2,6-Bis(functionalized) purines as metal-ion-binding surrogate nucleobases that enhance hybridization with unmodified 2′-O-methyl oligoribonucleotides

Abstract

The capacity of three different purine bases, viz. 2,6-bis(3,5-dimethylpyrazol-1-yl)purine, 2-(3,5-dimethylpyrazol-1-yl)adenine and 2,6-bis(2-acetyl-1-methylhydrazino)purine, to form metal-ion mediated base pairs with the native nucleobases has been examined. For this purpose, ribonucleosides derived from these bases were incorporated into an intrastrand or a 3'-terminal position of short 2'-O-methyl oligoribonucleotides and the hybridization properties of these base modified oligomers in the absence and presence of three different metal ions (Cu(2+), Zn(2+) and Pd(2+)) were studied by UV- and CD-spectrometry. The first two bases were found to stabilize short oligonucleotide duplexes when incorporated into the 3'-termini of both strands, even in the absence of divalent metal ions but especially in the presence of Cu(2+). The highest melting temperature determined for such a duplex was 71.8 °C, nearly 30 °C higher than the T(m) of the respective solely Watson-Crick paired duplex. Despite the dramatic stabilizing effect of the terminal metallo-base pairs, these short modified oligonucleotides retained sequence-selectivity for the internal Watson-Crick base pairs: two internal mismatches dropped the melting temperature to 10-11 °C. In an internal position, only 2,6-bis(3,5-dimethylpyrazol-1-yl)purine, which in the absence of metal ions was destabilizing, exhibited metal-ion-dependent stabilization of duplex formation with unmodified 2'-O-methyl oligoribonucleotides. The melting temperature in the presence of Cu(2+) was increased from 6 to 14 °C, depending on the identity of the opposite base.

Metal-Ion-Mediated Base Pairing between Natural Nucleobases and Bidentate 3,5-Dimethylpyrazolyl-Substituted Purine Ligands

“…To verify the hydrolytic stability of the modified oligonucleotides under conditions of the T m studies, the samples were analyzed by HPLC and ESI-MS before and after the measurement (3 cycles from 0 to 90°C and back). Oligonucleotides ON1p, ON1q, and ON1z proved to be stable in all the cases studied but the previously studied 8,11 ON1x had been converted almost quantitatively to the hydrolysis product ON1q in the presence of Cu 2+ (Scheme 3). In the presence of Zn 2+ or in the absence of divalent metal ions, on the other hand, no changes in the ON1x-containing samples were observed.…”

“…8 The greatest effect was observed when the artificial nucleoside was placed in a terminal position or flanked by mismatched base pairs, that is, with the most flexible structures studied. In the middle of a fully matched double helix, introduction of Cu 2+ barely overcame the destabilization caused by the bulky modified nucleoside.…”

Metal complexes of 6-pyrazolylpurine derivatives as models for metal-mediated base pairs

“…Chemical structures of 3,5-dimethylpyrazolyl-substituted derivatives of purine, adenine, and hypoxanthine (R = nucleic acid backbone) as previously reported by Lönnberg et al[32,33,35].…”

“…Most recently, Lönnberg et al have introduced a family of artificial purine-derived nucleosides that were designed with the aim of discriminating a natural nucleobase located opposite the artificial nucleoside [32][33][34][35]. Oligonucleotides capable of specifically recognizing short nucleic acid sequences are relevant in RNA interference and other therapeutic applications for the treatment of genetic disorders [36,37].…”

Recent advances in the synthesis and applications of 2,6-dipyrazolylpyridine derivatives and their complexes

“…24 The methods in eq 1 and eq 3 have also been used to prepare 1-bppym derivatives (Scheme 1), starting from the appropriate 2,6-dihaloor 2,6-di(hydrazino)-pyrimidine. 25,26 Chemical modification of 1-bpp itself can be achieved by 50 halogenation (Scheme 3). Mild electrophilic chlorination, bromination or iodination reagents lead to selective dihalogenation at the pyrazole C4 positions.…”