Boranophosphate-Modified Nucleic Acids as Biomolecular Probes: Synthesis, Substrate, and Antiviral Properties

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Cheek, Marcus Adrian

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In boranophosphate (BP) nucleotides, a borane (BH3) group is substituted for a non-bridging phosphoryl oxygen of a normal phosphate group, resulting in a class of modified isoelectronic DNA and RNA mimics that can modulate the reading and writing of genetic information. 5'-(α-P-borano)nucleoside triphosphates (NTPαBs) are good substrates or inhibitors for many viral RNA polymerases and reverse transcriptases (RT) when compared with natural nucleoside 5'-triphosphates (NTPs). A practical aspect of this coding phenomenon employs T7 bacteriophage DNA-dependent RNA polymerase (DdRP) to synthesize BP-modified RNA utilizing NTPαBs as monomeric substrates. Additionally, the α-P-borano modification can be used to probe the catalytic phosphoryl transfer mechanism used by viral polymerases, and possibly enhance existing anti-viral chain terminating nucleotides.

The primary goal of this dissertation is to better understand the effects of NTPαBs on the activity of viral polymerases. In the last decade several NTPαBs have been shown to be efficient and selective substrates for wild-type (wt) and, to a greater extent, HIV and MMLV drug-resistant viral reverse transcriptases. More recently NS5B, the Hepatitis C viral RNA-dependent RNA polymerase (HCV RdRP), is a viable target for nucleotide-based inhibition studies. Due to the similarities between the active sites of HIV-RT and HCV NS5B, it is therefore relevant to investigate the substrate properties of this unique modification. We investigated, for the first time, the inhibition kinetics of HCV NS5BΔ55 RdRP by two newly synthesized NTPαB analogs: 2'-O-methyladenosine 5'-(α-P-borano) triphosphate (2'-OMe ATPαB, 9a) and 3'-deoxyadenosine 5'-(α-P-borano) triphosphate (3'-dATPαB, 9b) and the steady state incorporation kinetics of ATPαB (51a). Our results showed that:

(1) Rp-2'-OMe ATPαB (9a) and Rp-3'-dATPαB (9b) exhibited a 3.5- and 16-fold lower IC50 respectively when compared with natural phosphate controls, suggesting greater inhibitory potency.

(2) Additionally 9a and 9b demonstrated a 5- and 21-fold lower inhibition constant (Ki) respectively when compared with the natural phosphate. Both compounds retained the competitive inhibition behavior of their parent nucleotides.

(3) HCV NS5BΔ55 preferred the Rp isomer of ATPαB (Vmax/Km = 0.095) over the natural ATP substrate (Vmax/Km = 0.057). None of the Sp isomers were substrates for HCV NS5BΔ55. We further concluded that wild-type (wt) HCV NS5B seems to discriminate against 3'-deoxy NTPs via lost interactions between the 3'-OH on the ribose and the active site residues, or lost intramolecular hydrogen bonding interactions between the 3'-OH and the pyrophosphate leaving group during phosphoryl transfer. The overall implications of this proof of concept study are that existing viral RdRP inhibitors could be retro-fitted with the boranophosphate modification to possibly increase potency.

This dissertation also explored the synthesis of anti-HIV-RT boranophosphate nucleotides which act through a chain terminating or mutagenic mechanism. 2'-3'-didehydro-2'-3'-dideoxythymidine 5'-(α-P-borano)-diphosphate (D4TDPαB, 30) was synthesized and later stereoselectively phosphorylated to yield the Rp-form of D4TTPαB (31). This was tested as a substrate in two multi-drug resistant forms of HIV-RT. Additionally, the NTPαB analogue of the mutagenic 5-aza-5,6-dihydro-2'-deoxycytidine (KP-1212-TPαB, 16) was synthesized with the eventual goal of inducing error catastrophe during viral genomic replication.

Lastly we detail the extraction and purification of gemcitabine (dFdC) from Gemzar® drug mixture using a derivatization method that produced a protected form of gemcitabine nucleoside. This protected gemcitabine was then used to synthesize gemcitabine 5'-triphosphate (dFdCTP, 42).






Cheek, Marcus Adrian (2011). Boranophosphate-Modified Nucleic Acids as Biomolecular Probes: Synthesis, Substrate, and Antiviral Properties. Dissertation, Duke University. Retrieved from


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