The conjugation of biomolecules by chemoselective oxime ligation is of great interest for the site-specific modification of proteins, peptides, nucleic acids, and carbohydrates. These conjugations proceed optimally at a reaction pH of 4-5, but some biomolecules are not soluble or stable under these conditions. Aniline can be used as a nucleophilic catalyst to enhance the rate of oxime formation, but even in its presence, the reaction rate at neutral pH can be slower than desired, particularly at low reagent concentrations and/or temperature. Recently, alternative catalysts with improved properties were reported, including anthranilic acid derivatives for small molecule ligations, as well as m-phenylenediamine at high concentrations for protein conjugations. Here, we report that p-substituted anilines containing an electron-donating ring substituent are superior catalysts of oxime-based conjugations at pH 7. One such catalyst, p-phenylenediamine, was studied in greater detail. This catalyst was highly effective at neutral pH, even at the low concentration of 2 mM. In a model oxime ligation using aminooxy-functionalized PEG, catalysis at pH 7 resulted in a 120-fold faster rate of protein PEGylation as compared to an uncatalyzed reaction, and 19-fold faster than the equivalent aniline-catalyzed reaction. p-Phenylenediamine (10 mM) was also an effective catalyst under acidic conditions and was more efficient than aniline throughout the pH range 4-7. This catalyst allows efficient oxime bioconjugations to proceed under mild conditions and low micromolar concentrations, as demonstrated by the PEGylation of a small protein.

High-throughput screening of National Cancer Institute libraries of synthetic and natural compounds identified the vinylogous ureas 2-amino-5, 6,7,8-tetrahydro-4H-cyclohepta[b] thiophene-3-carboxamide (NSC727447) and N-[3-(aminocarbonyl)-4,5-dimethyl-2-thienyl]-2-furancarboxamide (NSC727448) as inhibitors of the ribonuclease H (RNase H) activity of HIV-1 and HIV-2 reverse transcriptase (RT).A Yonetani-Theorell analysis demonstrated that NSC727447, and the active-site hydroxytropolone RNase H inhibitor β-thujaplicinol were mutually exclusive in their interaction with the RNase H domain. Mass spectrometric protein footprinting of the NSC727447 binding site indicated that residues Cys280 and Lys281 in helix I of the thumb subdomain of p51 were affected by ligand binding. Although DNA polymerase and pyrophosphorolysis activities of HIV-1 RT were less sensitive to inhibition by NSC727447, protein footprinting indicated that NSC727447 occupied the equivalent region of the p66 thumb. Sitedirected mutagenesis using reconstituted p66/p51 heterodimers substituted with natural or nonnatural amino acids indicates that altering the p66 RNase H primer grip significantly affects inhibitor sensitivity. NSC727447 thus represents a novel class of RNase H antagonists with a mechanism of action differing from active site, diva-lent metal-chelating inhibitors that have been reported.Although an absolute requirement for reverse transcriptase (RT)-associated ribonuclease H (RNase H) activity for human immunodeficiency virus (HIV) replication was documented almost two decades ago (1,2), development of potent and selective RNase H inhibitors has been surprisingly slow compared with the nucleoside and non-nucleoside DNA polymerase inhibitors currently in clinical use. Recently, however,4), diketo acids (5,6), and dihydroxytropolones (7) have shown promise by specifically inhibiting RNase H activity of HIV-1 and HIV-2 RT, and in some instances acting synergistically with clinically approved RT inhibitors. The preliminary crystal structure of an N-hydroxyimide bound to the RNase H domain of HIV-1 RT (4) suggests that it sequesters the divalent metal cofactor, laying the foundation for rational design of improved inhibitors. Increasing the diversity of RNase H © 2008 American Chemical Society * Corresponding author, slegrice@ncifcrf.gov.. Supporting Information Available:This material is free of charge via the Internet. In order to examine the NSC727447 binding site, we performed mass spectrometric protein footprinting based on biotin modification of exposed lysine residues in the free protein and the protein-inhibitor complex (8-10). Cys280 and Lys281, located in helix I of the thumb subdomain, were protected from modification by inhibitor binding. Proximity between the p51 thumb subdomain and the p66 RNase H domain implies that inhibitor binding adjacent to the catalytic center affects either divalent metal coordination or positioning of the nucleic acid substrate in the active site. Although DNA polymerase activity was less sensi...

Reverse transcriptase (RT) of the human immunodeficiency virus (HIV) possesses DNA polymerase and ribonuclease (RNase) H activities. Although the nucleic acid binding cleft separating these domains can accommodate structurally-diverse duplexes, it is currently unknown whether regular DNA/RNA hybrids can simultaneously contact both active sites. In this study we demonstrate that ligands capable of trapping the 3’-end of the primer at the polymerase active site affect specificity of RNase H cleavage without altering the efficiency of the reaction. Experiments under single turnover conditions reveal that complexes with a bound nucleotide substrate show specific RNase H cleavage at template position -18, while complexes with the pyrophosphate analogue foscarnet show a specific cut at position -19. This pattern is indicative for post- and pre-translocated conformations. The data are inconsistent with models postulating that the substrate toggles between both active sites, such that the primer 3’-terminus is disengaged from the polymerase active site when the template is in contact with the RNase H active site. In contrast, our findings provide strong evidence to suggest that the nucleic acid substrate can engage both active sites at the same time. As a consequence, the bound and intact DNA/RNA hybrid can restrict access of RNase H active site inhibitors. We have mapped the binding site of the recently discovered inhibitor β-thujaplicinol between the RNase H active site and Y501 of the RNase H primer grip and show that the inhibitor is unable to bind to a pre-formed RT-DNA/RNA complex. In conclusion, the bound nucleic acid substrate, and in turn, active DNA synthesis can represent an obstacle to RNase H inhibition with compounds that bind to the RNase H active site.

Vinylogous ureas 2-amino-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxamide and N-[3-(aminocarbonyl)-4,5-dimethyl-2-thienyl]-2-furancarboxamide (compounds 1 and 2, respectively) were recently identified to be modestly potent inhibitors of the RNase H activity of HIV-1 and HIV-2 reverse transcriptase (RT). Both compounds shared a 3-CONH 2 -substituted thiophene ring but were otherwise structurally unrelated, which prevented a precise definition of the pharmacophore. We have therefore examined a larger series of vinylogous ureas carrying amide, amine, and cycloalkane modifications of the thiophene ring of compound 1. While cycloheptane-and cyclohexane-substituted derivatives retained potency, cyclopentane and cyclooctane substitutions eliminated activity. In the presence of a cycloheptane ring, modifying the 2-NH 2 or 3-CONH 2 functions decreased the potency. With respect to compound 2, vinylogous ureas whose dimethylthiophene ring contained modifications of the 2-NH 2 and 3-CONH 2 functions were investigated. 2-NH 2 -modified analogs displayed potency equivalent to or enhanced over that of compound 2, the most active of which, compound 16, reflected intramolecular cyclization of the 2-NH 2 and 3-CONH 2 groups. Molecular modeling was used to define an inhibitor binding site in the p51 thumb subdomain, suggesting that an interaction with the catalytically conserved His539 of the p66 RNase H domain could underlie inhibition of RNase H activity. Collectively, our data indicate that multiple functional groups of vinylogous ureas contribute to their potencies as RNase H inhibitors. Finally, single-molecule spectroscopy indicates that vinylogous ureas have the property of altering the reverse transcriptase orientation on a model RNA-DNA hybrid mimicking initiation plus-strand DNA synthesis.Current success in treating HIV infection and AIDS can be attributed to effective combination antiretroviral therapy involving a cocktail of inhibitors directed primarily against the retroviral protease and reverse transcriptase (RT) (22). Inhibition of RT function is achieved either directly, by incorporating chain-terminating nucleoside derivatives (nucleoside RT inhibitors [NRTIs]), or indirectly, by nonnucleoside RT inhibitors (NNRTIs) that occupy a hydrophobic pocket at the base of the p66 thumb to interrupt the chemical step of DNA synthesis (25)