A P(V)-Platform for Oligonucleotide Synthesis

Abstract

<div><div><div><p>The early promise of gene-based therapies is currently being realized at an accelerated pace with over 155 active clinical trials for antisense compounds and multiple FDA-approved oligonucleotide therapeutics. Fundamental advances in this area are vital and present an unprecedented opportunity to both address disease states that have been resistant to other common modalities and improve the significant sustainability challenges associated with production of these complex molecules on a commercial scale. The advent of phosphoramidite coupling chemistry and solid-phase synthesis 40 years ago democratized oligonucleotide synthesis to the scientific community, paving the way for many of these stunning developments. The reliability and generality of this approach for the preparation of native phosphate-diesters is attributed to the high reactivity of phosphorus when in the P(III)-oxidation state versus the desired P(V), as it enables rapid P-heteroatom bond formation. However, the growing demand for more diverse phosphorus-based linkages has challenged the limits of this technology. For example, the phosphorothioate (PS) linkage, which stabilizes oligonucleotides towards nuclease cleavage, is universally employed in current oligonucleotide therapeutics but is generally incorporated in racemic form. Stereodefined PS oligonucleotides may have desirable biological and physical properties but are accessed with difficulty using phosphoramidite chemistry. Here we report a flexible and efficient [P(V)]-based platform that can install a wide variety of phosphate linkages at will into oligonucleotides. This approach uses readily accessible reagents and can efficiently install not only stereodefined or racemic thiophosphates, but can install any combination of (S, R or rac)-PS with native phosphodiester (PO2) and phosphorodithioate (PS2) linkages into DNA and other modified nucleotides. Importantly this platform easily accesses this diversity under a standardized coupling protocol with sustainably prepared, stable, P(V) reagents.</p></div></div></div>

Mild and Chemoselective Phosphorylation of Alcohols Using a Ψ-Reagent

“…The recently disclosed P(V)-based Ψ-platform for the construction of P-linkages has been applied to the simplified synthesis of an ever-growing, diverse range of compounds such as cyclic dinucleotides, 12,13 stereopure antisense oligonucleotides, 12 methylphosphonates, 14 chiral phosphines, 14 DNA, 15 and protein bioconjugates, 16 complex alkaloids, 17 and fully chemically modified oligonucleotides using a commercial automated synthesizer. 18 As part of the ongoing Ψ-platform development, Ψ O (1) was identified as a suitable reagent for forging phosphodiester bonds. 18 This Letter builds on those findings to highlight how the chemoselective nature of Ψreagents can be leveraged to access phosphates from alcohols in a mild, scalable, and operationally simple (one-pot) fashion across a wide range of alcohol substrates.…”

“…18 As part of the ongoing Ψ-platform development, Ψ O (1) was identified as a suitable reagent for forging phosphodiester bonds. 18 This Letter builds on those findings to highlight how the chemoselective nature of Ψreagents can be leveraged to access phosphates from alcohols in a mild, scalable, and operationally simple (one-pot) fashion across a wide range of alcohol substrates.…”