Modulation of excited state intramolecular proton transfer and intramolecular charge transfer pathways of symmetrical azines through micellar medium.

The photophysical studies of fluorescent probes in micellar medium can give a better insight about their interaction with biological membranes. This study attempts to access the photophysical properties of the dual emitting azine based probe diethylamino salicylidene azine dimer (DEASAD) in micellar media. DEASAD showed dual charge transfer emission due to the presence of open enol (480 nm) and closed enol (510 nm) forms in polar protic solvents. Upon increasing the concentration of ionic surfactants, there is a significant increase in the emission intensity of both the enol forms of DEASAD until premicellar concentration. After micellization, occurrence of a new anomalous keto form emission through excited state intramolecular proton transfer (ESIPT) was observed around 530 nm in ionic micelles and its intensity changes depend on the micellar surface charge. The emission studies revealed the position and interaction of DEASAD with the charge of micellar stern layer as confirmed through interaction of metal ion with the probe and control molecules with and without ESIPT and ICT moieties. In contrast, the new anomalous longer wavelength keto form of DEASAD emission was absent in neutral micelles like Triton X-100.

Synergistic dynamics of photoionization and photoinduced electron transfer probed by laser flash photolysis and ultrafast fluorescence spectroscopy.

Photoionization (PI) and photoinduced electron transfer (PET) dynamics of coumarin 450 (C450) in micelles were investigated in the time domains of micro to femtoseconds using steady-state and time-resolved absorption and fluorescence spectroscopy. The PI of C450 occurs inside the micelles leads to the formation of C450 cation radical (CR) and hydrated electron, which is characterized by the respective transient absorption. The PI of C450 is monophotonic in nature and the yield is dependent on the charge of the micelles. The observation of amine CR in the transient absorption confirms the PET from amine to the excited state of C450 in micelles, which results in the quenching of both fluorescence intensity and lifetime. The decrease in femtosecond fluorescent decay of C450 and the absence of transient C450 radical anion in the presence of amine implies that the concerted ultrafast PET promoted PI and PET to the C450 CR-electron pair. The decrease in the time constant for the formation of relaxed state in the presence of amines is due to the ultrafast PET to the C450 CR-electron pair, which prevents the formation of a relaxed state through recombination at a longer time scale. In the present investigation, the recombination dynamics of the CR-electron pair is justified as one of the origins of the slow solvation in micelles. The influence of amine concentration on the decay of C450 CR indicates ET reaction between C450 CR and amine, which is further confirmed by the bleach recovery of C450 ground state in the presence of amine.

"Turn-on" unsymmetrical azine based fluorophore for the selective detection of diethylchlorophosphate via photoinduced electron transfer to intramolecular charge transfer pathway.

The detection of chemical warfare agents (CWAs) in a highly selective, sensitive and speedy manner is essential for public safety in the case of terrorist attacks and achieving this is a challenging task. This study involves in developing a new unsymmetrical azine based fluorophore 4-((E)-(((E)-2-methoxybenzylidene)hydrazono)methyl)benzonitrile[A1] which shows high selectivity and sensitivity to the nerve agent mimic molecule, diethylchlorophosphate (DCP) through fluorescence switch on mechanism. In a fascinating manner, DCP sensing by A1 operates via solvent dependent optical output mechanisms. In the absence of DCP, the fluorescence of A1 was in the off state through photoinduced electron transfer process. In the presence of DCP, a nucleophilic substitution reaction occurs at the imine nitrogen is closer to the anisole moiety that results in the formation of a new intramolecular charge transfer state along with fluorescence enhancement. In acetonitrile, A1 shows 1763-fold fluorescence enhancement in the presence of DCP with a detection limit of 9.86 nM. In Acetonitrile/water (2:8) mixture, protonation at the imine nitrogen leads to 1188-fold fluorescence enhancement. The sensing mechanisms are confirmed by both experimental and time dependent density functional theoretical studies.

Simple C3-symmetric triaminoguanidine-triphenylamine conjugate as an efficient colorimetric sensor for Cu(II) and fluorescent sensor for Fe(III) ions.

The design and construction of novel C3-symmetric triaminoguanidine-triphenylamine conjugate (L) has been demonstrated and it displays positive solvatochromic behaviour with an increase in solvent polarity. The probe L acts as a selective colorimetric sensor for Cu2+ ions over other metal ions. Further, it shows high selective and sensitive detection of Fe3+ ions through turn-on fluorescence response. Moreover, the detection limits for Cu2+ and Fe3+ ions were found to be within the allowable range of the World Health Organisation (30 µM). The real-time application of the probe was showed by paper strip experiments as well as detection of Fe3+ ions in pharmaceutical tablets.

Synthesis and Characterization of Magnetic Superadsorbent Fe3O4-PEG-Mg-Al-LDH Nanocomposites for Ultrahigh Removal of Organic Dyes.

Considering the huge demands for economical and reliable eco-remediation applications, the goal of the present work is to synthesize cost-effective and functionally efficient magnetic layered nanocomposite adsorbents for the effective adsorption of dyes followed by easy separation from wastewater. This would ensure good reusability of adsorbents without altering its adsorption capacity in a relatively short time manner. To achieve this, different molecular weights of polyethylene glycol (PEG)-modified Fe3O4 combined with Mg-Al-layered double hydroxides (MAN-LDH) were synthesized and characterized using powder X-ray diffraction, Fourier transform infrared, scanning electron microscopy, transmission electron microscopy, thermogravimetric analysis, differential thermal analysis, energy-dispersive X-ray, and inductively coupled plasma optical emission spectroscopy. The efficacy of various adsorption parameters for the removal of methyl orange (MO) from water using Fe3O4-PEG-Mg-Al-LDH (FPL) adsorbents with different molecular weights of PEG (2FPL, 4FPL, and 6FPL) were investigated, and the results were compared. The maximum adsorption capacities of 2FPL, 4FPL, and 6FPL for MO were found to be 775.19, 826.44, and 833.33 mg/g, respectively. Detailed adsorption studies confirm that the higher adsorption capacity of 6FPL is due to the fast exchange of anions (NO3 -) by MO in the interlayers of MAN-LDH, larger surface area, hydrogen bonding, and electrostatic interaction between adsorbate and adsorbent. The thermodynamic data indicate that the adsorption behavior is spontaneous and endothermic in nature. The reusability of all FPL adsorbents is observed to be excellent. The MAN-LDH recoated after the 31st-cycle nanocomposites show a recovery of 100% adsorption efficiency, similar to the freshly prepared 6FPL. Such systematic studies greatly help in advancing the applications of newly functionalized nanomaterials toward eco-remediation approaches.

D-π-A azine based AIEgen with solvent dependent response towards a nerve agent.

We developed a D-π-A based unsymmetrical azine molecule 4-((E)-((E)-(4-(dipropylamino)benzylidene)hydrazono)methyl)benzonitrile [DPBN] and studied its optical and aggregation induced emission properties. The DPBN molecule shows good aggregation induced emission (AIE) behaviour with 1157-fold fluorescence enhancement in the aggregated state. In addition to that, both colorimetric as well as fluorometric sensing studies revealed that DPBN selectively detects diethylchlorophosphate (DCP), a potent nerve agent. Interestingly, DPBN shows solvent dependent optical output in the presence of DCPvia two different mechanisms. In the monomer state, it shows red shifted fluorescence enhancement along with color change from colorless to orange color via the formation of a new intramolecular charge transfer state in pure tetrahydrofuran (THF). In the aggregated state, DPBN shows blue shifted emission with fluorescence enhancement in THF-water mixture by protonation at the amine nitrogen centre. Thus, DPBN can be used as a diagnostic measure to selectively detect nerve agents like DCP. This study also paves the way for further development of molecular probes for nerve agents that would represent immense implications in various fields of chemistry and biology.

Photonic modulation of epidermal growth factor receptor halts receptor activation and cancer cell migration.

Epidermal growth factor receptor (EGFR) plays a key role in regulating cell survival, proliferation and migration, and its overexpression and activation has been correlated with cancer progression. Cancer therapies targeting EGFR have been applied in the clinic with some success. We show, by confocal microscopy analysis, that illumination of adenocarcinomic human alveolar basal epithelial cells (Human A549-EGFR biosensor cell line) with 280 nm at irradiance levels up to 20 times weaker than the Ultraviolet B (UVB) solar output for short periods of time (15-45 minutes) prevents epidermal growth factor-mediated activation of EGFR located on the cell membrane, preventing or reducing cellular disaggregation, formation of filopodia and cell migration. This effect of Ultraviolet (UV) light illumination was confirmed further in a functional scratch assay, and shown to be more effective than that of a specific EGFR-signaling inhibitor. This new photonic approach may be applicable to the treatment of various types of cancer, alone or in combination with other therapies.

Piezoflurochromism and Aggregation Induced Emission Properties of 9, 10-bis (trisalkoxystyryl) Anthracene Derivatives.

We report the synthesis of trisalkoxy substituted 9, 10-bis styrylanthracene derivatives (C8-ant and C12-ant) by Heck coupling with very good yield and their photophysical properties. Both C8-ant and C12-ant exhibit aggregation induced emission (AIE), mechnoflurochromism and thermochromism. Trisubstituted 9, 10-distyrylanthracene molecules having all the luminescent properties in a single molecule are first of its kind.

Development of elastin-like recombinamer films with antimicrobial activity.

In the present work we explored the ABP-CM4 peptide properties from Bombyx mori for the creation of biopolymers with broad antimicrobial activity. An antimicrobial recombinant protein-based polymer (rPBP) was designed by cloning the DNA sequence coding for ABP-CM4 in frame with the N-terminus of the elastin-like recombinamer consisting of 200 repetitions of the pentamer VPAVG, here named A200. The new rPBP, named CM4-A200, was purified via a simplified nonchromatographic method, making use of the thermoresponsive behavior of the A200 polymer. ABP-CM4 peptide was also purified through the incorporation of a formic acid cleavage site between the peptide and the A200 sequence. In soluble state the antimicrobial activity of both CM4-A200 polymer and ABP-CM4 peptide was poorly effective. However, when the CM4-A200 polymer was processed into free-standing films high antimicrobial activity against Gram-positive and Gram-negative bacteria, yeasts and filamentous fungi was observed. The antimicrobial activity of CM4-A200 was dependent on the physical contact of cells with the film surface. Furthermore, CM4-A200 films did not reveal a cytotoxic effect against both normal human skin fibroblasts and human keratinocytes. Finally, we have developed an optimized ex vivo assay with pig skin demonstrating the antimicrobial properties of the CM4-A200 cast films for skin applications.

Photonic activation of plasminogen induced by low dose UVB.

Activation of plasminogen to its active form plasmin is essential for several key mechanisms, including the dissolution of blood clots. Activation occurs naturally via enzymatic proteolysis. We report that activation can be achieved with 280 nm light. A 2.6 fold increase in proteolytic activity was observed after 10 min illumination of human plasminogen. Irradiance levels used are in the same order of magnitude of the UVB solar irradiance. Activation is correlated with light induced disruption of disulphide bridges upon UVB excitation of the aromatic residues and with the formation of photochemical products, e.g. dityrosine and N-formylkynurenine. Most of the protein fold is maintained after 10 min illumination since no major changes are observed in the near-UV CD spectrum. Far-UV CD shows loss of secondary structure after illumination (33.4% signal loss at 206 nm). Thermal unfolding CD studies show that plasminogen retains a native like cooperative transition at ~70 ºC after UV-illumination. We propose that UVB activation of plasminogen occurs upon photo-cleavage of a functional allosteric disulphide bond, Cys737-Cys765, located in the catalytic domain and in van der Waals contact with Trp761 (4.3 Å). Such proximity makes its disruption very likely, which may occur upon electron transfer from excited Trp761. Reduction of Cys737-Cys765 will result in likely conformational changes in the catalytic site. Molecular dynamics simulations reveal that reduction of Cys737-Cys765 in plasminogen leads to an increase of the fluctuations of loop 760-765, the S1-entrance frame located close to the active site. These fluctuations affect the range of solvent exposure of the catalytic triad, particularly of Asp646 and Ser74, which acquire an exposure profile similar to the values in plasmin. The presented photonic mechanism of plasminogen activation has the potential to be used in clinical applications, possibly together with other enzymatic treatments for the elimination of blood clots.

Modulating the structure of EGFR with UV light: new possibilities in cancer therapy.

The epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptor tyrosine kinases. EGFR is activated upon binding to e.g. epidermal growth factor (EGF), leading to cell survival, proliferation and migration. EGFR overactivation is associated with tumor progression. We have previously shown that low dose UVB illumination of cancer cells overexpressing EGFR prior to adding EGF halted the EGFR signaling pathway. We here show that UVB illumination of the extracellular domain of EGFR (sEGFR) induces protein conformational changes, disulphide bridge breakage and formation of tryptophan and tyrosine photoproducts such as dityrosine, N-formylkynurenine and kynurenine. Fluorescence spectroscopy, circular dichroism and thermal studies confirm the occurrence of conformational changes. An immunoassay has confirmed that UVB light induces structural changes in the EGF binding site. A monoclonal antibody which competes with EGF for binding sEGFR was used. We report clear evidence that UVB light induces structural changes in EGFR that impairs the correct binding of an EGFR specific antibody that competes with EGF for binding EGFR, confirming that the 3D structure of the EGFR binding domain suffered conformational changes upon UV illumination. The irradiance used is in the same order of magnitude as the integrated intensity in the solar UVB range. The new photonic technology disables a key receptor and is most likely applicable to the treatment of various types of cancer, alone or in combination with other therapies.

A thermostable Salmonella phage endolysin, Lys68, with broad bactericidal properties against gram-negative pathogens in presence of weak acids.

Resistance rates are increasing among several problematic Gram-negative pathogens, a fact that has encouraged the development of new antimicrobial agents. This paper characterizes a Salmonella phage endolysin (Lys68) and demonstrates its potential antimicrobial effectiveness when combined with organic acids towards Gram-negative pathogens. Biochemical characterization reveals that Lys68 is more active at pH 7.0, maintaining 76.7% of its activity when stored at 4°C for two months. Thermostability tests showed that Lys68 is only completely inactivated upon exposure to 100°C for 30 min, and circular dichroism analysis demonstrated the ability to refold into its original conformation upon thermal denaturation. It was shown that Lys68 is able to lyse a wide panel of Gram-negative bacteria (13 different species) in combination with the outer membrane permeabilizers EDTA, citric and malic acid. While the EDTA/Lys68 combination only inactivated Pseudomonas strains, the use of citric or malic acid broadened Lys68 antibacterial effect to other Gram-negative pathogens (lytic activity against 9 and 11 species, respectively). Particularly against Salmonella Typhimurium LT2, the combinatory effect of malic or citric acid with Lys68 led to approximately 3 to 5 log reductions in bacterial load/CFUs after 2 hours, respectively, and was also able to reduce stationary-phase cells and bacterial biofilms by approximately 1 log. The broad killing capacity of malic/citric acid-Lys68 is explained by the destabilization and major disruptions of the cell outer membrane integrity due to the acidity caused by the organic acids and a relatively high muralytic activity of Lys68 at low pH. Lys68 demonstrates good (thermo)stability properties that combined with different outer membrane permeabilizers, could become useful to combat Gram-negative pathogens in agricultural, food and medical industry.

Kinetics of cyclobutane thymine dimer splitting by DNA photolyase directly monitored in the UV.

CPD photolyase uses light to repair cyclobutane pyrimidine dimers (CPDs) formed between adjacent pyrimidines in UV-irradiated DNA. The enzyme harbors an FAD cofactor in fully reduced state (FADH(-)). The CPD repair mechanism involves electron transfer from photoexcited FADH(-) to the CPD, splitting of its intradimer bonds, and electron return to restore catalytically active FADH(-). The two electron transfer processes occur on time scales of 10(-10) and 10(-9) s, respectively. Until now, CPD splitting itself has only been poorly characterized by experiments. Using a previously unreported transient absorption setup, we succeeded in monitoring cyclobutane thymine dimer repair in the main UV absorption band of intact thymine at 266 nm. Flavin transitions that overlay DNA-based absorption changes at 266 nm were monitored independently in the visible and subtracted to obtain the true repair kinetics. Restoration of intact thymine showed a short lag and a biexponential rise with time constants of 0.2 and 1.5 ns. We assign these two time constants to splitting of the intradimer bonds (creating one intact thymine and one thymine anion radical T(∘-)) and electron return from T(∘-) to the FAD cofactor with recovery of the second thymine, respectively. Previous model studies and computer simulations yielded various CPD splitting times between < 1 ps and < 100 ns. Our experimental results should serve as a benchmark for future efforts to model enzymatic photorepair. The technique and methods developed here may be applied to monitor other photoreactions involving DNA.

Effect of the bases flanking an abasic site on the recognition of nucleobase by amiloride.

BACKGROUND: We explain here the various non-covalent interactions which are responsible for the different binding modes of a small ligand with DNA. METHODS: The combination of experimental and theoretical methods was used. RESULTS: The interaction of amiloride with thymine was found to depend on the bases flanking the AP site and different binding modes were observed for different flanking bases. Molecular modeling, absorption studies and binding constant measurements support for the different binding patterns. The flanking base dependent recognition of AP site phosphates was investigated by (31)P NMR experiments. The thermodynamics of the ligand-nucleotide interaction was demonstrated by isothermal titration calorimetry. The emission behavior of amiloride was found to depend on the bases flanking the AP site. Amiloride photophysics in the context of AP-site containing DNA is investigated by time-dependent density functional theory. CONCLUSIONS: Flanking bases affect the ground and excited electronic states of amiloride when binding to AP site, which causes flanking base-dependent fluorescence signaling. GENERAL SIGNIFICANCE: The various noncovalent interactions have been well characterized for the determination of nucleic acid structure and dynamics, and protein-DNA interactions. However, these are not clear for the DNA-small molecule interactions and we believe that our studies will bring a new insight into such phenomena.

NBD-based green fluorescent ligands for typing of thymine-related SNPs by using an abasic site-containing probe DNA.

The binding behavior of green fluorescent ligands, derivatives of 7-nitrobenzo-2-oxa-1,3-diazole (NBD), with DNA duplexes containing an abasic (AP) site is studied by thermal denaturation and fluorescence experiments. Among NBD derivatives, N(1)-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)propane-1,3-diamine (NBD-NH(2)) is found to bind selectively to the thymine base opposite an AP site in a DNA duplex with a binding affinity of 1.52 x 10(6) M(-1). From molecular modeling studies, it is suggested that the NBD moiety binds to thymine at the AP site and a protonated amino group tethered to the NBD moiety interacts with the guanine base flanking the AP site. Green fluorescent NBD-NH(2) is successfully applied for simultaneous G>T genotyping of PCR amplification products in a single cuvette in combination with a blue fluorescent ligand, 2-amino-6,7-dimethyl-4-hydroxypteridine (diMe-pteridine).

DNA repair by photolyase: a novel substrate with low background absorption around 265 nm for transient absorption studies in the UV.

CPD photolyase enzymatically repairs the major UV-induced lesion in DNA, the cyclobutane pyrimidine dimer (CPD), by photoreversion of the damage reaction. An enzyme-bound reduced flavin (FADH(-)) cofactor functions as photosensitizer. Upon excitation, it transiently transfers an electron to the CPD, triggering scission of the interpyrimidine bonds. After repair completion, the electron returns to the flavin to restore its functional reduced form. A major difficulty for time-resolved spectroscopic monitoring of the enzymatic repair reaction is that absorption changes around 265 nm accompanying pyrimidine restoration are obscured by the strong background absorption of the nondimerized bases in DNA. Here we present a novel substrate for CPD photolyase that absorbs only weakly around 265 nm: a modified thymidine 10-mer with a central CPD and all bases, except the one at the 3' end, replaced by 5,6-dihydrothymine which virtually does not absorb around 265 nm. Repair of this substrate by photolyases from Anacystis nidulans and from Escherichia coli was compared with repair of two conventional substrates: a 10-mer of unmodified thymidines containing a central CPD and an acetone-sensitized thymidine 18-mer that contained in average six randomly distributed CPDs per strand. In all cases, the novel substrate was repaired with an efficiency very similar to that of the conventional substrates (quantum yields in the order of 0.5 upon excitation of FADH(-)). Flash-induced transient absorption changes at 267 nm could be recorded on a millisecond time scale with a single subsaturating flash and yielded very similar signals for all three substrates. Because of its low background absorption around 265 nm and the defined structure, the novel substrate is a promising tool for fast and ultrafast transient absorption studies on pyrimidine dimer splitting by CPD photolyase.

Quantum yield measurements of short-lived photoactivation intermediates in DNA photolyase: toward a detailed understanding of the triple tryptophan electron transfer chain.

The light-dependent DNA repair enzyme photolyase contains a unique evolutionary conserved triple tryptophan electron transfer chain (W382-W359-W306 in photolyase from E. coli) that bridges the approximately 15 A distance between the buried flavin adenine dinucleotide (FAD) cofactor and the surface of the protein. Upon excitation of the semireduced flavin (FADH(o)), electron transfer through the chain leads to formation of fully reduced flavin (FADH(-); required for DNA repair) and oxidation of the most remote tryptophan residue W306, followed by its deprotonation. The thus-formed tryptophanyl radical W306(o)(+) is reduced either by an extrinsic reductant or by reverse electron transfer from FADH(-). Altogether the kinetics of these charge transfer reactions span 10 orders of magnitude, from a few picoseconds to tens of milliseconds. We investigated electron transfer processes in the picosecond-nanosecond time window bridging the time domains covered by ultrafast pump-probe and "classical" continuous probe techniques. Using a recent dedicated setup, we directly show that virtually no absorption change between 300 ps and 10 ns occurs in wild-type photolyase, implying that no charge recombination takes place in this time window. In contrast, W306F mutant photolyase showed a partial absorption recovery with a time constant of 0.85 ns. In wild-type photolyase, the quantum yield of FADH(-) W306(o)(+) was found at 19 +/- 4%, in reference to the established quantum yield of the long-lived excited state of [Ru(bpy)(3)](2+). With this yield, the optical spectrum of the excited state of FADH(o) can be constructed from ultrafast spectroscopic data; this spectrum is dominated by excited state absorption extending from below 450 to 850 nm. The new experimental results, taken together with previous data, allow us to propose a detailed kinetic and energetic scheme of the electron transfer chain.

Use of ruthenium dyes for subnanosecond detector fidelity testing in real time transient absorption.

Transient absorption spectroscopy is a powerful tool for the study of photoreactions on time scales from femtoseconds to seconds. Typically, reactions slower than approximately 1 ns are recorded by the "classical" technique; the reaction is triggered by an excitation flash, and absorption changes accompanying the reaction are recorded in real time using a continuous monitoring light beam and a detection system with sufficiently fast response. The pico- and femtosecond region can be accessed by the more recent "pump-probe" technique, which circumvents the difficulties of real time detection on a subnanosecond time scale. This is paid for by accumulation of an excessively large number of shots to sample the reaction kinetics. Hence, it is of interest to extend the classical real time technique as far as possible to the subnanosecond range. In order to identify and minimize detection artifacts common on a subnanosecond scale, like overshoot, ringing, and signal reflections, rigorous testing is required of how the detection system responds to fast changes of the monitoring light intensity. Here, we introduce a novel method to create standard signals for detector fidelity testing on a time scale from a few picoseconds to tens of nanoseconds. The signals result from polarized measurements of absorption changes upon excitation of ruthenium complexes {[Ru(bpy)(3)](2+) and a less symmetric derivative} by a short laser flash. Two types of signals can be created depending on the polarization of the monitoring light with respect to that of the excitation flash: a fast steplike bleaching at magic angle and a monoexponentially decaying bleaching for parallel polarizations. The lifetime of the decay can be easily varied via temperature and viscosity of the solvent. The method is applied to test the performance of a newly developed real time transient absorption setup with 300 ps time resolution and high sensitivity.