Molecular mechanics studies on the conformations of 2',3'-dideoxy-2',3'-didehydroguanine nucleoside, D4G

Conformational energy calculations have been presented on guanine nucleoside in which the furanose ring is replaced by 2',3'-dideoxy-2',3'-didehydrofuran using molecular mechanics and conformational analysis. Conformational energies have been evaluated using the MM2 and AMBER94 force field parameters at two different dielectric constants. The results are presented in terms of isoenergy contours in the conformational space of the glycosidic (chi) and C4'-C5' (gamma) bonds torsions. In general, the chi-gamma interrelationships differ from the corresponding plots for unmodified nucleosides and nucleotides, reported previously. Consistency of the calculated preferred conformations with the x-ray data is sensitive to the force field employed.     

Enzymatic properties of the unnatural beta-L-enantiomers of 2',3'-dideoxyadenosine and 2',3'-didehydro-2',3'-dideoxyadenosine

The beta-L-enantiomers of 2',3'-dideoxyadenosine and 2',3'-didehydro-2',3'-dideoxyadenosine have been stereospecifically synthesized. In an attempt to explain the previously reported antiviral activities of these compounds, their enzymatic properties were studied with respect to adenosine kinase, deoxycytidine kinase, adenosine deaminase, and purine nucleoside phosphorylase. Adenosine deaminase was strictly enantioselective and favored beta-D-ddA and beta-D-d4A, whereas adenosine kinase and purine nucleoside phosphorylase had no apparent substrate properties for the D- or L-enantiomers of beta-ddA or beta-d4A. Human deoxycytidine kinase showed a remarkable inversion of the expected enantioselectivity, with beta-L-ddA and beta-L-d4A having better substrate efficiencies than their corresponding beta-D-enantiomers. Our results demonstrate the potential of beta-L-adenosine analogues as antiviral agents and suggest that deoxycytidine kinase has a strategic importance in their cellular activation.     

Synthesis and biological properties of the four optical isomers of 5-o-carboranyl-2',3'-didehydro-2',3'-dideoxyuridine

The four isomers of the 5-o-carboranyl-2',3'-didehydro-2',3'-dideoxyuridine (d4CU) were synthesized and their antiviral activity and cytotoxicity in normal and cancer human cells determined. Coupling of silylated 5-o-carboranyluracil with the protected D/L 2,3-dideoxy-2-phenylselenenylribosylacetates provided after oxidative elimination and deprotection, the desired compounds. The presence of the electron deficient 5-o-carboranyl moiety on uracil influenced the yield of the various isomers. In general, the compounds demonstrated weak anti-human immunodeficiency virus activity in primary human lymphocytes. No marked difference in the biological profile was noted for the various optical isomers, suggesting that the high lipophilicity of these nucleosides imparted by the carboranyl moiety overrides stereochemical considerations in the 2',3'-didehydro-2',3'-dideoxyaglycon moiety.     

Diastereomeric specificity of 2',3'-cyclic nucleotide 3'-phosphodiesterase

The diastereomers of adenosine and uridine 2',3'-cyclic phosphorothioates were tested as substrates for 2',3'-cyclic nucleotide 3'-phosphodiesterase from bovine brain. The enzyme cleaves the Sp (or exo) diastereomers efficiently, whereas the Rp (or endo) diastereomers are resistant to hydrolysis, even after long incubation. As the enzyme exhibits strong substrate inhibition the precise determination of kinetic parameters posed problems, particularly with phosphorothioates. The stereoselectivity of this enzyme is opposite to that of RNase T1 and RNase A and thus could be a useful complement in determination of the configuration of nucleoside 2',3'-cyclic phosphorothioates resulting from hydrolysis reactions of unknown stereochemical course.     

Structural characterization of activation 'intermediate 2' on the pathway to human gastricsin

The crystal structure of an activation intermediate of human gastricsin has been determined at 2.4 A resolution. The human digestive enzyme gastricsin (pepsin C) is an aspartic proteinase that is synthesized as the inactive precursor (zymogen) progastricsin (pepsinogen C or hPGC). In the zymogen, a positively-charged N-terminal prosegment of 43 residues (Ala 1p-Leu 43p; the suffix 'p' refers to the prosegment) sterically prevents the approach of a substrate to the active site. Zymogen conversion occurs in an autocatalytic and stepwise fashion at low pH through the formation of intermediates. The structure of the non-covalent complex of a partially-cleaved peptide of the prosegment (Ala 1p-Phe 26p) with mature gastricsin (Ser 1-Ala 329) suggests an activation pathway that may be common to all gastric aspartic proteinases.     

Anti-hepatitis B virus activity and metabolism of 2',3'-dideoxy-2',3'-didehydro-beta-L(-)-5-fluorocytidine

2',3'-Dideoxy-2',3'-didehydro-beta-L(-)-5-fluorocytidine [L(-)Fd4C] was found to be at least 10 times more potent than beta-L-2',3'-dideoxy-3'-thiacytidine [L(-)SddC; also called 3TC, or lamivudine]against hepatitis B virus (HBV) in culture. Its cytotoxicity against HepG2 growth in culture was also greater than that of L(-)SddC (3TC). There was no activity of this compound against mitochondrial DNA synthesis in cells at concentrations upto 10 microM. The dynamics of recovery of virus from the medium of cells pretreated with equal drug concentrations were slower with L(-)Fd4C than with L(-)SddC (3TC). L(-)Fd4C could be metabolized to mono-, di-, and triphosphate forms. The degree of L(-)Fd4C phosphorylation to the 5'-triphosphate metabolite was higher than the degree of L(-)SddC (3TC) phosphorylation when equal extracellular concentrations of the two drugs were used. The apparent K(m) of L(-)Fd4C phosphorylated metabolites formed intracellularly was higher than that for L(-)SddC (3TC). This may be due in part to a difference in the behavior of L(-)Fd4C and L(-)SddC (3TC) towards cytosolic deoxycytidine kinase. Furthermore, L(-)Fd4C 5'-triphosphate was retained longer within cells than L(-)SddC (3TC) 5-triphosphate. L(-)Fd4C 5'-triphosphate inhibited HBV DNA polymerase in competition with dCTP with a Ki of 0.069 +/- 0.015 microM. Given the antiviral potency and unique pharmacodynamic properties of L(-)Fd4C, this compound should be considered for development as an expanded-spectrum anti-HBV drug.     

Effects of 2',3'-dideoxyinosine on Toxoplasma gondii cysts in mice

The activity against Toxoplasma gondii of 2',3' dideoxyinosine (ddI), an anti-human immunodeficiency virus drug, was examined in an in vitro and in vivo study. Cell cultures infected with a strain known to cause chronic infections were used to show the dose-dependent effect of this drug compared with spiramycin and sulfadiazine. When a dose of 4 microg/ml was used, no infected THP-1 cells or parasites were found after 60 h of incubation. An electron-microscopic study confirmed that after 12 h at 1 microg/ml, the few parasites observed were severely altered. The treatment of chronically infected mice 3 months postinfection showed that a 30-day treatment with 2 mg of ddI/ml induced a significant reduction in the number of T. gondii cysts in the cerebral tissue. These cysts were not viable, as confirmed by immunofluorescence and reinfection experiments. These experiments suggest a possible role for ddI in the treatment of toxoplasmosis, and this possibility deserves further investigation.     

Pharmacokinetics and distribution over the blood-brain barrier of zalcitabine (2',3'-dideoxycytidine) and BEA005 (2', 3'-dideoxy-3'-hydroxymethylcytidine) in rats, studied by microdialysis

Microdialysis was applied to sample the unbound drug concentration in the extracellular fluid in brain and muscle of rats given zalcitabine (2',3'-dideoxycytidine; n = 4) or BEA005 (2', 3'-dideoxy-3'-hydroxymethylcytidine; n = 4) (50 mg/kg of body weight given subcutaneously). Zalcitabine and BEA005 were analyzed by high-pressure liquid chromatography with UV detection. The maximum concentration of zalcitabine in the dialysate (Cmax) was 31.4 +/- 5. 1 microM (mean +/- standard error of the mean) for the brain and 238. 3 +/- 48.1 microM for muscle. The time to Cmax was found to be from 30 to 45 min for the brain and from 15 to 30 min for muscle. Zalcitabine was eliminated from the brain and muscle with half-lives 1.28 +/- 0.64 and 0.85 +/- 0.13 h, respectively. The ratio of the area under the concentration-time curve (AUC) (from 0 to 180 min) for the brain and the AUC for muscle (AUC ratio) was 0.191 +/- 0.037. The concentrations of BEA005 attained in the brain and muscle were lower than those of zalcitabine, with Cmaxs of 5.7 +/- 1.4 microM in the brain and 61.3 +/- 12.0 microM in the muscle. The peak concentration in the brain was attained 50 to 70 min after injection, and that in muscle was achieved 30 to 50 min after injection. The half-lives of BEA005 in the brain and muscle were 5.51 +/- 1.45 and 0.64 +/- 0.06 h, respectively. The AUC ratio (from 0 to 180 min) between brain and muscle was 0.162 +/- 0.026. The log octanol/water partition coefficients were found to be -1.19 +/- 0.04 and -1.47 +/- 0.01 for zalcitabine and BEA005, respectively. The degrees of plasma protein binding of zalcitabine (11% +/- 4%) and BEA005 (18% +/- 2%) were measured by microdialysis in vitro. The differences between zalcitabine and BEA005 with respect to the AUC ratio (P = 0.481), half-life in muscle (P = 0.279), and level of protein binding (P = 0.174) were not statistically significant. The differences were statistically significant in the case of the half-life in the brain (P = 0.032), clearance (P = 0.046), volume of distribution (P = 0.027) in muscle, and octanol/water partition coefficient (P = 0.019).     

Dual implication of 2',3'-cyclic nucleotide 3' phosphodiesterase as major autoantigen and C3 complement-binding protein in the pathogenesis of multiple sclerosis

Multiple sclerosis (MS) is characterized by intra-blood-brain barrier immunoglobulin synthesis that persists lifelong. Subcellular fractionation and two-dimensional electrophoresis were used in conjunction with immune precipitation and immunoblotting to identify antigenic determinants for this immunoglobulin. We report that 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNP), a protein associated with oligodendrocyte/myelin membranes, also present in lymphocytes and retina, is one major target for the humoral response. Antibodies to CNP are detected in sera of 74% of MS patients. The antibodies are IgM and are present in serum in high titer as well as in cerebrospinal fluid. The antibody response is temporally persistent, consistent with systemic immune activation and persistent antigenic stimulation. Moreover, CNP is isolated as an immune complex from MS brain. CNP is expressed as two isoforms, with CNPII identical to CNPI but with a 20-amino acid extension at the amino terminus of CNPII; however, the antibody response is exclusively restricted to CNPI. In contrast, both isoforms bind the C3 complement, providing a plausible mechanism in MS central nervous system (CNS) for opsonization of myelin membrane CNP, mediated via the C3 receptor, and phagocytosis of CNP-Ig immune complexes, mediated by membrane Ig Fc receptors of macrophages and CNS microglia.     

6,6"-二甲基-4'-苯基-2,2':6',2"-三联吡啶与铅显色反应的分光光度研究

用作者合成的新显色剂6,6"-二甲基-4'-苯基-2,2':6',2"-三联吡啶(TPY)分光光度法测定痕量铅,在pH5.0～6.0的HAc-NaAc介质中,该试剂与铅形成稳定络合物,其最大吸收波长在375nm处,Pb2+:TPY＝3:4,表观摩尔吸光系数ε＝5.71×104,铅量在0～25μg/25mL范围内服从比尔定律,用于纯铜试样的分析,结果满意.     

Bi2O3、Dy2O3掺杂对8YSZ导电性能的影响

用柠檬酸溶胶-凝胶法制备了分别掺Bi2O3和Dy2O3的8YSZ前驱体凝胶,凝胶在500℃预烧,压制成圆片状后在1 300℃煅烧2 h。分别研究Bi2O3和Dy2O3的不同掺量对试样的致密度、电导率的影响。结果表明Bi2O3可促进试样的烧结,在300～400℃掺Bi2O3可提高试样的电导率,在400～700℃掺Bi2O3使试样的电导率下降。在8YSZ试样中掺Dy2O3后,试样的致密度、电导率均降低。     

Pharmacokinetics and tissue distribution of (+/-)-3'-azido-2',3'-dideoxy-5'-O-(2-bromomyristoyl)thymidine, a prodrug of 3'-azido-2',3'-dideoxythymidine (AZT) in mice

The in-vivo biodistribution and pharmacokinetics in mice of 3'-azido-2',3'-dideoxythymidine (1, AZT), 2-bromomyristic acid (2) and their common prodrug, (+/-)-3'-azido-2',3'-dideoxy-5'-O-(2-bromomyristoyl)thymidine (3) are reported. The objectives of the work were to enhance the anti-human immunodeficiency virus and anti-fungal effects of 1 and 2 by improving their delivery to the brain and liver. The pharmacokinetics of AZT (beta t1/2 (elimination, or beta-phase, half-life) = 112.5 min; AUC (area under the plot of concentration against time) = 29.1 +/- 2.9 micromol g(-1) min; CL (blood clearance) = 10.5 +/- 1.1 mL min(-1) kg(-1)) and its ester prodrug (3, beta t1/2 = 428.5 min; AUC = 17.3 +/- 4.7 micromol g(-1) min; CL = 17.6 +/- 4.8 mL min(-1) kg(-1) were compared after intravenous injection of equimolar doses (0.3 mmol kg(-1)) via the tail vein of Balb/c mice (25-30 g). The prodrug was rapidly converted to AZT in-vivo, but plasma levels of AZT (peak concentration 0.17 micromol g(-1)) and AUC (12.3 micromol min g(-1)) were lower than observed after AZT administration (peak concentration 0.36 micromol g(-1); AUC 29.1 micromol min g(-1). The prodrug also accumulated rapidly in the liver immediately after injection, resulting in higher concentrations of AZT than observed after administration of AZT itself (respective peak concentrations 1.11 and 0.81 micromol g(-1); respective AUCs 42.5 and 12.7 micromol min g(-1)). Compared with doses of AZT itself, 3 also led to significantly higher brain concentration of AZT (25.7 compared with 9.8 nmol g(-1)) and AUCs (2.8 compared with 1.4 micromol min g(-1)). At the doses used in this study the antifungal agent 2-bromomyristic acid was measurable in plasma and brain within only 2 min of injection. Hepatic concentrations of 2-bromomyristic acid were higher for at least 2 h after dosing with 3 than after dosing with the acid itself. In summary, comparative biodistribution studies of AZT and its prodrug showed that the prodrug led to higher concentrations of AZT in the brain and liver. Although the prodrug did not result in measurably different concentrations of 2-bromomyristic acid in the blood and brain, it did lead to levels in the liver which were higher than those achieved by dosing with the acid itself.     

Preparation of 2'-thio-2'-deoxycytidine 2':3'-phosphorothioate

The synthesis of a novel ribonucleotide analog 2'-thio-2' deoxycytidine 2':3'-O,S-phosphorothioate is described. In the first step, 2,2'-anhydro 1-beta-D-arabinosylcytosine was thiophosphorylated by the action of dithiophosphate, a process which gave predominantly the 3'-O-phosphorothioate isomer. An intramolecular displacement reaction led to the formation of the title compound. Structure and reactivity of this thioanalog differ substantially from 2':3'-CMP.