Synthesis of 6-Alkynylated Purine-Containing DNA via On-Column Sonogashira Coupling and Investigation of Their Base-Pairing Properties

Synthetic unnatural base pairs have been proven to be attractive tools for the development of DNA-based biotechnology. Our group has very recently reported on alkynylated purine–pyridazine pairs, which exhibit selective and stable base-pairing via hydrogen bond formation between pseudo-nucleobases in the major groove of duplex DNA. In this study, we attempted to develop an on-column synthesis methodology of oligodeoxynucleotides (ODNs) containing alkynylated purine derivatives to systematically explore the relationship between the structure and the corresponding base-pairing ability. Through Sonogashira coupling of the ethynyl pseudo-nucleobases and CPG-bound ODNs containing 6-iodopurine, we have demonstrated the synthesis of the ODNs containing three (N)Pu derivatives ((N)Pu1, (N)Pu2, (N)Pu3) as well as three (O)Pu derivatives ((O)Pu1, (O)Pu2, (O)Pu3). The base-pairing properties of each alkynylated purine derivative revealed that the structures of pseudo-nucleobases influence the base pair stability and selectivity. Notably, we found that (O)Pu1 bearing 2-pyrimidinone exhibits higher stability to the complementary (N)Pz than the original (O)Pu, thereby demonstrating the potential of the on-column strategy for convenient screening of the alkynylated purine derivatives with superior pairing ability.