Steady-state and time-resolved investigations of a crown thioether conjugated with methylacridinium and its complexes with metal ions.

The crown thioether 9-[4-(4,7,10,13-tetrathia-1-azacyclopentadecyl]phenyl-N-methylacridinium perchlorate (TCMA) was synthesized and characterized with the aim to verify its ability to interact selectively with metal ions and substantiate the possibility to detect easily the presence of heavy metals in fluid samples. The spectroscopic properties of TCMA, alone and in the presence of metal ions, were therefore studied in polar solvents (MeCN and H(2)O); in particular, steady-state UV-Vis spectrophotometric and fluorimetric techniques were used together with transient absorption spectroscopy with fs time resolution to investigate the spectral and dynamic properties of the lowest excited singlet state of TCMA and of TCMA/metal ion complexes. The absorption in the Vis region is characterized by a charge-transfer nature with the methylacridinium moiety acting as the electron-acceptor and the anilic group as the electron-donor. No emission from the S(1) was detected both in MeCN and H(2)O, while a small S(2)→ S(0) fluorescence emission (λ(max) = 485 nm and ϕ(F) = 0.0011) was detected in water. Time-resolved measurements with fs resolution of TCMA in MeCN have shown that the relaxed S(1) state is reached ∼0.6 ps after the laser pulse, while the S(1)→ S(0) time constant is 3.7 ps. Among the investigated metal ions, only Fe(3+) (in MeCN) and Hg(2+) (in MeCN and H(2)O) were able to form stable complexes (association constant, K(ass) = 1-11 × 10(4) M(-1)) with TCMA. The S(1) state of the TCMA/M(n+) complexes emits with low quantum yield (ϕ(F) = 0.0023-0.014) and decays with time constants much longer than TCMA itself, at least in the case of TCMA/Hg(2+) in MeCN. This study showed that TCMA is a good candidate for colorimetric/fluorimetric sensing of Hg(2+) in aqueous media owing to its high selectivity towards metal ions.

Photophysics of aromatic thiourea derivatives and their complexes with anions. Fast and ultrafast spectroscopic investigations.

In order to collect detailed information on the interaction mechanism between fluorescent thiourea derivatives and anions, 9-[4-(trifluoromethyl)phenylthioureidomethyl]anthracene (1) and the corresponding 10-cyanoanthracene derivative (2) were synthesized and investigated in DMSO and MeCN by using absorption and emission steady state techniques, both in the absence and in the presence of different anions (AcO(-), H(2)PO(4)(-), HSO(4)(-), and Br(-)). A wide examination of the mechanism of anion recognition was also performed by time-resolved transient absorption spectroscopy, with nano- and femto-second time resolution. A complete picture of the excited state deactivation pathways of 1 and 2, where the main operative processes were fluorescence, intersystem crossing and internal conversion, was obtained. Even if steady-state measurements suggest that 1 and 2 selectively interact in the ground state with the anions H(2)PO(4)(-) and AcO(-), time-resolved investigations demonstrate that the substrates are able to complex all the four anions. The photophysics of such complexes was fully characterized. The anions mainly modify the lifetime of the lowest excited singlet state and, especially in the cases of H(2)PO(4)(-) and AcO(-), the efficiencies of fluorescence emission and triplet formation. In particular, no evidence was found of further deactivation processes such as photoinduced electron transfer, photodissociation, and photoionization.

Characterization of excited states of quinones and identification of their deactivation pathways.

Femtosecond time-resolved transient absorption was used to investigate the properties of the excited states of p-benzoquinone derivatives: ubiquinone 0 (UQ(0)), thermoquinone (TQ), and tetramethylbenzoquinone (TMBQ). To elucidate details of the deactivation pathways of quinones, experiments were carried out in solvents with different polarity by exciting the samples at 400 and 266 nm. The measurements carried out upon excitation centered at 400 nm pointed out that the S(1) --> T(1) intersystem crossing (ISC) is operative only for TMBQ, where the T(2)(pi,pi*) state is likely below S(1). For UQ(0) and TQ, this decay process is not efficient because of the high energy of T(2) located above S(1). Instead, the lowest triplet state of UQ(0) was detected upon excitation at 266 nm, showing the involvement of an upper excited singlet state (S(n)) in the ISC process.

Photophysical properties of quinolizinium salts and their interactions with DNA in aqueous solution.

The photophysical properties of three quinolizinium salts (naphto[2,1-b]quinolizinium bromide (Q2), naphto[1,2-b]quinolizinium bromide (Q3), and indolo[2,3-b]quinolizinium tetrafluoroborate (HI)) in fluid media and their interactions with DNA were investigated by steady-state and by nanosecond and femtosecond time-resolved techniques. The main decay pathways of the excited singlet state S(1), fluorescence, intersystem crossing, and internal conversion, were characterized in terms of quantum yields and rate constants. The lowest triplet state of the quinolizinium salts is able to sensitize singlet oxygen in rather high efficiency (phi(Delta) = 0.4 to 0.5 in MeCN). The complexes between quinolizinium salts and DNA formed in the ground state were characterized in terms of lifetimes and decay channels to give more details of the mechanism of photoinduced DNA strand break.

Photophysical properties of quinones and their interaction with the photosynthetic reaction centre.

Photophysical properties of tetramethyl-1,4-benzoquinone (TMBQ) and 2,6-dimethoxy-1,4-benzoquinone (DMOBQ) in solution and their interactions with the photosynthetic reaction centre (RC) isolated from the photosynthetic bacterium Rhodobacter sphaeroides have been investigated in this work. For these two benzoquinone derivatives an efficient ISC process which leads to the population of the lowest triplet state of the molecules upon direct excitation was observed. The presence of RC does not alter the properties of the triplet state of DMOBQ suggesting that interactions are negligible; on the other side RC efficiently quenched the T1 state of TMBQ. The behavior is rationalized in terms of redox potentials of quinones and kinetic characteristics of their transients.

Photophysical properties and photobiological behavior of amodiaquine, primaquine and chloroquine.

This article describes the results of a coupled photophysical and photobiological study aimed at understanding the phototoxicity mechanism of the antimalarial drugs amodiaquine (AQ), primaquine (PQ) and chloroquine (CQ). Photophysical experiments were carried out in aqueous solutions by steady-state and time-resolved spectrometric techniques to obtain information on the different decay pathways of the excited states of the drugs and on the transient species formed upon laser irradiation. The results showed that all three drugs possess very low fluorescence quantum yields (10(-2)-10(-4)). Laser flash photolysis experiments proved the occurrence of photoionization processes leading to the formation of a radical cation in all three systems. In the case of AQ the lowest triplet state was also detected. Together with the photophysical properties the photobiological properties of the antimalarial drugs were investigated under UV irradiation, on various biological targets through a series of in vitro assays. Phototoxicity on mouse 3T3 fibroblast and human keratinocyte cell lines NCTC-2544 was detected for PQ and CQ but not for AQ. In particular, PQ- and CQ-induced apoptosis was revealed by the externalization of phosphatidylserine. Furthermore, upon UV irradiation, the drugs caused significant variations of the mitochondrial potential (Deltapsi(mt)) measured by flow cytometry. The photodamages produced by the drugs were also evaluated on proteins, lipids and DNA. The combined approaches were useful in understanding the mechanism of phototoxicity induced by these antimalarial drugs.

DNA cleavage induced by photoexcited antimalarial drugs: a photophysical and photobiological study.

The interactions and the photosensitizing activity of three antimalarial drugs quinine (Q), mefloquine (MQ) and quinacrine (QC) toward DNA was studied. Evidences obtained by absorption and emission spectroscopy and by linear dichroism measurements indicate that these derivatives bind the macromolecule with a high affinity (binding constants Ka approximately 10(5) M(-1)). The absorption characteristics of the drugs changed markedly by addition of DNA and their fluorescence was quenched with rate constants higher than that of diffusion. The geometry of binding involves predominantly the intercalation into the double helix. The DNA photocleavage properties of antimalarials was investigated using plasmid DNA as a model, at different [drug]/ [DNA] ratios. The results indicate that mainly MQ and Q are able to induce significant photodamage to DNA. In particular the marked effect of the former drug is evidenced after treatment of photosensitized DNA by two base excision repair enzymes, formamydo-pyrimidine glycosilase (Fpg) and Endonuclease III (Endo III). From a mechanistic point of view, experiments carried out in different experimental conditions indicate that these drugs photoinduce DNA damage through singlet oxygen and/or radical cation production. These findings are further supported by the determination of two photoproducts of 2'-deoxyguanosine, which are diagnostic for Type I and Type II pathways, namely 2,2-diamino(2-deoxy-beta-D-erythro-pentofuranosyl)-4-amino-5(2H)-oxazolone and (R,S)4-hydroxy-8-oxo-4,8-dihydro-2'-deoxyguanosine (4-OH-8-oxo-dGuo). Laser flash photolysis experiments carried out in the presence of DNA indicates that the excitation produces mainly the triplet state for Q and the triplet and radical cation for QC. Moreover the singlet and triplet states and radical cations of the drugs are quenched by 2'-deoxyguanosine monophosphate. The absorbances of these transients decrease with increasing DNA concentration.

Photophysical and photobiological behavior of antimalarial drugs in aqueous solutions.

This article describes the results of a combined photophysical and photobiological study aimed at understanding the phototoxicity mechanism of the antimalarial drugs quinine (Q), quinacrine (QC) and mefloquine (MQ). Photophysical experiments were carried out in aqueous solutions by stationary and time-resolved fluorimetry and by laser flash photolysis to obtain information on the various decay pathways of the excited states of the drugs and on transient species formed on irradiation. The results obtained showed that fluorescence and intersystem crossing account for all the adsorbed quanta for Q and MQ (quantum yield of about 0.1 and 0.9, respectively) and only for 24% in the case of QC, which has a negligible fluorescence quantum yield (0.001). Laser flash photolysis experiments evidenced, for QC and MQ, the occurrence of photoionization processes leading to the formation of the radical cations of the drugs. The effects of tryptophan and histidine on the excited states and transient species of the three drugs were also investigated. In parallel, the photoactivity of the antimalarial drugs was investigated under UV irradiation on various biological targets through a series of in vitro assays in the presence and in the absence of oxygen. Phototoxicity on 3T3 cultured fibroblasts and lipid photoperoxidation were observed for all the drugs. The photodamage produced by the drugs was also evaluated on proteins by measuring the photosensitized cross-linking of spectrin. The combined approaches were proven to be useful for understanding the mechanism of phototoxicity induced by the antimalarial drugs.

Photophysical and phototoxic properties of the antibacterial fluoroquinolones levofloxacin and moxifloxacin.

Two antibacterial fluoroquinolones, levofloxacin and moxifloxacin, were investigated to evaluate their photophysical properties and to explore the mechanism of their phototoxicity. Photophysical experiments were carried out in aqueous solution by stationary and time-resolved fluorimetry, and by laser flash photolysis, to obtain information on the various decay pathways of the excited states of the drugs and on transient species formed upon irradiation. The results obtained show that levofloxacin is able to photosensitize red blood cell lysis in an oxygen-independent way and induce a high decrease in cell viability after UVA irradiation, although to a lesser degree than the racemic mixture ofloxacin. Moxifloxacin, which is an 8-MeO-substituted fluoroquinolone, is less phototoxic than the other compounds. Cellular phototoxicity was inhibited by the addition of superoxide dismutase, catalase, and free radical and hydroxyl radical scavengers (BHA, GSH, mannitol, and DMTU), indicating the involvement of superoxide anion and/or a radical mechanism in their cytotoxicity. A good correlation was observed between lipid peroxidation, protein photodamage, and cellular phototoxicity, indicating that test compounds exert their toxic effects mainly in the cellular membrane. Experiments carried out on pBR322 DNA show that these derivatives do not significantly photocleave DNA directly, but single-strand breaks were evidenced after treatment of photosensitized DNA by two base-excision-repair enzymes, and Endo III.

Intramolecular directional förster resonance energy transfer at the single-molecule level in a dendritic system.

We report on the directional Förster resonance energy transfer (FRET) process taking place in single molecules of a first (T1P4) and a second (T2P8) generation of a perylenemonoimide (P)-terrylenediimide (T)-based dendrimer in which the chromophores are separated by rigid polyphenylene arms. At low excitation powers, single-molecule detection and spectroscopy of T1P4 and T2P8 dendrimers point to a highly efficient directional FRET from P donors to the central T acceptor, optical excitation at 488 nm resulting in exclusively acceptor emission in the beginning of the detected fluorescence intensity. Donor emission is seen only upon the bleaching of the acceptor. High-resolution time-resolved single-molecule fluorescence data measured with a microchannel plate photomultiplier reveal, for T2P8, a broad range of FRET rates as a result of a broad range of distances and orientations experienced by the donor-acceptor dendrimers when immobilized in a polymer matrix. Single-molecule data from T2P8 on 488 nm excitation are indicative for the presence, after terrylenediimide bleaching, of a P-P excited dimer characterized by a broad emission spectrum peaking around 600 nm and by fluctuating fluorescence decay times. At high excitation powers, single T1P4 and T2P8 molecules display simultaneous emission from both donor and acceptor chromophores. The effect, called "exciton blockade", occurs due to the presence of multiple excitations in a single molecule.

Photosensitization of DNA strand breaks by three phenothiazine derivatives.

The interaction and the photosensitizing activity of three phenothiazine derivatives, fluphenazine hydrochloride (FP), thioridazine hydrochloride (TR), and perphenazine (PP), toward DNA were studied. Evidences obtained from various spectroscopic studies such as fluorimetric and linear dichroism measurements indicate that these derivatives bind to the DNA at least in two ways: intercalation and external stacking on the DNA helix, depending on their relative concentrations. Irradiation of supercoiled plasmid DNA in the presence of these phenothiazines leads to single strand breaks. The DNA photocleavage appears to be due to externally bound molecules rather than to those intercalated. The highest photocleavage activity was observed with PP and TR whereas FP was less efficient. The efficiency of the photocleavage in aerated and deaerated solutions does not change thus indicating that an involvement of singlet oxygen can be excluded. Primer extension analysis of plasmid DNA irradiated in the presence of phenothiazines indicates that photocleavage of DNA occurs predominantly at Gua and Cyt residues. Laser flash experiments carried out in the presence of 2'-deoxyguanosine 5'-monophosphate reveal an efficient electron transfer between the nucleotide and the radical cations produced by photoionization of the phenothiazines. In the presence of DNA, an electron transfer process takes place within the laser pulse from the lowest singlet state of phenothiazines to the DNA bases; the time-resolved measurements showed that the back-electron transfer is a negligible decay pathway for the charged species.