Why is HSO5- so effective against bacteria? Insights into the mechanisms of Escherichia coli disinfection by unactivated peroxymonosulfate.

This study examined the antimicrobial efficacy of peroxymonosulfate (PMS) against bacteria, using Escherichia coli (E. coli) as a model organism. Our investigation delineates the complex mechanisms exerted by unactivated PMS. Thus, an initial redox reaction between PMS and the target biomolecules of bacteria generates SO4•- as the pivotal reactive species for bacterial inactivation; to a lesser extent, •OH, 1O2, or O2•- may also participate. Damage generated during oxidation was identified using an array of biochemical techniques. Specifically, redox processes are promoted by PMS and SO4•- targets and disrupt various components of bacterial cells, predominantly causing extracellular damage as well as intracellular lesions. Among these, external events are the key to cell death. Finally, by employing gene knockout mutants, we uncovered the role of specific gene responses in the intracellular damage induced by radical pathways. The findings of this study not only expand the understanding of PMS-mediated bacterial inactivation but also explain the ten-fold higher effectiveness of PMS than that reported for H2O2. Hence, we provide clear evidence that unactivated PMS solutions generate SO4•- in the presence of bacteria, and consequently, should be considered an effective disinfection method.

BASHY Dyes Are Highly Efficient Lipid Droplet-Targeting Photosensitizers that Induce Ferroptosis through Lipid Peroxidation.

Ferroptosis is an iron-dependent lipid-peroxidation-driven mechanism of cell death and a promising therapeutic target to eradicate cancer cells. In this study, we discovered that boronic acid-derived salicylidenehydrazone (BASHY) dyes are highly efficient singlet-oxygen photosensitizers (PSs; ΦΔ up to 0.8) that induce ferroptosis triggered by photodynamic therapy. The best-performing BASHY dye displayed a high phototoxicity against the human glioblastoma multiform U87 cell line, with an IC50 value in the low nanomolar range (4.40 nM) and a remarkable phototoxicity index (PI > 22,700). Importantly, BASHY dyes were shown to accumulate in lipid droplets (LDs) and this intracellular partition was found to be essential for the enhanced phototoxicity and the induction of ferroptosis through lipid peroxidation. The safety and phototoxicity of this platform were validated using an in vivo zebrafish model (Danio rerio).

Synthesis of Bis(amino acids) Containing the Styryl-cyclobutane Core by Photosensitized [2+2]-Cross-cycloaddition of Allylidene-5(4H)-oxazolones.

The irradiation of 2-aryl-4-(E-3'-aryl-allylidene)-5(4H)-oxazolones 1 with blue light (456 nm) in the presence of [Ru(bpy)3](BF4)2 (bpy = 2,2'-bipyridine, 5% mol) gives the unstable cyclobutane-bis(oxazolones) 2 by [2+2]-photocycloaddition of two oxazolones 1. Each oxazolone contributes to the formation of 2 with a different C=C bond, one of them reacting through the exocyclic C=C bond, while the other does so through the styryl group. Treatment of unstable cyclobutanes 2 with NaOMe/MeOH produces the oxazolone ring opening reaction, affording stable styryl-cyclobutane bis(amino acids) 3. The reaction starts with formation of the T1 excited state of the photosensitizer 3[Ru*(bpy)3]2+, which reacts with S0 of oxazolones 1 through energy transfer to give the oxazolone T1 state 3(oxa*)-1, which is the reactive species and was characterized by transient absorption spectroscopy. Measurement of the half-life of 3(oxa*)-1 for 1a, 1b and 1d shows large values for 1a and 1b (10-12 µs), while that of 1d is shorter (726 ns). Density functional theory (DFT) modeling displays strong structural differences in the T1 states of the three oxazolones. Moreover, study of the spin density of T1 state 3(oxa*)-1 provides clues to understanding the different reactivity of 4-allylidene-oxazolones described here with respect to the previously reported 4-arylidene-oxazolones.

Engineering the BASHY Dye Platform toward Architectures with Responsive Fluorescence.

A set of nine boronic-acid-derived salicylidenehydrazone (BASHY) complexes has been synthesized in good to very good chemical yields in a versatile three-component reaction. In an extension to previous reports on this dye platform, the focus was put on the electronic modification of the "vertical" positions of the salicylidenehydrazone backbone. This enabled the observation of fluorescence quenching by photoinduced electron transfer (PeT), which can be reverted by the addition of acid in organic solvent (OFF-ON fluorescence switching). The resulting emission is observed in the green-to-orange spectral region (maxima at 520-590 nm). In contrast, under physiological pH conditions in water, the PeT process is inherently decativated, thereby enabling the observation of fluorescence in the red-to-NIR region (maxima at 650-680 nm) with appreciable quantum yields and lifetimes. The latter characteristic supported the application of the dyes in fluorescence lifetime imaging (FLIM) of live A549 cells.

Stereoselective, Ruthenium-Photocatalyzed Synthesis of 1,2-Diaminotruxinic Bis-amino Acids from 4-Arylidene-5(4H)-oxazolones.

The irradiation of (Z)-2-phenyl-4-aryliden-5(4H)-oxazolones 1 in deoxygenated CH2Cl2 at 25 °C with blue light (465 nm) in the presence of [Ru(bpy)3](BF4)2 (5% mole ratio) as a triplet photocatalyst promotes the [2+2] photocycloaddition of the C═C bonds of the 4-arylidene moiety, thus allowing the completely regio- and stereoselective formation of cyclobutane-bis(oxazolone)s 2 as single stereoisomers. Cyclobutanes 2 have been unambiguously characterized as the µ-isomers and contain two E-oxazolones coupled in an anti-head-to-head form. The use of continuous-flow techniques in microreactors allows the synthesis of cyclobutanes 2 in only 60 min, compared with the 24-48 h required in batch mode. Ring opening of the oxazolone heterocycle in 2 with a base affords the corresponding 1,2-diaminotruxinic bis-amino esters 3, which are also obtained selectively as µ-isomers. The ruthenium complex behaves as a triplet photocatalyst, generating the reactive excited state of the oxazolone via an energy-transfer process. This reactive excited state has been characterized as a triplet diradical 3(E/Z)-1* by laser flash photolysis (transient absorption spectroscopy). This technique also shows that this excited state is the same when starting from either (Z)- or (E)-oxazolones. Density functional theory calculations show that the first step of the [2+2] cycloaddition between 3(E/Z)-1* and (Z)-1 is formation of the C(H)-C(H) bond and that (Z) to (E) isomerization takes place at the 1,4-diradical thus formed.

Visible Light-Gated Organocatalysis Using a RuII -Photocage.

The light-gated organocatalysis via the release of 4-N,N-dimethylaminopyridine (DMAP) by irradiation of the [Ru(bpy)2 (DMAP)2 ]2+ complex with visible light was investigated. As model reaction the acetylation of benzyl alcohols with acetic anhydride was chosen. The pre-catalyst releases one DMAP molecule on irradiation at wavelengths longer than 455 nm. The photochemical process was characterized by steady-state irradiation and ultrafast transient absorption spectroscopy. The latter enabled the observation of the 3 MLCT state and the spectral features of the penta-coordinated intermediate [Ru(bpy)2 (DMAP)]2+ . The released DMAP catalyzes the acetylation of a wide range of benzyl alcohols with chemical yields of up to 99 %. Control experiments revealed unequivocally that it is the released DMAP which takes the role of the catalyst.

Assessing physical properties of amphoteric fluoroquinolones using phosphorescence spectroscopy.

The self-association of fluoroquinolones (FQ) in water would play a relevant role in their translocations across lipid membranes. Triplet excited states of these drugs have been shown as reporters of FQ self-association using laser flash photolysis technique. A study using low-temperature phosphorescence technique was performed with quinolone derivatives such as enoxacin (ENX), norfloxacin (NFX), pefloxacin (PFX), ciprofloxacin (CPX, ofloxacin (OFX), nalidixic acid (NLA), pipemidic acid (PPA) and piromidic acid (PRA) to explore emission changes associated with self-associations and to shed some light on the triplet excited state energy (ET) discrepancies described in the literature for most of these drugs. The emissions obtained at 77 K in buffered aqueous medium revealed that the amphoteric nature of the quinolones CPX, NFX, PFX, ENX, OFX and PPA must generate their self-associations because a redshift of their phosphorescence maxima is produced by FQ concentrations increases. Hence, this effect was not observed for NLA and PRA or when all quinolones were analysed using ethanol or ethylene glycol aqueous mixtures as glassed solvents. Interestingly, the presence of these organic mixtures produced a blue-shift in the phosphorescence emission maximum of each FQ. Additionally, laser flash photolysis experiments with PRA and the amphoteric quinolone PPA, compounds with the same skeleton but different peripheral substituent, confirm the expected correlations between the amphoteric nature of compounds and their self-associations in aqueous media because the excimer generation was only detected for PPA. Now, the discrepancies described in the literature for the ET of FQs can be understood considering that changes of medium polarity or proticity as well as the temperature can considerably modify their ET values. Thereby, low-temperature phosphorescence technique, is an effective way to detect molecular self-associations and surrounding changes in quinolones that opens the possibility to evaluate these effects in other drug families.

Chemical tuning for potential antitumor fluoroquinolones.

Phototoxic effects of 6,8 dihalogenated quinolones confers to this type of molecules a potential property as photochemotherapeutic agents. Two photodehalogenation processes seem to be involved in the remarkable photoinduced cellular damage. In this context, a new 6,8 dihalogenated quinolone 1 (1-methyl-6,8-difluoro-4-oxo-7-aminodimethyl-1,4-dihydroquinoline-3-carboxylic acid) was synthetized looking for improving the phototoxic properties of fluoroquinolones (FQ) and to determine the role of the photodegradation pathways in the FQ phototoxicity. With this purpose, fluorescence emissions, laser flash photolysis experiments and photodegradation studies were performed with compound 1 using 1-ethyl-6,8-difluoro-4-oxo-7-aminodimethyl-1,4-dihidroquinoline-3-carboxylic acid (2) and lomefloxacin (LFX) as reference compounds. The shortening of alkyl chain of the N(1) of the quinolone ring revealed a lifetime increase of the reactive aryl cation generated from photolysis of the three FQ and a significant reduction of the FQ photodegradation quantum yield. The fact that these differences were smaller when the same study was done using a hydrogen donor solvent (ethanol-aqueous buffer, 50/50 v/v) evidenced the highest ability of the reactive intermediate arising from 1 to produce intermolecular alkylations. These results were correlated with in vitro 3T3 NRU phototoxicity test. Thus, when Photo-Irritation-Factor (PIF) was determined for 1, 2 and LFX using cytotoxicity profiles of BALB/c 3T3 fibroblasts treated with each compound in the presence and absence of UVA light, a PIF more higher than 30 was obtained for 1 while the values for 2 and LFX were only higher than 8 and 10, respectively. Thereby, the present study illustrates an approach to modulate the photosensitizing properties of FQ with the purpose to improve the chemotherapeutic properties of antitumor quinolones. Moreover, the results obtained in this study also evidence that the key pathway responsible for the phototoxic properties associated with dihalogenated quinolones is the aryl cation generation.

Electronic and Functional Scope of Boronic Acid Derived Salicylidenehydrazone (BASHY) Complexes as Fluorescent Dyes.

A series of boronic acid derived salicylidenehydrazone (BASHY) complexes was prepared and photophysically characterized. The dye platform can be modified by (a) electronic tuning along the cyanine-type axis via modification of the donor-acceptor pair and (b) functional tuning via the boronic acid residue. On the one hand, approach (a) allows the control of photophysical parameters such as Stokes shift, emission color, and two-photon-absorption (2PA) cross section. The resulting dyes show emission light-up behavior in nonpolar media and are characterized by high fluorescence quantum yields (ca. 0.5-0.7) and brightness (ca. 35000-40000 M-1 cm-1). Moreover, the 2PA cross sections reach values in the order of 200-300 GM. On the other hand, the variation of the dye structure through the boronic acid derived moiety (approach (b)) enables the functionalization of the BASHY platform for a broad spectrum of potential applications, ranging from biorelevant contexts to optoelectronic materials. Importantly, this functionalization is generally electronically orthogonal with respect to the dye's photophysical properties, which are only determined by the electronic structure of the cyanine-type backbone (approach (a)). Rare exceptions to this generalization are the presence of redox-active residues (such a triphenylamine or pyrene). Finally, the advantageous photophysics is complemented by a significant photostability.

Drug-DNA complexation as the key factor in photosensitized thymine dimerization.

The crucial role of photosensitizer@DNA complexation in the formation of cyclobutane pyrimidine dimers (CPDs) has been demonstrated using femtosecond and nanosecond transient absorption and emission measurements in combination with in vitro DNA damage assays. This finding opens the door to re-evaluate the mechanisms involved in CPDs photosensitized by other chemicals.

Biradical vs singlet oxygen photogeneration in suprofen-cholesterol systems.

Cholesterol (Ch) is an important lipidic building block and a target for oxidative degradation, which can be induced via free radicals or singlet oxygen ((1)O2). Suprofen (SP) is a nonsteroidal anti-inflammatory drug that contains the 2-benzoylthiophene (BZT) chromophore and has a π,π* lowest triplet excited state. In the present work, dyads (S)- and (R)-SP-α-Ch (1 and 2), as well as (S)-SP-ß-Ch (3) have been prepared from ß- or α-Ch and SP to investigate the possible competition between photogeneration of biradicals and (1)O2, the key mechanistic steps in Ch photooxidation. Steady-state irradiation of 1 and 2 was performed in dichloromethane, under nitrogen, through Pyrex, using a 400 W medium pressure mercury lamp. The spectral analysis of the separated fractions revealed formation of two photoproducts 4 and 5, respectively. By contrast, under the same conditions, 3 did not give rise to any isolable Ch-derived product. These results point to an intramolecular hydrogen abstraction in 1 and 2 from the C7 position of Ch and subsequent C-C coupling of the generated biradicals. Interestingly, 2 was significantly more photoreactive than 1 indicating a clear stereodifferentiation in the photochemical behavior. Transient absorption spectra obtained for 1-3 were very similar and matched that described for the SP triplet excited state (typical bands with maxima at ca. 350 nm and 600 nm). Direct kinetic analysis of the decay traces at 620 nm led to determination of triplet lifetimes that were ca. 4.1 µs for 1 and 2 and 5.8 µs for 3. From these data, the intramolecular quenching rate constants in 1 and 2 were determined as 0.78 × 10(5) s(-1). The capability of dyads 1-3 to photosensitize the production of singlet oxygen was assessed by time-resolved near infrared emission studies in dichloromethane using perinaphthenone as standard. The quantum yields (ΦΔ) were 0.52 for 1 and 2 and 0.56 for 3. In conclusion, SP-α-Ch dyads are unique in the sense that they can be used to photogenerate both biradicals and singlet oxygen, thus being able to initiate Ch oxidation from their triplet excited states following either of the two competing mechanistic pathways.

Analysis of mebendazole binding to its target biomolecule by laser flash photolysis.

Mebendazole (MBZ) and related anticancer benzimidazoles act binding the ß-subunit of Tubulin (TU) before dimerization with α-TU with subsequent blocking microtubule formation. Laser flash photolysis (LFP) is a new tool to investigate drug-albumin interactions and to determine binding parameters such as affinity constant or population of binding sites. The aim of this study was to evaluate the interactions between the nonfluorescent mebendazole (MBZ) and its target biomolecule TU using this technique. Before analyzing the MBZ@TU complex it was needed to determine the photophysical properties of MBZ triplet excited state ((3)MBZ(⁎)) in different media. Hence, (3)MBZ(⁎) showed a transient absorption spectrum with maxima at 520 and 375 nm and a lifetime much longer in acetonitrile (12.5 µs) than in water (260 ns). The binding of MBZ to TU produces a greater increase of the lifetime of (3)MBZ(⁎) (25 µs). This fact and the strong electron acceptor capability observed for (3)MBZ* evidence that MBZ must not be located close to any electron donor amino acid of TU such as its tryptophan or cysteine residues. Adding increasing amounts of MBZ to aqueous TU was determined the MBZ-TU binding constant (2.0 ± 0.5 × 10(5)M(-1) at 298K) which decreased with increasing temperature. The LFP technique has proven to be a powerful tool to analyze the binding of drug-TU systems when the drug has a detectable triplet excited state. Results indicate that LFP could be the technique of choice to study the interactions of non-fluorescent drugs with their target biomolecules.

Steric shielding vs. σ-π orbital interactions in triplet-triplet energy transfer.

The influence of non-covalent σ-π orbital interactions on triplet-triplet energy transfer (TTET) through tuning of the donor excitation energy remains basically unexplored. In the present work, we have investigated intermolecular TTET using donor moieties covalently linked to a rigid cholesterol (Ch) scaffold. For this purpose, diaryl ketones of π,π* electronic configuration tethered to α- or ß-Ch were prepared from tiaprofenic acid (TPA) and suprofen (SUP). The obtained systems TPA-α-Ch, TPA-ß-Ch, SUP-α-Ch and SUP-ß-Ch were submitted to photophysical studies (laser flash photolysis and phosphorescence), in order to delineate the influence of steric shielding and σ-π orbital interactions on the rate of TTET to a series of energy acceptors. As a matter of fact, fine tuning of the donor triplet energy significantly modifies the rate constants of TTET in the absence of diffusion control. The experimental results are rationalized by means of theoretical calculations using first principles methods based on DFT as well as molecular dynamics.

Understanding of the photoallergic properties of fluoroquinolones: photoreactivity of lomefloxacin with amino acids and albumin.

Although the phototoxic and photoallergic properties of fluoroquinolone antibiotics (FQ) are remarkable, the mechanisms involved in these processes are not completely understood. For this reason, it is considered worthwhile to study in detail the photochemical interactions of lomefloxacin (LFX) and its N-acetyl derivative ALFX, two 6,8-dihalogenated fluoroquinolones, with the most abundant protein in human plasma (human serum albumin, HSA) to analyze their covalent binding. Fluorescence measurements and laser flash photolysis experiments performed in this work have revealed that N-acetylation of the LFX piperazinyl moiety produces an important increase of the drug affinity to albumin. Thus, while the association constant (Ka) for the LFX···HSA complex is below 10(3) M(-1), the Ka for the HSA···ALFX complex resulted in ca. 5 × 10(3) M(-1). Interestingly, LFX is mainly located at site I of HSA, while ALFX shows no preference for site I or II. A high reactivity between the aryl cations generated from (A)LFX dehalogenation and Trp and Tyr together with the generation of covalent adducts between the FQ and these amino acids was observed. However, the interactions between the FQ singlet excited state and albumin in FQ···HSA complexes seem to be the key process of FQ covalent binding to albumin. Moreover, our findings have shown a correlation between the photobinding properties of dihalogenated fluoroquinolones to HSA and their FQ···HSA association constants.

Seeking the mechanism responsible for fluoroquinolone photomutagenicity: a pulse radiolysis, steady-state, and laser flash photolysis study.

The mechanism responsible for the remarkable photomutagenicity of fluoroquinolone (FQ) antibiotics remains unknown. For this reason, it was considered worthwhile to study in detail the interactions between DNA and a dihalogenated FQ such as lomefloxacin (LFX; one of the most photomutagenic FQs) and its N-acetyl derivative ALFX. Studies of photosensitized DNA damage by (A)LFX, such as formation of DNA single-strand breaks (SSBs), together with pulse radiolysis, laser flash photolysis, and absorption and fluorescence measurements, have shown the important effects of the cationic character of the piperazinyl ring on the affinity of this type of drug for DNA. Hence, the formation of SSBs was detected for LFX, whereas ALFX and ciprofloxacin (a monofluorated FQ) needed a considerably larger dose of light to produce some damage. In this context, it was determined that the association constant (Ka) for the binding of LFX to DNA is ca. 2×10(3)M(-1), whereas in the case of ALFX it is only ca. 0.5×10(3)M(-1). This important difference is attributed to an association between the cationic peripheral ring of LFX and the phosphate moieties of DNA and justifies the DNA SSB results. The analysis of the transient species detected and the photomixtures has allowed us to establish the intermolecular processes involved in the photolysis of FQ in the presence of DNA and 2'-deoxyguanosine (dGuo). Interestingly, although a covalent binding of the dihalogenated FQ to dGuo occurs, the photodegradation of FQ…DNA complexes did not reveal any significant covalent attachment. Another remarkable outcome of this study was that (A)LFX radical anions, intermediates required for the onset of DNA damage, were detected by pulse radiolysis but not by laser flash photolysis.

Size-controlled photochemical synthesis of niobium nanoparticles.

The size of photochemically-prepared niobium nanoparticles (NbNP) can be controlled by varying the concentration of the photoinitiator in the reaction mixture. The particles, which may be metallic in nature, are readily oxidized upon air exposure to form stable niobium(v) oxide nanoparticles (NbONP) that act as strong Brønsted acids.

Behavior of drug excited states within macromolecules: binding of colchicine and derivatives to albumin.

The aqueous solutions of colchicine (COL) and analogues such as colchiceine (CEI), and N-deacetylcolchiceine (DCEI) have been studied by laser flash photolysis (LFP) with and without the presence of human serum albumin (HSA) to determine the possible interactions between the drugs and the albumin. When irradiation of these drugs was performed in aqueous media, transient absorption species were not detected. However, triplet excited states of CEI and DCEI were detected when the experiments were carried out in the presence of albumin. Contrary to expectations, in the case of COL, no transient absorption species was observed. A deep study of COL triplet excited-state properties has revealed that intersystem crossing quantum yield (φ(ISC)) decreases from organic media such as dichloromethane to water with φ(ISC) values ca. 0.035 and <0.001 respectively. This fact together the inappreciable reactivity of (3)COL with tyrosine and tryptophan agree with the unbinding of COL to HSA. This study will show for the first time a way to determine the association constant (K(a)) using the LFP technique. Thus, for CEI and DCEI binding to HSA, K(a) values of 8 ± 3 × 10(4) M(-1) and 2 ± 1 × 10(4) M(-1) were obtained, respectively. Moreover, it was also established that these drugs are mainly placed into site II of the albumin using this technique. Therefore, this study validates the LFP as a useful methodology to study the interactions of COL and its analogues with serum albumins, and consequently it could be applied to others proteins such as tubulin.

Photoreactivity of fluoroquinolones: nature of aryl cations generated in water.

The nature of stabilized aryl cations generated from photodehalogenations of fluoroquinolones in aqueous media has been studied by comparing the photophysical and photochemical behavior of lomefloxacin (LFX) and its N(4')-acetylated form (ALFX). Photoproduct studies, laser flash photolysis, and emission measurements have shown that this small peripheral modification produces important changes in the properties of the singlet aryl cations generated. Also, in basic medium, a new photodehalogenation pathway for 6,8-dihalogenated fluoroquinolones has been observed.

Triplet excimers of fluoroquinolones in aqueous media.

Generation of triplet eximers of 6-fluoro-7-piperazinyl-quinolone-3-carboxylic acids (FQs) have been detected in aqueous media using laser flash photolysis (LFP). These transient species (SS) are generated by self-quenching reactions of FQ triplet excited states such as pefloxacin (PFX), norfloxacin (NFX), the N-acetylated form of NFX (ANFX), and its methyl ester (EANFX) with their ground states. In this context, self-quenching rate constants in the range of (1-7) × 10(8) M(-1) s(-1) were determined. The triplet excimers show transient absorption spectra with λ(max) ca. 710 nm for SS(NFX), 740 nm for SS(PFX), and 620 nm for SS(ANFX) and E(ANFX), which are red-shifted with respect to their predecessors triplet excited states. These excimers can be also observed in the presence of phosphate buffer (PB). Experiments performed with NFX and ANFX at different PB concentrations showed that deprotonation processes are not involved in the generation of SS. The triplet multiplicity of the FQ excimers was confirmed by energy transfer reactions with naproxen. The correlation between fluorescence, intersystem crossing, excimer and photodegradation quantum yields of (A)NFX indicated that FQ self-quenching reactions are mainly a deactivation pathway. On the other hand, generation of FQ radical anions absorbing at λ(max) ca. 620 nm has been observed by an efficient electron transfer reaction from Trp to NFX, PFX, and ANFX (rate constants ca. 1 × 10(9) M(-1) s(-1)).