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.

Photochemical and Photodynamical Properties of Sulfur-Substituted Nucleic Acid Bases.

Sulfur-substituted nucleobases (a.k.a., thiobases) are among the world's leading prescriptions for chemotherapy and immunosuppression. Long-term treatment with azathioprine, 6-mercaptopurine and 6-thioguanine has been correlated with the photoinduced formation of carcinomas. Establishing an in-depth understanding of the photochemical properties of these prodrugs may provide a route to overcoming these carcinogenic side effects, or, alternatively, a basis for developing effective compounds for targeted phototherapy. In this review, a broad examination is undertaken, surveying the basic photochemical properties and excited-state dynamics of sulfur-substituted analogs of the canonical DNA and RNA nucleobases. A molecular-level understanding of how sulfur substitution so remarkably perturbs the photochemical properties of the nucleobases is presented by combining experimental results with quantum-chemical calculations. Structure-property relationships demonstrate the impact of site-specific sulfur substitution on the photochemical properties, particularly on the population of the reactive triplet state. The value of fundamental photochemical investigations for driving the development of ultraviolet-A chemotherapeutics is showcased. The most promising photodynamic agents identified thus far have been investigated in various carcinoma cell lines and shown to decrease cell proliferation upon exposure to ultraviolet-A radiation. Overarching principles have been elucidated for the impact that sulfur substitution of the carbonyl oxygen has on the photochemical properties of the nucleobases.

Dithionated Nucleobases as Effective Photodynamic Agents against Human Epidermoid Carcinoma Cells.

Sulfur-substituted nucleobases (i.e., thiobases) are a prospective class of compounds for clinical and cosmetic topical phototherapies. Recent investigations of several thiobases have revealed the ultrafast and efficient population of reactive triplet states upon ultraviolet-A (UVA) irradiation and the subsequent generation of singlet oxygen in high yield. In this contribution, we examine the photosensitizing activities of three of the most promising thiobase derivatives discovered to date: 2,4-dithiothymine, 2,4-dithiouracil, and 2,6-dithiopurine. These derivatives are shown to decrease the proliferation of human epidermoid carcinoma cells by up to 63 % in vitro, only upon activation with a low dose of UVA radiation (5 J cm-2 ). The generation of reactive oxygen species plays a minor role in the mode of action, suggesting these dithiobases may be effective within oxygen-deficient environments. Importantly, the photosensitized activity correlates with the magnitude of the triplet lifetime, which should guide the molecular design of next-generation photodynamic agents.

HnRNP A1 Alters the Structure of a Conserved Enterovirus IRES Domain to Stimulate Viral Translation.

Enteroviruses use a type I Internal Ribosome Entry Site (IRES) structure to facilitate protein synthesis and promote genome replication. Type I IRES elements require auxiliary host proteins to organize RNA structure for 40S ribosomal subunit assembly. Heterogeneous nuclear ribonucleoprotein A1 stimulates enterovirus 71 (EV71) translation in part through specific interactions with its stem loop II (SLII) IRES domain. Here, we determined a conjoined NMR-small angle x-ray scattering structure of the EV71 SLII domain and a mutant that significantly attenuates viral replication by abrogating hnRNP A1 interactions. Native SLII adopts a locally compact structure wherein stacking interactions in a conserved 5'-AUAGC-3' bulge preorganize the adjacent helices at nearly orthogonal orientations. Mutating the bulge sequence to 5'-ACCCC-3' ablates base stacking in the loop and globally reorients the SLII structure. Biophysical titrations reveal that the 5'-AUAGC-3' bulge undergoes a conformational change to assemble a functional hnRNP A1-RNA complex. Importantly, IRES mutations that delete the bulge impair viral translation and completely inhibit replication. Thus, this work provides key details into how an EV71 IRES structure adapts to hijack a cellular protein, and it suggests that the SLII domain is a potential target for antiviral therapy.

2-Thiouracil intersystem crossing photodynamics studied by wavelength-dependent photoelectron and transient absorption spectroscopies.

Single-atom substitution within a natural nucleobase-such as replacing oxygen by sulfur in uracil-can result in drastic changes in the relaxation dynamics after UV excitation. While the photodynamics of natural nucleobases like uracil are dominated by pathways along singlet excited states, the photodynamics of thiobases like 2-thiouracil populate the triplet manifold with near unity quantum yield. In the present study, a synergistic approach based on time-resolved photoelectron spectroscopy (TRPES), time-resolved absorption spectroscopy (TRAS), and ab initio computations has been particularly successful at unraveling the underlying photophysical principles and describing the dissimilarities between the natural and substituted nucleobases. Specifically, we find that varying the excitation wavelength leads to differences between gas-phase and condensed-phase experimental results. Systematic trends are observed in the intersystem crossing time constants with varying excitation wavelength, which can be readily interpreted in the context of ab initio calculations performed both in vacuum and including solvent effects. Thus, the combination of TRPES and TRAS experiments with high-level computational techniques allows us to characterize the topology of the potential energy surfaces defining the relaxation dynamics of 2-thiouracil in both gas and condensed phases, as well as investigate the accessibility of conical intersections and crossings, and potential energy barriers along the associated relaxation coordinates.

Decoding the Molecular Basis for the Population Mechanism of the Triplet Phototoxic Precursors in UVA Light-Activated Pyrimidine Anticancer Drugs.

The photosensitization of DNA by thionucleosides is a promising photo-chemotherapeutic treatment option for a variety of malignancies. DNA metabolization of thionated prodrugs can lead to cell death upon exposure to a low dose of UVA light. The exact mechanisms of thionucleoside phototoxicity are still not fully understood. In this work, we have combined femtosecond broadband transient absorption experiments with state-of-the-art molecular simulations to provide mechanistic insights into the ultrafast and efficient population of the triplet state in the UVA-activated pyrimidine anticancer drug 4-thiothymine. The triplet state is thought to act as a precursor to DNA lesions and the reactive oxygen species responsible for 4-thiothymine photocytotoxicity. The electronic-structure and mechanistic results presented in this contribution reveal key molecular design criteria that can assist in developing alternative chemotherapeutic agents that may overcome some of the primary deficiencies of classical photosensitizers.

Can a Six-Letter Alphabet Increase the Likelihood of Photochemical Assault to the Genetic Code?

In 2014, two unnatural nucleosides, d5SICS and dNaM, were shown to selectively base pair and replicate with high fidelity in a modified strain of E. coli, thus effectively expanding its genetic alphabet from four to six letters. More recently, a significant reduction in cell proliferation was reported in cells cultured with d5SICS, and putatively with dNaM, upon exposure to brief periods of near-visible radiation. The photosensitizing properties of the lowest-energy excited triplet state of both d5SICS and dNaM were implicated in their cytotoxicity. Importantly, however, the excited-state mechanisms by which near-visible excitation populates the triplet states of d5SICS and dNaM are currently unknown. In this study, steady-state and time-resolved spectroscopies are combined with quantum-chemical calculations in order to reveal the excited-state relaxation mechanisms leading to efficient population of the triplet states in these unnatural nucleosides in solution. It is shown that excitation of d5SICS or dNaM with near-visible light leads overwhelmingly to ultrafast population of their triplet states on the femtosecond time scale. The results presented in this work lend strong support to the proposal that photoexcitation of these unnatural nucleosides can accelerate oxidatively generated damage to DNA and other biomolecules within the cellular environment.

The origin of efficient triplet state population in sulfur-substituted nucleobases.

Elucidating the photophysical mechanisms in sulfur-substituted nucleobases (thiobases) is essential for designing prospective drugs for photo- and chemotherapeutic applications. Although it has long been established that the phototherapeutic activity of thiobases is intimately linked to efficient intersystem crossing into reactive triplet states, the molecular factors underlying this efficiency are poorly understood. Herein we combine femtosecond transient absorption experiments with quantum chemistry and nonadiabatic dynamics simulations to investigate 2-thiocytosine as a necessary step to unravel the electronic and structural elements that lead to ultrafast and near-unity triplet-state population in thiobases in general. We show that different parts of the potential energy surfaces are stabilized to different extents via thionation, quenching the intrinsic photostability of canonical DNA and RNA nucleobases. These findings satisfactorily explain why thiobases exhibit the fastest intersystem crossing lifetimes measured to date among bio-organic molecules and have near-unity triplet yields, whereas the triplet yields of canonical nucleobases are nearly zero.

Unintended Consequences of Expanding the Genetic Alphabet.

The base pair d5SICS·dNaM was recently reported to incorporate and replicate in the DNA of a modified strain of Escherichia coli, thus making the world's first stable semisynthetic organism. This newly expanded genetic alphabet may allow organisms to store considerably more information in order to translate proteins with unprecedented enzymatic activities. Importantly, however, there is currently no knowledge of the photochemical properties of d5SICS or dNaM-properties that are central to the chemical integrity of cellular DNA. In this contribution, it is shown that excitation of d5SICS or dNaM with near-visible light leads to efficient trapping of population in the nucleoside's excited triplet state in high yield. Photoactivation of these long-lived, reactive states is shown to photosensitize cells, leading to the generation of reactive oxygen species and to a marked decrease in cell proliferation, thus warning scientists of the potential phototoxic side effects of expanding the genetic alphabet.

Internal conversion and intersystem crossing pathways in UV excited, isolated uracils and their implications in prebiotic chemistry.

The photodynamic properties of molecules determine their ability to survive in harsh radiation environments. As such, the photostability of heterocyclic aromatic compounds to electromagnetic radiation is expected to have been one of the selection pressures influencing the prebiotic chemistry on early Earth. In the present study, the gas-phase photodynamics of uracil, 5-methyluracil (thymine) and 2-thiouracil-three heterocyclic compounds thought to be present during this era-are assessed in the context of their recently proposed intersystem crossing pathways that compete with internal conversion to the ground state. Specifically, time-resolved photoelectron spectroscopy measurements evidence femtosecond to picosecond timescales for relaxation of the singlet (1)ππ* and (1)nπ* states as well as for intersystem crossing to the triplet manifold. Trapping in the excited triplet state and intersystem crossing back to the ground state are investigated as potential factors contributing to the susceptibility of these molecules to ultraviolet photodamage.

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.

Electronic and structural elements that regulate the excited-state dynamics in purine nucleobase derivatives.

The excited-state dynamics of the purine free base and 9-methylpurine are investigated using experimental and theoretical methods. Femtosecond broadband transient absorption experiments reveal that excitation of these purine derivatives in aqueous solution at 266 nm results primarily in ultrafast conversion of the S2(ππ*) state to the vibrationally excited (1)nπ* state. Following vibrational and conformational relaxation, the (1)nπ* state acts as a doorway state in the efficient population of the triplet manifold with an intersystem crossing lifetime of hundreds of picoseconds. Experiments show an almost 2-fold increase in the intersystem crossing rate on going from polar aprotic to nonpolar solvents, suggesting that a solvent-dependent energy barrier must be surmounted to access the singlet-to-triplet crossing region. Ab initio static and surface-hopping dynamics simulations lend strong support to the proposed relaxation mechanism. Collectively, the experimental and computational results demonstrate that the accessibility of the nπ* states and the topology of the potential energy surfaces in the vicinity of conical intersections are key elements in controlling the excited-state dynamics of the purine derivatives. From a structural perspective, it is shown that the purine chromophore is not responsible for the ultrafast internal conversion in the adenine and guanine monomers. Instead, C6 functionalization plays an important role in regulating the rates of radiative and nonradiative relaxation. C6 functionalization inhibits access to the (1)nπ* state while simultaneously facilitating access to the (1)ππ*(La)/S0 conical intersection, such that population of the (1)nπ* state cannot compete with the relaxation pathways to the ground state involving ring puckering at the C2 position.

Photochemistry of nucleic acid bases and their thio- and aza-analogues in solution.

The steady-state and time-resolved photochemistry of the natural nucleic acid bases and their sulfur- and nitrogen-substituted analogues in solution is reviewed. Emphasis is given to the experimental studies performed over the last 3-5 years that showcase topical areas of scientific inquiry and those that require further scrutiny. Significant progress has been made toward mapping the radiative and nonradiative decay pathways of nucleic acid bases. There is a consensus that ultrafast internal conversion to the ground state is the primary relaxation pathway in the nucleic acid bases, whereas the mechanism of this relaxation and the level of participation of the (1)πσ*, (1) nπ*, and (3)ππ* states are still matters of debate. Although impressive research has been performed in recent years, the microscopic mechanism(s) by which the nucleic acid bases dissipate excess vibrational energy to their environment, and the role of the N-glycosidic group in this and in other nonradiative decay pathways, are still poorly understood. The simple replacement of a single atom in a nucleobase with a sulfur or nitrogen atom severely restricts access to the conical intersections responsible for the intrinsic internal conversion pathways to the ground state in the nucleic acid bases. It also enhances access to ultrafast and efficient inter-system crossing pathways that populate the triplet manifold in yields close to unity. Determining the coupled nuclear and electronic pathways responsible for the significantly different photochemistry in these nucleic acid base analogues serves as a convenient platform to examine the current state of knowledge regarding the photodynamic properties of the DNA and RNA bases from both experimental and computational perspectives. Further investigations should also aid in forecasting the prospective use of sulfur- and nitrogen-substituted base analogues in photochemotherapeutic applications.

2,4-Dithiothymine as a potent UVA chemotherapeutic agent.

Substitution of both oxygen atoms in the exocyclic carbonyl groups of the thymine chromophore by sulfur atoms results in a remarkable redshift of its absorption spectrum from an absorption maximum at 267 nm in thymidine to 363 nm in 2,4-dithiothymine (ΔE = 9905 cm(-1)). A single sulfur substitution of a carbonyl group in the thymine chromophore at position 2 or 4 results in a significantly smaller redshift in the absorption maximum, which depends sensitively on the position at which the sulfur atom is substituted, varying from 275 nm in 2-thiothymine to 335 nm in 4-thiothymidine. Femtosecond transient absorption spectroscopy reveals that excitation of 2,4-dithiothymine at 335 or 360 nm leads to the ultrafast population of the triplet state, with an intersystem crossing lifetime of 180 ± 40 fs­the shortest intersystem crossing lifetime of any DNA base derivative studied so far in aqueous solution. Surprisingly, the degree and position at which the sulfur atom is substituted have important effects on the magnitude of the intersystem crossing rate constant, showing a 1.2-, 3.2-, and 4.2-fold rate increases for 2-thiothymine, 4-thiothymidine, and 2,4-dithiothymine, respectively, relative to that of thymidine, whereas the triplet yield increases 60-fold to near unity, independent of the site of sulfur atom substitution. While the natural thymine monomers owe their high degree of photostability to ultrafast internal conversion to the ground state and low triplet yields, the near-unity triplet yields in the thiothymine series account for their potent photosensitization properties. Nanosecond time-resolved luminescence spectroscopy shows that 4-thiothymidine and 2,4-dithiothymine are efficient singlet oxygen generators, with singlet oxygen quantum yields of 0.42 ± 0.02 and 0.46 ± 0.02, respectively, in O2-saturated acetonitrile solution. Taken together, these photophysical measurements strongly suggest that 2,4-dithiothymine can act as a more effective UVA chemotherapeutic agent than the currently used 4-thiothymidine, especially in deeper-tissue chemotherapeutic applications.

Communication: the dark singlet state as a doorway state in the ultrafast and efficient intersystem crossing dynamics in 2-thiothymine and 2-thiouracil.

Femtosecond broadband transient absorption experiments are reported for 2-thiothymine and 2-thiouracil in aqueous buffer solution and in acetonitrile. It is shown that the S1(nπ*) state acts as a doorway state in the ultrafast and efficient population of the T1(ππ*) state upon 316 nm excitation. A sequential kinetic model is presented to explain the excited-state dynamics in 2-thiothymine and 2-thiouracil upon UVA excitation: S2(ππ*) → S1(nπ*) → T1(ππ*). The experimental results are also used to scrutinize the excited-state relaxation pathways recently predicted for 2-thiouracil at the CASPT2//CASSCF level of theory [G. Cui and W. Fang, J. Chem. Phys. 138, 044315 (2013)]. The efficient population of the T1(ππ*) state for both 2-thiothymine and 2-thiouracil in a few hundreds of femtoseconds lends further support to the emerging idea that thiobase derivatives exhibit photo-toxic properties that can be effectively harnessed in photo-chemotherapeutic applications.