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Dr. Chris Meier

Universität Hamburg

May 10th, 2019 – Spring Seminar

Time and Location: Noon in Meyerhoff Chemistry, Room 120

Host: Dr. Kathy Seley-Radtke


“How to get highly polar Nucleoside Analogue Triphosphates into cells – an Impossible Task? – No!”

Over the last decades a variety of nucleoside analogues were applied clinically in antiviral chemotherapy. However, quite often the antiviral potency of the nucleoside analogues is limited due to the lack of intracellular phosphorylation into the triphosphorylated forms by cellular kinases. This problem cannot be solved by using the phosphorylated nucleosides due to their high polarity which prevent an efficient cell membrane passage. An option to overcome this hurdle is the use of lipophilic precursors of nucleotides, which are able to pass the cell membrane and deliver the corresponding nucleotides intracellularly (pronucleotides). In the past we developed nucleoside mono- (cycloSal-system) and nucleoside diphosphate prodrug approaches (DiPPro-approach). Of course, the final aim should be to develop nucleoside triphosphate prodrugs because the delivered triphosphate is the direct acting inhibitor of the viral polymerases. In contrast, mono- or diphosphate delivery systems are still dependent on the forward phosphorylation into the triphosphate. So far, no example of such a prodrug system has been reported. In our work, d4TTP prodrugs with different aliphatic masking units have been synthesized via two different routes based on phosphoramidite or H-phosphonate chemistry. Our triphosphate delivery system is comprised of enzymatically cleavable system is comprised of enzymatically cleavable masking groups (acyloxybenzyl-moieties) which are covalently attached to the -phosphate group of the nucleoside triphosphate. The target prodrug compounds were obtained in yields up to 85%. Chemical hydrolysis studies, pig liver esterase studies, enzymatic cleavage in CEM/0 cell extract, primer extension assays, PCR assays, whole-cell incubations and antiviral HIV tests will be discussed and proved the successful delivery of nucleoside triphosphates. This new TriPPPro-concept will open up unknown possibilities not only in Medicinal Chemistry but also in Chemical Biology.(1-3) In a next step, a prodrug approach in which the -phosphate of NTPs is modified by two different groups was developed. One of these groups represents a cleavable masking group (acyloxybenzyl group) while the second group is a non-cleavable alkyl or ketobenzyl residue. The compounds showed a very high stability towards dephosphorylation as compared to d4TTP in cell extract hydrolysis studies. In antiviral assays, the compounds are potent inhibitors of HIV-1 and HIV-2 in cultures of infected thymidine kinase-deficient CD4+ T-cells (CEM/TK–). Primer extension assays using HIV’s reverse transcriptase and different cellular human DNA-polymerases showed that the new compounds act as a substrate for RT. In contrast, the γ-modified NTPs were found to be non-substrates for cellular DNA-polymerases β and γ. This opens a chance for improving the selectivity of a triphosphate derivative to act in infected but not in non-infected cells.