Omission of column washing operations in the solid phase synthesis of oligonucleotides

Abstract

Oligonucleotides are generally manufactured via a solid-phase nucleotide elongation process utilizing a four-step or three-step reaction cycle consisting of detritylation, coupling, sulfurization/oxidation and optional capping. Following each step, the solid support is rinsed with solvent to remove residual reagent and to prepare the growing oligonucleotide chain for the next synthetic manipulation. Reported herein is an investigation of the impact of column-washing operations on the quality of oligonucleotide synthesis in the three-step cycle process. Also described is a more sustainable synthetic protocol without column-washing after the coupling and sulfurization steps that provides material of similar quality compared to material from standard washing protocols. This improvement allows for an approximate 70% reduction in wash solvent use for a typical 18 to 20-mer oligonucleotide. For every kilogram of an oligonucleotide synthesized, >300 L of organic solvents are saved and Process Mass Intensity (PMI) for synthesis steps is decreased from >890 to ∼530.