An Improved PEG‐Linked Solid Support for Minimizing Process‐Related Impurities During Solid‐Phase Synthesis of DNA and RNA Sequences

Abstract

The preparation of controlled pore glass (CPG) supports, functionalized with several hexaethylene glycol spacers, to alleviate the problems associated with the porosity of commercial CPG supports is described in this article. The pore size of CPG restricts the diffusion of reagents to the leader nucleoside embedded in porous supports; this inhibits efficient solid-phase syntheses of DNA and RNA sequences and, by default, the purity of those sequences through formation of a shorter than full-length oligonucleotide. Functionalization of a CPG support with five hexaethylene glycol spacers led to a 42% reduction in processrelated impurities contaminating oligonucleotide sequences, compared to that obtained using the commercial long-chain alkylamine (LCAA) CPG support.

An Alternate Process for the Solid‐Phase Synthesis and Solid‐Phase Purification of Synthetic Nucleic Acid Sequences

“…On the basis of these observations, the CPG support was modified through the addition of multiple hexaethylene glycol spacers; this led to a significant reduction (by up to 40%) of the level of shorter-than-full-length polynucleotide sequences (Grajkowski et al, 2020(Grajkowski et al, , 2021 when compared to that measured using unmodified commercial CPG supports. Additional manufacturing process-related impurities contaminating synthetic DNA and RNA sequences are created while processing the nucleic acid sequences.…”

“…Intriguingly, about 60% of all shorter‐than‐full‐length polynucleotide sequences are produced during the first five solid‐phase synthesis cycles, possibly caused by crowding of the nascent nucleic acid biopolymer; this can interfere with the free diffusion of reagents within the pores of the commercial controlled‐pore glass (CPG) solid support during early synthesis cycles, given that little truncation of the biopolymer occurred during the final cycles of synthesis. On the basis of these observations, the CPG support was modified through the addition of multiple hexaethylene glycol spacers; this led to a significant reduction (by up to 40%) of the level of shorter‐than‐full‐length polynucleotide sequences (Grajkowski et al., 2020, 2021) when compared to that measured using unmodified commercial CPG supports. Additional manufacturing process–related impurities contaminating synthetic DNA and RNA sequences are created while processing the nucleic acid sequences.…”

Use of Arabinonucleosides for Development and Implementation of a Novel 2′‐O‐Protecting Group for Efficient Solid‐Phase Synthesis and 2′‐O‐Deprotection of RNA Sequences

“…Other factors affecting the purity, safety, and efficacy of DNA or RNA sequences have to do with incomplete removal of amine‐protecting groups from the nucleobases and/or alkylation of the imide function of thymine or uracil, presumably occurring during the removal of the 2‐cyanoethyl phosphate‐protecting groups under basic conditions. Although these inadequacies have been recently mitigated through the implementation of an improved PEG‐linked solid support (Grajkowski et al., 2020; Grajkowski, Takahashi, Cawrse, & Beaucage, 2021), the synthesis and deprotection of RNA sequences is more complex because the 2ʹ‐hydroxyl of ribonucleosides requires protection during synthesis and orthogonal deprotection conditions after synthesis. Indeed, the 2ʹ‐hydroxyl‐protecting group must be fully stable under the basic conditions employed for nucleobase and phosphate deprotection and be cleaved under conditions that will not induce RNA chain cleavage while maintaining complete integrity of the RNA sequence.…”