A 5′→3′ Phosphoramidite Approach for the Synthesis of Phosphorodiamidate and Thiophosphoramidate Morpholino Oligonucleotides and Their Chimera

The article outlines a scalable, streamlined 5′→3′ phosphoramidite approach for the synthesis of phosphorodiamidate and thiophosphoramidate morpholino oligonucleotides using 5′-morpholino phosphoramidites. The synthesis of two different subclasses of these 5′-morpholino phosphoramidites – tert-butyl protected 5′-tBu-morpholino phosphoramidites, and 2-cyanoethyl protected 5′-CE-morpholino phosphoramidites have been demonstrated. Use of these two subclasses of 5′-morpholino phosphoramidites have been expounded for the synthesis of PMOs and TMOs, respectively in automated DNA synthesizer, including a 19-mer T PMO. The synthesis is featured by its efficiency and operational simplicity in terms of complete compatibility with automated DNA synthesizer as the synthesis uses controlled pored glass as its support of choice and all the steps are almost similar as that of conventional DNA/ RNA synthesis. Standard amide-based nucleobase protecting groups have been used for the synthesis and no extra steps are involved, making the method commercially viable for the scale-up of PMO synthesis. Interestingly, this 5′→3′phosphoramidite approach provides with an opportunity to access various biologically relevant chimeric morpholino oligonucleotides like PMO-TMO and TMO-PMO chimeras for the first time.