# Stereocontrolled Synthesis of Dimethylamino Phosphorochloridate Monomers toward Stereopure Phosphorodiamidate Morpholino Oligonucleotides

## TL;DR

This paper presents a new method to synthesize PMOs with controlled stereochemistry, improving their physicochemical and biological properties.

## Contribution

A stereocontrolled synthesis method for PMO monomers using oxazaphospholidine derivatives is developed.

## Key findings

- A highly stereoselective synthesis of phosphorochloridate monomers was achieved (dr = 93:7–97:3).
- The stereochemistry of PMO dimers was confirmed using nuclear magnetic resonance analysis.
- The method enables practical synthesis of stereopure PMOs for further property studies.

## Abstract

As antisense oligonucleotides, phosphorodiamidate morpholino
oligonucleotides
(PMOs) exhibit excellent properties. However, they possess two stereoisomers
for each phosphorus atom, and these stereoisomers exhibit different
physicochemical and biological properties. In this study, we developed
a stereocontrolled synthesis method of dimethylamino phosphorochloridate
monomers, which was used for a practical synthesis method of PMOs,
from oxazaphospholidine derivatives. However, the condensation of
a 5′-oxazaphospholidine derivative with an amine under acidic
conditions is challenging because the resulting phosphoramidite intermediate
can be activated under such conditions. To address this challenge,
in the proposed synthesis method, a morpholino nucleoside 5′-oxazaphospholidine
derivative was condensed with a phenol derivative with a low pK

a
value under acidic conditions.
Subsequently, the resulting aryl phosphite was reacted with dimethylamine
to yield a phosphoramidite, thereby liberating the phenol derivative
as a leaving group. Chlorination of the phosphoramidite yielded a
phosphorochloridate monomer in a highly stereoselective manner (dr
= 93:7–97:3). Subsequently, the resulting chloridate monomer
was stereospecifically condensed with the amino group of the morpholino
nucleoside. The stereochemistry of the phosphorodiamidate morpholino
dimers was unambiguously determined by nuclear magnetic resonance
analysis. The results of this study facilitate the synthesis of stereocontrolled
PMOs and the elucidation of their properties.