Theme 1
This theme has two main goals: to explore peptide-oligonucleotide conjugates, and to improve key aspects of the oligonucleotide synthesis workflow, focusing initially on the protecting groups and solvents employed.
Theme 1.1 A direct-to-biology screening platform to enable high-throughput synthesis and screening of oligonucleotide-cell penetrating peptide (ON-CPP) conjugates.
Current work is focusing on the purification and analysis of a model cell-penetrating peptide (CPP) and a negative control peptide for conjugation to an oligonucleotide (ON).
Optimisation is ongoing to establish suitable native chemical ligation (NCL) conditions to enable attachment of a series of azido PEG-linkers to the CPP. Disulfide formation has been a recurring challenge when handling the CPPs, and so current synthetic efforts have focused on the reduction of the disulfide bond prior to conjugation of the azido PEG-linker. Upon identification of suitable NCL conditions, miniaturisation of the reaction will be explored.
Following this, the CPP-linker conjugate will be attached to an ON of choice via an ynamine-mediated click reaction. Synthesis of the desired ynamine phosphoramidite has now been completed and it is ready to be attached to the ON via SPOS. Upon completion of the 5’-capped ynamine ON synthesis, optimisation of the CPP-ON conjugation can begin.
Theme 1.2 Towards greener solvents through mechanistic understanding
Investigation of phosphoramidite coupling kinetics are ongoing. Following on from work studying the measurement of the rate of reaction between A-, G-, C- and U-containing 2’-fluorinated phosphoramidites and activators DCI, ETT and BTT by 19F NMR spectroscopy, a method to monitor the reaction by in situ IR spectroscopy is being developed. Assay development is ongoing to identify reaction conditions which allow observation of the reaction progress at synthetically relevant concentrations with the addition of a desiccant to remove competing water from the reaction mixture. Suitable model nucleophiles are being explored as surrogates for solid-supported sugars. Initial experiments indicate that the reaction is fast at these higher concentrations, and the method is still under development.
Theme 1.3 Alternative 5′ protecting groups with enhanced atom economy
The methoxyisopropyl (MIP) group is being investigated as a lower molecular weight alternative to the ubiquitous 5′ DMT protecting group. Our work has shown the MIP group to be stable to tetrazole as an activator, but unstable to more commonly implemented contemporary activators (BTT, ETT, and DCI). The MIP group has previously been shown to be stable towards other steps in the oligonucleotide synthetic sequence. A 5′-MIP protected phosphoramidite has been synthesised and will be trialled on a SPOS system to investigate stability under solid-phase coupling conditions. Alongside this, kinetic studies will be carried out on the deprotection of this alternative protecting group.