Base pairing of anhydrohexitol nucleosides with 2,6-diaminopurine, 5-methylcytosine and uracil as base moiety

V. Boudou 0 L. Kerremans 0 B. De Bouvere 0 E. Lescrinier 0 G. Schepers 0 R. Busson 0 A. Van Aerschot 0 P. Herdewijn 0 0 Laboratory of Medicinal Chemistry, Rega Institute for Medical Research, Katholieke Universiteit Leuven , Minderbroedersstraat 10, B-3000 Leuven, Belgium Hexitol nucleic acids (HNAs) with modified bases (5-methylcytosine, 2,6-diaminopurine or uracil) were synthesized. The introduction of the 5-methylcytosine base demonstrates that N-benzoylated 5-methylcytosylhexitol occurs as the imino tautomer. The base pairing systems (G:CMe, U:D, T:D and U:A) obey Watson-Crick rules. Substituting hT for hU, hCMe for hC and hD for hA generally leads to increased duplex stability. In a single case, replacement of hC by hCMe did not result in duplex stabilization. This sequence-specific effect could be explained by the geometry of the model duplex used for carrying out the thermal stability study. Generally, polypurine HNA sequences give more stable duplexes with their RNA complement than polypyrimidine HNA sequences. This observation supports the hypothesis that, besides changes in stacking pattern, the difference in conformational stress between purine and pyrimidine nucleosides may contribute to duplex stability. Introduction of hCMe and hD in HNA sequences further increases the potential of HNA to function as a steric blocking agent. - Duplex stability of dsDNA and dsRNA can be increased by modifications of the carbohydrate moiety, the base moiety or the internucleoside linkage. Hexitol nucleic acid (HNA) is an example of how sugar modifications may influence duplex stability in a beneficial way (13). The two most studied base modifications, leading to an increase in hybridization strength, are the replacement of cytosine by 5-methylcytosine and the replacement of adenine by 2-aminoadenine (or diaminopurine) (4). Introduction of a methyl group in the 5 position of uracil and cytosine bases increases hydrophobic interactions between the 5-methylpyrimidines and neighbouring bases, generally resulting in more stable complexes (57). It has been observed, however, that the 5-methyl group may sometimes reduce the cooperativity of the duplex melting process (5) and may induce a conformational transition of oligonucleotides in solution (58). Three WatsonCrick hydrogen bonds can be formed between the diaminopurine base and a regular uracil (thymine) base and this is the basis for the expected and observed higher stability of duplexes when adenine is replaced by 2,6-diaminopurine (D). However, such replacement may lead as well to duplex stabilization as to destabilization or may end up in no effect at all on thermal stability (912), mainly depending on the influence of the 2-amino group on the geometry of hydrogen bonding, hydration of the minor groove, helical conformational transitions, groove width and base stacking pattern. HNAs hybridize sequence-selectively and very strongly with natural nucleic acids. Until now, only two WatsonCrick base pairing schemes have been evaluated, i.e. G-C and A-T base pairs. To further investigate the influence of base modifications on HNA-containing complexes, we incorporated uracil (U), 5-methylcytosine (CMe) and 2,6-diaminopurine (D) modified hexitol nucleosides into regular HNA sequences. The influence on the thermal stability of HNA:DNA, HNA:RNA and dsHNA hybrids was evaluated. This study led to the conclusion that, even with these modified and unnatural bases, HNA obeys the general WatsonCrick pairing rules and that replacement of D for A and CMe for C may further increase the potential of HNA to function as a steric blocker. Synthesis of protected hexitol nucleosides with modified bases All experiments were carried out using instrumentations and manipulations as described previously (2,3,13). [BH2]+ stands for protonated base. 1H NMR and 13C NMR data of all compounds are available as supplementary material. 1,5-Anhydro-4,6-O-benzylidene-2,3-dideoxy-2-(uracil-1-yl)-D-arabino-hexitol (2). A suspension of 2.24 g (20 mmol) of uracil and 152 mg of LiH in DMF (150 ml) was heated at 120 C for 1 h, after which a solution of 3.9 g (10 mmol) of 1,5-anhydro-4,6-O-benzylidene-3-deoxy-2-O-(p-toluenesulfonyl)-D-ribo-hexitol (13) (1) in DMF (20 ml) was added. The reaction mixture was stirred for 24 h at 120 C, H2O (1 ml) was added and the mixture was evaporated at reduced pressure. The residue was diluted with brine (300 ml) and extracted with EtOAc (3 times). The combined organic layer was dried and evaporated. The title compound was obtained in 57% yield (3.5 g, 10.6 mmol) after column chromatographic purification (EtOAc/hexane 80:20). LSIMS (THGLY): m/z 331 [MH]+, 113 [BH2]+. Elemental analysis. Calculated for C17H18N2O5: C, 61.81; H, 5.49; N, 8.48. Found: C, 62.24; H, 5.62; N, 8.64. 1,5-Anhydro-2,3-dideoxy-2-(uracil-1-yl)-D-arabino-hexitol (3). A solution of 2.20 g (6.67 mmol) of 1,5-anhydro-4,6-O-benzylidene2,3-dideoxy-2-(uracil-1-yl)-D-arabino-hexitol in 100 ml of 80% aqueous HOAc was stirred overnight at room temperature and for an additional 2 h at 60 C. Acetic acid was evaporated and the residual oil was purified by column chromatography (CH2Cl2/MeOH 90:10). The title compound was obtained in 75% yield (1.21 g, 5 mmol). LSIMS (THGLY): m/z 243 [MH]+, 113 [BH2]+. Elemental analysis. Calculated for C10H14N2O5: C, 49.58; H, 5.83; N, 11.56. Found: C, 49.57; H, 5.82; N, 11.47. 1,5-Anhydro-2,3-dideoxy-2-(uracil-1-yl)-6-O-monomethoxytrityl-Darabino-hexitol (4). A solution of 2.0 g (8.3 mmol) of 1,5anhydro-2,3-dideoxy-2-(uracil-1-yl)-D-arabino-hexitol and 3.95 g (12.8 mmol) of monomethoxytrityl chloride in 40 ml of pyridine was stirred overnight at room temperature. The reaction mixture was diluted with 200 ml of saturated NaHCO3 solution and extracted three times with CH2Cl2. The organic layer was dried, evaporated, co-evaporated with toluene and purified by column chromatography (CH2Cl2/MeOH 98:2). Yield 3.34 g (6.5 mmol, 81%) LSIMS (THGLY/NaOAc): m/z 559 [M+2Na-H]+. Elemental analysis. Calculated for C30H30N2O6H2O: C, 67.64; H, 6.06; N, 5.26. Found: C, 67.56; H, 5.87; N, 5.22. 1,5-Anhydro-2,3-dideoxy-2-(5-methylcytosin-1-yl)-D-arabino-hexitol (7). A mixture of 1.7 g (5 mmol) of 1,5-anhydro-4,6-O-benzylidene-2,3-dideoxy-2-(thymin-1-yl)-D-arabino-hexitol (1,2), POCl3 (1 ml) and 1,2,4-triazole (2.96 g) in 120 ml of pyridine was stirred at room temperature for 4 h. The reaction mixture was cooled to 0 C and ammonia gas was bubbled through the mixture for 10 min. The reaction mixture was further stirred for 10 min at room temperature, evaporated and co-evaporated with toluene (3 times). The principal reaction product was isolated by silica gel chromatography (CH2Cl2/MeOH 95.5) giving 1.3 g (3.8 mmol, 80% yield) of an oil. The compound was identified after removal of the benzylidene protecting group. Hereto, the oil was dissolved in 80% HOAc (100 ml) and heated for 5 h at 80 C. The mixture was evaporated, co-evaporated with toluene (3 tim (...truncated)