Shortwave infrared polymethine dyes for bioimaging: ultrafast relaxation dynamics and excited-state decay pathways.

Excited-state relaxation in two prototypical shortwave infrared (SWIR) polymethine dyes developed for bioimaging, heptamethine chromenylium Chrom7 and flavylium Flav7, is studied by means of femtosecond transient absorption with broadband ultraviolet-to-SWIR probing complemented by steady-state and time-resolved fluorescence and phosphorescence measurements. The relaxation processes of the dyes in dichloromethane are resolved with sub-100 fs temporal resolution using SWIR, near-IR, and visible photoexcitation. Different population members of the ground-state inhomogeneous ensemble are found to equilibrate via skeletal deformation changes with time constants of 90 fs and either 230 fs (Chrom7) and 350 fs (Flav7) followed by slower evolution matching the 1-ps timescale of diffusive solvation dynamics. Molecules excited into high-lying singlet electronic states (Sn) by visible excitation repopulate with time constants of 400 fs (Chrom7) and 450 fs (Flav7) the corresponding first excited singlet S1 states, which decay within several hundreds of picoseconds in dichloromethane and chloroform solvents. Vibrational relaxation in S1 for both Chrom7 and Flav7 in dichloromethane occurs with time constants of 350 and 800 fs for excess of vibrational energy of ∼1000 and 10 000 cm-1 deposited by near-IR and visible excitation, respectively. Two competing non-radiative processes are present in S1: temperature-independent internal conversion, and thermally-activated twisting about a carbon-carbon bond of the conjugated chain, which is substantial at room temperature but essentially nonreactive, producing traces of isomer product. Intersystem crossing in S1, and thus the triplet quantum yield, is minor. The importance of absorption bands from the excited S1 state in applications requiring high-intensity excitation conditions is discussed.

Recovering true FRET efficiencies from smFRET investigations requires triplet state mitigation.

Single-molecule fluorescence resonance energy transfer (smFRET) methods employed to quantify time-dependent compositional and conformational changes within biomolecules require elevated illumination intensities to recover robust photon emission streams from individual fluorophores. Here we show that outside the weak-excitation limit, and in regimes where fluorophores must undergo many rapid cycles of excitation and relaxation, non-fluorescing, excitation-induced triplet states with lifetimes orders of magnitude longer lived than photon-emitting singlet states degrade photon emission streams from both donor and acceptor fluorophores resulting in illumination-intensity-dependent changes in FRET efficiency. These changes are not commonly taken into consideration; therefore, robust strategies to suppress excited state accumulations are required to recover accurate and precise FRET efficiency, and thus distance, estimates. We propose both robust triplet state suppression and data correction strategies that enable the recovery of FRET efficiencies more closely approximating true values, thereby extending the spatial and temporal resolution of smFRET.

Triple-Dearomative Photocycloaddition: A Strategy to Construct Caged Molecular Frameworks.

An unprecedented caged 2H-benzo-dioxo-pentacycloundecane framework was serendipitously obtained in a single transformation via triple-dearomative photocycloaddition of chromone esters with furans. This caged structure was generated as part of an effort to access a tricyclic, oxygen-bridged intermediate enroute to the dihydroxanthone natural product nidulalin A. Reaction scope and limitations were thoroughly investigated, revealing the ability to access a multitude of synthetically challenging caged scaffolds in a two-step sequence. Photophysical studies provided key mechanistic insights on the process for formation of the novel caged scaffold.

Environmental Markers of Plastics and Microplastics.

The slow reaction rates to chemical and photochemical degradation are well-known properties of plastics. However, large plastic surfaces exposed to environmental conditions release particles and compounds that affect ecosystems and human health. The aim of this work was to identify compounds associated with the degradation of polyethylene (PE), polystyrene (PS), and polyvinyl chloride (PVC) microplastics (markers) on silica and sand and evaluate their use to screen microplastics on natural sand. Products were identified by using targeted and untargeted LC-HRMS analysis. All polymers underwent chemical oxidation on silica. PE released dicarboxylic acids (HO2C-(CH2)n-CO2H (n = 4-30), while PS released cis/trans-chalcone, trans-dypnone, 3-phenylpropiophenone, and dibenzoylmethane. PVC released dicarboxylic acids and aromatic compounds. Upon irradiation, PE was stable while PS released the same compounds as under chemical oxidation but at lower yields. Under the above condition, PVC generated HO2C-[CH2-CHCl]n-CH2-CO2H and HO2C-[CH2-CHCl]n-CO2H (n = 2-19) dicarboxylic acids. The same products were detected on sand but at a lower concentration than on silica due to better retention within the pores. Detection of markers of PE and PS on natural sand allowed us to screen microplastics by following a targeted analysis. Markers of PVC were not detected before or after thermal/photo-oxidation due to the low release of compounds and limitations associated with surface exposure/penetration of radiation.

Photochemical [2+4]-Dimerization Reaction from the Excited State.

Aryl-maleimides undergo a novel [2+4]-photodimerization instead of the expected [2+2]-photodimerization under both direct irradiation with visible light and under sensitized energy transfer conditions. This new excited state reactivity in aryl-maleimides is deciphered through photochemical, photophysical, and spectroscopic studies. The stereochemistry of the photodimer depends on the type of non-bonding interactions prevalent during photodimerization which is in turn dictated by the substituents on the maleimide ring. More importantly, the stereochemistry of the photodimer formed is complementary to the product observed under thermal conditions.

Thioxanthone Dioxide Triplet States Have Low Oxygen Quenching Rate Constants.

With few exceptions, triplet excited states of organic molecules, 3M1, are quenched by ground state molecular oxygen, O2(X3Σg-), with rate constants kq greater than ∼109 M-1 s-1 in fluid solutions. If the energy of the triplet state is above 94 kJ/mol, then such quenching can result in the sensitized production of singlet oxygen, O2(a1Δg). In the interaction between 3M1 and O2(X3Σg-), the magnitudes of both kq and the yield of the O2(a1Δg) depend appreciably on mixing with the M-O2 charge-transfer state. Here, we report that triplet states of several thioxanthen-9-one-10,10-dioxide derivatives have unusually low kq values (as low as ∼1 × 108 M-1 s-1) but have quantum yields for the photosensitized production of O2(a1Δg) that approach unity. Because these molecules possess high oxidation potentials (∼3.5 V vs SCE), we suggest that charge transfer character in the 3M1-O2(X3Σg-) encounter complex is reduced, thereby lowering kq while maintaining high O2(a1Δg) yields. These results provide important experimental support for existing models for the quenching of organic molecule excited states by O2(X3Σg-).

Chemoselective light-induced reactivity of ß-enaminones.

The irradiation of ß-enaminones, generated in situ from cyclic 1,3-diketones and activated alkenes leads to polyheterocyclic skeletons. The photoproduct chemoselectivity depends on the type of cyclic 1,3-diketones employed viz., 2-acetylcyclopentanone and 2-acetylcyclohexanone. The observed chemoselectivity was rationalized based on the Dieckmann-Kon rule.

Thieno[3,4-d]pyrimidin-4(3H)-thione: an effective, oxygenation independent, heavy-atom-free photosensitizer for cancer cells.

All-organic, heavy-atom-free photosensitizers based on thionation of nucleobases are receiving increased attention because they are easy to make, noncytotoxic, work both in the presence and absence of molecular oxygen, and can be readily incorporated into DNA and RNA. In this contribution, the DNA and RNA fluorescent probe, thieno[3,4-d]pyrimidin-4(1H)-one, has been thionated to develop thieno[3,4-d]pyrimidin-4(3H)-thione, which is nonfluorescent and absorbs near-visible radiation with about 60% higher efficiency. Steady-state absorption and emission spectra are combined with transient absorption spectroscopy and CASPT2 calculations to delineate the electronic relaxation mechanisms of both pyrimidine derivatives in aqueous and acetonitrile solutions. It is demonstrated that thieno[3,4-d]pyrimidin-4(3H)-thione efficiently populates the long-lived and reactive triplet state generating singlet oxygen with a quantum yield of about 80% independent of solvent. It is further shown that thieno[3,4-d]pyrimidin-4(3H)-thione exhibits high photodynamic efficacy against monolayer melanoma cells and cervical cancer cells both under normoxic and hypoxic conditions. Our combined spectroscopic, computational, and in vitro data demonstrate the excellent potential of thieno[3,4-d]pyrimidin-4(1H)-thione as a heavy-atom-free PDT agent and paves the way for further development of photosensitizers based on the thionation of thieno[3,4-d]pyrimidine derivatives. Collectively, the experimental and computational results demonstrate that thieno[3,4-d]pyrimidine-4(3H)-thione stands out as the most promising thiobase photosensitizer developed to this date.

Unraveling the Photophysical Characteristics, Aromaticity, and Stability of π-Extended Acene-Quinodimethyl Thioamides†.

Poly-aromatic systems that contain quinodimethyl (QDM) units are appealing for several photonic and spintronic applications owing to the unique electronic structure, aromaticity, and spin state(s) of the QDM ring. Herein, we report the synthesis and characterization of novel QDM-based chromophores 1-3, which exhibit unique photo-excited behavior and aromaticity. Extending the aromatic core with a biphenyl/phenanthryl- and a pyrrolo-fragment led to reducing the optoelectronic bandgap and modulating the photophysics QDM 1-3. Yet, QDM 2 and 3 suffer from "aromaticity imbalance" and become relatively unstable compared to the parent compound QDM 1. Further assessment of local aromaticity using computational tools revealed that the pseudo-quinoidal ring B is the main driving force allowing to easily populate the excited triplet state of these chromophores. The present study provides complementary guidelines for designing novel non-classical poly-aromatic systems.

Modulating Photochemical Properties to Enhance the Stability of Electronically Dimmable Eye Protection Devices†.

The study evaluates compatibility of stabilizers with dye doped liquid crystal (LC) scaffolds that are used in electronically dimmable materials. The photodegradation of the materials was investigated and suitable stabilizers were evaluated to slow the degradation process. Various types of benzotriazole-based stabilizers were evaluated for stabilizing the liquid crystals. Based on spin trapping experiments, radicals generated upon UV exposure is likely responsible for the degradation of the system. The radical generation is competitively inhibited by the addition of stabilizers.

Discovery of SARS-CoV-2 antiviral synergy between remdesivir and approved drugs in human lung cells.

SARS coronavirus 2 (SARS-CoV-2) has caused an ongoing global pandemic with significant mortality and morbidity. At this time, the only FDA-approved therapeutic for COVID-19 is remdesivir, a broad-spectrum antiviral nucleoside analog. Efficacy is only moderate, and improved treatment strategies are urgently needed. To accomplish this goal, we devised a strategy to identify compounds that act synergistically with remdesivir in preventing SARS-CoV-2 replication. We conducted combinatorial high-throughput screening in the presence of submaximal remdesivir concentrations, using a human lung epithelial cell line infected with a clinical isolate of SARS-CoV-2. This identified 20 approved drugs that act synergistically with remdesivir, many with favorable pharmacokinetic and safety profiles. Strongest effects were observed with established antivirals, Hepatitis C virus nonstructural protein 5A (HCV NS5A) inhibitors velpatasvir and elbasvir. Combination with their partner drugs sofosbuvir and grazoprevir further increased efficacy, increasing remdesivir's apparent potency > 25-fold. We report that HCV NS5A inhibitors act on the SARS-CoV-2 exonuclease proofreader, providing a possible explanation for the synergy observed with nucleoside analog remdesivir. FDA-approved Hepatitis C therapeutics Epclusa® (velpatasvir/sofosbuvir) and Zepatier® (elbasvir/grazoprevir) could be further optimized to achieve potency and pharmacokinetic properties that support clinical evaluation in combination with remdesivir.

Phenacyl Bromide as a Single-Component Photoinitiator: Photoinduced Step-Growth Polymerization of N-Methylpyrrole and N-Methylindole.

The synthesis of step-growth polymers by photoinduced methods is a challenging issue in synthetic chemistry. Here, we report a single component near UV responsive photopolymerization system for step-growth polymerization of N-methylpyrrole (MPyr) and N-methylindole (MIn) by using phenacyl bromide (PAB). The obtained high molecular weight conjugated polymers were characterized by spectral and chromatographic methods. Detailed laser flash photolysis and spectroscopic studies revealed that polymerization proceeds by successive photoinduced cleavage of PAB followed by electron transfer, proton release and coupling processes. After photolysis, chain growth continues also in daylight or dark by acidic oxidation. The structural features of the polymers were investigated. PAB single component photoinitiator enables an efficient, rapid, room temperature step-growth polymerization process of MPyr and MIn that can be applied to other conjugated monomers.

2-Oxopurine Riboside: A Dual Fluorescent Analog and Photosensitizer for RNA/DNA Research.

There is significant interest in developing suitable nucleoside analogs exhibiting high fluorescence and triplet yields to investigate the structure, dynamics, and binding properties of nucleic acids and promote selective photosensitized damage to DNA/RNA, respectively. In this study, steady-state, laser flash photolysis, time-resolved IR luminescence, and femtosecond broad-band transient absorption spectroscopies are combined with quantum chemical calculations to elucidate the excited-state dynamics of 2-oxopurine riboside in aqueous solution and to investigate its prospective use as a fluorescent or photosensitizer analog. The Franck-Condon population in the S1 (ππ*) state decays through a combination of solvent and conformational relaxation to its minimum in 1.9 ps. The population trapped in the 1ππ* minimum bifurcates to either fluoresce or intersystem cross to the triplet manifold within ca. 5 ns, while another fraction of the population decays nonradiatively to the ground state. It is demonstrated that 2-oxopurine riboside exhibits both high fluorescent (48%) and significant triplet (between 10% and 52%) yields, leading to a yield of singlet oxygen generation of 10%, making this nucleoside analog a dual fluorescent and photosensitizer analog for DNA and RNA research.

Combination of antiviral drugs inhibits SARS-CoV-2 polymerase and exonuclease and demonstrates COVID-19 therapeutic potential in viral cell culture.

SARS-CoV-2 has an exonuclease-based proofreader, which removes nucleotide inhibitors such as Remdesivir that are incorporated into the viral RNA during replication, reducing the efficacy of these drugs for treating COVID-19. Combinations of inhibitors of both the viral RNA-dependent RNA polymerase and the exonuclease could overcome this deficiency. Here we report the identification of hepatitis C virus NS5A inhibitors Pibrentasvir and Ombitasvir as SARS-CoV-2 exonuclease inhibitors. In the presence of Pibrentasvir, RNAs terminated with the active forms of the prodrugs Sofosbuvir, Remdesivir, Favipiravir, Molnupiravir and AT-527 were largely protected from excision by the exonuclease, while in the absence of Pibrentasvir, there was rapid excision. Due to its unique structure, Tenofovir-terminated RNA was highly resistant to exonuclease excision even in the absence of Pibrentasvir. Viral cell culture studies also demonstrate significant synergy using this combination strategy. This study supports the use of combination drugs that inhibit both the SARS-CoV-2 polymerase and exonuclease for effective COVID-19 treatment.

Intramolecular Charge Transfer in the Azathioprine Prodrug Quenches Intersystem Crossing to the Reactive Triplet State in 6-Mercaptopurine.

The thiopurine prodrugs 6-mercaptopurine and azathioprine are among the world's essential medications for acute lymphoblastic leukemia, immunosuppression and several autoimmune conditions. Thiopurine prodrugs are efficient UVA absorbers and singlet oxygen generators and the long-term treatment with these prodrugs correlates with a high incidence of sunlight-induced skin cancer in patients. In this contribution, we show that the electronic relaxation mechanisms and photochemical properties of azathioprine are remarkably different from those of 6-mercaptopurine upon absorption of UVA radiation. UVA excitation of 6-mercaptopurine results in nearly 100% triplet yield and up to 30% singlet oxygen generation, whereas excitation of azathioprine with UVA leads to triplet yields of 15-3% depending on pH of the aqueous solution and <1% singlet oxygen generation. While photoexcitation of 6-mercaptopurine and other thiopurine prodrugs can facilitate oxidatively generated cell damage, azathioprine's poor photosensitization ability reveals the use of interchromophoric charge-transfer interactions for the molecular design of photostable prodrugs exhibiting a remarkable reduction in photocytotoxic side effects before drug metabolization.

Novel Dual-Organelle-Targeting Probe (RCPP) for Simultaneous Measurement of Organellar Acidity and Alkalinity in Living Cells.

Many organelles, such as lysosomes and mitochondria, maintain a pH that is different from the cytoplasmic pH. These pH differences have important functional ramifications for those organelles. Many cellular events depend upon a well-compartmentalized distribution of H+ ions spanning the membrane for the optimal function. Cells have developed a variety of mechanisms that enable the regulation of organelle pH. However, the measurement of organellar acidity/alkalinity in living cells has remained a challenge. Currently, most existing probes for the estimation of intracellular pH show a single -organelle targeting capacity. Such probes provide data that fails to comprehensively reveal the pathological and physiological roles and connections between mitochondria and lysosomes in different species. Mitochondrial and lysosomal functions are closely related and important for regulating cellular homeostasis. Accordingly, the design of a single fluorescent probe that can simultaneously target mitochondria and lysosomes is highly desirable, enabling a better understanding of the crosstalk between these organelles. We report the development of a novel fluorescent sensor, rhodamine-coumarin pH probe (RCPP), for detection of organellar acidity/alkalinity. RCPP simultaneously moves between mitochondrion and lysosome subcellular locations, facilitating the simultaneous monitoring of pH alterations in mitochondria and lysosomes.

Combination of Antiviral Drugs to Inhibit SARS-CoV-2 Polymerase and Exonuclease as Potential COVID-19 Therapeutics.

SARS-CoV-2 has an exonuclease-based proofreader, which removes nucleotide inhibitors such as Remdesivir that are incorporated into the viral RNA during replication, reducing the efficacy of these drugs for treating COVID-19. Combinations of inhibitors of both the viral RNA-dependent RNA polymerase and the exonuclease could overcome this deficiency. Here we report the identification of hepatitis C virus NS5A inhibitors Pibrentasvir and Ombitasvir as SARS-CoV-2 exonuclease inhibitors. In the presence of Pibrentasvir, RNAs terminated with the active forms of the prodrugs Sofosbuvir, Remdesivir, Favipiravir, Molnupiravir and AT-527 were largely protected from excision by the exonuclease, while in the absence of Pibrentasvir, there was rapid excision. Due to its unique structure, Tenofovir-terminated RNA was highly resistant to exonuclease excision even in the absence of Pibrentasvir. Viral cell culture studies also demonstrate significant synergy using this combination strategy. This study supports the use of combination drugs that inhibit both the SARS-CoV-2 polymerase and exonuclease for effective COVID-19 treatment.

In vitro antiviral activity of the anti-HCV drugs daclatasvir and sofosbuvir against SARS-CoV-2, the aetiological agent of COVID-19.

BACKGROUND: Current approaches of drug repurposing against COVID-19 have not proven overwhelmingly successful and the SARS-CoV-2 pandemic continues to cause major global mortality. SARS-CoV-2 nsp12, its RNA polymerase, shares homology in the nucleotide uptake channel with the HCV orthologue enzyme NS5B. Besides, HCV enzyme NS5A has pleiotropic activities, such as RNA binding, that are shared with various SARS-CoV-2 proteins. Thus, anti-HCV NS5B and NS5A inhibitors, like sofosbuvir and daclatasvir, respectively, could be endowed with anti-SARS-CoV-2 activity. METHODS: SARS-CoV-2-infected Vero cells, HuH-7 cells, Calu-3 cells, neural stem cells and monocytes were used to investigate the effects of daclatasvir and sofosbuvir. In silico and cell-free based assays were performed with SARS-CoV-2 RNA and nsp12 to better comprehend the mechanism of inhibition of the investigated compounds. A physiologically based pharmacokinetic model was generated to estimate daclatasvir's dose and schedule to maximize the probability of success for COVID-19. RESULTS: Daclatasvir inhibited SARS-CoV-2 replication in Vero, HuH-7 and Calu-3 cells, with potencies of 0.8, 0.6 and 1.1 µM, respectively. Although less potent than daclatasvir, sofosbuvir alone and combined with daclatasvir inhibited replication in Calu-3 cells. Sofosbuvir and daclatasvir prevented virus-induced neuronal apoptosis and release of cytokine storm-related inflammatory mediators, respectively. Sofosbuvir inhibited RNA synthesis by chain termination and daclatasvir targeted the folding of secondary RNA structures in the SARS-CoV-2 genome. Concentrations required for partial daclatasvir in vitro activity are achieved in plasma at Cmax after administration of the approved dose to humans. CONCLUSIONS: Daclatasvir, alone or in combination with sofosbuvir, at higher doses than used against HCV, may be further fostered as an anti-COVID-19 therapy.

Uncovering New Excited State Photochemical Reactivity by Altering the Course of the De Mayo Reaction.

An unprecedented and previously unknown photochemical reactivity of 1,3-dicarbonyl compounds is observed with amino-alkenes leading to dihydropyrans. This novel photochemical reactivity changes the established paradigm related to the De Mayo reaction between 1,3-dicarbonyl compounds and alkenes. This new reaction allows convenient access to the Marmycin core in a single step from commercially available reactants. The origin and scope of this new photoreaction is detailed with preliminary photophysical and mechanistic investigations.

Molecular Engineering of Chromophores to Enable Triplet-Triplet Annihilation Upconversion.

Triplet-triplet annihilation upconversion (TTA-UC) is an unconventional photophysical process that yields high-energy photons from low-energy incident light and offers pathways for innovation across many technologies, including solar energy harvesting, photochemistry, and optogenetics. Within aromatic organic chromophores, TTA-UC is achieved through several consecutive energy conversion events that ultimately fuse two triplet excitons into a singlet exciton. In chromophores where the singlet exciton is roughly isoergic with two triplet excitons, the limiting step is the triplet-triplet annihilation pathway, where the kinetics and yield depend sensitively on the energies of the lowest singlet and triplet excited states. Herein we report up to 40-fold improvements in upconversion quantum yields using molecular engineering to selectively tailor the relative energies of the lowest singlet and triplet excited states, enhancing the yield of triplet-triplet annihilation and promoting radiative decay of the resulting singlet exciton. Using this general and effective strategy, we obtain upconversion yields with red emission that are among the highest reported, with remarkable chemical stability under ambient conditions.