Synthesis and polymerase incorporation of 5'-amino-2',5'-dideoxy-5'-N-triphosphate nucleotides.
ABSTRACT: Owing to the markedly increased reactivity of amino functional groups versus hydroxyls, the 5'-amino-5'-deoxy nucleoside and nucleotide analogs have proven widely useful in biological, pharmaceutical and genomic applications. However, synthetic procedures leading to these analogs have not been fully explored, which may possibly have limited the scope of their utility. Here we describe the synthesis of the 5'-amino-2',5'-dideoxy analogs of adenosine, cytidine, guanosine, inosine and uridine from their respective naturally occurring nucleosides via the reduction of 5'-azido-2',5'-dideoxy intermediates using the Staudinger reaction, and the high yield conversion of these modified nucleosides and 5'-amino-5'-deoxythymidine to the corresponding 5'-N-triphosphates through reaction with trisodium trimetaphosphate in the presence of tris(hydroxymethyl)aminomethane (Tris). We also show that each of these nucleotide analogs can be efficiently incorporated into DNA by the Klenow fragment of Escherichia coli DNA polymerase I when individually substituted for its naturally occurring counterpart. Mild acid treatment of the resulting DNA generates polynucleotide fragments that arise from specific cleavage at each modified nucleotide, providing a sequence ladder for each base. Because the ladders are generated after the extension, the corresponding products may be manipulated by enzymatic and/or purification processes. The potential utility of this extension-cleavage procedure in genomic sequence analysis is discussed.
Project description:Derivatives of 3-amino-3,6-dideoxyhexoses are widespread in Nature. They are part of the repeating units of lipopolysaccharide O-antigens, of the glycan moiety of S-layer (bacterial cell surface layer) glycoproteins and also of many antibiotics. In the present study, we focused on the elucidation of the biosynthesis pathway of dTDP-alpha-D-Quip3NAc (dTDP-3-acetamido-3,6-dideoxy-alpha-D-glucose) from the Gram-positive, anaerobic, thermophilic organism Thermoanaerobacterium thermosaccharolyticum E207-71, which carries Quip3NAc in its S-layer glycan. The biosynthesis of dTDP-alpha-D-Quip3NAc involves five enzymes, namely a transferase, a dehydratase, an isomerase, a transaminase and a transacetylase, and follows a pathway similar to that of dTDP-alpha-D-Fucp3NAc (dTDP-3-acetamido-3,6-dideoxy-alpha-D-galactose) biosynthesis in Aneurinibacillus thermoaerophilus L420-91(T). The ORFs (open reading frames) of interest were cloned, overexpressed in Escherichia coli and purified. To elucidate the enzymatic cascade, the different products were purified by HPLC and characterized by NMR spectroscopy. The initiating reactions catalysed by the glucose-1-phosphate thymidylyltransferase RmlA and the dTDP-D-glucose-4,6-dehydratase RmlB are well established. The subsequent isomerase was shown to be capable of forming a dTDP-3-oxo-6-deoxy-D-glucose intermediate from the RmlB product dTDP-4-oxo-6-deoxy-D-glucose, whereas the isomerase involved in the dTDP-alpha-D-Fucp3NAc pathway synthesizes dTDP-3-oxo-6-deoxy-D-galactose. The subsequent reaction steps of either pathway involve a transaminase and a transacetylase, leading to the specific production of nucleotide-activated 3-acetamido-3,6-dideoxy-alpha-D-glucose and 3-acetamido-3,6-dideoxy-alpha-D-galactose respectively. Sequence comparison of the ORFs responsible for the biosynthesis of dTDP-alpha-D-Quip3NAc revealed homologues in Gram-negative as well as in antibiotic-producing Gram-positive bacteria. There is strong evidence that the elucidated biosynthesis pathway may also be valid for LPS (lipopolysaccharide) O-antigen structures and antibiotic precursors.
Project description:An unknown amino sugar, U-7, which had been detected in the hydrolysate of the polysaccharide fraction (F-A) of Pseudomonas aeruginosa P14 lipopolysaccharide, was isolated from the hydrolysate of whole cells of this micro-organism and converted into the N-acetyl derivative (U-7NAc). On the basis of i.r.-absorption spectrometry, 13C-n.m.r. and 1H-n.m.r. spectroscopy and mass spectrometry, the structure of compound U-7NAc was identified as 2-acetamido-3-amino-2,3-dideoxyhexofuranurono-6,3-lactam. The configuration of compound U-7NAc was then unequivocally identified as 2-acetamido-3-amino-2,3-dideoxy-D-glucofuranurono-6,3-lactam by comparing the synthetic and natural compounds. Compound U-7 and synthetic 2,3-diamino-2,3-dideoxy-D-glucofuranurono-6,3-lactam showed the same behaviour on chromatography. G.l.c.--mass-spectral analyses of fraction F-A and synthetic 2,3-diacetamido-2,3-dideoxy-D-glucuronic acid after methanolyses and trimethylsilylations showed the presence of the same derivative. It was concluded that the amino sugar U-7 was produced from the 2,3-diacetamido-2,3-dideoxy-D-glucuronic acid residue present in fraction F-A.
Project description:L-nucleoside analogs represent an important class of small molecules for treating both viral infections and cancers. These pro-drugs achieve pharmacological activity only after enzyme-catalyzed conversion to their tri-phosphorylated forms. Herein, we report the crystal structures of human deoxycytidine kinase (dCK) in complex with the L-nucleosides (-)-beta-2',3'-dideoxy-3'-thiacytidine (3TC)--an approved anti-human immunodeficiency virus (HIV) agent--and troxacitabine (TRO)--an experimental anti-neoplastic agent. The first step in activating these agents is catalyzed by dCK. Our studies reveal how dCK, which normally catalyzes phosphorylation of the natural D-nucleosides, can efficiently phosphorylate substrates with non-physiologic chirality. The capability of dCK to phosphorylate both D- and L-nucleosides and nucleoside analogs derives from structural properties of both the enzyme and the substrates themselves. First, the nucleoside-binding site tolerates substrates with different chiral configurations by maintaining virtually all of the protein-ligand interactions responsible for productive substrate positioning. Second, the pseudo-symmetry of nucleosides and nucleoside analogs in combination with their conformational flexibility allows the L- and D-enantiomeric forms to adopt similar shapes when bound to the enzyme. This is the first analysis of the structural basis for activation of L-nucleoside analogs, providing further impetus for discovery and clinical development of new agents in this molecular class.
Project description:<h4>Background</h4>Antiretroviral drug discovery and formulation design will facilitate viral clearance in infectious reservoirs. Although progress has been realized for selected hydrophobic integrase and nonnucleoside reverse transcriptase inhibitors, limited success has been seen to date with hydrophilic nucleosides. To overcome these limitations, hydrophobic long-acting drug nanoparticles were created for the commonly used nucleoside reverse transcriptase inhibitor, lamivudine (2',3'-dideoxy-3'-thiacytidine, 3TC).<h4>Methods</h4>A 2-step synthesis created a slow-release long-acting hydrophobic 3TC. Conjugation of 3TC to a fatty acid created a myristoylated prodrug which was encased into a folate-decorated poloxamer 407. Both in vitro antiretroviral efficacy in human monocyte-derived macrophages and pharmacokinetic profiles in mice were evaluated for the decorated nanoformulated drug.<h4>Results</h4>A stable drug formulation was produced by poloxamer encasement that improved monocyte-macrophage uptake, antiretroviral activities, and drug pharmacokinetic profiles over native drug formulations.<h4>Conclusions</h4>Sustained release of long-acting antiretroviral therapy is a new therapeutic frontier for HIV/AIDS. 3TC depot formation in monocyte-derived macrophages can be facilitated through stable subcellular internalization and slow drug release.
Project description:Porcine pancreatic elastase (PPE) was crystallized under new sulfate-free conditions containing 0.3 M NaCl and 50 mM tris(hydroxymethyl)aminomethane-HCl at pH 7.0. The crystal structure determined at 1.5 angstroms resolution had a unique conformation in four regions which contained loop portions. A chloride ion was bound near the catalytic triad instead of the sulfate ion in PDB entry 1qnj, a typical PPE crystal structure. However, the chloride ion did not affect the configuration of the catalytic triad. A tris(hydroxymethyl)aminomethane (Tris) molecule was bound to the S4 and S5 subsites in place of the adjacent molecule in the 1qnj crystal and played a significant role in the structural change of the region. The distortion in this region may subsequently have induced conformational changes in the other three regions. The fact that Tris and these four regions make a diagonal line in the ac plane may have affected the crystal-packing contraction along the a and c axes in the crystal compared with the typical crystal.
Project description:The pathogenic bacteria Pseudomonas aeruginosa and Bordetella pertussis contain in their outer membranes the rare sugar 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid. Five enzymes are required for the biosynthesis of this sugar starting from UDP-N-acetylglucosamine. One of these, referred to as WlbB, is an N-acetyltransferase that converts UDP-2-acetamido-3-amino-2,3-dideoxy-d-glucuronic acid (UDP-GlcNAc3NA) to UDP-2,3-diacetamido-2,3-dideoxy-d-glucuronic acid (UDP-GlcNAc3NAcA). Here we report the three-dimensional structure of WlbB from Bordetella petrii. For this analysis, two ternary structures were determined to 1.43 A resolution: one in which the protein was complexed with acetyl-CoA and UDP and the second in which the protein contained bound CoA and UDP-GlcNAc3NA. WlbB adopts a trimeric quaternary structure and belongs to the LbetaH superfamily of N-acyltransferases. Each subunit contains 27 beta-strands, 23 of which form the canonical left-handed beta-helix. There are only two hydrogen bonds that occur between the protein and the GlcNAc3NA moiety, one between O(delta1) of Asn 84 and the sugar C-3' amino group and the second between the backbone amide group of Arg 94 and the sugar C-5' carboxylate. The sugar C-3' amino group is ideally positioned in the active site to attack the si face of acetyl-CoA. Given that there are no protein side chains that can function as general bases within the GlcNAc3NA binding pocket, a reaction mechanism is proposed for WlbB whereby the sulfur of CoA ultimately functions as the proton acceptor required for catalysis.
Project description:Prebiotic phosphorylation of (pre)biological substrates under aqueous conditions is a critical step in the origins of life. Previous investigations have had limited success and/or require unique environments that are incompatible with subsequent generation of the corresponding oligomers or higher-order structures. Here, we demonstrate that diamidophosphate (DAP)-a plausible prebiotic agent produced from trimetaphosphate-efficiently (amido)phosphorylates a wide variety of (pre)biological building blocks (nucleosides/tides, amino acids and lipid precursors) under aqueous (solution/paste) conditions, without the need for a condensing agent. Significantly, higher-order structures (oligonucleotides, peptides and liposomes) are formed under the same phosphorylation reaction conditions. This plausible prebiotic phosphorylation process under similar reaction conditions could enable the systems chemistry of the three classes of (pre)biologically relevant molecules and their oligomers, in a single-pot aqueous environment.
Project description:<h4>Objective</h4>It is so difficult to formulate human growth hormone (hGH) in a solution with high stability and new drug delivery system (NDDs) due to physiochemical instability. The purpose of this study was to investigate the possibility of using Tris as a hGH stabilizer.<h4>Materials and methods</h4>In this experimental study, the role of tris(hydroxymethyl)aminomethane (Tris) was evaluated as a hGH stabilizing agent in phosphate buffer, as a practical aqueous solution and a media to release NDDs. Highperformance liquid chromatography (HPLC) and enzyme-linked immune sorbent assay (ELISA) were applied to investigate the stability of hGH in solutions and dynamic light scattering (DLS) was used to measure the effect of Tris on the hydrodynamic size of hGH in aqueous solutions. Ultra violet (UV) spectrophotometry was used to check the hGH spectrum. In computational study, formation of ligand-protein complex of the Tris-hGH, and the intermolecular interactions between Tris and hGH were studied by molecular docking modeling.<h4>Results</h4>The results demonstrated that Tris at the optimum concentration, increases hGH stability in aqueous solutions. Also, molecular docking modeling confirmed that amino acid residues such as tyrosine (Tyr), proline (Pro), glutamic acid (Glu), aspartic acid (Asp), leucine (Leu), and phenylalanine (Phe) in hGH structure, were linked with Tris as a ligand.<h4>Conclusion</h4>It seems that interactions between hGH and Tris are the most important reason for increment of the physicochemical stability of hGH in aqueous solutions containing Tris.
Project description:Purine nucleoside phosphorylase (PNP) catalyzes the synthesis and phosphorolysis of purine nucleosides, interconverting nucleosides with their corresponding purine base and ribose-1-phosphate. While PNP plays significant roles in human and pathogen physiology, we are interested in developing PNP as a catalyst for the formation of nucleoside analog drugs of clinical relevance. Towards this aim, we describe the engineering of human PNP to accept 2',3'-dideoxyinosine (ddI, Videx((R))) as a substrate for phosphorolysis using a combination of site-directed mutagenesis and directed evolution. In human PNP, we identified a single amino acid, Tyr-88, as a likely modulator of ribose selectivity. RosettaLigand was employed to calculate binding energies for substrate and substrate analog transition state complexes for single mutants of PNP where Tyr-88 was replaced with another amino acid. In parallel, these mutants were generated by site-directed mutagenesis, expressed and purified. A tyrosine to phenylalanine mutant (Y88F) was predicted by Rosetta to improve PNP catalytic activity with respect to ddI. Kinetic characterization of this mutant determined a 9-fold improvement in k(cat) and greater than 2-fold reduction in K(M). Subsequently, we used directed evolution to select for improved variants of PNP-Y88F in Escherichia coli cell extracts resulting in an additional 3-fold improvement over the progenitor strain. The engineered PNP may form the basis for catalysts and pathways for the biosynthesis of ddI.
Project description:The antiviral efficacies and cytotoxicities of 2',3'- and 4'-substituted 2',3'-didehydro-2',3'-dideoxycytidine analogs were evaluated. All compounds were tested (i) against a wild-type human immunodeficiency virus type 1 (HIV-1) isolate (strain xxBRU) and lamivudine-resistant HIV-1 isolates, (ii) for their abilities to inhibit hepatitis B virus (HBV) production in the inducible HepAD38 cell line, and (iii) for their abilities to inhibit bovine viral diarrhea virus (BVDV) production in acutely infected Madin-Darby bovine kidney cells. Some compounds demonstrated potent antiviral activities against the wild-type HIV-1 strain (range of 90% effective concentrations [EC(90)s], 0.14 to 5.2 micro M), but marked increases in EC(90)s were noted when the compounds were tested against the lamivudine-resistant HIV-1 strain (range of EC(90)s, 53 to >100 micro M). The beta-L-enantiomers of both classes of compounds were more potent than the corresponding beta-D-enantiomers. None of the compounds showed antiviral activity in the assay that determined their abilities to inhibit BVDV, while two compounds inhibited HBV production in HepAD38 cells (EC(90), 0.25 micro M). The compounds were essentially noncytotoxic in human peripheral blood mononuclear cells and HepG2 cells. No effect on mitochondrial DNA levels was observed after a 7-day incubation with the nucleoside analogs at 10 micro M. These studies demonstrate that (i) modification of the sugar ring of cytosine nucleoside analogs with a 4'-thia instead of an oxygen results in compounds with the ability to potently inhibit wild-type HIV-1 but with reduced potency against lamivudine-resistant virus and (ii) the antiviral activity of beta-D-2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine against wild-type HIV-1 (EC(90), 0.08 micro M) and lamivudine-resistant HIV-1 (EC(90) = 0.15 micro M) is markedly reduced by introduction of a 3'-fluorine in the sugar (EC(90)s of compound 2a, 37.5 and 494 micro M, respectively).