ABSTRACT
Several types of isopolar modified oligothymidylates and oligoadenylates (15 mers) with the phosphonate -O-P-CH2-O- internucleotide linkage were prepared. The modified oligonucleotides were subjected to the study of their hybridization properties, resistance against nucleases, and the ability to elicit RNase H activity.
Subject(s)
Oligonucleotides, Antisense/chemical synthesis , Organophosphonates/chemistry , Adenosine Monophosphate/chemistry , Cross-Linking Reagents/chemistry , Nucleic Acid Hybridization , Oligonucleotides, Antisense/chemistry , Oligonucleotides, Antisense/metabolism , Ribonuclease H/metabolism , Thymidine Monophosphate/chemistryABSTRACT
In order to identify inhibitors of various drug-resistant forms of the human immunodeficiency virus protease (HIV PR), we have designed and synthesized pseudopeptide libraries with a general structure Z-mimetic-Aa1-Aa2-NH2. Five different chemistries for peptide bond replacement have been employed and the resulting five individual sublibraries tested with the HIV PR and its drug-resistant mutants. Each mutant contains amino acid substitutions that have previously been shown to be associated with resistance to protease inhibitors, including Ritonavir, Indinavir, and Saquinavir. We have mapped the subsite preferences of resistant HIV PR species with the aim of selecting a pluripotent pharmaceutical lead. All of the enzyme species in this study manifest clear preference for an L-Glu residue in the P2' position. Slight, but significant, differences in P3' subsite specificity among individual resistant PR species have been documented. We have identified three compounds, combining the most favorable features of the inhibitor array, that exhibit low-nanomolar or picomolar Ki values for all three mutant PR species tested.
Subject(s)
Drug Resistance , HIV Protease/chemistry , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacokinetics , Combinatorial Chemistry Techniques , Drug Design , Kinetics , Models, Molecular , Mutation , Peptide Library , Substrate SpecificityABSTRACT
The mechanisms of reactions causing irreversible inhibition of the activity of enzymes when irradiated in the presence of the recently developed alpha-methyl-6-nitroveratryl-based photolinker [Holmes CP. J. Org. Chem. 1997; 62: 2370-2380] have been investigated. Several experiments based on the interaction of the photolinker with model peptides or n-butylamine have been accomplished. A complexity of products, resulting from the side reactions competing with the 'normal' photocleavage of the linker, have been found. The amino and thiol groups of the molecules present in the solvents upon irradiation were recognized as having a major influence on the course of photolysis. Some of these side products resulting from the interaction with amines were identified and the mechanisms by which they can be generated are discussed. The mechanism of the interaction of the thiol groups present in peptides or proteins with the photolinker is unclear and it remains to be further elucidated. It was found that the undesirable effects are favored by a basic pH and are largely reduced by a slightly acidic pH, together with the presence of dithiothreitol. Significant positive effects of dithiothreitol have been observed on the rate as well as the yield of the photocleavage. These results demonstrate that the use of photolabile linkers in biological media can be accompanied by undesired effects, which can be largely reduced by choosing appropriate conditions and additives.
Subject(s)
Enzyme Inhibitors/chemistry , Enzymes/chemistry , Photosensitizing Agents/chemistry , Resins, Plant/chemistry , Animals , Male , Peptides/chemistry , Photolysis , RatsABSTRACT
Parallel simultaneous synthesis of fifty linear peptides has been carried out in order to compare in detail two promising methodologies of simultaneous multiple peptide synthesis (SMPS): the "T bag" method, utilizing 4-methyl-benzhydrylamine resin (MeBHA), and synthesis on derivatized Fmoc-Gly-O-cotton fabric strips. The basic set of experiments, which utilizes identical Fmoc/Bu(t) strategy for both approaches, shows that the peptides synthesized on cotton are superior in purity to those synthesized using T bags. In experiments utilizing Boc/Bzl strategy in T bags, the purities of peptides were higher than in the case of peptides synthesized in T bags by Fmoc/Bu(t) strategy, and comparable with the purities achieved in synthesis performed on cotton. The lower yields on cotton are caused by mechanical losses in the given experimental arrangement.
