Synthesis and recognition by DNA polymerases of a reactive nucleoside, 1-(2-deoxy-beta-D-erythro-pentofuranosyl)-imidazole-4-hydrazide

Abstract

We report the synthesis of a new nucleoside, 1-(2-deoxy-beta-D-erythro-pentofuranosyl)-imidazole-4-hydrazide (dY(NH2)) as a reactive monomer for DNA diversification. The 5'-triphosphate derivative (dY(NH2)TP, 1) was evaluated in vitro as a substrate for several DNA polymerases. Primer extension reactions showed that dYNH2TP was well tolerated by KF (exo(-)) and Vent (exo-) DNA polymerases. One dYNH2MP was incorporated opposite each canonical base with an efficiency depending on the template base (A approximately T > G > C). Significant elongation after YNH2 incorporation was observed independently of the YNH2:N base pair formed. When the nucleobase YNH2 was incorporated into synthetic oligodeoxynucleotides via the phosphoramidite derivative 11, it directed the insertion of natural bases as well as itself. The mutagenicity of dYNH2TP was evaluated by PCR amplification using Vent (exo-) DNA polymerase. The triphosphate dY(NH2)TP was preferentially incorporated as a dATP or dGTP analogue and led to misincorporations at frequencies of approximately 2 x 10(-2) per base per amplification. A high proportion of transversions with a large distribution of all possible mutations was obtained. The reactivity of the nucleobase YNH2 within a template with several aldehydes was demonstrated.

3-Phosphono-l-alanine as pyrophosphate mimic for DNA synthesis using HIV-1 reverse transcriptase

“…However, in the case of compounds 6-8, ZnCl 2 was added to increase the reactivity of the nucleoside phosphorimidazolides 10 and the compounds were purified by column chromatography followed by preparative HPLC to remove the EDTA-Zn 2+ complex. The ability of the phosphoramidate analogues (1)(2)(3)(4)(5) to function as substrate for HIV-1 RT was investigated by the gel-based single nucleotide incorporation assay (Fig. 4).…”

“…Most of these triphosphate mimics are obtained by modifying the bridging O-atoms of the triphosphate moiety 2 or using non-standard nucleobases. 3 In previous studies, we have demonstrated that the pyrophosphate moiety of nucleoside triphosphate can be replaced by leaving groups whose structures are not based on phosphate chemistry. For example, some selected amino acid derivatives could be considered as mimics of the pyrophosphate moiety of deoxyadenosine triphosphate.…”

A Novel Versatile Phosphoramidite Building Block for the Synthesis of 5'- and 3'-Hydrazide Modified Oligonucleotides

“…Next to immobilization, hydrazone formation-based methods are widely used for conjugating ONs and various reporter molecules, [4,10,12,19,27,28] including markers, dyes, and peptides. Most often 5'-modified ONs are obtained through post-synthetic introduction of the hydrazide function, and they were reported to be stable in solution.…”

“…For non-terminal modifications, the synthesis of a nucleoside analogue containing a hydrazide derived imidazole residue as a nucleobase was reported. [27] It can be incorporated into ONs both chemically and enzymatically. Conjugation with aldehydes permits the synthesis of a large variety of novel nucleobases in a single step.…”

“…The coupling reaction of this hydrazide moiety with several aromatic aldehydes was reported to proceed very fast (in several minutes) under the conditions chosen (at pH 4) with high yield. [27] In summary, hydrazide-functionalized ONs are undoubtedly very promising compounds, which clearly will find more and more applications in the near future. At the present time, most known methods propose to the inclusion of the hydrazide moiety into ON post-synthetically.…”