Thiopurines Analogues with Additional Ring: Synthesis, Spectroscopic Properties, and Anticancer Potency.

Purine scaffolds constitute a starting point for the synthesis of numerous chemotherapeutics used in treating cancer, viruses, parasites, as well as bacterial and fungal infections. In this work, we synthesized a group of guanosine analogues containing an additional five-membered ring and a sulfur atom at the C-9 position. The spectral, photophysical, and biological properties of the synthesized compounds were investigated. The spectroscopic studies revealed that a combination of the thiocarbonyl chromophore and the tricyclic structure of guanine analogues shifts the absorption region above 350 nm, allowing for selective excitation when present in biological systems. Unfortunately, due to the low fluorescence quantum yield, this process cannot be used to monitor the presence of these compounds in cells. The synthesized compounds were evaluated for their effect on the viability of human cervical carcinoma (HeLa) and mouse fibroblast (NIH/3T3) cells. It was found that all of them display anticancer activity. In vitro studies were preceded by in silico ADME and PASS analyses, which confirmed that the designed compounds are promising candidates for anticancer agents.

Unusual photophysical properties of a new tricyclic derivative of thiopurines in terms of potential applications.

The thio analogues of purine bases have been found to possess notable biological and pharmacological capabilities and have an important role to play as anticancer and immunosuppressive drugs. In this work a new tricyclic analogue of guanosine containing sulfur was synthesized, in particular, DTEG (2',3',5'-tri-O-acetyl-6,9-dithioethanoguanosine). Although there is promise for thiopurine derivatives for biomedical applications, there are some liabilities in regard to their exposure to light. As a preliminary survey for such difficulties with DTEG, this work looks into spectral and photophysical processes of DTEG using time-resolved and steady-state optical excitation. In contrast to other thiopurines, which have long-lived triplets, DTEG is shown to have a short-lived triplet making it less dangerous for singlet-oxygen sensitization. Even in anaerobic solutions, its photoreactivity is negligible. These various unusual photochemical properties of DTEG are consistent with DTEG being very promising as an alternative drug to the currently used 6-thiopurines. DTEG also has some interesting photophysical behavior that is distinct from other thioketones. Although thioketones have an unusual fluorescence violating Kasha's Rule and emitting from the second excited singlet state, DTEG does this also, but, in addition, it shows dual fluorescence by emitting from its first excited singlet as well. The assignments of the nature of these excited states are supported by DFT results. This theory and associated kinetic analysis show quantitatively that the dual fluorescence is, in part, tied to the relatively fast S2 to S1 internal conversion compared to other S2 decays and, in part, tied to the relatively slow nonradiative decay of S1 itself.

Controlled, Sunlight-Driven Reversible Cycloaddition of Multiple Singlet Oxygen Molecules to Anthracene-Containing Trianglimine Macrocycles.

Controlled release of singlet oxygen is of interest not only to chemists, but also to biologists and medics involved in cancer therapy. Two chiral polyaza macrocyclic compounds and their corresponding endoperoxides have been synthesized. These peroxides exhibit high temperature stability, up to 80 °C. Detailed studies on their structure, including X-ray analysis as well as NMR, UV-VIS ECD spectroscopy and theoretical calculations, combined with photochemical measurements indicate that their high stability is related to the arrangement of oxygen atoms in a conformationally stable macrocyclic ring. Despite the change of carbon hybridization from sp2 to sp3 at the 9 and 10 positions of the anthracene units, the macrocyclic skeleton of the obtained compounds does not change its conformation. The obtained endoperoxides can be formed and release singlet oxygen by irradiation with UV light of 365 and 275 nm, respectively. Release of the oxygen does not degrade the macrocyclic structure.

Universal and strain specific structure features of segment 8 genomic RNA of influenza A virus-application of 4-thiouridine photocrosslinking.

RNA structure in the influenza A virus (IAV) has been the focus of several studies that have shown connections between conserved secondary structure motifs and their biological function in the virus replication cycle. Questions have arisen on how to best recognize and understand the pandemic properties of IAV strains from an RNA perspective, but determination of the RNA secondary structure has been challenging. Herein, we used chemical mapping to determine the secondary structure of segment 8 viral RNA (vRNA) of the pandemic A/California/04/2009 (H1N1) strain of IAV. Additionally, this long, naturally occurring RNA served as a model to evaluate RNA mapping with 4-thiouridine (4sU) crosslinking. We explored 4-thiouridine as a probe of nucleotides in close proximity, through its incorporation into newly transcribed RNA and subsequent photoactivation. RNA secondary structural features both universal to type A strains and unique to the A/California/04/2009 (H1N1) strain were recognized. 4sU mapping confirmed and facilitated RNA structure prediction, according to several rules: 4sU photocross-linking forms efficiently in the double-stranded region of RNA with some flexibility, in the ends of helices, and across bulges and loops when their structural mobility is permitted. This method highlighted three-dimensional properties of segment 8 vRNA secondary structure motifs and allowed to propose several long-range three-dimensional interactions. 4sU mapping combined with chemical mapping and bioinformatic analysis could be used to enhance the RNA structure determination as well as recognition of target regions for antisense strategies or viral RNA detection.

Radiation Induced One-Electron Oxidation of 2-Thiouracil in Aqueous Solutions.

Oxidative damage to 2-thiouracil (2-TU) by hydroxyl (•OH) and azide (●N3) radicals produces various primary reactive intermediates. Their optical absorption spectra and kinetic characteristics were studied by pulse radiolysis with UV-vis spectrophotometric and conductivity detection and by time-dependent density functional theory (TD-DFT) method. The transient absorption spectra recorded in the reactions of •OH with 2-TU depend on the concentration of 2-TU, however, only slightly on pH. At low concentrations, they are characterized by a broad absorption band with a weakly pronounced maxima located at λ = 325, 340 and 385 nm, whereas for high concentrations, they are dominated by an absorption band with λmax ≈ 425 nm. Based on calculations using TD-DFT method, the transient absorption spectra at low concentration of 2-TU were assigned to the ●OH-adducts to the double bond at C5 and C6 carbon atoms (3●, 4●) and 2c-3e bonded ●OH adduct to sulfur atom (1…●OH) and at high concentration of 2-TU also to the dimeric 2c-3e S-S-bonded radical in neutral form (2●). The dimeric radical (2●) is formed in the reaction of thiyl-type radical (6●) with 2-TU and both radicals are in an equilibrium with Keq = 4.2 × 103 M-1. Similar equilibrium (with Keq = 4.3 × 103 M-1) was found for pH above the pKa of 2-TU which involves admittedly the same radical (6●) but with the dimeric 2c-3e S-S bonded radical in anionic form (2●-). In turn, ●N3-induced oxidation of 2-TU occurs via radical cation with maximum spin location on the sulfur atom which subsequently undergoes deprotonation at N1 atom leading again to thiyl-type radical (6●). This radical is a direct precursor of dimeric radical (2●).

Converging Fate of the Oxidation and Reduction of 8-Thioguanosine.

Thione-containing nucleobases have attracted the attention of the scientific community for their application in oncology, virology, and transplantology. The detailed understanding of the reactivity of the purine derivative 8-thioguanosine (8-TG) with reactive oxygen species (ROS) and free radicals is crucial for its biological relevance. An extensive investigation on the fate of 8-TG under both reductive and oxidative conditions is here reported, and it was tested by employing steady-state photooxidation, laser flash photolysis, as well as γ-radiolysis in aqueous solutions. The characterization of the 8-TG T1 excited state by laser flash photolysis and the photooxidation experiments confirmed that singlet oxygen is a crucial intermediate in the formation of the unexpected reduced product guanosine, without the formation of the usual oxygenated sulfinic or sulfonic acids. Furthermore, a thorough screening of different radiolytic conditions upon γ-radiation afforded the reduced product. These results were rationalized by performing control experiments in the predominant presence of each reactive species formed by radiolysis of water, and the mechanistic pathway scenario was postulated on these bases.

Carboxyl groups of citric acid in the process of complex formation with bivalent and trivalent metal ions in biological systems.

Binary complexes of citric acid (H3L - protonated form, H2L and HL - partly protonated forms, L - fully deprotonated) with d- and f-electron metal ions were investigated. The studies have been performed in aqueous solution using the potentiometric method with computer analysis of the data, electron paramagnetic resonance, infrared, visible as well as luminescence spectroscopies. The overall stability constants of the complexes were determined. Analysis of the equilibrium constants of the reactions and spectroscopic data has allowed determination of the type of coordination and effectiveness of the carboxyl groups in the process of complex formation. On the basis of potentiometric titration for d-electron were found dimeric and monomeric type of complexes and for f-electron four type of complexes: MHL, ML, ML(OH) and ML(OH)2.

Mechanism and dynamics of intramolecular triplet state decay of 1-propyl-4-thiouracil and its α-methyl-substituted derivatives studied in perfluoro-1,3-dimethylcyclohexane.

The absorption, phosphorescence and phosphorescence excitation spectra, phosphorescence quantum yields, and T(1) excited state lifetimes of four 4-thiouracil derivatives were measured for the first time in chemically inert and very weakly interacting perfluoro-1,3-dimethylcyclohexane at room temperature. The set of the 4-thiouracil derivatives comprises 1-propyl-4-thiouracil (PTU) and the related compounds having a methyl substituent at the position α to the thiocarbonyl group, namely 1-propyl-4-thiothymine (PTT), 1,3-dimethyl-4-thiouracil (DMTU), and 1-methyl-3-trideuteriomethyl-4-thiouracil ([D(3)]DMTU). Quantitative information on the intramolecular decay of the T(1) excited state of the four 4-thiouracil derivatives is presented, and the mechanism and dynamics of this process are discussed. In the absence of self quenching and solvent induced deactivation, the T(1) decay of the four 4-thiouracil derivatives was dominated by intramolecular nonradiative processes (NR). The values of the rate constant k(NR) in DMTU and [D(3)]DMTU are about 4 times larger than that in PTT and about 3 times larger than that in PTU. The reasons for the enhanced nonradiative rate constant in DMTU are discussed. It is concluded that the faster rate of the nonradiative processes in DMTU is related to a larger contribution from mixing of the T(2) (nπ*) state into the lowest energy T(1) (ππ*) state, as compared to the analogous coupling in PTU and PTT. This conclusion is supported by ab initio calculations performed at the EOM-CC2/aug-cc-pVDZ level of theory. The energy spacing between the T(2) (nπ*) and T(1) (ππ*) states is estimated to be about 500, 1100, and 2000 cm(-1) for DMTU, PTU, and PTT, respectively. Among the three compounds in question, the predicted energy spacing is thus the smallest for DMTU.

Photoinduced fluorescent cross-linking of 5-chloro- and 5-fluoro-4-thiouridines with thymidine.

Two highly fluorescent, thermally stable diastereomeric photoadducts, 3a,b, are formed when either 5-chloro-4-thiouridine, 1, or 5-fluoro-4-thiouridine, 2, are photoexcited with 366 nm UV light in the presence of thymidine (T). 5-Fluoro-4-thiouridine, 2, exhibits photoreactivity much higher than that of the 5-chloro derivative 1. In both cases the photoreaction is very clean, leading to highly eficient conversion of the 5-halogeno-4-thiouridine (1, 2) and T to photoadducts 3a,b. The identity and structure of 3a was confirmed using mass spectrometry and 2-D NMR. The epimeric relationship of 3a,b was established by UV circular dichroism spectroscopy. The geometry of the fluorescent photoadduct is consistent with formation of an interstrand cross-link in a DNA duplex if 1 or 2 is flanked by T in an opposite strand.

Photochemical reactions of 4-thiouridine disulfide and 4-benzylthiouridine--the involvement of the 4-pyrimidinylthiyl radical.

The reactions of a disulfide and a benzylsulfide derived from 4-thiouridine were studied in aqueous acetonitrile using stationary and laser flash photolysis methods. Irradiation of the compounds results in specific cleavage of the S-S bond in the disulfide and the S-CH(2) bond in the sulfide. Identical pyrimidine-derived intermediates were observed in the transient absorption spectra (lambda(max) = 420 nm, epsilon(max) approximately 2500 M(-1) cm(-1)) recorded for both compounds in laser flash photolysis experiments. The intermediate was identified as the 4-pyrimidinylthiyl radical. Irradiation of the disulfide in the absence of oxygen gives 4-thiouridine while the sulfide under identical conditions produced, additionally, 3-benzyl-4-thiouridine as a stable photoproduct. The formation of the latter photoproduct provides evidence for the existence of the N-centered 4-thioxopyrimidynyl radical formed from the initially produced S-centered (thiyl) radical. The 4-thiouridine is formed from the radicals generated in the primary photochemical step by an H abstraction reaction from the solvent (acetonitrile) or from additives (alcohols) that were purposely added. Interestingly, in contrast to the benzylsulfide, the photoreaction of the disulfide is quenched by molecular oxygen with the concomitant formation of uridine. However it appears that uridine is not produced as a result of the reaction of the radicals with oxygen. A mechanism is proposed for the photochemical transformations of the disulfide and benzylsulfide derived from 4-thiouridine. The proposed mechanism is based on the structures of the identified stable photoproducts, the values of the photoreaction quantum yields determined under differing irradiation conditions, and the flash photolysis results.

Solvent effects on the redox properties of thioethers.

The one-electron reduction potential of the radical cations of thioanisole (1), benzyl methyl sulfide (2) and 2-hydroxyethyl benzyl sulfide (3) in water, formamide, acetonitrile, acetone, 1,1,1,3,3,3-hexafluoropropan-2-ol, methanol and 2-propanol was investigated by cyclic voltammetry. For comparison the one-electron reduction potentials in water were also measured using pulse radiolysis. The redox potential is strongly influenced by the nature of the solvent and the solvent sensitivity increases with charge localization. The present results have been used to evaluate solvent effects in view of the Kamlet-Taft relationship. The Kamlet-Taft expression quantitatively describes the solvent effects on the redox properties of 1-3 and gives the relative importance of the different solvent properties. The dominating contribution to the solvent effects is the solvent dipolarity/polarizability pi*, whereas alpha appears to be of minor importance. Furthermore, the relationship between the pi* and reduction potential of radical cations of 1-3 appear to be linear. It was also possible to find the same trend between the solvent dipole moment and peak potential of 1-3. These facts indicate that the nature of solvation is mainly nonspecific.

Generation of thiyl radicals by the photolysis of 5-iodo-4-thiouridine.

The photochemistry of 2',3',5'-tri-O-acetyl-5-iodo-4-thiouridine (3) in deoxygenated 1:1 CH(3)CN-H(2)O pH 5.8 (phosphate buffer) solution has been studied by means of steady-state and nanosecond laser flash photolysis methods. Under steady-state irradiation (lambda > or = 334 nm), the stable photoproducts were iodide ion, 2',3',5'-tri-O-acetyl-4-thiouridine (4), and two disulfides. The disulfides were the symmetrical bis-(2',3',5'-tri-O-acetyl-5-iodo-4-thiouridine) (5) and unsymmetrical 6, which contains both 4-thiouridine and 5-iodo-4-thiouridine residues. The formation of the dehalogenated photoproduct suggests that C(5)-I bond cleavage is a primary photochemical step. Attempts to scavenge the resulting C(5)-centered radical by suitable addends, bis-(N-alpha-acetyl)cystine-bis-N-ethylamide or benzene, were unsuccessful. Analysis of the photoproducts formed under these conditions showed that the S-atom is the reactive center. The photoproduct 4, obtained by irradiation of 3 in CD(3)CN-H(2)O, followed by reversed-phase HPLC isolation using nonlabeled eluents, did not contain deuterium. An analogous experiment performed in CH(3)CN-D(2)O gave deuterated product 4-d with 88% of the deuterium incorporated at C(5). Transient absorption observed upon laser excitation (lambda= 308 nm) of 3 was assigned to the 4-uridinylthiyl radical on the basis of the similarity of this spectrum with that obtained upon laser photolysis of the disulfide: bis-(2',3',5'-tri-O-acetyl-4-thiouridine) 14. On the basis of the results of steady-state and laser photolysis studies, a mechanism of the photochemical reaction of 3 is proposed. The key mechanistic step is a transformation of the C(5)-centered radical formed initially by C(5)-I bond cleavage into a long-lived S-centered radical via a 1,3-hydrogen shift. Theoretical calculations confirmed that the long-lived S-centered radical is the most stable radical derived from the 4-thiouracil residue.