YOGESH S. SANGHVI
Rasayan Inc., 2802 Crystal Ridge Road, Encinitas, CA 92024-6615, USA

Abstract

This review highlights the central role and importance of P(III) phosphoramidite chemistry in oligonucleotide synthesis. The choice of orthogonal protecting groups and synthesis of amidites from nucleoside building-blocks is described. Details of various analytical protocols used to access the quality and purity of the amidites is included. Appropriate instruction for storage and handling of these sensitive molecules is also summarized. A discussion on the hot-topic of cost-of-goods in relation to key suppliers is presented. The incredible versatility and scalability of phosphoramidite has fueled the extraordinary growth of synthetic oligonucleotides for both diagnostic and therapeutic applications.

INTRODUCTION

Today a researcher can order any number of short synthetic oligonucleotides from multiple suppliers at a reasonable cost and have it delivered the next day. This advancement is primarily due to the robustness, reproducibility and affordability of assembling oligonucleotides using the phosphoramidite approach. It was the pioneering work first reported in 1981 by Beaucage and Caruthers that became the cornerstone for the successful utility of P(III) phosphoramidite chemistry in automated synthesis of oligonucleotides (1). There are numerous applications of synthetic oligonucleotides in the diagnostic, therapeutic, nanomaterials, primers for PCR/sequencing and molecular/structural biology experiments. Given the broad scope of nucleic acids chemistry, this article provides a focused overview of various phosphoramidite building-blocks currently in use for the manufacturing of therapeutic oligonucleotides (2).
Phosphoramidites are key components for the synthesis of both unmodified and modified oligonucleotides. Figure 1 shows general scheme for oligonucleotide synthesis where the central role of 2’-deoxynucleoside phosphoramidite