Synthetic studies of chemical probes for i) DNA, ii) RNA polymerases and iii) tropomyosin receptor kinase

DNA polymerase β (DNA pol β) is responsible for DNA repair mechanisms that are involved with gap-filling DNA synthesis in the single nucleotide base excision repair (BER) pathways. BER is very important in maintaining healthy cells because it removes the damaged base, avoiding further mutations. On another hand, RNA polymerases are involved in the transcription process, where a single-stranded mRNA is created from a dsDNA chain, in combination with a diverse number of general transcription factors. This process is crucial for cell growth and differentiation and therefore studying the mechanism of action of these enzymes is of extreme importance. The functionality and structure of both DNA pol β and RNA polymerase II (RNA pol II), as well as their fidelity mechanism and the use of these enzymes as potential cancer therapeutic targets, is explored in detail in the introductory chapter, Chapter 1. ❧ In a complementary manner, Chapter 2 is focused on the synthesis of β,γ-CHF dCTP probes for DNA pol β. A series of β,γ-CXY dNTP compounds have been extensively synthesized by the McKenna lab over the years in order to provide an accessible tool kit of dNTPs to study how DNA pol β cleaves the bisphosphonate moiety of the nucleotide depending on the different CXY derivatives they possess. The diastereomers of β,γ-CHF dCTP were synthesized, analyzed and both kinetically and structurally studied. When observing similar electronegative effects of CXY, it can be seen that the dihalo derivatives display a lower kₚₒₗ when compared with the monohalo and non-halo line (Figure 2.3). ❧ In addition, Chapter 2 further explores the use of small molecule inhibitors to target the lyase domain of DNA pol β. Previous studies have shown that pamoic acid has lyase inhibitory properties, therefore we aimed to synthesize modified pamoic acid derivatives, which possess a phosphorus moiety, and test their inhibitory effect. Compound 8 from Generation 2 (Scheme 2.4) was successfully synthesized and characterized and shown to inhibit the lyase domain of DNA pol β at a concentration of 500 μM (Figure 2.12). ❧ Moreover, the synthesis of β,γ-CXY UTP probes to study the mechanism and fidelity of RNA pol II is analyzed in Chapter 3. This unique enzyme possesses a well-organized network, using a crucially conserved motif called the trigger loop. β,γ-CCI₂ UTP, β,γ-CCI₂ ATP, β,γ-CHCl UTP and β,γ-CHCl ATP were synthesized and the individual diastereomers β,γ-CHCl-1 and β,γ-CHCl-2 UTP were successfully isolated (Scheme 3.2). According to in vitro transcription assays, β,γ-CCI₂ UTP showed a lower incorporation rate when compared with the fluorine derivative, but higher than he methylene version (Figure 3.6). The optimal coupling step conditions were also examined. ❧ Lastly, Chapter 4 is focused on the solid-phase synthesis of the bisphosphonate conjugate 1Aa to target Tropomyosin receptor kinase B and C (TrkB and TrkC) in the inner ear. Recently, our group has published preliminary data regarding the use of a bisphosphonate-linked TrkB agonist as a delivery method to reach the cochlea. 1Aa was synthesized over 12 steps, 7 of them being in solid-phase support with an overall yield of 16% (Scheme 4.1). 1Aa promotes spiral ganglion neurite outgrowth in vitro and also bolsters the regeneration of cochlear ribbon synapses in vitro (Figures 4.4 and 4.5). Further studies involve the synthesis of the bisphosphonate counterpart, RIS-1Aa, and its effect on the spiral ganglion neurite outgrowth and the regeneration of cochlear ribbon synapses.