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.


Introduction
Cancer is one of major killers in modern society. The fundamental cause for cancer incidence is DNA damage and mutation [1]. An agent or treatment that can cause DNA damage can also be used as a therapeutic means by destroying cancerous cells. This, in fact, is the principle underlying chemotherapy and radiotherapy. However, often such treatments are either too toxic (in the case of chemotherapy) or too powerful (in the case of radiotherapy) and thus indiscriminate, also causing harm to normal cells. Clearly, improved treatments for cancer patients are urgently required. Recently we reported that 4-thiothymidine (thioT), an analogue of the naturally occurring nucleoside thymidine, can be incorporated into cellular DNA and activated by UVA light to kill cells [2,3]. These findings offered a new therapeutic approach to cancer treatment. This mild and synergistic approach has the advantage over conventional therapies, that it can target proliferating cells more selectively.
The presence of a thiocarbonyl group in 2 is evident from the appearance of NH and thiocarbonyl carbon signals in the 1 H and 13 C-NMR spectra respectively. Exchangeable signals in the δ 12.68-13.10 ppm range in the low-field part of 1 H-NMR spectra, attributable to the N-H protons, suffort the structures of molecules 2. The appearance of only one carbon signal in the δ C 185.30-190.70 ppm region (characteristic for a thiocarbonyl group) together with an intense absorption band at 332-344 nm in the UV spectra ( Figure 1), confirm the presence of the thiocarbonyl moieties in compounds 2.
It is interesting to note that the 1 H chemical shifts of the imino proton (NH) in all the thionucleosides 2 are substantially higher (at around 13 ppm) than those of the parent nucleosides 1 that resonate at δ 11.2-11.83 ppm ( Table 1). This difference offers a valuable NMR window to detect the imino proton of thionucleosides, as in general there are no signals from normal nucleosides appearing at such a low field. In addition these NH-proton signals are exchangeable and readily identifiable by D 2 O exchange experiments. Therefore these would also be a good marker in NMR studies of 4-thionucleosides and their corresponding DNAs. 13 C-NMR: All the 13 C signals were assigned using the standard procedure or DEPT protocols or 2-D NMR spectroscopy where necessary. A careful inspection of the 13 C-NMR data ( Table 2) provides useful structural information. As the modification occurs on the base, this is also reflected in the 13 C chemical shifts of the base. There are four carbons on the base. The carbon at C-2-position (i.e., 2-C) is substantially away from the modified positions (4 and 5), thus the chemical shifts of C-2 vary little. The most notable differences between the 13 C-NMR spectra of 1 and 2 are the chemical shifts of the carbon at the 4-position (i.e., 4-C), and a characteristic downfield shift (from δ C around 160 to δ C around 190 ppm) was observed. This could be due to increased conjugation by the replacement of oxygen with sulfur at the 4-position. Interestingly, 2-thio-2'-deoxynucleosides differ only slightly while 4-thio-2'-deoxynucleosides exhibit somewhat large differences [8]. As shown in Figure 1, the introduction of a sulfur atom at the 4-position in the nucleosides shifts their UV spectra towards longer wavelength with maxima at 330 to 340 nm. In contrast, the UV spectra of 2-thio-2'-deoxyuridine analogues are similar to those of their un-modified nucleosides. For instance the UV λ max of 2-thio-2'-deoxyuridine is at 273 nm (in H 2 O) [17] which is only slightly changed from that of its parent compound 1a (UV λ max at 262 nm). Due to the energy level of lone pair electrons of 3p in sulfur is higher than that of lone pair electrons of 2p in oxygen, the requied energy of the n→π * transition for a thiocarbonyl is lower than that for a carbonyl. Although the spectra of 2-thionucleosides containing C=S bonds would be expected to shift to longer wavelengths relative to those containing C=O bonds, the thiocarbonyl in 2-position is attached to two nitrogens, so the UV absorption wavelength should also move back to shorter wavelengths. Overall, in the end there is a little change in the UV absorption maxima for the 2-thionucleoside analogues. This is also one of the reasons why 2-thionucleoside analogues are not exploited as useful agents for UVA-induced DNA damage. .
As the major factor in 13 C chemical shift is paramagnetic shielding, that is different from diamagnetic shielding in 1 H chemical shift, thus the shielding constant σ for 13 C spectrum is generally expressed as below [19]: where C is the light speed; m and e are respectively for the mass of atom and number of electron; the term {r −3 } 2p is the mean inverse cube of the distance from the nucleus for the carbon 2p atomic orbit; This term accounts for the charge density effect in the paramagnetic term since increasing charge density leads to an expansion of the orbital and thereby a reduction in σ p ; ΔE represents the excitation energy of average electron to the low lying excited state; Σ B goes over all atoms; Q is bond order; Q AA is contribution of electron density of 2p orbital in nucleus; Q AB is bond order between nucleus and bond which relate to nucleus.
Comparing the carbonyl oxygen group in a nucleoside with the thiocarbonyl sulfur group in a thionucleoside would lead to the prediction that σ p should be more for the latter if the {r −3 } 2p term dominates since there is a less contribution from electronegativity on sulfur, the deshielding of carbon atom is strong and the signal carbon resonate moves to lower field such as the 13 C-NMR spectra of δ C = 159.28-163.75 ppm for C=O in compound 1 and δ C = 179.86-190.70 ppm for C=S in compound 2.
Obviously, the equation shows that the ‫|‬σ p ‫|‬ dependence is mainly affected by the excitation energy of an average electron ΔE to paramagnetic shielding under consideration; that is, the bigger ‫|‬σ p ‫,|‬ the stronger paramagnetic shielding, and the atom will resonate at lower field. Therefore, σ p depends upon ΔE. In addition, it has been shown by Figgis et al. [20] that organic carbonyl oxygen shieldings correlate approximately with λ max n→π* values (i.e., the inverse of the excitation energy for the lowest electronic transition). For C=O, it is n→π * absorption, the absorption of UV is 280 nm, (ΔE) −1 which is bigger makes ‫|‬σ p ‫|‬ bigger, the deshielding of the carbon atom is stronger, and the signal carbon resonance moves to lower field. For C=S, it is also n→π * , the absorption of UV moves towards longer wavelength, approaching to 400 nm, the intensity of absorption in C=S is bigger than that in C=O, so the wave of absorption is longer. For 13 C-NMR, the C=S resonance is downfield in comparison with that in a C=O. Since the C=C double bond carries a polar group, the electron distribution is thus displaced. This displacement is usually understood as a combination of inductive effects and conjugative effects.
There is the electron excited state as n-π* or π-π* in molecules where C=C double bond attached a polar group when highly electronegative atoms like halogen are near these sigma σ electrons. The highly electronegative atoms attract these sigma σ electrons toward themselves and away from the spinning nuclei. These would affect on the deshielding and an NMR signal to be generated would move further downfield. Table 2 shows that the 5-C signals of the fluorine-modified deoxyuridine 1c and its thioanalogue 2c are substantially different from other halogen-modified compounds 1d, 1e, 1f and their 4-thioanalogues 2d, 2e, 2f respectively. Figure 2 plots 13 C chemical shifts of C-5 against with the electronegativity of the 5-substitutents. The greater the electronegativity of atom or group, the lower the electron density around C-X and the further downfield the chemical shift. When linked with fluorine atom, the 5-C has the highest values, namely 142.03 ppm (for 1c) and 147.23 ppm (for 2c). This could be ascribed to the extremely high electronegativity of fluorine. F, Cl, Br and I are each more electronegative than the H atom, it could be anticipated that, when the H atom of C-5 are replaced by these substituents, the 13 C resonance would be progressively shiftded to much lower field. Obviously, the electron-withdrawing inductive effects of the 5-substituent do play an important role for F, Cl and methyl group. However when a substituent at the 5-position is I or Br, the chemical shift of the 5-C is even lower than that of those 5-C bearing H-or CH 3 group. The 13 C resonance of C-5 for BrdU (5-bromo-2'-deoxyuridine) and IdU (5-iodo-2'-deoxyuridine) is displaced to higher field relative to that of dU (2'-doxyuridine) by 6 ppm for BrdU and by 32.49 ppm for IdU. Clearly the electron-withdrawing effect alone is not enough to explain those. This unusual effect could be explained by "heavy atom effect" which invariably cause shifts upfield [21]. When a carbon atom is attached by heavy halogen atom (such as Br or I), the diamagnetic interactions arising from more electron charge within Br or I atoms may exhibit an abnormally high 13 C shielding and cause the chemical shift of 13 C resonance to upfield.
On the other hand, the circulating π electrons are in the delocalized pyrimidine ring. These π electrons create a magnetic field that is parallel to the lines of force of the external magnetic field. This will aid the external field (a deshielding effect) and allow the NMR signal generation to occur at a lower magnetic field strength setting. For example, the carbons at the 6-position of 1c and 2c have reduced values (δ C 126.44 ppm for 1c and δ C 118.16 ppm for 2c). These results can be explained by the conjugative effects which operate in the π-system and have π-donor group for p orbital in Cl, Br, I, causing an upfield shift. Therefore, as the conjugation to p orbital of Cl and Br elements usually has a smaller effect on C=C than that to p orbital of I element, so the signal of C-6 of iodo-substituted nucleosides (1f and 2f) have high values than other halogen-substituted analogues.
Such a shift suggests possible changes of base-pairing properties compared with their parent nucleosides. The cause of this lower field shift may also reflect the longer conjugation of the ring system of thio-nucleosides. This is further evidenced from their longer wavelength absorption in their UV spectra (Figure 1). In general, deoxyuridine nucleosides have absorption maxima at 260-285 nm arising from their π→π * transitions. Although several deoxyuridines containing an X group are found to have increased UV maxima and 4-thioanalogues have further increased UV maxima, there is still a need for new compounds absorbing at even long-wavelengths. We developed a method by replacing the oxygen atom by sulfur to produce UVA-sensitive thio-analogues as shown in Scheme 1 (above). The replacement of an oxygen atom at the 4-position by selenium in uridine derivatives produces UVA-sensitive 4-seleno-uridine and its analogues [22]. The UV absorption spectrum for 4-seleno-2'-deoxyuridine was compared to that of 2'-deoxyuridine and 4-thio-2'-deoxyuridine. As expected, its absorption maximum was found at longer wavelength [368 nm (in H 2 O)] than those of 2'-deoxyuridine and 4-thio-2'-deoxyuridine. This is due to the fact the energy level of the lone pair on selenium is higher than those on a sulfur atom or an oxygen atom, (ΔE) which is smaller, leads to longer wavelength absorption. Therefore the 4-selenonucleosides would be more sensitive toward UVA light than 4-oxy-and 4-thio-nucleosides. This UVA absorption property should provide a potential to specially target molecules of seleno-modified DNA with UVA light at 368 nm.
Recently, much effort was directed to study the photophysical and photochemical properties of uridine derivatives that contain both C=O and C=S fragments. In the series of 4-thio-2'-deoxyuridines, it was found that the presence of the C=S function in the molecules dominated their photochemistry in aqueous solution [2,8,23,24]. According to the idea, 4-selenouridine derivatives that contain X groups (where X = halogen) will shift to much longer UV wavelengths than that of thiocarbonyl groups and could be particularly useful as potential UVA-induced anticancer agents.
In summary, the thiation of nucleosides and related compounds has been extensively studied in the past, the UVA-sensitive 4-thio-2'deoxyuridine and its derivatives have never been studied in detail by NMR and UV spectroscopy. We have now established the identities of the thio products derived from carbonyl products in NMR and UV spectroscopy.

General
1 H-Nuclear magnetic resonance (NMR) spectra and 13 C-NMR spectra were recorded using a JEOL LA300 spectrometer at 300 and 75 MHz, respectively. 1 H-NMR and 13 C-NMR spectra were determined in DMSO-d 6 solution and chemical shifts are quoted in parts per million (p.p.m.) from tetramethylsilane as internal standard. Ultraviolet spectra were recorded with a Philips PU 8700 UV/Vis spectrophotometer for 5-substituted 4-thio-2'-deoxyuridine derivative (50 mM in CH 3 CN).
1,2,4-Triazole (6.8 g, 98.4 mmol) was suspended in anhydrous CH 3 CN (80 mL) at 0 °C. POCl 3 (2.1 mL), then triethylamine (16 mL) were added slowly. After 1 h, the 3',5'-bis-(trimethylsilyl)-5-substituted-2'-deoxyuridine derivative (6.7 mmol) in CH 3 CN (30 mL) was added over 30 min. Then the solution was stirred for 16 h at room temperature and the reaction was monitored by TLC (solvent: 50:50 n-pentane/diethyl ether). After the starting material was converted into a new compound with lower R f , the reaction mixture was filtered, diluted with ethyl acetate (160 mL) and washed with saturated aqueous NaHCO 3 (150 mL), then twice with 150 mL of saturated aqueous NaCl. The organic layer was dried over anhydrous Na 2 SO 4 and the solvent evaporated. The residue was dried by repeated evaporation of a toluene solution to give product, then used in the next step.

Conclusions
5-Substituted-4-thio-2'-deoxyuridines can be effectively prepared from its parent nucleosides and have distinctive NMR and UV properties that can be used for easy monitoring and exploited as potential UVA-induced anticancer agents.