Excited States of Thio-2'-deoxyuridine Bearing an Extended π-Conjugated System: 3',5'-Di-O-acetyl-5-phenylethynyl-4-thio-2'-deoxyuridine.

A new thio-2'-deoxyuridine with an extended π-conjugated group was successfully synthesized: 3',5'-di-O-acetyl-5-phenylethynyl-4-thio-2'-deoxyuridine. The thio-2'-deoxyuridine derivative has a large red-shifted absorption band in the UVA region and also shows fluorescence, a rare photo-property among thionucleobases/thionucleosides. The triplet-triplet absorption spectrum and the rate constants (the intrinsic decay rate constant of the triplet state, the self-quenching rate constant, and the quenching rate constant of the triplet state by an oxygen molecule) of the thio-2'-deoxyuridine were obtained by transient absorption spectroscopy. The quantum yield of intersystem crossing and the quantum yield of singlet molecular oxygen formation (ϕΔ) under an oxygen atmosphere were also determined. The Ï•Δ value of the new thio-2'-deoxyuridine was found to be substantially higher than all reported values of other thio-2'-deoxyribonucleosides in low oxygen concentrations similar to cancer cell environments. The fluorescence quantum yield depended on the excitation wavelength, revealing certain photochemical reactions in the higher excited singlet states. However, when excited into the higher excited state with non-resonant two-photon absorption, the Ï•Δ of the thio-2'-deoxyuridine derivative was found to remain sufficiently large. These findings should be very useful for the development of thio-2'-deoxyribonucleoside-based pharmaceuticals as DNA-specific photosensitizers for photochemotherapy.

Simultaneous Two-Photon Absorption of the Thioguanosine Analogue 2',3',5'-Tri-O-acetyl-6,8-dithioguanosine with Its Potential Application to Photodynamic Therapy.

2',3',5'-Tri-O-acetyl-6,8-dithioguanosine (taDTGuo) is an analogue of nucleosides and currently under investigation as a potential agent for photodynamic therapy (PDT). Excitation by simultaneous two-photon absorption of visible or near-infrared light would provide an efficient PDT for deep-seated tumors. The two-photon absorption spectrum of taDTGuo was obtained by optical-probing photoacoustic spectroscopy (OPPAS). A two-photon absorption band corresponding to the S5 ← S0 transition was observed at 556 nm, and the two-photon absorption cross-section σ(2) was determined to be 26 ± 3 GM, which was much larger than that of other nucleobases and nucleosides. Quantum chemical calculations suggested that the large σ(2) value of taDTGuo was responsible for large transition dipole moments and small detuning energy resulting from the thiocarbonyl group at 6- and 8-positions. This is the first report on two-photon absorption spectra and cross-sections of thionucleoside analogues, which could be used to develop a more specific PDT for cancers in deep regions.

Characteristics of the excited triplet states of thiolated guanosine derivatives and singlet oxygen generation.

Thioguanine is sensitive to UVA light and generates singlet molecular oxygen (1O2*) when exposed to UVA. Three thioguanosine derivatives, 2',3',5'-tri-O-acetyl-6-thioguanosine (ta6TGuo), 2',3',5'-tri-O-acetyl-8-thioguanosine (ta8TGuo), and 2',3',5'-tri-O-acetyl-6,8-dithioguanosine (taDTGuo) were explored photophysically and photochemically. Nanosecond transient absorption and time-resolved near-infrared emission measurements were carried out to investigate the characteristics of their excited triplet states in acetonitrile solution. The quantum yield of intersystem crossing (ΦISC), the intrinsic decay rate constant (k0), the quenching rate constant by 3O2 (kq) and the self-quenching rate constant (kSQ) of their triplet states were all determined. From the precise analysis of the quantum yield of 1O2* generation (ΦΔ) against the concentration of dissolved molecular oxygen, the fraction of the triplet states quenched by dissolved oxygen which gives rise to 1O2* formation (SΔ) was successfully obtained with high accuracy. The ΦΔ values at low oxygen concentrations reveal that these thioguanosines, particularly taDTGuo, can still effectively generate 1O2* at low molecular oxygen concentrations like carcinomatous microenvironments. These findings indicate that taDTGuo would perform well as a potential agent for photo-induced cancer therapies.

NMR studies on 4-thio-5-furan-modified and 4-thio-5-thiophene-modified nucleosides.

Systematic NMR characterization of 4-thio-5-furan-pyrimidine nucleosides or 4-thio-5-thiophene-pyrimidine nucleosides (ribonucleosides and 2'-deoxynucleosides) was performed. All proton and carbon signals of 4-thio-5-thiophene-ribouridine and related analogues were unambiguously assigned. The orientations of the base (4-thiouridine or its deoxy analogue) relative to the ring (furan or thiophene) are explored by a NMR approach and further supported by X-ray crystallographic studies. The procedures presented here would be applicable to other modified nucleosides and nucleotides. Copyright © 2016 John Wiley & Sons, Ltd.

Excited state properties of 5-fluoro-4-thiouridine derivative†.

The excited state properties of thionated 5-fluorouridine (2',3',5'-tri-O-acetyl-5-fluoro-4-thiouridine; ta5F4TUrd), synthesized with Lawesson's reagent, have been intensively investigated with nanosecond transient absorption spectroscopy, time-resolved thermal lensing, near-infrared emission, and quantum chemical calculation. The intrinsic triplet lifetime of ta5F4TUrd was determined to be 4.2 ± 0.7 µs in acetonitrile, and the formation quantum yield of the excited triplet state was as large as 0.79 ± 0.01 . The quenching rate constants of the triplet ta5F4TUrd by the dissolved oxygen molecule and by the self-quenching process were found to be nearly equal to the diffusion-controlled rate of acetonitrile. The quantum yield of the singlet molecular oxygen produced through energy transfer between the triplet ta5F4TUrd and the dissolved oxygen, Φ Δ , was successfully determined to be 0.61 ± 0.02 under the oxygen-saturated condition. From the oxygen concentration dependence of the Φ Δ value, the fraction of triplet ta5F4TUrd quenched by dissolved oxygen which gives rise to the 1 O2 * formation, S Δ , was successfully obtained to be 0.78 ± 0.01 , which was the largest among the thionucleobases and the thionucleosides reported so far. This could be due to the lower energy and/or the ππ* character of the triplet state.

Identification of potentially cytotoxic lesions induced by UVA photoactivation of DNA 4-thiothymidine in human cells.

Photochemotherapy-in which a photosensitizing drug is combined with ultraviolet or visible radiation-has proven therapeutic effectiveness. Existing approaches have drawbacks, however, and there is a clinical need to develop alternatives offering improved target cell selectivity. DNA substitution by 4-thiothymidine (S(4)TdR) sensitizes cells to killing by ultraviolet A (UVA) radiation. Here, we demonstrate that UVA photoactivation of DNA S(4)TdR does not generate reactive oxygen or cause direct DNA breakage and is only minimally mutagenic. In an organotypic human skin model, UVA penetration is sufficiently robust to kill S(4)TdR-photosensitized epidermal cells. We have investigated the DNA lesions responsible for toxicity. Although thymidine is the predominant UVA photoproduct of S(4)TdR in dilute solution, more complex lesions are formed when S(4)TdR-containing oligonucleotides are irradiated. One of these, a thietane/S(5)-(6-4)T:T, is structurally related to the (6-4) pyrimidine:pyrimidone [(6-4) Py:Py] photoproducts induced by UVB/C radiation. These lesions are detectable in DNA from S(4)TdR/UVA-treated cells and are excised from DNA more efficiently by keratinocytes than by leukaemia cells. UVA irradiation also induces DNA interstrand crosslinking of S(4)TdR-containing duplex oligonucleotides. Cells defective in repairing (6-4) Py:Py DNA adducts or processing DNA crosslinks are extremely sensitive to S(4)TdR/UVA indicating that these lesions contribute significantly to S(4)TdR/UVA cytotoxicity.

Systematic assignment of NMR spectra of 5-substituted-4-thiopyrimidine nucleosides.

Unambiguous characterization of 5-substituted-4-thiopyrimidine nucleosides (ribonucleosides and 2'-deoxynucleosides) was performed using NMR spectroscopy. Assignments of all proton and carbon signals of 5-bromo-4-thiouridine and related nucleosides were systematically carried out and firmly established by COSY and HMQC techniques. The NMR data of various 4-thiopyrimidine nucleosides are compared, and the key contributing factors discussed. The approach presented here is applicable to other modified nucleosides and nucleotides, as well as nucleobases.

Guanine sulphinate is a major stable product of photochemical oxidation of DNA 6-thioguanine by UVA irradiation.

The DNA of patients taking the immunosuppressant and anticancer drugs azathioprine or 6-mercaptopurine contains 6-thioguanine (6-TG). The skin of these patients is selectively sensitive to ultraviolet A radiation (UVA) and they suffer an extremely high incidence of sunlight-induced skin cancer with long-term treatment. DNA 6-TG interacts with UVA to generate reactive oxygen species, which oxidize 6-TG to guanine sulphonate (G(SO3)). We suggested that G(SO3) is formed via the reactive electrophilic intermediates, guanine sulphenate (G(SO)) and guanine sulphinate (G(SO2)). Here, G(SO2) is identified as a significant and stable UVA photoproduct of free 6-TG, its 2'-deoxyribonucleoside, and DNA 6-TG. Mild chemical oxidation converts 6-TG into G(SO2), which can be further oxidized to G(SO3)-a stable product that resists further reaction. In contrast, G(SO2) is converted back to 6-TG under mild conditions. This suggests that cellular antioxidant defences might counteract the UVA-mediated photooxidation of DNA 6-TG at this intermediate step and ameliorate its biological effects. In agreement with this possibility, the antioxidant ascorbate protected DNA 6-TG against UVA oxidation and prevented the formation of G(SO3).

NMR and UV studies of 4-thio-2'-deoxyuridine and its derivatives.

5-Substituted-4-thio-2'-deoxyuridine nucleosides have been chemically synthesized and studied by NMR and UV spectroscopy. The results have been analyzed and discussed in connection with the previous data. The imino proton signal and the carbon signal of the thiocarbonyl group in the 5-substituted-4-thio-2'-deoxyuridines were found to be at much lower field, offering a potential for monitoring these modified bases at the DNA level. All 4-thionucleosides have strong absorptions at around 340 nm and consequently would be useful as potential UVA-induced anticancer agents.

Novel DNA lesions generated by the interaction between therapeutic thiopurines and UVA light.

The therapeutic effect of the thiopurines, 6-thioguanine (6-TG), 6-mercaptopurine, and its prodrug azathioprine, depends on the incorporation of 6-TG into cellular DNA. Unlike normal DNA bases, 6-TG absorbs UVA radiation, and UVA-mediated photochemical damage of DNA 6-TG has potentially harmful side effects. When free 6-TG is UVA irradiated in solution in the presence of molecular oxygen, reactive oxygen species are generated and 6-TG is oxidized to guanine-6-sulfonate (G(SO3)) and guanine-6-thioguanine in reactions involving singlet oxygen. This conversion is prevented by antioxidants, including the dietary vitamin ascorbate. DNA G(SO3) is also the major photoproduct of 6-TG in DNA and it can be selectively introduced into DNA or oligonucleotides in vitro by mild chemical oxidation. Thermal stability measurements indicate that G(SO3) does not form stable base pairs with any of the normal DNA bases in duplex oligonucleotides and is a powerful block for elongation by Klenow DNA polymerase in primer extension experiments. In cultured human cells, DNA damage produced by 6-TG and UVA treatment is associated with replication inhibition and provokes a p53-dependent DNA damage response.

Unambiguous structural elucidation of base-modified purine nucleosides using NMR.

A general and unambiguous approach has been developed for structural elucidation of modified purine nucleosides using NMR spectroscopy. Systematic assignment of proton and carbon signals of modified nucleosides was firmly established by COSY and the anomerism of the glycosidic linkage of synthetic nucleosides clearly elucidated by NOESY experiments. Characteristic properties of 15N-isotopic labelling at specific positions of nucleosides were also employed for structural studies. The reported approach is applicable to other modified nucleosides and nucleotides, as well as nucleobases.

Thiothymidine plus low-dose UVA kills hyperproliferative human skin cells independently of their human papilloma virus status.

The thymidine analogue 4-thiothymidine (S(4)TdR) is a photosensitizer for UVA radiation. The UV absorbance spectrum of S(4)TdR and its incorporation into DNA suggests that it might act synergistically with nonlethal doses of UVA to selectively kill hyperproliferative or cancerous skin cells. We show here that nontoxic concentrations of S(4)TdR combine with nonlethal doses of UVA to kill proliferating cultured skin cells. Established cell lines with a high fraction of proliferating cells were more sensitive than primary keratinocytes or fibroblasts to apoptosis induction by S(4)TdR/UVA. Although S(4)TdR plus UVA treatment induces stabilization of p53, cell death, as measured by apoptosis or clonal survival, occurs to a similar extent in both p53 wild-type and p53-null backgrounds. Furthermore, different types of human papilloma virus E6 proteins, which protect against UVB-induced apoptosis, have little effect on killing by S(4)TdR/UVA. S(4)TdR/UVA offers a possible therapeutic intervention strategy that seems to be applicable to human papilloma virus-associated skin lesions.

Absorption Characteristics and Quantum Yields of Singlet Oxygen Generation of Thioguanosine Derivatives.

6-Thioguanine (1a) is considered to be photochemotherapeutic due to its specific characteristics of photosensitivity to UVA light and singlet molecular oxygen generation. To extend its phototherapeutic ability, two related thioguanines, 8-thioguanine (2a) and 6,8-dithioguanine (3a), have been designed and explored. Since the solubility of these thioguanines in dehydrated organic solvents is too poor to study, their triacetyl-protected ribonucleosides, that is, 2',3',5'-tri-O-acetyl-6-thioguanosine (1c), 2',3',5'-tri-O-acetyl-8-thioguanosine (2c) and 2',3',5'-tri-O-acetyl-6,8-dithioguanosine (3c) were prepared and investigated. The absorption maxima of 1c, 2c and 3c in acetonitrile were found at longer wavelengths than that of unthiolated guanosine (4c). Especially, 3c has the longest wavelength for absorption maximum and the highest value in terms of molar absorption coefficient among all thionucleobases and thionucleosides reported. These absorption properties were also well reproduced by quantum chemical calculations. Quantum yields of singlet oxygen generation of 2c and 3c were determined by near-infrared emission measurements to be as large as that of 1c. These results suggest that the newly synthesized thioguanosines, in particular 3c, can be further developed as a potential photosensitive agent for light-induced therapies.

Acid Dissociation Equilibrium and Singlet Molecular Oxygen Quantum Yield of Acetylated 6,8-Dithioguanosine in Aqueous Buffer Solution.

2',3',5'-Tri- O-acetyl-6,8-dithioguanosine (taDTGuo) is a nucleoside derivative of drug 6-thioguanine and under further development as a potential photochemotherapeutic agent due to its desirable properties of photosensitivity to UVA light and singlet molecular oxygen generation. The photochemical characteristics of taDTGuo under biological conditions (namely in aqueous solution) were intensively investigated by the steady-state absorption and emission, time-resolved near-infrared emission measurements, and quantum chemical calculations. taDTGuo was found to be held in sequential acid dissociation equilibria within pH 3.79-11.93. With the global fitting analysis of the absorption spectra at various pHs, two p Ka values of the equilibria were determined to be 7.02 ± 0.01 and 9.79 ± 0.01. Quantum chemical calculations suggested that its mono- and dianionic species in the ground state should be 1-imide anionic form (N1-taDTGuo-) and 1,7-di-imide anionic form (taDTGuo2-). taDTGuo generates a singlet molecular oxygen effectively and has pH-dependent quantum yields. In conclusion, taDTGuo would be very useful as a potent agent for photochemotherapy under certain carcinomatous pH conditions.

Triplet formation of 4-thiothymidine and its photosensitization to oxygen studied by time-resolved thermal lensing technique.

Excited-state dynamics of 4-thiothymidine (S4-TdR) and its photosensitization to molecular oxygen in solution with UVA irradiation were investigated. Absorption and emission spectra measurements revealed that UVA photolysis of S4-TdR gives rise to a population of T1(pipi*), following S2(pipi*) --> S1(npi*) internal conversion. In transient absorption measurement, the 355 nm laser photolysis gave broad absorption (380-600 nm) bands of triplet S4-TdR. The time-resolved thermal lensing (TRTL) signal of S4-TdR containing the thermal component due to decay of triplet S4-TdR was clearly observed by the 355 nm laser excitation. The quantum yield for S1 --> T1 intersystem crossing was estimated to be unity by a triplet quenching experiment with potassium iodide. In the presence of molecular oxygen, the photosensitization from triplet S4-TdR gave rise to singlet oxygen O2 (1Deltag) with a quantum yield of 0.50. Therapeutic implications of such singlet oxygen formation are discussed.

Post-synthetic and site-specific modification of endocyclic nitrogen atoms of purines in DNA and its potential for biological and structural studies.

Site-specific modification of the N1-position of purine was explored at the nucleoside and oligomer levels. 2'-deoxyinosine was converted into an N1-2,4-dinitrophenyl derivative 2 that was readily transformed to the desired N1-substituted 2'-deoxyinosine analogues. This approach was used to develop a post-synthetic method for the modification of the endocyclic N1-position of purine at the oligomer level. The phosphoramidite monomer of N1-(2,4-dinitrophenyl)-2'-deoxyinosine 9 was prepared from 2'-deoxyinosine in four steps and incorporated into oligomers using an automated DNA synthesizer. The modified base, N1-(2,4-dinitrophenyl)-hypoxanthine, in synthesized oligomers, upon treatment with respective agents, was converted into corresponding N1-substituted hypoxanthines, including N1-15N-hypoxanthine, N1-methylhypoxanthine and N1-(2-aminoethyl)-hypoxanthine. These modified oligomers can be easily separated and high purity oligomers obtained. Melting curve studies show the oligomer containing N1-methylhypoxanthine or N1-(2-aminoethyl)-hypoxanthine has a reduced thermostability with no particular pairing preference to either cytosine or thymine. The developed method could be adapted for the preparation of oligomers containing mutagenic N1-beta-hydroxyalkyl-hypoxanthines and the availability of the rare base-modified oligomers should offer novel tools for biological and structural studies.

The effect of S-substitution at the O6-guanine site on the structure and dynamics of a DNA oligomer containing a G:T mismatch.

The effect of S-substitution on the O6 guanine site of a 13-mer DNA duplex containing a G:T mismatch is studied using molecular dynamics. The structure, dynamic evolution and hydration of the S-substituted duplex are compared with those of a normal duplex, a duplex with S-substitution on guanine, but no mismatch and a duplex with just a G:T mismatch. The S-substituted mismatch leads to cell death rather than repair. One suggestion is that the G:T mismatch recognition protein recognises the S-substituted mismatch (GS:T) as G:T. This leads to a cycle of futile repair ending in DNA breakage and cell death. We find that some structural features of the helix are similar for the duplex with the G:T mismatch and that with the S-substituted mismatch, but differ from the normal duplex, notably the helical twist. These differences arise from the change in the hydrogen-bonding pattern of the base pair. However a marked feature of the S-substituted G:T mismatch duplex is a very large opening. This showed considerable variability. It is suggested that this enlarged opening would lend support to an alternative model of cell death in which the mismatch protein attaches to thioguanine and activates downstream damage-response pathways. Attack on the sulphur by reactive oxygen species, also leading to cell death, would also be aided by the large, variable opening.

Two-deoxyglucose-induced long-term potentiation in slices of rat dentrate gyrus.

In keeping with previous observations in the CA1 and the somatosensory neocortex of the brain of rat, 20-min applications of 2-deoxy-D-glucose (2DG; 10 mM, replacing glucose) induced a long-term potentiation (LTP)-like enhancement of field excitatory synaptic potentials (fEPSPs) in the dentate region of hippocampal slices. The effects of 2DG were not identical at synapses of medial and lateral perforant paths (MPP and LPP). At MPP synapses, there was no post-2DG early depression of fEPSPs and the potentiation reached +78.6 +/- 5.7 % (+/- standard error of the mean) 40 min after the return to glucose. In the presence of 50 microM D-amino-phosphono valerate (APV; an N-methyl-D-aspartate [NMDA] receptor antagonist), a marked post-2DG depression appeared and the subsequent LTP was reduced to +34.7 +/- 2.8 % (for both 2DG- and APV-treatment P<0.001 by ANOVA-2W). At LPP synapses, even under control conditions, there was a sharp post-2DG depression followed by LTP (+62.2 +/- 5.7 %) and APV had little effect on either the post-2DG depression or LTP, reducing the latter by only 24 % [the 2DG treatment was very significant (P<0.001) but not the APV treatment]. Thus, 2DG evokes both NMDAR-dependent and -independent components of LTP in the perforant pathways. In view of these findings, the consumption of 2DG could have significant effects on synaptic plasticity and cognitive function.

Ambiguous coding is required for the lethal interaction between methylated DNA bases and DNA mismatch repair.

The thiopurine 6-thioguanine (S6G) is used to treat acute leukaemia. Its cytotoxic effect requires an active DNA mismatch repair (MMR) system. S6G is incorporated into DNA where a small fraction undergoes in situ conversion to S6-thiomethylguanine (S6meG). After replication, S6meG-containing base pairs interact with MMR. This interaction is ultimately lethal and MMR-defective cells are resistant to S6G. Here, we report that growing human cells extensively incorporate the thiopyrimidine nucleoside 4-thiothymidine (S4TdR) into their DNA. The incorporated thiopyrimidine (S4T) can also undergo facile S-methylation to 4-thiomethylthymine (S4meT). The rate of methylation of S4TdR in model substrates is similar to that for the conversion of S6G to S6meG indicating that the DNA of cells grown in S4TdR will contain significant levels of S4meT. Despite this, S4TdR is not associated with MMR-related cell death. We demonstrate that, in contrast to S6meG, neither DNA S4T nor S4meT codes ambiguously. S4T retains the coding properties of unmodified T, whereas S4meT behaves like a normal cytosine and exclusively directs the incorporation of guanine. The preferred S4meT:G base pair is also a poor substrate for binding by the hMutSalpha mismatch recognition factor. We suggest that the ability of S4meT to produce a structurally acceptable base pair during replication underlies the absence of MMR-related death in cells treated with S4TdR.

UVA photoactivation of DNA containing halogenated thiopyrimidines induces cytotoxic DNA lesions.

Photochemotherapy, the combination of a photosensitiser and ultraviolet (UV) or visible light, is an effective treatment for skin conditions including cancer. The high mutagenicity and non-selectivity of photochemotherapy regimes warrants the development of alternative approaches. We demonstrate that the thiopyrimidine nucleosides 5-bromo-4-thiodeoxyuridine (SBrdU) and 5-iodo-4-thiodeoxyuridine (SIdU) are incorporated into the DNA of cultured human and mouse cells where they synergistically sensitise killing by low doses of UVA radiation. The DNA halothiopyrimidine/UVA combinations induce DNA interstrand crosslinks, DNA-protein crosslinks, DNA strand breaks, nucleobase damage and lesions that resemble UV-induced pyrimidine(6-4)pyrimidone photoproducts. These are potentially lethal DNA lesions and cells defective in their repair are hypersensitive to killing by SBrdU/UVA and SIdU/UVA. DNA SIdU and SBrdU generate lethal DNA photodamage by partially distinct mechanisms that reflect the different photolabilities of their C-I and C-Br bonds. Although singlet oxygen is involved in photolesion formation, DNA SBrdU and SIdU photoactivation does not detectably increase DNA 8-oxoguanine levels. The absence of significant collateral damage to normal guanine suggests that UVA activation of DNA SIdU or SBrdU might offer a strategy to target hyperproliferative skin conditions that avoids the extensive formation of a known mutagenic DNA lesion.