Herein we report the synthesis of N⁸-glycosylated 8-aza-deoxyguanosine (N⁸-8-aza-dG) and 8-aza-9-deaza-deoxyguanosine (N⁸-8-aza-9-deaza-dG) nucleotides and their base pairing properties with 5-methyl-isocytosine (d-isoC^Me), 8-amino-deoxyinosine (8-NH₂-dI), 1-N-methyl-8-amino-deoxyinosine (1-Me-8-NH₂-dI), 7,8-dihydro-8-oxo-deoxyinosine (8-Oxo-dI), 7,8-dihydro-8-oxo-deoxyadenosine (8-Oxo-dA), and 7,8-dihydro-8-oxo-deoxyguanosine (8-Oxo-dG), in comparison with the d-isoC^Me:d-isoG artificial genetic system. As demonstrated by T_m measurements, the N⁸-8-aza-dG:d-isoC^Me base pair formed less stable duplexes as the C:G and d-isoC^Me:d-isoG pairs. Incorporation of 8-NH₂-dI versus the N⁸-8-aza-dG nucleoside resulted in a greater reduction in T_m stability, compared to d-isoC^Me:d-isoG. Insertion of the methyl group at the N¹ position of 8-NH₂-dI did not affect duplex stability with N⁸-8-aza-dG, thus suggesting that the base paring takes place through Hoogsteen base pairing. The cellular interpretation of the nucleosides was studied, whereby a lack of recognition or mispairing of the incorporated nucleotides with the canonical DNA bases indicated the extent of orthogonality in vivo. The most biologically orthogonal nucleosides identified included the 8-amino-deoxyinosines (1-Me-8-NH₂-dI and 8-NH₂-dI) and N⁸-8-aza-9-deaza-dG. The 8-oxo modifications mimic oxidative damage ahead of cancer development, and the impact of the MutM mediated recognition of these 8-oxo-deoxynucleosides was studied, finding no significant impact in their in vivo assay.

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