Synthesis of morpholino monomers, chlorophosphoramidate monomers, and solid-phase synthesis of short morpholino oligomers

Phosphorodiamidate morpholino oligomers (PMOs) are a highly capable class of synthetic antisense oligonucleotides that are used to study gene functions in in vitro and in vivo models. This unit describes the synthesis of exocyclic-amine-protected 7′-hydroxy and 7′-chlorophosphoramidate-activated morpholino monomers of A, T, G, and C, together with their incorporation into short PMO oligomers by solid-phase synthesis. Starting from ribonucleosides, the exocyclic-amine-protected 7′-hydroxy monomers are prepared following a modified Summerton protocol, which consists of a periodate cleavage/Schiff base formation/reduction cycle. The exocyclic amine protections are installed at a later stage (except G) to avoid the use of costly exocyclic-amine-protected counterparts that give control over protecting group manipulation. The 7′-hydroxy monomers with N-Trit/N-MMTr are then converted to the 7′-chlorophosphoramidate morpholino monomers in one step employing a combination of lithium bromide and DBU. These chlorophosphoramidate monomers are finally assembled by solid-support synthesis to obtain the short PMO oligomers.