Polymerases have a structurally highly conserved negatively charged amino acid motif that is strictly required for Mg 2+ cation-dependent catalytic incorporation of (d)NTP nucleotides into nucleic acids. Based on these characteristics, a nucleoside monophosphonate scaffold, α-carboxy nucleoside phosphonate (α-CNP), was designed that is recognized by a variety of polymerases. Kinetic, biochemical, and crystallographic studies with HIV-1 reverse transcriptase revealed that α-CNPs mimic the dNTP binding through a carboxylate oxygen, two phosphonate oxygens, and base-pairing with the template. In particular, the carboxyl oxygen of the α-CNP acts as the potential equivalent of the α-phosphate oxygen of dNTPs and two oxygens of the phosphonate group of the α-CNP chelate Mg 2+ , mimicking the chelation by the β-and γ-phosphate oxygens of dNTPs. α-CNPs (i) do not require metabolic activation (phosphorylation), (ii) bind directly to the substrate-binding site, (iii) chelate one of the two active site Mg 2+ ions, and (iv) reversibly inhibit the polymerase catalytic activity without being incorporated into nucleic acids. In addition, α-CNPs were also found to selectively interact with regulatory (i.e., allosteric) Mg 2+ -dNTP-binding sites of nucleos(t)ide-metabolizing enzymes susceptible to metabolic regulation. α-CNPs represent an entirely novel and broad technological platform for the development of specific substrate active-or regulatory-site inhibitors with therapeutic potential. The polymerization of nucleotides by Escherichia coli DNA polymerase I represents a general model for catalytic action of nucleic acid polymerases (SI Appendix, Fig. S1) (1, 2). According to this model, there is a universal role for the Mg 2+ cation to interact with three phosphate oxygens of dNTP. The highly conserved consensus motifs in every polymerase active site consist of either aspartate or glutamate residues that chelate Mg 2+ through three additional coordination bonds during polymerization (2, 3). The crucial role of the metal cofactor and structurally conserved active site architecture in polymerases has also been demonstrated by validating Mg 2+ as a target for the design of antiviral drugs, not only against HIV RT but also, among others, against HIV integrase, HIV ribonuclease H (RNase H), and influenza-encoded endonuclease (4, 5). Hence, it should be feasible to design a universal but simplified (d)NTP mimic that binds efficiently to a wide variety of DNA/RNA polymerases.It was hypothesized that a universal nucleoside triphosphate mimic should contain three major indispensable entities: (i) a nucleobase part (i.e., to achieve optimal Watson-Crick basepairing with the template overhang), (ii) a replacement of the triphosphate moiety that should enable efficient Mg 2+ -directed coordination, and (iii) a variable linker between the nucleobase and the modified triphosphate to mimic the pentose entity present in natural (d)NTPs. For the triphosphate part, we chose an α-carboxy phosphonate entity that is chemically stable in physiolog...

The synthesis of the first series of a new class of nucleoside phosphonate analogues is described. Addition of a carboxyl group at the α position of carbocyclic nucleoside phosphonate analogues leads to a novel class of potent HIV reverse transcriptase (RT) inhibitors, α-carboxy nucleoside phosphonates (α-CNPs). Key steps in the synthesis of the compounds are Rh-catalyzed O-H insertion and Pd-catalyzed allylation reactions. In cell-free assays, the final products are markedly inhibitory against HIV RT and do not require phosphorylation to exhibit anti-RT activity, which indicates that the α-carboxyphosphonate function is efficiently recognized by HIV RT as a triphosphate entity, an unprecedented property of nucleoside monophosph(on)ates.

Relatively little is known about how gold nanoparticles (GNP) might interact in vivo with aquatic organisms. Gold nanoparticles (GNP) of defined average diameter may be synthesized and used to challenge test organisms held in aquaria. The blue mussel, Mytilus edulis, is a popular sentinel species in environmental toxicology. This chapter describes how mussels exposed to GNP (average diameter ~5 nm) may be dissected and extracts of digestive gland can be analyzed for oxidative stress. Protein thiols are labeled with 5'-iodoacetamide-fluorescein (IAF), and proteins are separated in one-dimensional electrophoresis. After scanning for IAF-associated fluorescence, gels are stained with colloidal coomassie. A ratio of fluorescence to protein stain is calculated revealing that thiol oxidation increases with GNP treatment.