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.
Thiobases are established compounds in the WHO list of top 100 essential medicines for the treatment of leukemia, autoimmune conditions, and immunosuppression. Although they are highly effective medications, long‐term treatment of patients is associated with a 10‐fold increase in basal cell carcinomas and a 65‐ to 250‐fold increase in squamous cell carcinomas. These side effects are induced by the exposure to sunlight, which originates from the ability of the thiobases to absorb UVA radiation. An in‐depth understanding of their photochemistry can provide a route to overcoming these side effects or, alternatively, a basis for developing effective compounds for phototherapy.
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.
6-Thioguanine, an immunosuppressant and anticancer prodrug, has been shown to induce DNA damage and cell death following exposure to UVA radiation. Its metabolite, 6-thioguanosine, plays a major role ...in the prodrug's overall photoreactivity. However, 6-thioguanine itself has proven to be cytotoxic following UVA irradiation, warranting further investigation into its excited-state dynamics. In this contribution, the excited-state dynamics and photochemical properties of 6-thioguanine are studied in aqueous solution following UVA excitation at 345 nm in order to provide mechanistic insight regarding its photochemical reactivity and to scrutinize whether N9-glycosylation modulates its phototoxicity in solution. The experimental results are complemented with time-dependent density functional calculations that include solvent dielectric effects by means of a reaction-field solvation model. UVA excitation results in the initial population of the S₂(ππ*) state, which is followed by ultrafast internal conversion to the S₁(nπ*) state and then intersystem crossing to the triplet manifold within 560 ± 60 fs. A small fraction (ca. 25%) of the population that reaches the S₁(nπ*) state repopulates the ground state. The T₁(ππ*) state decays to the ground state in 1.4 ± 0.2 μs under N₂-purged conditions, using a 0.2 mM concentration of 6-thioguanine, or it can sensitize singlet oxygen in 0.21 ± 0.02 and 0.23 ± 0.02 yields in air- and O₂-saturated solution, respectively. This demonstrates the efficacy of 6-thioguanine to act as a Type II photosensitizer. N9-glycosylation increases the rate of intersystem crossing from the singlet to triplet manifold, as well as from the T₁(ππ*) state to the ground state, which lead to a ca. 40% decrease in the singlet oxygen yield under air-saturated conditions. Enhanced vibronic coupling between the singlet and triplet manifolds due to a higher density of vibrational states is proposed to be responsible for the observed increase in the rates of intersystem crossing in 6-thioguanine upon N9-glycosylation.
Special issue on nucleic acid photophysics Crespo‐Hernández, Carlos E.
Photochemistry and photobiology,
March/April 2024, 2024-03-00, 20240301, Volume:
100, Issue:
2
Journal Article
Gene expression in higher eukaryotic cells orchestrates interactions between thousands of RNA-binding proteins (RBPs) and tens of thousands of RNAs
. The kinetics by which RBPs bind to and dissociate ...from their RNA sites are critical for the coordination of cellular RNA-protein interactions
. However, these kinetic parameters have not been experimentally measured in cells. Here we show that time-resolved RNA-protein cross-linking with a pulsed femtosecond ultraviolet laser, followed by immunoprecipitation and high-throughput sequencing, allows the determination of binding and dissociation kinetics of the RBP DAZL for thousands of individual RNA-binding sites in cells. This kinetic cross-linking and immunoprecipitation (KIN-CLIP) approach reveals that DAZL resides at individual binding sites for time periods of only seconds or shorter, whereas the binding sites remain DAZL-free for markedly longer. The data also indicate that DAZL binds to many RNAs in clusters of multiple proximal sites. The effect of DAZL on mRNA levels and ribosome association correlates with the cumulative probability of DAZL binding in these clusters. Integrating kinetic data with mRNA features quantitatively connects DAZL-RNA binding to DAZL function. Our results show how kinetic parameters for RNA-protein interactions can be measured in cells, and how these data link RBP-RNA binding to the cellular function of RBPs.
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.
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.
Mechanism unraveled! By combining femtosecond transient absorption spectroscopy and high‐level molecular simulations, the mechanism by which long‐lived triplet excited states in 4‐thiothymine are populated has been characterized (see figure). The triplet state is believed to be responsible for the phototoxic properties of 4‐thiothymine following activation by UVA light.
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.
Thymine Dimerization in DNA Is an Ultrafast Photoreaction Schreier, Wolfgang J; Schrader, Tobias E; Koller, Florian O ...
Science (American Association for the Advancement of Science),
02/2007, Volume:
315, Issue:
5812
Journal Article
Peer reviewed
Open access
Femtosecond time-resolved infrared spectroscopy was used to study the formation of cyclobutane dimers in the all-thymine oligodeoxynucleotide (dT)₁₈ by ultraviolet light at 272 nanometers. The ...appearance of marker bands in the time-resolved spectra indicates that the dimers are fully formed ~1 picosecond after ultraviolet excitation. The ultrafast appearance of this mutagenic photolesion points to an excited-state reaction that is approximately barrierless for bases that are properly oriented at the instant of light absorption. The low quantum yield of this photoreaction is proposed to result from infrequent conformational states in the unexcited polymer, revealing a strong link between conformation before light absorption and photodamage.
Damage to RNA from ultraviolet radiation induces chemical modifications to the nucleobases. Unraveling the excited states involved in these reactions is essential; however, investigations aimed at ...understanding the electronic-energy relaxation pathways of the RNA nucleotide uridine 5′-monophosphate (UMP) have not received enough attention. In this Letter, the excited-state dynamics of UMP is investigated in aqueous solution. Excitation at 267 nm results in a trifurcation event that leads to the simultaneous population of the vibrationally excited ground state, a long-lived 1nπ* state, and a receiver triplet state within 200 fs. The receiver state internally converts to the long-lived 3ππ* state in an ultrafast time scale. The results elucidate the electronic relaxation pathways and clarify earlier transient absorption experiments performed for uracil derivatives in solution. This mechanistic information is important because long-lived nπ* and ππ* excited states of both singlet and triplet multiplicities are thought to lead to the formation of harmful photoproducts.
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