Threofuranosyl-(3',2')-nucleic acid (TNA) is an artificial genetic polymer in which the natural five carbon ribose sugar found in RNA has been replaced with an unnatural four-carbon threose sugar. Despite a backbone repeat unit that is one bond shorter than the backbone repeat unit found in DNA and RNA, TNA is capable of forming stable anti-parallel Watson-Crick duplex structure with complimentary strands of DNA, RNA and TNA. This remarkable property, coupled with strong nuclease stability, makes TNA an attractive candidate for therapeutic and diagnostic applications. Due to commercial unavailability, TNA molecules are synthesized in lab. My project is to develop a streamlined and scalable approach for routinely synthesizing the TNA phosphoramidites and triphosphate monomers for solid-phase and polymerase mediated oligonucleotide synthesis, respectively. In our lab we have developed a new methodology for making TNA 2'-phosphoramidite which overcomes the drawbacks of low regioselectivity and lower yield of Eschenmoser’s original synthesis pathway.Introduction of different orthogonal protecting groups to the sugars as well as on the base moieties, reduce the effort of silica gel the purification because we obtained most of the product just by precipitation. Due to low reactivity and steric crowding of the secondary 3'-alcohol, it was difficult to synthesize the TNA-3'-triphosphates (tNTPs). Here I have developed a new synthetic methodology that can be able to synthesize tNTPs with high yield (~65-70%) and higher purity. We checked the reaction progress by analytical HPLC and purified all the tNTPs by a reverse phase semi-preparative HPLC. Template directed primer extension with engineered polymerase shows the efficiency of the newly formed tNTPs. The new synthetic method can be able to synthesize ~50 gram scale synthesis of TNA-phosohoramidite and ~50 miligram scale of tNTP which we can use for all the ongoing and future projects of our lab.

Developing a pipeline for generating TNA-phosphoramidite and TNA-triphosphate monomers for the in vitro evaluation of the properties of the polymers

253rd National Meeting (2017)

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