Thesis
Establishing synthetic methodology to prepare difluoromethylated oligonucleotides
- Creator
- Rights statement
- Awarding institution
- University of Strathclyde
- Date of award
- 2025
- Thesis identifier
- T17254
- Person Identifier (Local)
- 202069718
- Qualification Level
- Qualification Name
- Department, School or Faculty
- Abstract
- Oligonucleotide therapeutics (ONTs) are coming of age as a precision treatment for a wide variety of diseases. Through selective association with the Watson-Crick face of an RNA sequence, ONTs modulate gene expression by specifically inhibiting or promoting the expression of a protein of interest. ONTs act through a range of different mechanisms: steric block ONTs hinder the binding site of ribonucleoproteins, whereas siRNA and RNase H-activating ONTs recruit endogenous cellular machinery to cleave target mRNA sequences. However, oligonucleotides exhibit poor drug-like properties and often require extensive modification for therapeutic applications. Modification of the 2′-position of the ribose sugar is essential as a sterically demanding and/or electronegative substituent at this position favours a more RNA-like (2′-endo) conformation, which enhances binding to the target RNA strand. Additionally, these modifications confer stability against nuclease-mediated degradation. Difluoromethyl (CF2H) modifications are used extensively throughout medicinal chemistry due to their high lipophilicity and enzymatic stability, while also possessing the capacity to act as hydrogen bond donors. These factors render the difluoromethyl group a promising candidate as a bioisosteric replacement for multiple functional positions on the nucleoside scaffold. This thesis describes the synthesis of a range of novel difluoromethylated nucleosides and oligonucleotides. Chapter 1 introduces the chemical modification strategies employed in the design of current, clinically relevant ONTs and the potential of the difluoromethyl group to improve upon their current limitations. Previous strategies for nucleoside difluoromethylation will also be reviewed. Chapter 2 investigates the reactivity of pyrimidines with difluorocarbene, leading to Odifluoromethylation of the nucleobase. Further investigation and optimisation of this reaction yielded a suite of novel difluoromethylated nucleoside analogues, and their therapeutic potential as antimetabolites was explored. Chapter 3 documents a synthetic procedure to access 5′-O-difluoromethyl uridines via a copper-catalysed electrophilic difluoromethylation reaction. These conditions were also extended to therapeutically relevant 2′-modified pyrimidines and allowed for the synthesis of the corresponding phosphoramidite building blocks. Finally, these building blocks were incorporated into an oligonucleotide using solid-phase synthesis. Chapter 4 will explore methods for installing a difluoromethyl ether at the 2′-position of various nucleosides, highlighting the impracticalities of this approach for developing modified oligonucleotides. Optimisation of existing methodologies led to the direct difluoromethylation of the 2′-carbon in uridine, with a radical Negishi reaction yielding the best results. This nucleoside was then converted into the corresponding phosphoramidites, and its incorporation into oligonucleotides was demonstrated. Finally, Chapter 5 reflects on the work presented in this thesis and proposes experiments to investigate the biochemical and biophysical properties of these modified nucleosides and oligonucleotides.
- Advisor / supervisor
- Bush, Jacob T.
- Burley, Glenn A.
- Resource Type
- DOI
- Embargo Note
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File | 2025-05-29 | Embargo |