Fully Liquid Phase Oligonucleotide Synthesis

Representative oligonucleotides (oligos), a 21-mer siRNA strand and a 16-mer ASO-like gapmer, were synthesized by Nanostar Sieving, a membrane-assisted liquid phase oligo synthesis (LPOS) method, at a 10 mmol scale and 20 mM concentration in a single solvent. This process is the first total LPOS beyond 10-mer, i.e., the reagents and intermediates remain in the liquid phase from the very first reaction to isolation. At all times during the synthesis of the full-length product, the concentration of oligos within the Nanostar-10 reactor remained roughly constant. Both sequences were synthesized four times, twice using membrane sheets clamped flat in circular cells and twice using scalable spiral wound modules. Cleavage and deprotection continued to maintain the oligo in the liquid phase until the final lyophilization, when yields were calculated from the optical density, adjusting for chromatographic and mass spectral purity. Synthetic process mass intensities approaching those of solid phase oligo synthesis (SPOS) were achieved.