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2-THIOZEBULARINE: BASE MODIFIED NUCLEOSIDE FULLY CONSTRAINED IN C3'-endo CONFORMATION IN SOLUTION [Collection of Czechoslovak Chemical Communication](Collection of Czechoslovak Chemical Communications Via Acquire Media NewsEdge) 2-Thiopyrimidinone ribofuranoside (2-thiozebularine, s2 zeb) was synthesized by the adaptation of silyl method of N-glycosidic bond formation and using thionation of protected 2-oxonucleoside derivative (zebularine, zeb) with Lawesson reagent. The X-ray crystal structure of s2 zeb and NMR determined conformations of s2 zeb and zeb in solution were compared with structures of 2-thiouridine (s2U) and uridine (U). In the solid state s2 zeb molecule adopts conformation typical for ribonucleosides: C3'-endo C2'-exo twist type of ribofuranose pucker, anti of N-glycosidic bond and trans around C4'-C5' bond. In aqueous solution, however, almost 100% population of s2 zeb exhibits C3'-endo ribofuranose pucker. The population of N-conformer of s2 zeb is about 20% higher than for zeb (analogously to pair of s2U and U nucleosides) indicating similar influence of steric effect of bulky sulfur atom on stabilization of N-type ribose conformation. Interestingly, the absence of 4-carbonyl function in zeb and s2 zeb raises the population of C3'-endo conformation by about 30% in comparison to U and s2U as a result of significant anomeric effect. Additive action of both effects makes the 2-thiozebularine almost fully constrained in C3'-endo conformation in aqueous solution. Cytotoxic properties of s2 zeb are less pronounced in comparison to zebularine, with IC50 > 100 mM for HeLa and K562 cancer cells and for HUVEC non-cancerous cells. Keywords: 2-Thiozebularine; Zebularine; Modified nucleosides; Conformation of nucleosides; X-ray; NMR; Cytotoxity of nucleosides. 2-Thiozebularine (s2 zeb) is one of the 2-pyrimidinone nucleoside analogues in which carbonyl group at C-2 position of the heterobase moiety was replaced by thiocarbonyl function. First synthesis of 2-thiozebularine was described by Wightman and Holý in 1973 1. The family of 2-pyrimidinone nucleosides plays an important role among biologically active analogues of natural pyrimidine nucleosides and was intensively investigated. Parent 1-ß-D-ribofuranosylpyrimidin-2-one (zebularine, zeb) exhibits an antibacterial activity connected with its in vivo transformation into 1-(2'-deoxy- ß-D-ribofuranosyl)pyrimidin-2-one 5'-phosphate, a strong inhibitor of thymidylate synthetase2,3. Zebularine is also a strong cytidine deaminase inhibitor4,5 and effectively inhibits DNA methylation6,7. Besides of the wide spectrum of biological activity, 2-pyrimidinone nucleosides are important tools as model compounds in structural studies involving RNA, and nucleic acid-binding proteins. This is mainly because 2-pyrimidinone nucleosides lacking N3-amide hydrogen and carbonyl function at C4 position in heterobase moiety, offer entirely different possibilities for base pairing within RNA duplexes8,9. As the sulfur modification of pyrimidine nucleosides by the replacement of the C2-carbonyl group with C2-thiocarbonyl is known to have a great impact on their structural features, we are interested in 2-thiozebularine in the context of our structural studies on modified nucleosides with nonnatural bases10. In the present work we describe the results of our conformational studies of 2-thiozebularine (s2 zeb, 1, Fig. 1) in the solid state and in aqueous solution. The resolved crystal structure of 1 is compared to the published structures of zebularine, (zeb, 2)11, 2-thiouridine (s2U, 3)12 and uridine (U, 4)13. Conformational studies in aqueous solution were performed for 1 and also for not so far precisely determined conformation of 2 by means of high-resolution NMR methods and the results were compared with the known data for s2U 14 and U 15. Moreover, cytotoxic properties of s2 zeb and zeb as well as their 2'-deoxyribo analogues were determined by MTT assay in HeLa and K542 cancer cells and in HUVEC non-cancerous cells. EXPERIMENTAL Materials All reagents were commercially available. In particular, CH3CN was distilled from P2O5; MeOH from Mg and benzene from Na. SnCl4 was distilled from P2O5 under reduced pressure. All other reagents were carefully dried before use. Silylation of heterobase and the reaction of N-glycoside bond formation were performed under anhydrous conditions. For column chromatography Merck silica gel 60 (230-400 mesh) was used. TLC was performed on analytical silica plates (Kieselgel 60 F254/0.2 mm thickness). NMR spectra were obtained on Bruker DPX 250 MHz and Bruker Advance II Plus 700 MHz. 1H and 13C NMR chemical shifts are given in ppm (d-scale), coupling constants (J) in Hz. Mass spectra were obtained on Finnigan MAT 95 spectrometer. UV-data were obtained on HITACHI UV Vis U-2800A. 2-Trimethylsilylmercaptopyrimidine (5) To a suspension of 2-mercaptopyrimidine (6.2 g, 55 mmol) in anhydrous benzene (550 ml), trimethylsilyl chloride (9.6 ml, 68.8 mmol) and triethylamine (9.6 ml, 68.8 mmol) dissolved in 12 ml of anhydrous benzene were added dropwise and the mixture was stirred at room temperature for 7 days. After this time the resulting precipitate (Et3N.HCl) was filtered off with careful exclusion of moisture. The filtrate was evaporated to a small volume (15 ml) and the residue was distilled under reduced pressure (0.1 mm Hg, 105 °C) to afford silylated 2-mercaptopyrimidine 5 (7 g, 38 mmol; 69% yield). 2',3',5'-Tri-O-benzoyl-1-(ß-D-ribofuranosyl)-1,2-dihydropyrimidine-2-thione (7) and 2S-(2',3',5'-Tri-O-benzoyl-ß-D-ribofuranosyl)-2-thiopyrimidine (8) Trimethylsilyl derivative 5 (1.84 g, 10 mmol) in anhydrous acetonitrile (25 ml) was added to the solution of 1-O-acetyl-2',3',5'-tri-O-benzoyl-ß-D-ribofuranoside (6; 3.53 g, 7 mmol) in anhydrous acetonitrile (50 ml). Then a solution of SnCl4 (1.3 ml, 11 mmol) in 80 ml of acetonitrile was dropped with stirring under exclusion of moisture. After 40 min at room temperature, when the reaction was judged to be complete by TLC (chloroform-methanol 98:2), the mixture was concentrated to 2/3 of its volume, diluted with CH2Cl2 (100 ml) and neutralized by addition of saturated aqueous solution of NaHCO3 (100 ml). After filtration through a layer of Celite to remove the tin salts and repeated washings of the Celite with CH2Cl2, the layers were separated and the aqueous phase was extracted with 2 × 100 ml of CH2Cl2. The combined organic phase was washed with water (50 ml) and dried over MgSO4. The final solution was filtered off, evaporated in vacuo and the residue was purified by silica gel column chromatography (hexane/ethyl acetate gradient from 2:1 to 1:2 v/v) to give N-glycoside 7 (3.19 g, 5.74 mmol; isolated yield 82%) and S-glycoside 8 (0.39 g, 0.70 mmol; isolated yield 10%). N-Glycoside 7: RF 0.11 CHCl3-MeOH 98:2; 0.51 CH3COOC2H5. 1H NMR (250 MHz, CDCl3): 8.46 dd, 1 H, 3J(5,4) = 4.0, 4J(6,4) = 2.3 (H4); 8.42 dd, 1 H, 3J(6,5) = 6.7, 4J(6,4) = 2.3 (H6); 7.82-8.09 m, 6 H (Ar-H); 7.26-7.68 m, 9 H (Ar-H); 7.03 d, 1 H, 3J(1',2') = 2.0 (H1'); 6.46 dd, 1 H, 3J(5,4) = 4.0, 3J(5,6) = 6.7 (H5); 6.01 dd, 1 H, 3J(2',1') = 2.0, 3J(2',3') = 5.0 (H2'); 5.77 dd, 1 H, 3J(3',2') = 5.0, 3J(3',4') = 7.8 (H3'); 4.87-4.97 m, 2 H (H4',H5'); 4.65-4.72 m, 1 H (H5'). S-Glycoside 8: RF 0.47 CHCl3-MeOH 98:2; 0.64 CH3COOC2H5. 1H NMR (250 MHz, CDCl3): 8.56 d, 2 H, 3J(6,5) = 3J(4,5) = 4.9 (H4, H6); 7.30-8.13 m, 15 H (Ar-H); 7.03 t, 1 H, 3J(5,6) = 3J(5,4) = 4.9 (H5); 6.51 d, 1 H, 3J(1',2') = 3.8 (H1'); 6.04 dd, 1 H, 3J(2',3') = 5.0, 3J(2',1') = 3.8 (H2'); 5.96 dd, 1 H, 3J(3',2') = 5.0, 3J(3',4') = 5.9 (H-3'); 4.80 m, 1 H (H4'); 4.71 dd, 1 H, 2J(5',5') = 12.1, 3J(5',4') = 4.0 (H5'); 4.60 dd, 1 H, 2J(5',5') = 12.1, 3J(5',4') = 4.1 (H5'). 1-(ß-D-Ribofuranosyl)-1,2-dihydropyrimidine-2-thione (s2 zeb, 1) The benzoylated derivative 7 (800 mg, 1.44 mmol) was suspended in anhydrous MeOH (57 ml), slightly warmed and then the solution was allowed to cool down to the room temperature. Afterwards 1 M solution of MeONa in MeOH was added (1.4 ml). The mixture was stirred at room temperature for 30 min. After this time TLC analysis (15% MeOH in CHCl3) showed that the starting material was completely consumed. The mixture was worked up with Dowex (pyridine salt form) and after filtered off the resin, the remaining solution was evaporated in vacuo and coevaporated with toluene. The oily residue was dissolved in water (15 ml) and washed with diethyl ether (3 × 10 ml). The aqueous layer was frozen and lyophilized to give N-glycoside 1 (0.25 g, 1.02 mmol; yield 71%). 1H NMR (700 MHz, D2O): 8.82 dd, 1 H, 3J(6,5) = 6.8, 4J(6,4) = 2.2 (H6); 8.49 dd, 1 H, 3J(5,4) = 4.4, 4J(6,4) = 2.2 (H4); 7.09 dd, 1 H, 3J(5,4) = 4.4, 3J(5,6) = 6.8 (H5); 6.38 s, 1 H (H1'); 4.37 d, 1 H, 3J(2',3') = 4.9 (H2'); 4.22 ddd, 1 H, 3J(4',5') = 2.4, 3J(4',5') = 3.5, 3J(4',3') = 9.4 (H4'); 4.10 dd, 1 H, 3J(3',2') = 4.9, 3J(3',4') = 9.4 (H3'); 4.05 dd, 1 H, 3J(4',5') = 2.4, 2J(5',5') = 13.2 (H5'); 3.85 dd, 1 H, 3J(4',5') = 3.5, 2J(5',5') = 13.2 (H5'). 13C NMR (175 MHz, D2O): 179.76 (C2), 160.24 (C4), 145.69 (C6), 111.50 (C5), 95.93 (C1'), 83.55 (C4'), 74.54 (C2'), 67.26 (C3'), 59.23 (C5'). MS-FAB (m/z, %): 245.1 (100) [M + H]+, calculated for C9H12O4N2S: 244. UV-Spectrum (H2O): ?max 281 nm (e 15739), 216 nm (e 6630), 351 nm (e 2662). 2S-(ß-D-Ribofuranosyl)-2-thiopyrimidine (9) The benzoylated derivative 8 (100 mg, 0.18 mmol) was dissolved in saturated methanolic ammonia (10 ml) and the mixture was stirred at room temperature for 48 h. After this time the reaction was judged to be complete by TLC (20% MeOH in CHCl3) and the mixture was evaporated in vacuo. The residue was dissolved in water (10 ml) and washed with diethyl ether (3 × 10 ml) to remove the methyl benzoate and benzamide. The crude product was purified by silica gel column chromatography (CHCl3/MeOH gradient from 100 to 90%). The fraction containing product was evaporated, dissolved in water, frozen and lyophilized to give S-glycoside 9 (36 mg, 0.15 mmol; 82%). 1H NMR (250 MHz, D2O): 8.55 d, 2 H, 3J(4,5) = 3J(6,5) = 4.9 (H4, H6); 7.23 t, 1 H, 3J(5,6) = 3J(5,4) = 4.9 (H5); 5.89 d, 1 H, 3J(1',2') = 5.0 (H1'); 4.20-4.31 m, 2 H, 3J(3',4') = 9.4, 3J(2',1') = 5.0 (H3', H2'); 4.02-4.08 m, 1 H, 3J(4',5') = 3.7, 3J(4',5') = 4.9, 3J(4',3') = 9.4 (H4'); 3.70 dd, 1 H, 2J(5',5') = 12.5, 3J(5',4') = 3.7 (H5'); 3.61 dd, 1 H, 2J(5',5') = 12.5, 3J(5',4') = 4.9 (H5'). 13C NMR (62 MHz, D2O): 168.91 (C2), 158.15 (C6, C4), 118.32 (C5), 86.29 (C1'), 85.05 (C4'), 74.43 (C2'), 70.74 (C3'), 61.44 (C5'). MS-CI (m/z, %): 245.1 (100) [M + H]+, calculated for C9H12O4N2S : 244. UV-Spectrum (H2O): ?max 241.5 nm (e 10049). Transformation of 2',3',5'-Tri-O-benzoylzebularine to 2',3',5'-Tri-O-benzoyl-2-thiozebularine (7) with Lawesson Reagent To a solution of 0.25 g (0.46 mmol) 2',3',5'-tri-O-benzoyl-1-(ß-D-ribofuranosyl)-1,2-dihydropyrimidine- 2-one in anhydrous dioxane (11.5 ml) Lawesson reagent (0.25 g, 0.51 mmol) was added and the mixture was stirred at boiling point for 1.5 h. After this time the reaction was judged to be complete by TLC (2% MeOH in CHCl3) and 4.7 ml of water was added to the reaction mixture. After 30 min the mixture was evaporated and the residue was dissolved in 10 ml of CHCl3 and then washed with 2 × 20 ml of 5% aqueous solution of NaHCO3 and 2 × 20 ml of water. The combined organic phase was dried over MgSO4. The final solution was filtered off, evaporated in vacuo and the residue was purified by silica gel column chromatography (CH2Cl2/(CH3)2CO gradient from 100 to 95%) to give 89 mg of N-glycoside 7 (0.16 mmol; yield 35%). X-ray Structure Analysis Crystal data for 2-thiozebularine: C9H12N2O4S, M = 244.28, monoclinic, space group P21, a = 4.9452(1) Å, b = 7.6077(2) Å, c = 13.7185(4) Å, ß = 92.783(4)°, V = 515.50(2) Å3, Z = 2, Dx = 1.574 g cm-3, T = 293 K, µ = 0.315 mm-1, ? = 0.71073 Å, data/parameters = 1779/147; flack x = 0.06(5), final R1 = 0.0181. Crystals of 2-thiozebularine were obtained by slow evaporation from their methanolic solutions. The measurements of the crystals were performed on a SMART diffractometer with graphite-monochromated MoKa radiation (? = 0.71073 Å) at room temperature. The structures were solved by direct method and refine with SHELXTL 16. E-maps provided positions for all non-H-atoms. The full-matrix least-squares refinement was carried out on F2's using anisotropic temperature factors for all non-H-atoms. All C-bound H atoms were placed in idealized locations and refined using a riding model, with C-H = 0.93 Å and Uiso(H) = 1.2 Ueq(C). CCDC 818090 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge, CB2 1EZ, UK; fax: +44 1223 336033; or [email protected]). NMR Studies of Modified Nucleoside The one- and two-dimensional NMR experiments were recorded on 700.2 MHz spectrometer at 25 °C in D2O with DSS as the internal standards. The samples for the NMR measurements were prepared by dissolving 6-8 mg of the nucleosides in 0.6 ml of D2O. Spectra were processed by means of TopSpin 2.1 software (Bruker BioSpin). In the case of overlapping signals in 1H 1D-NMR spectra the DAISY (Bruker BioSpin) deconvolution procedure was applied. 1H-13C vicinal coupling constants were derived from the coupled 13C NMR spectra and verified by J-HMBC experiments17. NOESY spectra were recorded with 0.5, 1 and 2 s mixingtimes. Cells and MTT Cytotoxicity Assay The cytotoxicity experiments were carried out with two cancer cell lines HeLa (human cervix carcinoma) and K562 (human chronic myelogenous leukemia). The HeLa and K562 cells were cultured in RPMI 1640 medium supplemented with antibiotics and 10% fetal calf serum in a 5% CO2-95% air atmosphere. 7 × 103 cells were seeded on each well on 96-well plate (Nunc). 24 h later cells were exposed to the test compounds for another 24 or 48 h. Stock solutions (100 mM) of test compounds were freshly prepared in water (distilled, MiliQ). The final concentrations of the compounds tested in the cell cultures were 1, 1 × 10-2, 1 × 10-4 and 1 × 10-6 mM. The values of IC50 (the concentration of test compound required to reduce the cell survival fraction to 50% of the control) were calculated from dose-response curves and used as a measure of cellular sensitivity to a given treatment. The cytotoxicity of all compounds was determined by the MTT (3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide; Sigma, St. Louis, MO) assay as described previously18,19. Briefly, after 24 or 48 h of incubation with drugs, cells were treated with the MTT reagent and incubation was continued for 2 h. MTT-formazan crystals were dissolved in 20% SDS and 50% DMF at pH 4.7 and absorbance was read at 570 and 650 nm on an microplate reader FLUOstar Omega (BMG LABTECH). As a control (100% viability), cells grown in the presence of vehicle (1% DMSO) only were used. RESULT AND DISCUSSION Chemical Synthesis The general methodology of nucleosides synthesis by N-glycosidic bond formation was successfully applied for 2-thiopyrimidinone nucleoside. Using this methodology, 2-thiozebularine was obtained for the first time by Wightman and Holý, in the reaction of metal salts of 2-mercaptopyrimidine and 2,3,5-tri-O-benzoyl ribofuranosyl chloride1 to give a mixture of S- and N-glycosides. Further efficient conversion of S-glycoside to the N-isomer on treatment with tin tetrachloride raised the yield of desired per-benzoylated 2-thiozebularine to 68%. Final removal of benzoyl groups from N-riboside sugar moiety by alkaline methanolysis afforded the deprotected 2-thionucleoside in good overall yield. Exclusive formation of 2-mercaptopyrimidine N-riboside was reported by Niedballa and Vorbruggen using the silyl method of nucleoside synthesis20. Reaction of the silylated 2-mercaptopyrimidine with 1-O-acetyl-2,3,5- tri-O-benzoyl-ß-D-ribofuranose in the presence of SnCl4 afforded perbenzoylated 2-thiozebularine in very good yield, but the sugar deprotected nucleoside was not characterized20. For our conformational study 2-thiozebularine (s2 zeb, 1) was synthesized using the silyl method, according to Scheme 1. Condensation of silyl derivative of 2-mercaptopyrimidine 5 with 1-O-acetyl-2,3,5-tri-O-benzoyl-ß-D-ribofuranose in acetonitrile, catalyzed with SnCl4, gave the protected N-glycoside 7 and S-glycoside 8 in 82 and 10% of isolated yield, respectively. It is worth to note that deprotection of benzoyl groups of N-glycoside 7 with standard methanolic ammonia treatment led to nucleoside decomposition while the S-isomer 8 was stable under these conditions. Removal of the sugar protecting groups of 7 by transestrification with 0.1 M NaOMe in MeOH, following work-up with Dowex (in pyridinium salt form), quantitatively gave the title nucleoside 1. The purity and structural identity of s2 zeb and its S-isomer 9 were fully confirmed by TLC chromatography and spectral data (UV, MS and NMR). Additionally, the new approach for the synthesis of s2 zeb using transformation of zebularine derivative with Lawesson thionation reagent21 was elaborated. The starting 2',3',5'-tri-O-benzoyl-zebularine obtained according Vorbruggen procedure20 was treated with 1.1 molar excess of Lawesson reagent in dioxane at 101 °C for 1.5 h. Purification of the product by column chromatography afforded per-benzoylated 2-thiozebularine in 35% yield. Structural Analysis Conformational analysis of modified nucleosides was carried out based on parameters defined by Altona and Sundaralingam22,23. Three parameters describe the primary features of the conformation of a pyrimidine nucleoside: the glycosidic bond torsion angle ? (O4'-C1'-N1-C2) describes the orientation of the base relative to the furanose ring; the C4'-C5' torsion angle ? determines the orientation of the 5'-hydroxyl group relative to the furanose ring (angle O5'-C5'-C4'-C3'); and the furanose ring puckering is specified by the pseudorotational phase angle P. Two major furanose ring conformations are strongly preferred for the nucleosides: C3'-endo (North, N-type conformation) and C2'-endo (South, S-type conformation)22,23. X-ray Structure Analysis 2-Thiozebularine was crystallized from methanol solution to give yellow needles. The detailed structure of 1 was established by X-ray crystallography and required data were deposited at the CCDC database. Figure 2a presents the ORTEP drawings of 2-thiozebularine molecule in projections perpendicular to the plane passing through atoms C1', C4' and O4', showing conformational details of the molecule. Selected geometrical parameters of s2 zeb and related nucleosides: zeb, s2U and U, indispensable for conformations comparison and discussion are listed in Table I. The geometry of the 2-thiopyrimidinone heterobase moiety of s2 zeb is not remarkably changed in comparison to those of 2-pyrimidinone, 2-thiouracil or uracil residues in zeb 11, s2U 12, and U 13 structures. However, the presence of the bulky sulfur atom at C-2 position of heterobases influences the conformation of the modified nucleoside treated as a whole (Fig. 2a, Table I), as well as the hydrogen bonds pattern in the crystals (Fig. 2b, Table II). In the crystal structure of s2 zeb the sugar residue adopts C3'-endo/C2'-exo twist type N-conformation (3 2T), with phase angle of pseudorotation P = 6.7° and pseudorotation amplitude 40.4°. It is worth to note that in the structure of s2 zeb the sugar twist conformation is less symmetrical (more C3'-endo) than in zebularine molecule, for which nearly ideal twist conformation occurs. For s2 zeb, the C3' atom is displaced by 0.44 Å from the plane C1'-O1'-C4' on the same side as C5', while by 0.31 Å for zeb, whereas C2' is at a distance of 0.21 Å from this plane on the opposite side for s2 zeb and 0.32 Å for zeb. The orientation of the heterocyclic base relative to the sugar moiety in s2 zeb molecules (? = -168.3°) is in the typical anti range, similar to zeb, s2U and U structures (Table I). The conformation around the C4'-C5' ribose bond of s2 zeb is in trans arrangement, observed less frequently than gauche(+) in pyrimidine nucleosides having N-type sugar ring pucker23. The preference of 1 to exist as the C4'-C5' trans conformer comes from the intermolecular hydrogen bonding between 5'O-H sugar group and 2-thiocarbonyl function of another molecule (Table II), similarly to hydrogen bonds pattern in the crystals of s2U molecules12. It is important to underline that the absence of N-3 hydrogen donor and 4-carbonyl acceptor functions in the heterobase influences the molecular packing of s2 zeb in crystals. The observed net of intermolecular hydrogen bonds varies from those ones observed for pyrimidine nucleosides: s2U 12, and U 13. Comprehensive description of the hydrogen bonds in s2 zeb crystal structure is summarized in Table II. The architecture of crystals of s2 zeb (in the P21 space group) is based on helical molecular arrangements by only the "sugar-to-sugar" junctions, with the strongest O2'-H2'...O3' hydrogen bonds (Fig. 2b). Two remaining H-bonds, one strong (O3'-H3'...O5') and one weak (5'O -H5'...S2), are responsible for joining helices present in the crystal (Table II). It is interesting that the identified three hydrogen bonds are managed to form 11-membered rings with graph-set notation of [R3 3(11)] 24, the same as in the structure of s2U. NMR Conformational Analysis 1D and 2D NMR techniques were used to determine the solution conformations of 2-thiozebularine (s2 zeb) and zebularine (zeb). In general, nucleoside conformation in solution is characterized by the dominant sugar pucker and preferred glycosidic bond arrangement25-28. Conformation of the Sugar Moiety It was assumed that the sugar ring of nucleoside in solution exists as an equilibrium mixture of the two puckered forms: C2'-endo (S conformer) or C3'-endo (N conformer). The percentage of S and N conformers can be estimated based on the values 3JH1'-H2' and 3JH3'-H4' coupling constants according to the following equations: % C2'-endo = 100 JH1'-H2'/(JH1'-H2' + JH3'-H4') and % C3'-endo = 100 - % C2'-endo 25,26. The experimental 3JH-H coupling constants and the calculated populations of C2'-endo and C3'-endo conformers of s2 zeb and zeb in comparison to s2U and U are listed in Table III. Resonance signal of H1' proton for s2 zeb was observed as a single line with 1.85 Hz half-width. Assuming similar puckering for s2 zeb and zeb sugar rings and the same sum of values of J(H1',H2') and J(H3',H4') coupling constants are equal to 10 Hz 25,26, the coupling constant J(H1',H2') for s2 zeb was estimated at 0.5 Hz. A lack of splitting of H1' line was confirmed by simulation of the line shape for different coupling constants by means of DAISY software. Splitting of the 1.85 Hz half-width line was observed when coupling constant was higher than 1 Hz. On the basis of J(H1',H2') = 0.5 Hz the population of C3'-endo conformer was estimated as at least 95%. Complete pseudorotation analysis of s2 zeb and zeb was performed using the PSEUROT software (version 6.2)29. In this program, minimization of the differences between the experimental and calculated values of couplings is accomplished by non-linear Newton-Raphson minimization, while the quality of the fit is expressed by root-mean-square (rms) differences. Calculations for s2 zeb, based on three coupling constants J(H1',H2'), J(H2',H3') and J(H3',H4') were performed by means of the PSEUROT automated procedure which led to 2800 results. For further analysis were taken only data with rms below 0.55 and puckering amplitude 30-40°. For different values of J(H1',H2') (0.0, 0.5 and 1.0 Hz) the dominating was N conformer (99%) and the best results were achieved for J(H1',H2') equal to 1 Hz. The lowest rms 0.17 corresponds to N conformer with the angle of pseudorotation PN = 31° and puckering amplitude ?mN = 41°, when for the minor S conformer PS = 169° and ?mS = 31°. Analogous PSEUROT analysis of zebularine led to 89% population of N conformer (PN = 29°, ?mN = 36°, PS = 172°, ?mS = 38°, rms 0.00). Our results evidently confirm that heterobase modification strongly affects conformational characteristics of the ribose moiety (Table III). Structure of pyrimidine nucleobase influences the pentafuranose conformation mainly through the steric effects and steroelectronic interactions within O4'-C1'-N1 fragment (anomeric effect)30. The replacement of C2 oxygen atom in U by more sterically demanding sulfur atom causes an increase of N conformer population of s2U by about 20% (Table III). This feature is attributed to steric effects between the 2-tiocarbonyl group of nucleobase and 2'-hydroxyl group of the ribose ring31-33, although recent hybrid DFT and MP2 calculations showed that the distant electrostatic effects between 2'-OH and the 2-tiocarbonyl function may enhance the selectivity of the C3'-endo conformation of ribose in the 2-thiouridine molecule34. The same extent of increase of the C3'-endo population was observed when 2-carbonyl function of zeb was replaced by bulky 2-tiocarbonyl group in s2 zeb (Table III; 80% of N conformer for zeb, 100% for s2 zeb). Interestingly, an absence of the carbonyl oxygen at C4 in zeb and s2 zeb raises the population of the C3'-endo conformers by about 30% in comparison to U and s2U, respectively (Table III). This feature can be attributed to the presence of the strong anomeric effect caused by electron-deficient 2-pyrimidinone or 2-thiopyrimidinone heterobase. In the case of s2 zeb, strong steric and stereoelectronic effects are additive and therefore the N conformer population increases to almost 100%. It is an unique case where the structure of nucleobase forces the ribose conformation to almost exclusive C3'-endo pucker, without any sugar modification. Conformation Around the Glycosidic Bond The syn/anti conformation around the N-glycosidic bond was probed by means of vicinal carbon-proton couplings and NOE effects. Proton-coupled 13C spectra reveal carbon-proton couplings that indicate the syn/anti conformation according to the following relationships JC6-H1' > JC2-H1' = anti; JC2-H1' > JC6-H1' = syn 27,28,35,36. For example, the magnitudes of the corresponding J values JC6-H1' = 3.6 Hz and JC2-H1' = 2.4 Hz for uridine clearly confirm the preference of anti conformation for this nucleoside in solution36. In the case of 2-thiozebularine and zebularine the measured smaller values of JC2-H1' than JC6-H1 (1.8 vs 2.1 Hz for s2 zeb and 1.1 vs 2.6 Hz for zeb) evidently indicate domination of the anti conformation around their N-glycoside bond. The population of the anti conformer can be estimated from the equation % anti = [10 - (JC2-H1' + JC6-H1')/6.4] 31 and for s2 zeb and zeb nucleosides the contents of anti conformers are similar and equal to 95 and 98%, respectively. To confirm domination of anti conformation around N-glycosidic bond of s2 zeb and zeb the NOE measurements were performed. Results of 1D NOE experiments were inconclusive because resonance of heterobase H6 and H4 protons were to close for selective irradiation of diagnostic H6 one. Thus the NOESY experiments were performed. The cross-peaks were integrated and interproton distances were calculated from the relation ?A/?B = r6 B/r6 A 27,37, where ?A and ?B are NOEs measured for two pairs of protons A and B, and rA and rB are distances for these two pairs of protons. As reference the NOE for H6 and H5 protons was used as the distance between these protons is known and equal to 2.45 Å. For both nucleosides the relationship H6-H1' > H6-H3' was observed (3.2 > 2.7 Å and 3.2 > 2.6 Å for s2 zeb and zeb, respectively), characteristic for anti conformer domination. Conformation Around the C4'-C5' Bond The local conformation around the C4'-C5' bond was examined by analysis with three staggered forms: gauche(+), trans, and gauche(-), using coupling constants H5' and H5' protons with H4' 27,38. The stereospecific assignment of the H5' and H5' proton in 1H NMR spectra was based on the deshielding effect of the phosphate group on H5' and H5' in 3'-monophosphates of uridines39. It was shown that the H5' and H5' spectral region shows a similar characteristic spectral pattern: d(H5') > d(H5') and J4'-5' < J4'-5' in a number of other nucleosides and nucleotides25. Therefore, it appears reasonable to assume that for all these nucleosides where this pattern is observed the more shielded proton is assigned as H5'. The stereospecific assignments of H5' and H5' methylene protons in the 1H NMR spectra of s2 zeb and zeb and measurements of the J4'-5' and J4'-5' proton-proton coupling constants enabled the determination of the population of three exocyclic C4'-C5' rotamers gauche(+), trans and gauche(-)µ The results are given in Table IV. All of analyzed nucleosides exhibit preferentially gauche(+) conformation with the higher population of this rotamer for 2-thionucleosides. Cytotoxicity of Zebularine and 2-Thiozebularine and Their 2'-Deoxyanalogues A series of compounds: zebularine 2, 2-thiozebularine 1, 2'-deoxyzebularine 10 and 2-thio-2'-deoxyzebularine 11 was screened for cytotoxic properties against K562 (leukemia), HeLa (human cervix carcinoma) and HUVEC (normal) cells. The viability of cells was determined in the MTT assay at four different compound concentrations: 1, 1 × 10-2, 1 × 10-4 and 1 × 10-6 mM. Compounds 1, 10 and 11 were not toxic toward cells used in the study both after 24 and 48 h incubation time (IC50 > 1 mM). However, zebularine 2 showed limited toxicity toward K562 leukemia cancer cells only. Cytotoxicity of 2 seems to be time-dependent, as its concentration needed to induce cell death after 48 h (100 µM) is six-fold lower comparing to IC50 at 24 h (600 µM). CONCLUSIONS The s2 zeb nucleoside was synthesized in good overall yield by coupling of silyl derivative of 2-mercaptopyrimidine with 1-O-acetyl-2,3,5-tri-O-benzoyl- ß-D-ribofuranose in acetonitrile, catalyzed with SnCl4, and removal of the sugar protecting groups with NaOMe in MeOH, or alternatively, by the thionation of appropriate zebularine derivative with Lawesson reagent. X-ray analysis revealed that in solid state s2 zeb molecules adopt C3'-endo/ C2'-exo twist puckering of ribofuranose ring and anti of N-glycosidic bond conformation, generally consistent with those of zeb, s2U and U crystal structures. NMR spectroscopy analysis of s2 zeb and zeb conformations in solution and their comparison to reference s2U and U compounds evidently confirms that the sugar pucker can be steered by heterobase structure (from 53% of N population in uridine to almost 100% in 2-thiozebularine). The replacement of oxygen atom at C2 either of zeb or U by more sterrically demanding sulfur atom, leads to 20% increase of C3'-endo conformer population, as in s2 zeb and s2U. The lack of carbonyl oxygen at position 4, as in s2 zeb or zeb, gives the 30% increase of the C3'-endo conformer population due to the electron-withdrawing effect of modified heterobase on sugar moiety. In the case of s2 zeb both effects operate simultaneously and, therefore, the population of N conformer increases to almost 100%. 2-Thiozebularine is unique example of pyrimidine ribonucleoside which in aqueous solution is fully constrained in the C3'-endo conformation and this feature is exerted exclusively through nucleobase steric and stereoelectronic interactions with ribose ring. 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Katarzyna EBENRYTERa1, Stefan JANKOWSKIa2, Janina KAROLAK-WOJCIECHOWSKAb1, Andrzej FRUZINSKIb2, Julia KAZMIERCZAK-BARANSKAc1, Barbara NAWROTc2 and Elzbieta SOCHACKAa3,* a Institute of Organic Chemistry, Technical University of Lodz, Zeromskiego 116, 90-924 Lodz, Poland; e-mail: 1 [email protected], 2 [email protected], 3 [email protected] b Institute of General and Ecological Chemistry, Technical University of Lodz, Zeromskiego 116, 90-924 Lodz, Poland; e-mail: 1 [email protected], 2 [email protected] c Department of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies of the Polish Academy of Sciences, Sienkiewicza 112, 90-363 Lodz, Poland; e-mail: 1 [email protected], 2 [email protected] Received April 19, 2011 Accepted May 19, 2011 Published online August 24, 2011 This article is dedicated to Professor Antonín Holý on the occasion of his 75th birthday in recognition of his outstanding contribution to the area of nucleic acids chemistry. (c) 2011 Institute of Organic Chemistry and Biochemistry |