Development and synthesis of oligonucleotide building blocks and conjugates for therapeutic applications

Sammanfattning: The research and development of oligonucleotide therapeutics has been a topic of great importance in life sciences. The potential to treat rare genetic diseases can be demonstrated simply by highlighting the increasing amount of approved oligonucleotide-based drugs. However, the delivery and stability issues of oligonucleotides complicate the transition to clinic and hampers the development in the field. Numerous oligonucleotide modifications have been developed to improve their properties and to overcome these limitations. This thesis focuses on the development and preparation of selected compounds, which are designed to improve the oligonucleotide properties related to stability and delivery. The first chapter (papers I and II) presents the development of a versatile synthetic platform for oligonucleotide-conjugate synthesis. The linkers, specifically developed for oligonucleotide conjugation, are compatible with automated oligonucleotide synthesis conditions and enable the incorporation of e.g., biologically active moieties at any position of the oligonucleotide sequence. Therefore, this approach enables the preparation of constructs with tailored properties. The development and synthesis of orthogonal linkers is described in this chapter together with the preparation of several different oligonucleotide conjugates, bearing different biologically active moieties/modalities. Furthermore, stability studies are performed on one of the linkers to evaluate its potential to be used during automated oligonucleotide synthesis in the future. The second chapter (paper III) describes the upscaling and optimization for the synthesis of methyl-uridine and methyl-cytidine nucleosides bearing 2′-O-(N-(aminoethyl)carbamoyl)-methyl modification. Since this modification showed promising results in nuclease stability and cellular uptake, larger amounts of modified nucleosides are necessary for future evaluations. Therefore, the syntheses of 2′-modified MeC and MeU building blocks are developed and demonstrated at larger (up to 100 g) scales. The final nucleosides are prepared as phosphoramidites to allow for the direct incorporation into an oligonucleotide sequence during the automated oligonucleotide synthesis. Suitable work-up and alternative purification strategies to reduce the number of chromatographic steps are also explored in this chapter.