Phosphoramidite Platform for the Automated Synthesis of Morpholino Antisense Oligonucleotides and Their Chimeras

Abstract Phosphorodiamidate morpholino oligonucleotides (PMOs) are antisense oligonucleotides that modulate gene expression through a steric blocking mechanism. Despite their increasing use in antisense therapeutics, the synthesis of PMOs remains challenging, thus limiting their accessibility. As a result, a modular synthesis protocol for PMOs, compatible with DNA/RNA synthesizers, is highly anticipated to facilitate the development of next‐generation PMO chimeras incorporating other oligonucleotide backbones with proven therapeutic efficacy. In this article, we demonstrate a streamlined 5′→3′ phosphoramidite approach for synthesizing PMOs using 3′‐ N ‐MMTr (monomethoxytrityl)‐5′‐ t Bu (tert‐butyl)‐morpholino phosphoramidites and 3′‐ N ‐Tr(trityl)‐5′‐CE(cyanoethyl)‐morpholino phosphoramidites, enabling the synthesis of thiophosphoramidate (TMO) and phosphoramidate (MO) morpholino oligonucleotides. The phosphoramidite building blocks were synthesized from 5′‐OH morpholino monomers derived from commercially available ribonucleosides. The solid‐phase oligonucleotide synthesis was carried out in acetonitrile solvent on controlled pore glass (CPG) support using an automated DNA/RNA synthesizer, resulting in excellent overall yields. The synthesis process also incorporated regular amide protecting groups for the exocyclic amines of cytidine and adenosine, as well as dimethylformamidine for guanosine. The solid‐phase synthesis cycle consists of the following steps: (a) deblocking the 3′‐N protecting group of the morpholino ring, (b) neutralization, (c) coupling and oxidation of the 5′‐phosphoramidite (oxidative coupling), and (d) capping unreacted morpholino‐NH. Afterward, cleavage from the solid support is achieved through treatment with aqueous ammonia. This method provides a convenient and efficient approach for synthesizing various biologically relevant antisense oligonucleotide (ASO) designs, including PMO–TMO and PMO–MO chimeras, with reproducible and high yields. © 2025 Wiley Periodicals LLC. Basic Protocol 1 : Synthesis of 3′‐ N ‐Tr‐5′‐CE‐morpholino phosphoramidites Basic Protocol 2 : Synthesis of 3′‐N‐MMTr‐5′‐ t Bu ‐morpholino phosphoramidites Basic Protocol 3 : Solid phase synthesis of PMO, TMO, PMO–TMO, and other chimeras in an automated DNA synthesizer Basic Protocol 4 : HPLC purification of synthesized ONs