Development of method for large scale manufacturing of ON constructs

Abstract

Oligonucleotide therapeutics rapidly advanced in recent years, with several studies being conducted in the biological and medical fields. Therefore, it has become imperative to develop methodologies for the large-scale synthesis of oligonucleotides, with chemical modification to match the barriers that need to be overcome, namely instability, tissue delivery and affinity, immunogenicity and off-target effects. The primary objective of this research project is to synthesise linkers that can be used in oligonucleotide solid-phase synthesis, enabling conjugation between these oligonucleotides and biological macromolecules via click chemistry. The initial phase of the work involved the chemical synthesis of linkers, continuing ongoing studies in the host laboratory on amino and alkyne linkers. While the alkyne linkers enable conjugation with macromolecules through copper-catalysed azide-alkyne cycloaddition, the amino linker provides orthogonality through amide bond formation with biological entities, such as peptides. In addition, linkers based on dibenzoazacyclooctyne (DBCO) were investigated as DBCO can react with azide-handle biomolecules via copper-free click chemistry in biological conditions. The DBCO-based linker was designed using 5,6-dihydrodibenzo[b,f]azocine as key intermediate. This core structure can be acylated to yield the desired DBCO, followed by the reaction with amino acids or analogues. Given the requirement that the linkers undergo oligonucleotide solid-phase synthesis, a phosphoramidite moiety was incorporated into the linkers. This was achieved by introducing an aminodiol building block. This common scaffold contains an amine group for bonding with the click chemistry moiety via amide bond formation and two hydroxyl groups, one for phosphoramidite group introduction; other, DMTr-protected ensures compatibility with solid-phase oligonucleotide synthesis, enabling elongation of the oligonucleotide chain, as well as the incorporation of linkers at any position. The aminodiol building blocks studies in this project were a linear aminodiol, a cyclic aminodiol and the serinol. The second phase of the research project involved the comparison of the efficiency of the synthesized linkers in the oligonucleotide solid-phase conditions, as well as evaluating their behaviour during the handling processes after the synthesis, including cleavage, deprotection from the solid support, and purification. In addition, the conjugation of DBCO-containing oligonucleotides with biological molecules, such as peptides and monoclonal antibodies, was also investigated in this project