in the bloodstream and are always ready to attach to endothelial cells lining blood vessels and extravasate to enter tissues and organs to form a metastatic site. They show plastic phenotype and a small number of these cells undergo the epithelial-to-mesenchymal (EMT) program. De-differentiation and dissemination from the primary tumor is a basic prerequisite for colonization and growth of distant metastasis. Phenotypic and functional plasticity of cancer cells and the ability to adapt permanently to demanding conditions provide great challenge for identification and characterization of CTC´s from blood. Their clear identification and characterization is, however, also an important prerequisite to obtain valuable information for diagnosis and prognosis by downstream analytical methods. A novel platform for identification and morphological characterization of cancer cells in body fluids by digital methods is presented.

One of the most intriguing predictions of energy landscape models is the existence of non-exponential protein folding kinetics caused by hierarchical structures in the landscapes. Here we provide the strongest evidence so far of such hierarchy and determine the time constants and weights of the kinetic components of the suggested hierarchic energy landscape. To our knowledge, the idea of hierarchical folding energy barriers has never been tested over such a broad timescale. Refolding of yeast phosphoglycerate kinase was initiated from the guanidine-unfolded state by stopped-flow or manual mixing and monitored by tryptophan fluorescence from 1 ms to 15 min. The strategy to build a model that describes folding of yeast phosphoglycerate kinase was to start from the simplest paradigm and modify it stepwise to the necessary minimal extent after repeated comparisons with the experiments. We made no a priori assumptions about the folding landscape. The result was a hierarchic finite level landscape model that quantitatively describes the refolding of yeast phosphoglycerate kinase from 1 ms to 15 min. The early steps of the folding process happen in the upper region of the landscape, where the surface has a hierarchic structure. This leads to stretched kinetics in the early phase of the folding. The lower region of the energy landscape is dominated by a trap that reflects the accumulation of molten globule intermediate state. From this intermediate, the protein can reach the global energy minimum corresponding to the native state through a cross-barrier folding step.

There are proteins that are built of two structural domains and are deposited full-length in amyloid plaques formed in various diseases. In spite of the known differences in the mechanisms of folding of single- and multidomain proteins, no published studies can be found that address the role of the domain-domain interactions during misfolding and amyloid formation. By the discovery of the role of domain-domain interactions, here we provide important insight in the submolecular mechanism of amyloid formation. A model system based on yeast phosphoglycerate kinase was designed. This system includes the wild-type yeast phosphoglycerate kinase and single-tryptophan mutants of the individual N and C terminal domains and the complete protein. Electron microscopic measurements proved that amyloid fibrils grow from all mutants under identical conditions as for the wild-type protein. Misfolding and amyloid formation was followed in stopped-flow and manual mixing experiments on the 1 ms to 4 days timescale. Tryptophan fluorescence was used for selective detection of conformational changes accompanying the formation of the amyloidogenic intermediates and the growth of amyloid fibrils. The interactions between the polypeptide chains of the two domains direct the misfolding process from the early steps to the amyloid formation, and influence the final structure. The kinetics of misfolding is different for the individual domains, pointing to the significance of the amino acid sequence. Misfolding of the domains within the complete protein is synchronized indicating that domain-domain interactions direct the misfolding and amyloid formation mechanism.

Lipid-polymer conjugates with differing hydrophobic to hydrophilic dimensions were prepared. The polymer part consisted of hydrophilic poly(2-methyl-1,3-oxazolines) (POZO), whose chain length can be controlled by living polymerization methods. The lipid-polymer conjugates were incorporated into vesicles composed of L-α-dimyristoylphosphatidylcholine (DMPC) or L-α-dipalmitoylphosphatidylcholine (DPPC) in amounts of up to 25 mol%. The formation of domains within the lipid bilayer membrane is observed by atomic force microscopy (AFM), thermal analysis (DSC), and fluorescence spectroscopy. The synthetic design of the lipids allows control over the distribution of polymer chains on the vesicle surface.

The absorption and induced circular dichroism (icd) spectra of the α-CD:DMABN system in water solution at 20 °C evidenced the presence of 1∶1 and 2∶1 complexes. The β-CD:DMABN system was found to be characterized by 1∶1 and 2∶2 associates. The association constants and the absolute absorption and icd spectra of each species were determined. The structure of the complexes was determined by dynamic Monte Carlo (DMC) calculations including solvation effects and induced circular dichroism calculations.

Fluorescence correlation spectroscopy (FCS) was used to study the diffusion behavior of the glucocorticoid dexamethasone on the cell membrane of the mouse pituitary cell line AtT-20 to investigate the putative membrane glucocorticoid receptor (mGR). This putative membrane receptor is thought to mediate glucocorticoid effects independent from protein synthesis and the well-known intracellular glucocorticoid receptor (iGR), the so-called “nongenomic” effects. The high spatial resolution of the FCS technique allowed for focusing exclusively on the cell membrane; measurements were taken on living cells in solution after incubation with 60 nM fluoresceindexamethasone for 1 h without manipulation of the cells. FCS measurements were performed by moving the position of the laser focus in 1 μm steps through a single cell. In a scan through the cell a component typical for a receptor-bound ligand (diffusion constant 3 x 10-10 cm2 sec-1) becomes apparent in the autocorrelation function (ACF) at the position of the cell membrane whereas another component (diffusion constant 2 x 10-8 cm2 sec-1) typical for a ligand diffusing in the cytosol becomes predominant in the ACF curves inside the cell. After pre-incubation with unlabeled dexamethasone in 1000-fold excess the “receptor-typical” component on the cell membrane was absent. This result is the first direct evidence for the existence of a mGR on a living cell.

Fluorescence correlation spectroscopy (FCS) allows determining diffusion and relaxation properties of fluorescent molecules. It requires only extremely small amounts of sample, down to picomolar concentrations, in an effective analysis volume of a few femtoliters. In essence, FCS determines the autocorrelation of fluorescence fluctuations caused by single labeled molecules passing through the confocal volume of a microscope equipped with a suitable detector; it permits investigating interactions of (macro)molecules, even in single cells. We present an FCS protocol for exploring, under in vitro conditions, the dynamic processes that take place during the early steps of virus infection. We cover two important issues of rhinovirus research, the kinetics of directional RNA release, and virus-receptor interactions exemplified by using human rhinovirus type A2 (HRV-A2) as a model.

Fluorescence correlation spectroscopy (FCS) allows determining diffusion and relaxation properties of fluorescent molecules. It requires only extremely small amounts of sample, down to picomolar concentrations, in an effective analysis volume of a few femtoliters. In essence, FCS determines the autocorrelation of fluorescence fluctuations caused by single labeled molecules passing through the confocal volume of a microscope equipped with a suitable detector; it permits investigating interactions of (macro)molecules, even in single cells. We present an FCS protocol for exploring, under in vitro conditions, the dynamic processes that take place during the early steps of virus infection. We cover two important issues of rhinovirus research, the kinetics of directional RNA release, and virus-receptor interactions exemplified by using human rhinovirus type A2 (HRV-A2) as a model.

Supramolecular host–guest complexes formed between dye molecules, which are absorbing and fluorescing in the visible range, and cyclodextrin molecules are studied with the aim to generate a self-assembled nanostructured layer on a surface. Studies in solution show that an aminocoumarin dye, i.e. coumarin-6, binds strongly to β-cyclodextrin and yields fluorescent structures. The complex formation is substantiated by 17 nm blue shift of the fluorescence spectrum. The binding of thiolated cyclodextrin, 6-monodeoxy-6-monothio-β-cyclodextrin, to gold surface is then studied by induced change in the second-harmonic signal intensity generated from the surface. The saturation times of surface adsorption, as a measure of binding kinetics, are estimated for thiolated cyclodextrin as well as hexadecanethiol. The results support formation of self-assembled layers of the host molecules, which can serve as basis for the formation of supramolecular host–guest surface layers.

Many donor–acceptor substituted organic compounds show internal charge separation upon excitation. Within this class, the following compounds are studied as suitable candidates for molecules with high second-order nonlinear optical (NLO) response: 4- N , N -dimethylamino-benzonitrile (DMABN), 6-propanoyl-2-(dimethylamino)-naphthalene (PRODAN), and various 4-(4- N , N -dimethylamino-phenyl)-1,3-diphenyl-pyrazolo[3,4-b]quinolines (DMA-DPPQ). The configuration interaction method with single and double excitations (CISD) is used in combination with the sum over states (SOS) approach within the semiempirical AM1 parametrization to calculate the vector component of the hyperpolarizability along the dipole axis β μ . The influence of changing molecular geometry on the second-order nonlinear properties is calculated for various intramolecular relaxation coordinates. Solvent effects are explicitly studied for DMABN by a self-consistent reaction field (SCRF) model revealing an increasing NLO-effect with increasing solvent polarity. The hyperpolarizability is discussed in relationship to the charge transfer properties of the excited state. The calculated values are in good agreement with experimental data for both DMABN and PRODAN. DMA-DPPQ is found to be a good NLO chromophore only when increasing either the donor or acceptor strength. Its nonlinear response can be drastically increased by an additional strong electron acceptor group, e.g. a NO 2 -group, at the primary acceptor moiety.

The excited states of various di-alkyl-aminobenzonitriles (ABN) emitting dual fluorescence are investigated theoretically by a combination of density functional theory and multireference configuration interaction (DFT/MRCI). Experimentally, it was found that intramolecular charge transfer (ICT) emission increases on expense of the normal fluorescence with increasing n-alkyl chain length. The long wavelength ICT band becomes the predominant fluorescence band in diisopropylaminobenzonitrile (DIABN) even in an apolar environment. The calculated energy of both the planar and twisted ICT (PICT and TICT) state increases slightly with increasing chain length relative to the energy of the first excited state at optimized planar ground-state geometry. The good agreement of the calculated TICT state dipole moment with the experimental ICT state dipole moment leads us to the conclusion that the dual fluorescence of all linear substituted di-alkyl ABNs originate from a TICT state. The bulky system DIABN is twisted by 33° already in the ground state and complete decoupling of donor and acceptor units takes place without a significant energy barrier. In contrast, planarization toward the PICT state is energetically disfavored. Thus, the red-shifted fluorescence of DIABN is also assigned to emission out of the TICT state. © 2001 John Wiley & Sons, Inc. Int J Quantum Chem 84: 149–156, 2001

The structures, spectroscopic and photophysical properties of the inclusion complexes between 4-OH-biphenyl and cyclodextrins (CD) in water were studied theoretically and experimentally. The complex structures were predicted using a dynamic Monte Carlo procedure including solvation effects and analyzed via computed and experimentally determined circular dichroism. The interpretation of the induced circular dichroism spectra indicated the formation of stable 2:2 complexes between the probe and alpha-CD, while 1:1 structures account for the circular dichroism induced by beta- and gamma-CD. Fluorescence emission quantum yields and lifetimes, measured as a function of the CD concentration, confirmed the formation of this higher order complex with alpha-CD. Prototropic equilibration in the singlet excited state was found to be depressed in 2:2 complexes due to the hydrophobic environment of the OH groups, while it remained unperturbed in 1:1 complexes, where the substituent is exposed to the aqueous environment. Triplet-triplet absorption and triplet quenching data supported this interpretation. The photophysical properties of both the 1:1 and the 2:2 complexes are characterized by a significant reduction of the nonradiative decay rates.

Many enzymes occurring in nature like superoxide dismutase are systems rather too big to be accessible for vibrational and quantum chemical investigations. Thus, enzyme-mimetic model compounds consisting of a biological active metal centre surrounded by a macrocyclic ligand are used to shed light on binding properties of the active metal centre. Far- and mid-range IR spectroscopic investigations and a conformational analysis with the semi-empirical ZINDO/1 method of superoxide dismutase-mimetic complex Cu[TAAB] 2+ are performed (TAAB=[ b , f , j , n ][1,5,9,13]tetra-aza-cyclohexadecine (tetra-anhydroamino benzaldehyde)). A distorted tetrahedral copper(II) centre with slightly twisted phenyl subunits is determined as the most stable conformation. Calculated mid- and far-IR spectra are in good agreement with the experimental data and confirm the proposed structure. A harmonic normal-coordinate analysis is used to assign the vibrational modes of the observed spectra.

The photophysical properties of naphthalene were studied in aqueous solution (H2O and D2O), in some organic solvents (ethanol, hexane, and acetonitrile), and in complexes with the cyclodextrins (CDs) α-CD and β-CD, by means of absorption, steady-state and time-resolved fluorescence, circular dichroism, and triplet−triplet absorption spectroscopies. The structures of the CD inclusion complexes were computed using a dynamic Monte Carlo method. The main difference of the photophysics in the pure aqueous with respect to the organic media consists in a reduction of the fluorescence lifetime, τF, by a factor of about 2.5. Consideration of the triplet properties in aqueous and organic media led to the conclusion that this effect is most probably due to a corresponding increase of the intersystem crossing rate, induced by H2O or D2O. Inclusion of naphthalene in the CD hosts has the effect, at high CD concentration (>0.01 M), of increasing τF with respect to the aqueous medium, the value in α-CD being near to that...

Acid–base properties of four 3,5-dimethyl-1,7-diphenyl-bis-pyrazolo-[3,4-b; 4′,3′-e]-pyridine derivatives with different substituents in position 4, i.e. N,N-dimethylaminophenyl, phenyl, nitrophenyl, and methoxyphenyl (DMA-DMPP, H-DMPP, NO2-DMPP, and CH3O-DMPP) were investigated in nonaqueous solutions. Absorption and stationary fluorescence as well as time-resolved spectroscopy were applied as methods. The acidity of the amino group in DMA-DMPP which is preferentially protonated in the ground state, does practically not change in the first excited state. The second protonation step of DMA-DMPP and the protonation of the other compounds occurs at the heterocyclic subunit, in the ground state most probably on the pyridine nitrogen. The basicity of this nitrogen atom increases remarkably in the first excited state. Experimental results were compared with those of semiempirical calculations. The amino group as the preferential site of protonation before the pyridine nitrogen in the ground state was confirmed. According to quantum mechanical computations, one pyrazolo nitrogen is preferentially protonated in the first excited state. The protonation is accompanied by a geometrical relaxation leading to a planarized conformer with a lower total energy.

Potential photosynthetic models, porphyrin dyads with various acceptors, that are separated by a diazaphenylene spacer, are synthesized by a new strategy. The amidine and vinamidinium salts are coupled by pyrimidine rings. In one example (n=1 in the figure), excitation of a flavin acceptor results in energy transfer to the porphyrin singlet (≈60 %) and population of a charge-separated state (≈40 %), which relaxes to the ground state on a subnanosecond time scale.

The decay kinetics of triplet excited probes (meso-tetraphenylporphyrin or anthracene) in unilamellar phospholipid bilayer membranes was studied using nanosecond laser-induced transient absorption ...

Fluorescence properties of 1-heptanoylpyrene in homogeneous solutions and incorporated into liposome membranes have been measured. The fluorescence intensity of the alkanoylpyrene is small in neat hydrocarbon solutions (Qf = 0.006) but increases with increasing solvent polarity. On the contrary, Qf of the 1-alkylpyrene is larger by a factor of 10 in a hydrocarbon solvent and decreases slightly in polar solvents. In alcohols the fluorescence yield of 1-heptanoylpyrene becomes considerably larger in comparison to a nonhydroxylic environment and nearly identical to that of heptylpyrene in the same solvent. The fluorescence spectra shift bathochromically in that polar environment. The fluorescence properties of 1-heptanoylpyrene incorporated in a liposome membrane indicate that the probe molecules are well embedded within the hydrophobic core of the bilayer. Semiempirical calculations were performed for 1-ethanoylpyrene and 1-ethylpyrene as model compounds in order to analyze the influence of solvent polarity and of hydrogen bonding on excited state energies and fluorescence properties of 1-acyl derivatives of pyrene. Hydrogen-bonding increases the energy gap between the lowest singlets, which are nearly degenerated, and the 3π*-n triplet and reduced consequently the intersystem crossing efficiency. But also a change in the sequence of the excited 1π*-π states is concluded from experimental data and should contribute to the increase of the fluorescence yield upon hydrogen bonding.

The excited states of 4-(N,N-dimethylamino)benzonitrile (DMABN) and 4-aminobenzonitrile (ABN) are characterized by the similarity transformed equation-of-motion coupled-cluster method with single and double excitations (STEOM-CCSD). The long wavelength band of DMABN is assigned by STEOM-CCSD to an emission of a geometrically relaxed twisted intramolecular charge transfer (TICT) state. In contrast, no lowering in total energy upon twisting is found in ABN. The different behavior of the TICT states in ABN and DMABN can quite clearly be understood from the behavior upon twisting of the correlated ionization potentials and electron affinities from IP/EA-EOM-CCSD results that are obtained as a side product of STEOM-CCSD calculations. Inclusion of electron correlation is found to be crucial however, as the effect is largely lost at the Hartree−Fock (Koopmans') level of accuracy. The STEOM-CCSD results are similar to results from DFT/SCI calculations. In general they also agree well with results from previous CA...

Picosecond transient absorption spectra of 4-(N,N-dimethylamino)-benzonitrile (DMABN), 6-cyanobenzquinuclidine (CBQ), and benzquinuclidine (BQ) in polar and nonpolar solvents were measured for the first time at wavelengths

This paper presents a computational study on the formation of a molecular necklace formed by specific threading of cyclodextrins (CDs) on block copolymers. Structural as well as energetic principles for the selective complexation of α- and β-cyclodextrin with poly(ethylene oxide)–poly(propylene oxide) block copolymers (PEO–PPO) are elucidated considering a diblock copolymer of equimolecular composition (PEO)4–(PPO)4 as guest. A non-statistical distribution of CDs, i.e. α-CDs primarily located on the PEO chain and β-CDs on PPO blocks of the polymer, is based on a variety of structural features and energetic preferences considering both potential as well as solvation energies. This selectivity becomes already obvious considering 1:1 complexes between PEO and PPO monomers and the two CDs, but is increasingly evident when calculating higher order ensembles. Besides the host–guest interaction, docking between CDs themselves is an important, also non-statistical, prerequisite for the self-assembly of highly ordered tubes. The formation of intermolecular hydrogen bonds between adjacent CDs in a tubular aggregate gives an important contribution to the overall stability of the molecular necklace. The net effect, based on the preferential interaction between host and guest as well as between the host molecules themselves, results in the formation of a stable, highly ordered macromolecular, multicomponent aggregate.

The efficient fluorescence probe 6-propanoyl-2-(N,N-dimethylamino)naphthalene (PRODAN) shows a significant Stokes shift in polar solvents. Neither experimental nor theoretical studies performed up to now yield a definitive description of the character of the emissive state. Results of the recently developed DFT/SCI method are compared to semiempirical calculations with the spectroscopic ZINDO/S parametrization and the AM1/CISD Hamiltonian which was not parametrized for computation of spectroscopic properties. The most reliable results are obtained with the DFT/SCI and AM1/CISD approaches. The absorption spectrum of PRODAN is excellently reproduced by the DFT/SCI calculations, and the AM1/CISD results are superior to ZINDO/S. These results allow the assignment of the red-shifted fluorescence band to both emission of a highly polar dimethylamino twisted intramolecular charge transfer state (N-TICT) and a geometrically unchanged planar intramolecular charge transfer (PICT) state. A comparison with 2-(N,N-dim...

The photophysical properties of 10-decyl- and 10-propenyl-9-acridones, important emitters in electro- and chemiluminescence, are investigated in dependence of the solvent polarity and its hydrogen bonding ability at room temperature as well as in frozen matrix at 77 K. Fluorescence is inefficient in non-polar solvents at room temperature due to fast intersystem crossing but increases with solvent polarity due to the inversion of the S 1 (ππ ∗ ) and the T (nπ ∗ ) states. In the case of 9-[10-(1-propenyl)]-acridone the dependencies of the fluorescence spectrum and quantum yield on the ET(30) solvent parameters indicate that intermolecular hydrogen bonded solute-solvent complexes are formed in the excited state, inducing efficient non-radiative deactivation.

The photophysical properties of three newly synthesized pyrazoloquinolines, composed of N,N-dimethylaniline as donor subunit and various substituted forms of the acceptor pyrazoloquinoline (DPPQ), were investigated by absorption as well as by stationary and time resolved fluorescence spectroscopy. These compounds show generally highly efficient emission in nonpolar and medium polar solvents; the dipole moment of the emitting state increases and the quantum yield decreases with solvent polarity. These results are explained by state reversion in polar solvents: At low polarities emission originates from a state localized on the DPPQ moiety, whereas in the high-polarity regime the next excited state of charge transfer character, in which an electron is promoted from the amino nitrogen lone pair into an excited orbital of the DPPQ moiety, becomes the fluorescent state. This view is corroborated by semiempirical calculations including the solvent reaction field, low-temperature fluorescence measurements, and the observation of effects of protonation on the spectroscopic and photophysical properties.

The photophysics of 9,9‘-bianthryl (BA) were investigated by means of fluorescence spectroscopy, nanosecond transient-absorption spectroscopy, and semiempirical calculations. Fluorescence spectra and lifetimes were measured in more than 50 solvents in order to get a detailed picture of the solvent dependence. The results show that the fluorescence lifetime is constant in solvents of low polarity (D 5), thus providing direct proof of the charge-separated character of the fluorescent ...

Geometries, conformational energies and activation barriers for rotations about the single bonds connecting the various subunits of a new class of bulky electron donor–acceptor systems derived from the bis-pyrazolopyridine chromophore are characterized for several representatives by semiempirical methods (AM1 and PM3). For the dimethylaminophenyl derivative DMA–DMPP (4-(4′- N , N -dimethylaminophenyl)-3,5-dimethyl-1,7-diphenyl-bis-pyrazolo-[3,4-b;4′,3′e]pyridine) dual fluorescence was observed in polar, protic solvents and was discussed in the context of the TICT (twisted intramolecular charge transfer) hypothesis, first formulated to explain dual fluorescence of 4-( N , N -dimethylamino)benzonitrile (DMABN). Besides the parent H–DMPP molecule, derivatives substituted with different donor or acceptor groups were compared with respect to their optimum ground-state geometries.

4-( N , N -Dimethylamino)-benzonitrile (DMABN) is the classical model compound showing solvent-induced dual fluorescence, a phenomenon attributed to formation of an intramolecular charge-transfer state and concomitant rotational relaxation of the dimetylamino group about the amine–aryl single bond. This mechanism is supported by the fluorescence properties of various stereoselectively substituted derivatives of DMABN and, therefore, the ground-state properties of such a set of model compounds are studied as a basis for a detailed examination of the influence of substitution on excited-state relaxation pathways. The factors examined in this study are torsion about the single bond connecting the donor amino group to the aryl moiety, the amine wagging vibration and the bending of the cyano group (rehybridization). Ground-state geometries and electrostatic properties as well as the energetics of these modes are obtained from semiempirical AM1 calculations and are compared with Hartree–Fock ab initio results using 3-21G* and 6-21G** split valence basis sets and those obtained by the Becke3 density functional theory method with the Lee–Young–Parr correlation function and the D95** basis set.

4-(4′-N,N-dimethylaminophenyl)-3,5-dimethyl-1,7-diphenyl-bis-pyrazolo-[3,4-b;4′,3′e]-pyridine (DMA-DMPP), was recently synthesized as an example of a bulky electron donor-acceptor compound and was examined by spectroscopic methods. The molecule consists of several subunits of different redox properties and the observed behavior was thus interpreted by an intramolecular charge transfer model. This hypothesis is validated by semiempirical excited state calculations. They are focused on the coupling of intramolecular charge transfer to various relaxation modes, e.g. rotation about single bonds leading to decoupling π systems. Rotation about the single bond connecting the dimethylanilino and heteroaromatic subunit results as the most important relaxation coordinate. The two moieties non orthogonal in the ground state become perpendicularly oriented in the optimum excited state. In this conformation the dipole moment also becomes maximal. These results agree satisfactorily with the observation of a marked red shift of its fluorescence in polar solvents and dual fluorescence found in polar, protic environment.

6-Cyanobenzquinuclidine (CBQ) is widely assumed to be a good model to study twisted internal charge transfer (TICT), a hypothesis proposed to explain the appearance of solvent-induced dual fluorescence in 4-(N,N-dimethylamino)benzonitrile (DMABN) derivatives. This is due to the orthogonal arrangement of the nitrogen lone pair and the aromatic π orbitals in this cage amine and the exclusive appearance of long wavelength TICT fluorescence. Spectral and photophysical properties of CBQ are investigated by stationary and time-resolved fluorescence spectroscopy as a function of solvent polarity. The results are discussed in the context of conformational analysis studies performed for CBQ and benzquinuclidine by X-ray crystallography as well as by semiempirical calculations. It is shown that only one singlet excited state is emissive. Fluorescence spectra and intersystem crossing rates can be modeled as charge recombination processes, and excellent agreement between simulated and measured data is found. Rehybrid...

Contbrmational energies of PRODAN (6-Propionyl-2-(dimethylamino)-naphthalene) have been calculated by Hartree-Fock ab initio methods as a function of the twisting and wagging motion of the amino and the rotational motion of the propionyl group. In agreement with experimental data, PRODAN is found planar in its optimum geometry. The CIS method is used to compute molecular orbital energies, excitation energies and dipole moments in dependence of the rotational isomerization of the dimethylamino group. The calculations allow the peculiar fluorescence properties of PRODAN to be explained by the TICT model.

Molecular self-assembly is frequently found to generate higher-order functional structures in biochemical systems. One such example is the self-assembly of lipids in aqueous solution forming membranes, micelles, and vesicles; another is the dynamic formation and rearrangement of the cytoskeleton. These processes are often driven by local, short-range forces and therefore the dynamics is solely based on local interactions. In this paper, we introduce a cellular automata based simulation, the lattice molecular automaton, in which data structures, representing different molecular entities such as water and hydrophilic and hydrophobic monomers, share locally propagated force information on a hexagonal, two-dimensional lattice. The purpose of this level of description is the simulation of entropic and enthalpic flows in a microcanonical, molecular ensemble to gain insight about entropy-driven processes in molecular many-particle systems. Three applications are shown, i.e., modeling structural features of a polar solvent, cluster formation of hydrophobic monomers in a polar environment, and the self-assembly of polymers. Processes leading to phase separation on a molecular level are discussed. A thorough discussion of the computational details, advantages, and limitations of the lattice molecular automaton approach is given elsewhere [B. Mayer and S. Rasmussen (unpublished)]. {copyright} {ital 1997} {ital The American Physical Society}

Complexation trajectories and the variation ofinduced circular dichroism are calculated for thedocking of phenol and 2,4,6-trimethyl-phenol withβ-cyclodextrin. The results are compared toexperimental chirality data to elucidate themechanism of nonspecific molecular recognitionprocesses in aqueous solution. Large geometricalchanges along nearly isoenergetic Dynamic Monte Carlotrajectories show the conformational flexibility ofsuch host–guest systems. This proves diffuseintermolecular interactions, van der Waals orelectrostatic in nature, as the main contributions to thebinding energy. The number and position of the methylsubstituents of the guest reduces the complexityof the conformational space as the guest‘s positionbecomes fixed by steric constraints. The solvation free energy is calculated from thesolvent accessible surface area weighted byrespective atomic solvation parameters. Consideringthe solvation term in the dynamic simulationsrestricts the conformational flexibility of themacromolecular system. The relative importance ofvarious contributions to the solvation energy isdiscussed and it is shown that those terms arisingfrom the interaction of hydrophobic groups with theaqueous environment are essential for thedetermination of the complex structure. Consideringthese terms in the dynamic simulation model, the signand strength of the calculated rotatory strength isin perfect agreement with induced circular dichroismobtained from experimentally determined averagedspectra. The results demonstrate the accuracy of thegeometrical properties of host–guest systems obtainedfrom these simulations.

The fluorescence properties of several pyrazoloquinoline derivatives were performed by stationary as well as by time-resolved spectrosopy. Non-donor-substituted compounds show a high quantum yield; transition dipole moments of absorption and fluorescence as well as experimental and calculated lifetimes are in excellent agreement. The donor-substituted compound DMA-DPPQ exhibits a charge transfer fluorescence in polar solvents. Additionally, dual fluorescence appears in polar protic solvents. The nature of the charge transfer state is discussed with respect to the the TICT model.

The long chain Mannich bases, especially with the piperidine and morpholine groups, display very promising antimicrobial activity. In order to extend our knowledge on their impact on biological systems, we examined the interactions of the 5-pentadecyl-2-((piperidin-1-yl)methyl)phenol (PPDP) with model lipid membrane by means of differential scanning calorimetry (DSC) and fluorescence measurements. The small unilamellar vesicles of dipalmitoylophosphatidylcholine (DPPC) with different piperidine Mannich base concentration were investigated as a function of the increase of temperature. The phase separation accompanied by the rise of the transition enthalpy of both subcomponents, the increase of the function of the GP values of Laurdan versus the wavelength of excitation in the gel phase of PPDP/DPPC systems, and no remarkable differences in the fluorescence anisotropy of PPDP molecules in lipid environment for different mixtures of PPDP/DPPC was observed. Additionally, it was shown that PPDP itself interdigitated in solid state.

Upon infection, many RNA viruses reorganize their capsid for release of the genome into the host cell cytosol for replication. Often, this process is triggered by receptor binding and/or by the acidic environment in endosomes. In the genus Enterovirus, which includes more than 150 human rhinovirus (HRV) serotypes causing the common cold, there is persuasive evidence that the viral RNA exits single-stranded through channels formed in the protein shell. We have determined the time-dependent emergence of the RNA ends from HRV2 on incubation of virions at 56°C using hybridization with specific oligonucleotides and detection by fluorescence correlation spectroscopy. We report that psoralen UV crosslinking prevents complete RNA release, allowing for identification of the sequences remaining inside the capsid. We also present the structure of uncoating intermediates in which parts of the RNA are condensed and take the form of a rod that is directed roughly towards a two-fold icosahedral axis, the presumed RNA exit point. Taken together, in contrast to schemes frequently depicted in textbooks and reviews, our findings demonstrate that exit of the RNA starts from the 3′-end. This suggests that packaging also occurs in an ordered manner resulting in the 3′-poly-(A) tail becoming located close to a position of pore formation during conversion of the virion into a subviral particle. This directional genome release may be common to many icosahedral non-enveloped single-stranded RNA viruses., Author Summary Viral infection requires safe transfer of the viral genome from within the protective protein shell into the host cell's cytosol. For many viruses this happens after uptake into endosomes, where receptor-binding and/or the acidic pH trigger conformational modifications or disassembly of the shell, allowing the nucleic acids to escape. For example, common cold viruses are converted into subviral particles still containing the single-stranded positive sense RNA genome; subsequently, the RNA escapes into the cytoplasm, leaving behind empty capsids. We triggered this process by heating HRV2 to 56°C and found that 3′- and 5′-end emerged with different kinetics. Crosslinking prevented complete RNA egress and upon nuclease digestion only sequences derived from the 5′-end were protected. Part of the RNA remaining within the viral shell adopted a rod-like shape pointing towards one of the two-fold axes where the RNA is presumed to exit in single-stranded form. Egress thus commences with the poly-(A) tail and not with the genome-linked peptide VPg. This suggests that assembly and uncoating are well-coordinated to avoid tangling, kinetic traps, and/or simultaneous exit of the two RNA ends at different sites.

The influence of solvation on α-helix formation is studied for a 14-residue homoalanine and a 16-residue alanine-based polypeptide, in aqueous solution. Four variants of Dynamic Monte Carlo (DMC) simulations, using a multiple annealing procedure are performed. The results obtained under in vacuo conditions are compared to simulations considering either contributions to the solvation energy of non-favourable solvent interactions by hydrophobic groups, or the total solvation energy, both calculated by a continuum approximation. The highest average helix lengths are found when the total solvation energy (hydrophilic and hydrophobic contributions) is considered, although solvation counteracts the formation of compact peptide structures. The influence of solvation on helix formation is discussed.