Singlet oxygen generation on a superhydrophobic surface: Effect of photosensitizer coating and incident wavelength on 1O2 yields.

Photochemical generation of singlet oxygen (1O2) often relies on homogenous systems; however, a dissolved photosensitizer (PS) may be unsuitable for some applications because it is difficult to recover, expensive to replenish, and hazardous to the environment. Isolation of the PS onto a solid support can overcome these limitations, but implementation faces other challenges, including agglomeration of the solid PS, physical quenching of 1O2 by the support, photooxidation of the PS, and hypoxic environments. Here, we explore a superhydrophobic polydimethylsiloxane (SH-PDMS) support coated with the photosensitizer 5,10,15,20-tetrakis(pentafluorophenyl)-21H,23H-porphyrin (TFPP). This approach seeks to address the challenges of a heterogeneous system by using a support that exhibits low 1O2 physical quenching rates, a fluorinated PS that is chemically resistant to photooxidation, and a superhydrophobic surface that entraps a layer of air, thus preventing hypoxia. Absorbance and fluorescence spectroscopy reveal the monomeric arrangement of TFPP on SH-PDMS surfaces, a surprising but favorable characteristic for a solid-phase PS on 1O2 yields. We also investigated the effect of incident wavelength on 1O2 yields for TFPP in aqueous solution and immobilized on SH-PDMS and found overall yields to be dependent on the absorption coefficient, while the yield per absorbed photon exhibited wavelength independence, in accordance with Kasha-Vavilov's rule.

Role of curvature in acridone for 1 O2 oxidation of a natural product homoallylic alcohol: A novel iso-hydroperoxide intermediate.

A density functional theoretical (DFT) study is presented, implicating a 1 O2 oxidation process to reach a dihydrobenzofuran from the reaction of the natural homoallylic alcohol, glycocitrine. Our results predict an interconversion between glycocitrine and an iso-hydroperoxide intermediate [R(H)O+ -O- ] that provides a key path in the chemistry which then follows. Formations of allylic hydroperoxides are unlikely from a 1 O2 'ene' reaction. Instead, the dihydrobenzofuran arises by 1 O2 oxidation facilitated by a 16° curvature of the glycocitrine ring imposed by a pyramidal N-methyl group. This curvature facilitates the formation of the iso-hydroperoxide, which is analogous to the iso species CH2 I+ -I- and CHI2 + -I- formed by UV photolysis of CH2 I2 and CHI3 . The iso-hydroperoxide is also structurally reminiscent of carbonyl oxides (R2 C=O+ -O- ) formed in the reaction of carbenes and oxygen. Our DFT results point to intermolecular process, in which the iso-hydroperoxide's fate relates to O-transfer and H2 O dehydration reactions for new insight into the biosynthesis of dihydrobenzofuran natural products.

On the mechanism of visible-light sensitized photosulfoxidation of toluidine blue O.

We report on the formation of toluidine blue O (TBO) sulfoxide by a self-sensitized photooxidation of TBO. Here, the photosulfoxidation process was studied by mass spectrometry (MS) and discussed in the context of photodemethylation processes which both contribute to TBO consumption over time. Analysis of solvent effects with D2O, H2O, and CH3CN along with product yields and MS fragmentation patterns provided mechanistic insight into TBO sulfoxide's formation. The formation of TBO sulfoxide is minor and detectable up to 12% after irradiation of 3 h. The photosulfoxidation process is dependent on oxygen wherein instead of a type II (singlet oxygen, 1O2) reaction, a type I reaction involving TBO to reach the TBO sulfoxide is consistent with the results. Density functional theory results point to the formation of the TBO sulfoxide by the oxidation of TBO via transiently formed peroxyl radical or thiadioxirane intermediates. We discover that the TBO photosulfoxidation arises competitively with TBO photodemethylation with the latter leading to formaldehyde formation.

Bluer Phototruncation: Retro-Diels-Alder of Heptamethine Cyanine to Trimethine Cyanine through an Allene Hydroperoxide Intermediate.

The photoconversion of heptamethine to pentamethine cyanines and of pentamethine to trimethine cyanines was recently reported. Here, we report mechanistic studies and initial experimental evidence for a previously unexplored 4-carbon truncation reaction that converts the simplest heptamethine cyanine to the corresponding trimethine cyanine. We propose a DFT-supported model describing a singlet oxygen (1O2)-mediated formation of an allene hydroperoxide intermediate and subsequent 4-carbon loss through a retro-Diels-Alder process. Fluorescence and mass spectrometry measurements provide evidence of this direct conversion process. This 4-carbon truncation reaction adds to a growing body of cyanine reactivity and may provide an optical tool leading to a substantial blue-shift (Δλem) of ∼200 nm.

Superhydrophobic Tipped Antimicrobial Photodynamic Therapy Device for the In Vivo Treatment of Periodontitis Using a Wistar Rat Model.

Limited options exist for treatment of periodontitis; scaling and root planing (SRP) are not sufficient to eradicate P. gingivalis and the resulting inflammatory disease. Chlorhexidine (CHX), used as an adjuvant to SRP, may reduce bacterial loads but leads to pain and staining, while evidence for its efficacy is lacking. Antibiotics are effective but can lead to drug-resistance. The rising concern of antibiotic resistance limits the future use of this treatment approach. This study evaluates the efficacy of a novel superhydrophobic (SH) antimicrobial photodynamic therapy (aPDT) device as an adjuvant to SRP for the treatment of periodontitis induced in a Wistar rat in vivo model relative to CHX. The SH-aPDT device comprises an SH silicone rubber strip coated with verteporfin photosensitizer (PS), sterilized, and secured onto a tapered plastic optical fiber tip connected to a red diode laser. The superhydrophobic polydimethylsiloxane (PDMS) strips were fabricated by using a novel soluble template method that creates a medical-grade elastomer with hierarchical surface roughness without the use of nanoparticles. Superhydrophobicity minimizes direct contact of the PS-coated surface with bacterial biofilms. Upon insertion of the device tip into the pocket and energizing the laser, the device generates singlet oxygen that effectively targets and eliminates bacteria within the periodontal pocket. SH-aPDT treatment using 125 J/cm2 of red light on three consecutive days reduced P. gingivalis significantly more than SRP-CHX controls (p < 0.05). Clinical parameters significantly improved (p < 0.05), and histology and stereometry results demonstrated SH-aPDT to be the most effective treatment for improving healing and reducing inflammation, with an increase in fibroblast cells and extracellular matrix and a reduction in vascularization, inflammatory cells, and COX-2 expression. The SH-aPDT approach resulted in complete disease clearance assessed 30 days after treatment initiation with significant reduction of the periodontal pocket and re-formation of the junctional epithelium at the enamel-cementum junction. PS isolation on a SH strip minimizes the potential for bacteria to develop resistance, where the treatment may be aided by the oxygen supply retained within the SH surface.

Nitric Oxide Detection Using a Chemical Trap Method for Applications in Bacterial Systems.

Plant growth-promoting bacteria (PGPB) can be incorporated in biofertilizer formulations, which promote plant growth in different ways, such as fixing nitrogen and producing phytohormones and nitric oxide (NO). NO is a free radical involved in the growth and defense responses of plants and bacteria. NO detection is vital for further investigation in different agronomically important bacteria. NO production in the presence of KNO3 was evaluated over 1-3 days using eight bacterial strains, quantified by the usual Griess reaction, and monitored by 2,3-diaminonaphthalene (DAN), yielding 2,3-naphthotriazole (NAT), as analyzed by fluorescence spectroscopy, gas chromatography-mass spectrometry, and high-performance liquid chromatography. The Greiss and trapping reaction results showed that Azospirillum brasilense (HM053 and FP2), Rhizobium tropici (Br322), and Gluconacetobacter diazotrophicus (Pal 5) produced the highest NO levels 24 h after inoculation, whereas Nitrospirillum amazonense (Y2) and Herbaspirillum seropedicae (SmR1) showed no NO production. In contrast to the literature, in NFbHP-NH4Cl-lactate culture medium with KNO3, NO trapping led to the recovery of a product with a molecular mass ion of 182 Da, namely, 1,2,3,4-naphthotetrazole (NTT), which contained one more nitrogen atom than the usual NAT product with 169 Da. This strategy allows monitoring and tracking NO production in potential biofertilizing bacteria, providing future opportunities to better understand the mechanisms of bacteria-plant interaction and also to manipulate the amount of NO that will sustain the PGPB.

Shades of phototoxicity in fluorescent imaging agents (that are not supposed to be phototoxic)†.

This article is a highlight of the paper by Huang et al. in this issue of Photochemistry and Photobiology. It describes shades of phototoxicity in fluorescent imaging agents that are not intended to be phototoxic. Phototoxicity was assessed using a modified neutral red uptake (NRU) in vitro assay with mean photo-effects (MPE) for the fluorescent agents IRdye800, indocyanine green (ICG), proflavine, and methylene blue (MB), with comparisons to known phototoxic agents benzoporphyrin derivative (BPD) and rose bengal (RB). The experimental conditions were aimed to mimic clinical settings, using not only visible light, but also near-infrared light for insight to photosafety and deep tissue damage. Molecular mechanisms underlying the phototoxicities were not sought, but IRdye800 and ICG were mainly deemed to be safe, whereas proflavine and MB would require precautions since phototoxicity can overshadow their utility as fluorescent imaging agents.

Tuning the 1O2 Oxidation of a Phenol at the Air/Solid Interface of a Nanoparticle: Hydrophobic Surface Increases Oxophilicity.

Although silica surfaces have been used in organic oxidations for the production of peroxides, studies of airborne singlet oxygen at interfaces are limited and have not found widespread advantages. Here, with prenyl phenol-coated silica and delivery of singlet oxygen (1O2) through the gas phase, we uncover significant selectivity for dihydrofuran formation over allylic hydroperoxide formation. The hydrophobic particle causes prenyl phenol to produce an iso-hydroperoxide intermediate with an internally protonated oxygen atom, which leads to dihydrofuran formation as well as O atom transfer. In contrast, hydrophilic particles cause prenyl phenol to produce allylic hydroperoxide, due to phenol OH hydrogen bonding with SiOH surface groups. Mechanistic insight is provided by air/nanoparticle interfaces coated with the prenyl phenol, in which product yield was 6-fold greater on the hydrophobic nanoparticles compared to the hydrophilic nanoparticles and total rate constants (ASI-kT) of 1O2 were 13-fold greater on the hydrophobic vs hydrophilic nanoparticles. A slope intersection method was also developed that uses the airborne 1O2 lifetime (τairborne) and surface-associated 1O2 lifetime (τsurf) to quantitate 1O2 transitioning from volatile to non-volatile and surface boundary (surface···1O2). Further mechanistic insights on the selectivity of the reaction of prenyl phenol with 1O2 was provided by density functional theory calculations.

Synthesis, Characterization and Photocleavage of Bis-decyl Pteroic Acid: A Folate Derivative with Affinity to Biomembranes†.

Here, we provide mechanistic insight to the photocleavage of a compound in the folate family, namely pteroic acid. A bis-decyl chain derivative of pteroic acid was synthesized, structurally characterized and photochemically investigated. We showed that, like folic acid, pteroic acid and the decylated derivative undergo a photocleavage reaction in the presence of H2 O, while no reaction was observed in methanol solution. Furthermore, density functional theory calculations were carried out to predict relative stabilities of hypothetical mono-, bis- and tris-decylated pteroic acid derivatives to help rationalize the regioselectivity of the bis-decyl pteroic acid product. Additionally, the lipophilicity of the bis-decyl pteroic acid appears to confer a hydrophobic property enabling an interaction with biomembranes.

Practical Aspects in the Study of Biological Photosensitization Including Reaction Mechanisms and Product Analyses: A Do's and Don'ts Guide†.

The interaction of light with natural matter leads to a plethora of photosensitized reactions. These reactions cause the degradation of biomolecules, such as DNA, lipids, proteins, being therefore detrimental to the living organisms, or they can also be beneficial by allowing the treatment of several diseases by photomedicine. Based on the molecular mechanistic understanding of the photosensitization reactions, we propose to classify them in four processes: oxygen-dependent (type I and type II processes) and oxygen-independent [triplet-triplet energy transfer (TTET) and photoadduct formation]. In here, these processes are discussed by considering a wide variety of approaches including time-resolved and steady-state techniques, together with solvent, quencher, and scavenger effects. The main aim of this survey is to provide a description of general techniques and approaches that can be used to investigate photosensitization reactions of biomolecules together with basic recommendations on good practices. Illustration of the suitability of these approaches is provided by the measurement of key biomarkers of singlet oxygen and one-electron oxidation reactions in both isolated and cellular DNA. Our work is an educational review that is mostly addressed to students and beginners.

Singlet Oxygen's Potential Role as a Nonoxidative Facilitator of Disulfide S-S Bond Rotation†.

The role of singlet oxygen potentially mediating increased conformational flexibility of a disulfide was investigated. Density functional theory (DFT) calculations indicate that the singlet oxygenation of 1,2-dimethyldisulfane produces a peroxy intermediate. This intermediate adopts a structure with a longer S-S bond distance and a more planar torsional angle θ (C-S-S-C) compared with the nonoxygenated 1,2-dimethyldisulfane. The lengthened S-S bond enables a facile rotation about the torsional angle in the semicircle region 0° < θ < 210°, that is ~5 kcal mol-1 lower in energy than the disulfane. The peroxy intermediate bears nO → σS-S and nO → σ*S-S interactions that stabilize the S-O bond but destabilize the S-S bond, which contrasts with stabilizing nS → σ*S-S hyperconjugative effects in the disulfane S-S bond. Subsequent departure of O2 from the disulfane peroxy intermediate is reminiscent of peroxy intermediates which also expel O2 , yet facilitate cis-trans isomerizations of stilbenes, hexadienes, cyanines, and carotenes. "Non-oxidative" 1 O2 interactions with a variety of bond types are currently underappreciated. We hope to raise awareness of how these interactions can help elucidate the origins of molecular twisting.

Sensitized Photooxidation of Ortho-Prenyl Phenol: Biomimetic Dihydrobenzofuran Synthesis and Total 1O2 Quenching.

The sensitized photooxidation of ortho-prenyl phenol is described with evidence that solvent aproticity favors the formation of a dihydrobenzofuran [2-(prop-1-en-2-yl)-2,3-dihydrobenzofuran], a moiety commonly found in natural products. Benzene solvent increased the total quenching rate constant (kT ) of singlet oxygen with prenyl phenol by ~10-fold compared to methanol. A mechanism is proposed with preferential addition of singlet oxygen to prenyl site due to hydrogen bonding with the phenol OH group, which causes a divergence away from the singlet oxygen 'ene' reaction toward the dihydrobenzofuran as the major product. The reaction is a mixed photooxidized system since an epoxide arises by a type I sensitized photooxidation.

Evaluation of photosensitizer-containing superhydrophobic surfaces for the antibacterial treatment of periodontal biofilms.

Antimicrobial photodynamic therapy (aPDT) is a promising approach to control biofilms involved in periodontal diseases. However, certain challenges, such as staining of teeth, preferential interaction of photosensitizer (PS) with Gram-positive versus Gram-negative bacteria, and insufficient oxygen in hypoxic periodontal pockets have presented barriers to its use in the clinic. To overcome these challenges, a novel superhydrophobic (SH) film that generates airborne singlet oxygen has been developed. The SH-aPDT approach isolates the PS onto a topologically rough solid SH film on which channels allow air to diffuse to the PS surface, thus ensuring sufficient oxygen supply. Upon illumination, gas phase singlet oxygen (1O2) is produced and diffuses from the SH surface to the underlying biofilm. The killing efficacy was assessed as a function of transmitted fluence (17.9-89.5 J/cm2) and chorin e6 loading (96-1110 nmol/cm2) by counting of colony forming units, biofilm metabolism by XTT and confocal microscopy. The decrease in viability of both Gram-positive and Gram-negative bacteria in a multi-species biofilm was found to be linearly dependent on the fluence as well as the loading of the PS up to 71.6 J/cm2 when 1110 nmols/cm2 of chlorin e6 was used. A > 4.6 log bacterial reduction was observed under these conditions (p < 0.05). This novel SH-aPDT approach shows promise as an effective method to disinfect multi-species bacterial biofilms associated with periodontal disease and will be evaluated in animal models in future studies.

Probing the Transition State-to-Intermediate Continuum: Mechanistic Distinction between a Dry versus Wet Perepoxide in the Singlet Oxygen "Ene" Reaction at the Air-Water Interface.

A mechanistic study is reported for the reactions of singlet oxygen (1O2) with alkene surfactants of tunable properties. Singlet oxygen was generated either top-down (photochemically) by delivery as a gas to an air-water interface or bottom-up (chemically) by transport to the air-water interface as a solvated species. In both cases, reactions were carried out in the presence of 7-carbon (7C), 9-carbon (9C), or 11-carbon (11C) prenylsurfactants [(CH3)2C═CH(CH2)nSO3- Na+ (n = 4, 6, 8)]. Higher "ene" hydroperoxide regioselectivities (secondary ROOH 2 to tertiary ROOH 3) were reached in delivering 1O2 top-down through air as compared to bottom-up via aqueous solution. In the photochemical reaction, ratios of 2:3 increased from 2.5:1 for 7C, to 2.8:1 for 9C, and to 3.2:1 for 11C. In contrast, in the bubbling system that generated 1O2 chemically, the selectivity was all but lost, ranging only from 1.3:1 to 1:1. The phase-dependent regioselectivities appear to be correlated with the "ene" reaction with photochemically generated, drier 1O2 at the air-water interface vs those with wetter 1O2 from the bubbling reactor. Density functional theory-calculated reaction potential energy surfaces (PESs) were used to help rationalize the reaction phase dependence. The reactions in the gas phase are mediated by perepoxide transition states with 32-41 kJ/mol binding energy for C═C(π)···1O2. The perepoxide species, however, evolve to well-defined stationary structures in the aqueous phase, with covalent C-O bonds and 85-88 kJ/mol binding energy. The combined experimental and computational evidence points to a unique mechanism for 1O2 "ene" tunability in a perepoxide continuum from a transition state to an intermediate.

Computational Evidence for Tunneling and a Hidden Intermediate in the Biosynthesis of Tetrahydrocannabinol.

Quantum tunneling is computed for a reaction sequence that models the conversion of the ortho-quinone methide of cannabigerolic acid 1 to the decarboxylated product (-)-trans-Δ9-tetrahydrocannabinol (THC, 3). This calculation is the first to evaluate multidimensional tunneling in this sequence. Computations were carried out with POLYRATE and GAUSSRATE using B3LYP/6-31G(d,p) to examine the mechanism of THC 3 formation. The pentyl chain on THC 3 and its precursors were replaced with a methyl group to compute tunneling contributions to the rates of four separate steps: (i) initial Diels-Alder reaction of the quinone methide with the trisubstituted alkene end-group of the geranyl 1Z-CH3 to give 2Z-CH3, (ii) acid-catalyzed keto-enol tautomerization, which converts 2rZ-CH3 to 4rZ-CH3, (iii) carboxyl rotamerization converting 4rZ-CH3 to 4E-CH3, and (iv) decarboxylation that converts 4E-CH3 to 3-CH3. Tunneling contributions to the rate constants of steps (i)-(iv) are between 19 and 76% at 293 K. In step (ii), nonuniform changes in the zero-point vibrational energy along the reaction path created a shallow minimum in the 0 K free energy. It is a hidden intermediate because it is not a minimum on the potential energy surface and is detectable only when zero-point energy is taken into account along the reaction path. Predicted kinetic isotope effects would be experimentally observable at temperatures that are convenient to use. This is particularly relevant in the decarboxylation stage of the reaction sequence and has important implications because of its role in THC 3 formation.

Violet-blue Light Induces "Natural" Photodynamic Plasma Disinfection with Endogenous Sensitizers.

Naturally, endogenous porphyrins can provide sensitized disinfection power, and to photobiologists' delight, violet-blue light has potential virtues, but progress is needed before violet-blue light treatment can be used for microbe treatment of blood plasma, and yet safeguard against protein photooxidation. A report by Maclean et al. in this issue of Photochemistry & Photobiology on microbe reduction in blood plasma showing negligible competing protein photooxidation may bring that goal a step closer.

Retirement Issue Dedicated to Edward L. Clennan.

The enclosed is a special retirement issue of Photochemistry and Photobiology, dedicated to Edward L. Clennan. Papers are included by those with ties to Ed as former group members, collaborators, and colleagues. They cover a range of topics in photochemistry, photosensitization, photocatalysis, chemiluminescence, photorelease, helical compounds, and reactive intermediates, all of which are very fitting to Ed's contributions in fundamental science. His efforts include essential contributions to mechanisms of singlet oxygen in homogeneous and heterogeneous media, as well as photoinduced electron transfer reactions and photocyclizations to form helicenes. The latter topics also have potential use in organic electronic devices.

Defining the Basis of Cyanine Phototruncation Enables a New Approach to Single-Molecule Localization Microscopy.

The light-promoted conversion of extensively used cyanine dyes to blue-shifted emissive products has been observed in various contexts. However, both the underlying mechanism and the species involved in this photoconversion reaction have remained elusive. Here we report that irradiation of heptamethine cyanines provides pentamethine cyanines, which, in turn, are photoconverted to trimethine cyanines. We detail an examination of the mechanism and substrate scope of this remarkable two-carbon phototruncation reaction. Supported by computational analysis, we propose that this reaction involves a singlet oxygen-initiated multistep sequence involving a key hydroperoxycyclobutanol intermediate. Building on this mechanistic framework, we identify conditions to improve the yield of photoconversion by over an order of magnitude. We then demonstrate that cyanine phototruncation can be applied to super-resolution single-molecule localization microscopy, leading to improved spatial resolution with shorter imaging times. We anticipate these insights will help transform a common, but previously mechanistically ill-defined, chemical transformation into a valuable optical tool.