Electronic relaxation mechanism of 9-methyl-2,6-diaminopurine and 2,6-diaminopurine-2'-deoxyribose in solution.

Prolonged ultraviolet exposure results in the formation of cyclobutane pyrimidine dimers (CPDs) in RNA. Consequently, prebiotic photolesion repair mechanisms should have played an important role in the maintenance of the structural integrity of primitive nucleic acids. 2,6-Diaminopurine is a prebiotic nucleobase that repairs CPDs with high efficiency when incorporated into polymers. We investigate the electronic deactivation pathways of 2,6-diaminopurine-2'-deoxyribose and 9-methyl-2,6-diaminopurine in acetonitrile and aqueous solution to shed light on the photophysical and excited state properties of the 2,6-diaminopurine chromophore. Evidence is presented that both are photostable compounds exhibiting similar deactivation mechanisms upon the population of the S1 (ππ* La ) state at 290 nm. The mechanism involves deactivation through the C2- and C6-reaction coordinates and >99% of the excited state population decays through nonradiative pathways involving two conical intersections with the ground state. The radiative and nonradiative lifetimes are longer in aqueous solution compared to acetonitrile. While τ1 is similar in both derivatives, τ2 is ca. 1.5-fold longer in 2,6-diaminopurine-2'-deoxyribose due to a more efficient trapping in the S1 (ππ* La ) minimum. Therefore, 2,6-diaminopurine could have accumulated in significant quantities during prebiotic times to be incorporated into non-canonical RNA and play a significant role in its photoprotection.

The BR-body proteome contains a complex network of protein-protein and protein-RNA interactions.

Bacterial ribonucleoprotein bodies (BR-bodies) are non-membrane-bound structures that facilitate mRNA decay by concentrating mRNA substrates with RNase E and the associated RNA degradosome machinery. However, the full complement of proteins enriched in BR-bodies has not been defined. Here, we define the protein components of BR-bodies through enrichment of the bodies followed by mass spectrometry-based proteomic analysis. We find 111 BR-body-enriched proteins showing that BR-bodies are more complex than previously assumed. We identify five BR-body-enriched proteins that undergo RNA-dependent phase separation in vitro with a complex network of condensate mixing. We observe that some RNP condensates co-assemble with preferred directionality, suggesting that RNA may be trafficked through RNP condensates in an ordered manner to facilitate mRNA processing/decay, and that some BR-body-associated proteins have the capacity to dissolve the condensate. Altogether, these results suggest that a complex network of protein-protein and protein-RNA interactions controls BR-body phase separation and RNA processing.

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.

Insecticidal activity of essential oils from American native plants against Aedes aegypti (Diptera: Culicidae): an introduction to their possible mechanism of action.

Searching for new bioactive molecules to design insecticides is a complex process since pesticides should be highly selective, active against the vector, and bio-safe for humans. Aiming to find natural compounds for mosquito control, we evaluated the insecticidal activity of essential oils (EOs) from 20 American native plants against Aedes aegypti larvae using bioassay, biochemical, and in silico analyses. The highest larvicide activity was exhibited by EOs from Steiractinia aspera (LC50 = 42.4 µg/mL), Turnera diffusa (LC50 = 70.9 µg/mL), Piper aduncum (LC50 = 55.8 µg/mL), Lippia origanoides (chemotype thymol/carvacrol) (LC50 = 61.9 µg/mL), L. origanoides (chemotype carvacrol/thymol) (LC50 = 59.8 µg/mL), Hyptis dilatata (LC50 = 61.1 µg/mL), Elaphandra quinquenervis (LC50 = 61.1 µg/mL), and Calycolpus moritzianus (LC50 = 73.29 µg/mL) after 24 h. This biological activity may be related to the disruption of the electron transport chain through the mitochondrial protein complexes. We hypothesized that the observed EOs' effect is due to their major components, where computational approaches such as homology modeling and molecular docking may suggest the possible binding pose of secondary metabolites that inhibit the mitochondrial enzymes and acetylcholinesterase activity (AChE). Our results provided insights into the possible mechanism of action of EOs and their major compounds for new insecticide designs targeting the mitochondria and AChE activity in A. aegypti for effective and safe insecticide.

Photostability of 2,6-diaminopurine and its 2'-deoxyriboside investigated by femtosecond transient absorption spectroscopy.

Ultraviolet radiation (UVR) from the sun is essential for the prebiotic syntheses of nucleotides, but it can also induce photolesions such as the cyclobutane pyrimidine dimers (CPDs) to RNA or DNA oligonucleotide in prebiotic Earth. 2,6-Diaminopurine (26DAP) has been proposed to repair CPDs in high yield under prebiotic conditions and be a key component in enhancing the photostability of higher-order prebiotic DNA structures. However, its electronic relaxation pathways have not been studied, which is necessary to know whether 26DAP could have survived the intense UV fluxes of the prebiotic Earth. We investigate the electronic relaxation mechanism of both 26DAP and its 2'-deoxyribonucleoside (26DAP-d) in aqueous solution using steady-state and femtosecond transient absorption measurements that are complemented with electronic-structure calculations. The results demonstrate that both purine derivatives are significantly photostable to UVR. It is shown that upon excitation at 287 nm, the lowest energy 1ππ* state is initially populated. The population then branches following two relaxation coordinates in the 1ππ* potential energy surface, which are identified as the C2- and C6-relaxation coordinates. The population following the C6-coordinate internally converts to the ground state nonradiatively through a nearly barrierless conical intersection within 0.7 ps in 26DAP or within 1.1 ps in 26DAP-d. The population that follows the C2-relaxation coordinate decays back to the ground state by a combination of nonradiative internal conversion via a conical intersection and fluorescence emission from the 1ππ* minimum in 43 ps and 1.8 ns for the N9 and N7 tautomers of 26DAP, respectively, or in 70 ps for 26DAP-d. Fluorescence quantum yields of 0.037 and 0.008 are determined for 26DAP and 26DAP-d, respectively. Collectively, it is demonstrated that most of the excited state population in 26DAP and 26DAP-d decays back to the ground state via both nonradiative and radiative relaxation pathways. This result lends support to the idea that 26DAP could have accumulated in large enough quantities during the prebiotic era to participate in the formation of prebiotic RNA or DNA oligomers and act as a key component in the protection of the prebiotic genetic alphabet.

On the Photostability of Cyanuric Acid and Its Candidature as a Prebiotic Nucleobase.

Cyanuric acid is a triazine derivative that has been identified from reactions performed under prebiotic conditions and has been proposed as a prospective precursor of ancestral RNA. For cyanuric acid to have played a key role during the prebiotic era, it would have needed to survive the harsh electromagnetic radiation conditions reaching the Earth's surface during prebiotic times (≥200 nm). Therefore, the photostability of cyanuric acid would have been crucial for its accumulation during the prebiotic era. To evaluate the putative photostability of cyanuric acid in water, in this contribution, we employed density functional theory (DFT) and its time-dependent variant (TD-DFT) including implicit and explicit solvent effects. The calculations predict that cyanuric acid has an absorption maximum at ca. 160 nm (7.73 eV), with the lowest-energy absorption band extending to ca. 200 nm in an aqueous solution and exhibiting negligible absorption at longer wavelengths. Excitation of cyanuric acid at 160 nm or longer wavelengths leads to the population of S5,6 singlet states, which have ππ* character and large oscillator strengths (0.8). The population reaching the S5,6 states is expected to internally convert to the S1,2 states in an ultrafast time scale. The S1,2 states, which have nπ* character, are predicted to access a conical intersection with the ground state in a nearly barrierless fashion (ca. ≤ 0.13 eV), thus efficiently returning the population to the ground state. Furthermore, based on calculated spin-orbit coupling elements of ca. 6 to 8 cm-1, the calculations predict that intersystem crossing to the triplet manifold should play a minor role in the electronic relaxation of cyanuric acid. We have also calculated the vertical ionization energy of cyanuric acid at 8.2 eV, which predicts that direct one-photon ionization of cyanuric acid should occur at ca. 150 nm. Collectively, the quantum-chemical calculations predict that cyanuric acid would have been highly photostable under the solar radiation conditions reaching the Earth's surface during the prebiotic era in an aqueous solution. Of relevance to the chemical origin of life and RNA-first theories, these observations lend support to the idea that cyanuric acid could have accumulated in large quantities during the prebiotic era and thus strengthens its candidature as a relevant prebiotic nucleobase.

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.

Femtosecond intersystem crossing to the reactive triplet state of the 2,6-dithiopurine skin cancer photosensitizer.

Site-selected sulfur-substituted nucleobases are a class of all organic, heavy-atom-free photosensitizers for photodynamic therapy applications that exhibit excellent photophysical properties such as strong absorption in the ultraviolet-A region of the electromagnetic spectrum, near-unity triplet yields, and a high yield of singlet oxygen generation. Recent investigations on doubly thionated nucleobases, 2,4-dithiothymine, 2,4-dithiouracil, and 2,6-dithiopurine, demonstrated that these set of dithionated nucleobases outperform the photodynamic efficacy exhibit by 4-thiothymidine-the most widely studied singly substituted thiobase to date. Out of the three dithionated nucleobases, 2,6-dithiopurine was shown to be the most effective, exhibiting inhibition of cell proliferation of up to 63% when combined with a low UVA dose of 5 J cm-2. In this study, we elucidated the electronic relaxation pathways leading to the population of the reactive triplet state of 2,6-dithiopurine. 2,6-Dithiopurine populates the triplet manifold in less than 150 fs, reaching the nπ* triplet state minimum within a lifetime of 280 ± 50 fs. Subsequently, the population in the nπ* triplet state minimum internally converts to the long-lived ππ* triplet state within a lifetime of 3 ± 1 ps. The relatively slow internal conversion lifetime is associated with major conformational relaxation in going from the nπ* to ππ* triplet state minimum. A unity triplet yield of 1.0 ± 0.1 is measured.

Electronic Relaxation Pathways in Heavy-Atom-Free Photosensitizers Absorbing Near-Infrared Radiation and Exhibiting High Yields of Singlet Oxygen Generation.

Heavy-atom-free photosensitizers (HAF-PSs) based on thionation of carbonyl groups of readily accessible organic compounds are rapidly emerging as a versatile class of molecules. However, their photochemical properties and electronic relaxation mechanisms are currently unknown. Investigating the excited-state dynamics is essential to understand their benefits and limitations and to develop photosensitizers with improved photochemical properties. Herein, the photochemical and electronic-structure properties of two of the most promising HAF-PSs developed to date are revealed. It is shown that excitation of thio-4-(dimethylamino)naphthalamide and thionated Nile Red with near-infrared radiation leads to the efficient population of the triplet manifold through multiple relaxation pathways in hundreds of femtoseconds. The strong singlet-triplet couplings in this family of photosensitizers should enable a broad range of applications, including in photodynamic therapy, photocatalysis, photovoltaics, organic LEDs, and photon up-conversion.

Detection of the thietane precursor in the UVA formation of the DNA 6-4 photoadduct.

Notwithstanding the central biological role of the (6-4) photoadduct in the induction of skin cancer by sunlight, crucial mechanistic details about its formation have evaded characterization despite efforts spanning more than half a century. 4-Thiothymidine (4tT) has been widely used as an important model system to study its mechanism of formation, but the excited-state precursor, the intermediate species, and the time scale leading to the formation of the (6-4) photoadduct have remained elusive. Herein, steady-state and time-resolved spectroscopic techniques are combined with new and reported quantum-chemical calculations to demonstrate the excited state leading to the formation of the thietane intermediate, its rate, and the formation of the (6-4) photoadduct using the 5'-TT(4tT)T(4tT)TT-3' DNA oligonucleotide. Efficient, sub-1 ps intersystem crossing leads to the population of a triplet minimum of the thietane intermediate in as short as 3 ps, which intersystem crosses to its ground state and rearranges to form the (6-4) photoadduct.

Thionated organic compounds as emerging heavy-atom-free photodynamic therapy agents.

This minireview focuses on recent progress in developing heavy-atom-free photosensitizers based on the thionation of nucleic acid derivatives and other biocompatible organic compounds for prospective applications in photodynamic therapy. Particular attention is given to the use of thionated nucleobase derivatives as "one-two punch" photodynamic agents. These versatile photosensitizers can act as "Trojan horses" upon metabolization into DNA and exposure to activating light. Their incorporation into cellular DNA increases their selectivity and photodynamic efficacy against highly proliferating skin cancer tumor cells, while simultaneously enabling the use of low irradiation doses both in the presence and in the absence of molecular oxygen. Also reviewed are their primary photochemical reactions, modes of action, and photosensitization mechanisms. New developments of emerging thionated organic photosensitizers absorbing visible and near-infrared radiation are highlighted. Future research directions, as well as, other prospective applications of heavy-atom-free, thionated photosensitizers are discussed.

Photochemical relaxation pathways of S6-methylthioinosine and O6-methylguanosine in solution.

S6-Methylthioinosine and O6-methylguanosine are byproducts resulting from the enzymatic reactions of sulfur-substituted prodrugs in cells and from the interaction of alkylating agents with cellular DNA, respectively. Their photochemistry has not been investigated, and it is currently unknown whether light absorption by these byproducts may pose any threat to the cell. In this contribution, their photoinduced processes upon absorption of UVB radiation are reported using broadband transient absorption spectroscopy. Plausible electronic relaxation mechanisms are proposed for both biological molecules, which are supported by steady-state absorption and emission measurements, and by singlet and triplet vertical excitation energies performed on a large subset of ground-state optimized conformational isomers in solution. The results are compared to the body of knowledge gathered in the scientific literature about the light-induced processes in the sulfur-substituted and canonical purine monomers. In particular, it is shown that S6-methylation decreases the rate to populate the lowest-energy triplet state and blueshifts the ground-state absorption spectrum compared to those for the sulfur-substituted prodrugs and for the 6-thioguanosine metabolite. Similarly, O6-methylation decreases the rate of internal conversion to the ground state observed in the guanine monomers by more than 10-fold in acetonitrile and 40-fold in aqueous solution, while it redshifts the ground-state absorption spectrum. Collectively, this investigation provides relevant new insights about the relationship between structural modifications of the purine chromophore and the electronic relaxation mechanisms in this important group of biological molecules.

Excited-State Dynamics in O6-Methylguanosine: Impact of O6-Methylation on the Relaxation Mechanism of Guanine Monomers.

Absorption of ultraviolet radiation by DNA bases results in ultrafast internal conversion to the ground state, which minimizes photodamage. However, exogenous and endogenous alkylating agents present in the cellular environment can methylate the nucleobases in DNA. In particular, methylation of guanosine at the O6 position in DNA leads to the formation of the O6-methylguanosine adduct, which may alter the photostability of DNA. This contribution demonstrates that O6-methylation of guanosine red shifts its ground-state absorption spectrum and slows down the rate of internal conversion to the ground state by ∼40-fold in aqueous solution. The 40-fold decrease in the rate of excited-state decay increases the probability of photodamage within cellular DNA. It is proposed that the longer decay lifetime corresponds to relaxation of the excited-state population in O6-methylguanosine along a C6-puckered reaction coordinate in the 1ππ*(La) potential energy surface that runs parallel to an ultrafast internal conversion pathway along a C2-puckered coordinate.

The Triplet State of 6-thio-2'-deoxyguanosine: Intrinsic Properties and Reactivity Toward Molecular Oxygen.

Thiopurine prodrugs are currently among the leading treatment options for leukemia, immunosuppression, and arthritis. Patients undergoing long-term thiopurine treatment are at a higher risk of developing sunlight-induced skin cancers than the general population. This side effect originates from the cellular metabolization of thiopurine prodrugs to form 6-thio-2'-deoxyguanosine, which can absorb UVA radiation, populating its reactive triplet state and leading to oxidatively generated damage. However, the photo-oxidation mechanism is not fully understood. In this contribution, the oxidation potential and the adiabatic triplet energy of 6-thio-2'-deoxyguanosine are estimated computationally, whereas the intrinsic rate of triple-state decay and the rate constant for triplet quenching by molecular oxygen are determined using time-resolved spectroscopic techniques. A singlet oxygen quantum yield of 0.24 ± 0.02 is measured in aqueous solution (0.29 ± 0.02 in acetonitrile). Its magnitude correlates with the relatively low percentage of triplet-O2 collision events that generate singlet oxygen (SΔ = 37%). This behavior is rationalized as being due to the exergonic driving force for electron transfer between the triplet state of 6-thio-2'-deoxyguanosine and molecular oxygen (ΔGET = -69.7 kJ mol-1 ), resulting in the formation of a charge-transfer complex that favors nonradiative decay to the ground state over triplet energy transfer.