Oligonucleotides incorporating 7-deaza-2'-deoxyxanthosine (3) and 2'-deoxyxanthosine (1) were prepared by solid-phase synthesis using the phosphoramidites 6 -9 and 16 which were protected with allyl, diphenylcarbamoyl, or 2-(4-nitrophenyl)ethyl groups. Among the various groups, only the 2-(4nitrophenyl)ethyl group was applicable to 7-deazaxanthine protection being removed with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) by b-elimination, while the deprotection of the allyl residue with Pd 0 catalyst or the diphenylcarbamoyl group with ammonia failed. Contrarily, the allyl group was found to be an excellent protecting group for 2'-deoxyxanthosine (1). The base pairing of nucleoside 3 with the four canonical DNA constituents as well as with 3-bromo-1-(2-deoxy-b-D-erythro-pentofuranosyl)-1H-pyrazolo [3,4-d]pyrimidine-4,6-diamine (4) within the 12-mer duplexes was studied, showing that 7-deaza-2'-deoxyxanthosine (3) has the same universal base-pairing properties as 2'-deoxyxanthosine (1). Contrary to the latter, it is extremely stable at the N-glycosylic bond, while compound 1 is easily hydrolyzed under slightly acidic conditions. Due to the pK a values 5.7 (1) and 6.7 (3), both compounds form monoanions under neutral conditions (95% for 1; 65% for 3). Although both compounds form monoA C H T U N G T R E N N U N G anions at pH 7.0, pH-dependent T m measurements showed that the base-pair stability of 7-deaza-2'-deoxyxanthosine (3) with dT is pH-independent. This indicates that the 2-oxo group is not involved in base-pair formation.