A Unified Phosphoramidite\nPlatform for the Synthesis\nof Morpholino Oligonucleotides and Diverse Chimeric Backbones

Phosphorodiamidate Morpholino Oligonucleotides\n(PMOs)\nhave been\nwell established in the milieu of FDA-approved oligonucleotide-based\ndrugs in the past decade. Given their relevance in antisense therapeutics,\na DNA/RNA synthesizer-compatible modular synthesis protocol of PMOs\nis long awaited to explore next-generation PMO chimeras with other\ntherapeutically proven oligonucleotide backbones. Herein, we demonstrate\na streamlined 5′ → 3′phosphoramidite approach\nfor the synthesis of PMOs using <i>tert</i>-butyl-protected\n5′-<sup><i>t</i></sup><i>Bu-</i>morpholino\nphosphoramidites, which were synthesized from 5′–OH\nmorpholino monomers derived from commercially available ribonucleosides.\n2-Cyanoethyl (CE)-protected 5′-CE-morpholino phosphoramidites\nwere also synthesized to generate thiophosphoramidate (TMO) and phosphoramidate\n(MO) morpholino oligonucleotides. Full-length PMOs and TMOs in exceptional\noverall yields were obtained with operational simplicity and compatibility\nwith automated DNA/RNA synthesizers utilizing controlled pore glass\n(CPG) as the solid support and CH<sub>3</sub>CN as the solvent. Importantly,\nthis method has opened the possibility of designing various biologically\nrelevant ASO design modalities, such as PMO-TMO and PMO-MO, which\nwere inaccessible otherwise. Moreover, DNA nucleotides were also incorporated\nto generate PMO-psDNA and PMO–DNA using commercially available\n5′-DNA phosphoramidites. The biophysical properties of all\nsynthesized oligonucleotides were analyzed using UV melting and circular\ndichroism studies. The serum interaction profile and innate immune\nresponse of the PS-modified polythymidine oligonucleotides were analyzed\nand it was found that the mixed backbone oligonucleotides had a balanced\nprofile compared to fully charged or neutral extremities. Overall,\nthe synthesis is amenable to fast generation of various types of PMO\nchimeras for biological screening, which will expedite their therapeutic\nexploration and transition to clinic.