Preparation of 6,6,1',1',6',6'-hexadeutero sucrose

The preparation of 6,6,1',1',6',6'-hexadeutero sucrose is reported. The synthesis is based on a triple oxidation of a protected sucrose 6,1',6'-triol to the corresponding 6,1',6'-tricarboxylic acid or ester, followed by reduction with lithium aluminium deuteride. This triple oxidation could be achieved either using cat. TEMPO-NaOCl (to the acid) or PDC-Ac(2)O-t-BuOH (to the t-butyl carboxylic ester).     

Studies towards the large scale chemical synthesis of the precursors of ribonucleosides-3',4',5',5'-2H4 and -2',3',4',5',5'-2H5

A summary delineating the large scale synthetic studies to prepare labeled precursors of ribonucleosides-3',4',5',5'-2H4 and -2',3',4',5',5'-2H5 from D-glucose is presented. The recycling of deuterium-labeled by-products has been devised to give a high overall yield of the intermediates and an expedient protocol has been elaborated for the conversion of 3-O-benzyl-alpha,beta-D-allofuranose-3,4-d2 6 to 1-O-methyl-3-O-benzyl-2-O-t-butyldimethylsilyl-alpha,beta-D-ribofuranose-3,4,5,5'-d4 16 (precursor of ribonucleosides-3',4',5',5'-2H4) or to 1-O-methyl-3,5-di-O-benzyl-alpha,beta-D-ribofuranose-3,4,5,5'-d4 18 (precursor of ribonucleosides-3',4',5',5'-2H4).     

Synthesis and biological evaluation of 2',3'-didehydro-2',3'-dideoxy-9-deazaguanosine, a monophosphate prodrug and two analogues, 2',3'-dideoxy-9-deazaguanosine and 2',3'-didehydro-2',3'-dideoxy-9-deazainosine

2',3'-Didehydro-2',3'-dideoxy-9-deazaguanosine (1), its monophosphate prodrug (2), and two analogues, 2',3'-dideoxy-9-deazaguanosine (3) and 2',3'-didehydro-2',3'-dideoxy-9-deazainosine (4), have been synthesized from benzoylated 9-deazaguanosine (5). Basic hydrolysis of 5, selective protection of the 2-amino and 5'-hydroxy functions with isobutyryl and silyl groups, respectively, followed by reaction with thiocarbonyldiimidazole gave the cyclic thiocarbonate, which, upon reaction with triethyl phosphite, followed by deprotection, afforded 1. Treatment of 1 with phenyl methoxyalaninylphosphochloridate and N-methylimidazole gave 2. Catalytic hydrogenation of 1 gave 3. Hydrodediazoniation of 1 with tert-butyl nitrite and tris(trimethylsilyl)silane gave 4. Compounds 1-4 were found to be inactive against the human immunodeficiency virus and exhibited minimal to no cytotoxic activity against the L1210 leukemia, CCRF-CEM lymphoblastic leukemia, and B16F10 melanoma in vitro.     

Synthesis of 2',3'-dideoxy-2',3'-didehydro nucleosides via a serendipitous route

This paper describes a "green" synthesis of 2',3'-unsaturated 2',3'-dideoxynucleosides via an electrochemical reaction. Using this approach d4T, d4U, ddA and ddI can be synthesized in high yields.     

Synthesis and antioxidative activity of 3',4',6,7-tetrahydroxyaurone, a metabolite of Bidens frondosa

3',4',6,7-Tetrahydroxyaurone (1a), an aurone isolated from Bidens frondosa, and five analogues (1b-1f) were synthesized from pyrogallol in three steps. The antioxidative activity of 1a-1f was determined by the superoxide free radical and 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging methods.     

2',3'-didehydro-2',3'-dideoxy-5-chlorocytidine is a selective anti-retrovirus agent

2',3'-Didehydro-2',3'-dideoxy-5-chlorocytidine (D4CC) is, in contrast with 2',3'-dideoxy-5-chlorocytidine (ddClCyd) and 2',3'-didehydro-2',3'-dideoxy-5-chlorouridine (D4CU), a potent and selective inhibitor of the replication of human immunodeficiency virus (HIV) types 1 and 2, simian immunodeficiency virus (SIV) and simian AIDS related virus (SRV). D4CC is a poor inhibitor of the phosphorylation of [5-3H]2'-deoxycytidine (dCyd) by partially purified MT-4 cell dCyd kinase (Ki: 612 microM). The findings that (i) D4CC has little, if any, affinity for MT-4 cell Cyd/dCyd deaminase, (ii) D4CU is not antivirally active and (iii) the antiretroviral action of D4CC can be reversed by dCyd, but not dThd, indicate that D4CC is antivirally active as its Cyd metabolite (D4CC 5'-triphosphate) and does not need to be deaminated (to the corresponding Urd metabolite) to exert its antiretroviral action.     

Parallel helical domains in DNA branched junctions containing 5',5' and 3',3' linkages

The Holliday junction is a central intermediate in genetic recombination. It contains four strands of DNA that are paired into four double helical arms that flank a branch point. In the presence of Mg2+, the four arms are known to stack in pairs forming two helical domains whose orientations are antiparallel but twisted by about 60 degrees. The basis for the antiparallel orientation of the domains could be either junction structure or the effect of electrostatic repulsion between domains. To discriminate between these two possibilities, we have constructed and characterized an analogue, called a bowtie junction, in which one strand contains a 3',3' linkage at the branch point, the strand opposite it contains a 5',5' linkage, and the other two strands contain conventional 3',5' linkages. Electrostatic effects are expected to lead to an antiparallel structure in this system. We have characterized the molecule in comparison with a conventional immobile branched junction by Ferguson analysis and by observing its thermal transition profile; the two molecules behave virtually identically in these assays. Hydroxyl radical autofootprinting has been used to establish that the unusual linkages occur at the branch point and that the arms stack to form the same domains as the conventional junction. Cooper-Hagerman gel mobility analyses have been used to determine the relative orientations of the helical domains. Remarkably, we find them to be closer to parallel than to antiparallel, suggesting that the preferred structure of the branch point dominates over electrostatic repulsion. We have controlled for the number of available bonds in the branch point, for gel concentration, and for the role of divalent cations. This finding suggests that control of branch point structure alone can lead to parallel domains, which are generally consistent with recombination models derived from genetic data.     

Synthesis, topoisomerase I inhibition and antitumor cytotoxicity of 2,2':6',2"-, 2,2':6',3"- and 2,2':6',4"-terpyridine derivatives

For the development of new anticancer agents, 2,2':6',2"-, 2,2':6',3"- and 2,2':6',4"-terpyridine derivatives were designed and evaluated for their topoisomerase I inhibitory activity and antitumor cytotoxicity. Structure-activity relationship studies indicated that 2,2':6',2"-terpyridine derivatives were highly cytotoxic toward several human tumor cell lines, whereas 2,2':6',3"- and 2,2':6',4"-terpyridine derivatives were potent topoisomerase I inhibitors.     

Interaction of mammalian deoxyribonuclease V, a double strand 3' to 5' and 5' to 3' exonuclease, with deoxyribonucleic acid polymerase-beta from the Novikoff hepatoma

Novikoff hepatoma stimulatory factor IV has been resolved from the DNA polymerase-beta on a single-stranded DNA-cellulose column and then purified to > 95% homogeneity on hydroxylapatite. A single band of Mr = 12,000 is found on sodium dodecyl sulfate-polyacrylamide gels. Addition of factor IV to a DNA synthesis reaction causes (i) an increase in initial velocity, (ii) a prolongation of linear synthesis, and (iii) an increase in extent of incorporation. In the absence of factor IV, the reaction reaches a plateau in approximately 1 h. Factor IV, added at this point, causes resumption of synthesis with kinetics similar to when factor IV was present from the start. When factor IV is present, synthesis is followed by DNA degradation, indicating nuclease activity. Factor IV is shown to be an exonuclease which hydrolyzes double-stranded substrates in both the 3' to 5' and 5' to 3' directions at similar rates. Factor IV interacts with the 3.3 S beta-polymerase forming an aggregate sedimenting at 4.1 S and containing both polymerase and exonuclease activities. Analysis of fractions containing a beta-polymerase . exonuclease complex on polyacrylamide gels suggests a stoichiometry of 1:1. The exonuclease shows a strong preference for double-stranded substrates and is most active on poly(dA-dT). It can hydrolyze chains containing either a 3'- or 5'-phosphoryl or a 5'- or 3'-hydroxyl terminus. The product of digestion is predominantly 5'-nucleoside monophosphates. The enzyme cannot hydrolyze di- or trinucleotides, lacks RNase-H activity, and will not liberate thymine dimers from UV-irradiated DNA. The exonuclease has an alkaline pH optimum and requires a divalent cation. Since the properties of this exonuclease are unlike those of previously described mammalian DNases, we have named this enzyme mammalian DNase V.     

Flexibility of 3',5' deoxyribonucleoside diphosphates

In 3',5' deoxyribonucleoside diphosphates, in addition to the nature of the base and the sugar puckering, there are six single bond rotations. However, from the analysis of crystal structure data on the constituents of nucleic acids, only three rotational angles, that are about glycosyl bond, about C4'-C5' and about C3'-O3' bonds, are flexible. For a given sugar puckering and a base, potential energy calculations using non-bonded, electrostatic and torsional functions were carried out by varying the three torsion angles. The energies are represented as isopotential energy surfaces. Since the availability of the real-time color graphics, it is possible to analyse these isopotential energy surfaces. The calculations were carried out for C3' exo and C3' endo puckerings for deoxyribose and also for four bases. These calculations throw more light not only on the allowed regions for the three rotational angles but also on the relationships among them. The dependence of base and the puckering of the sugar on these rotational angles and thereby the flexibility of the 3',5' deoxyribonucleoside diphosphates is discussed. From our calculations, it is now possible to follow minimum energy path for interconversion among various conformers.     

Synthesis and evaluation of 2',4',6'-trihydroxychalcones as a new class of tyrosinase inhibitors

In this study, we synthesized a series of hydroxychalcones and examined their tyrosinase inhibitory activity. The results showed that 2',4',6'-trihydroxychalcone (1), 2,2',3,4',6'-pentahydroxychalcone (4), 2',3,4,4',5,6'-hexahydroxychalcone (5), 2',4',6'-trihydroxy- 3,4-dimethoxychalcone (9) and 2,2',4,4',6'-pentahydroxychalcone (15) exhibited high inhibitory effects on tyrosinase with respect to l-tyrosine as a substrate. By the structure-activity relationship study, it was suggested that the 2',4',6'-trihydroxyl substructure in the chalcone skeleton were efficacious for the inhibition of tyrosinase activity. And also, the catechol structure on B-ring of chalcones was not advantageous for the inhibitory potency. Furthermore, 15 (IC(50)=1microM) was found to show the highest activity out of a set of 15 hydroxychalcones, even better than both 2,2',4,4'-tetrahydroxychalcone (13, IC(50)=5microM) and kojic acid (16, IC(50)=12microM), which were known as potent tyrosinase inhibitors. Kinetic study revealed that 15 acts as a competitive inhibitor of tyrosinase with K(i) value of 3.1microM.     

Axially dissymmetric (R)-(+)-5,5',6,6',7,7',8,8' octahydro-[1,1']binaphthyldiimine chiral salen type-ligands for copper-catalyzed asymmetric aziridination

Axially dissymmetric chiral diimine ligand 2 was prepared from the reaction of (R)-(+)-5,5',6,6',7,7',8,8'-octahydro-[1,1']binaphthyl-2,2'-diamine 1 with 2,6-dichlorobenzaldehyde. The catalytic asymmetric aziridination of alkenes was examined using this novel chiral ligand. Excellent enantioselective aziridination of cinnamates was achieved using C(2)-symmetric chiral ligand 2.     

Potent induction of rat liver microsomal, drug-metabolizing enzymes by 2,3,3',4,4',5-hexabromobiphenyl, a component of fireMaster

The multistep synthesis and purification of 2,3,3',4,4',5-hexabromobiphenyl (HBBp) is described. Capillary gas chromatography revealed that HBBp comprises 0.05% of the industrial polybrominated biphenyl (PBB) mixture, fireMaster BP-6 (lot 7062). When administered to immature male Wistar rats, HBBp caused a dose-dependent increase in (a) the activity of benzo[a]pyrene (B[a]P) hydroxylase (AHH) and 4-chlorobiphenyl (4-CBP) hydroxylase and (b) the concentration of cytochrome P-450. Sodium dodecyl sulfate (SDS)-gel electrophoresis indicated that these increases in cytochrome P-450 and cytochrome P-450-dependent monooxygenase activities were accompanied by a dose-dependent intensification of a protein of relative molecular weight (Mr) 55 000 which comigrated with the major 3-methylcholanthrene(MC)-inducible form of cytochrome P-450 (i.e., cytochrome P-448). Like MC, but in contrast to phenobarbitone (PB), HBBp competitively displaced 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin ([3H]-TCDD) from the cytosolic protein thought to be the receptor for cytochrome P-448 induction. The results indicate that HBBp is a potent inducer of cytochrome P-448 and as such is the third MC-type inducer identified in fireMaster BP-6.     

(2')3',5'-Bisphosphate nucleotidase

(2')3',5'-Bisphosphate nucleotidase has been prepared in electrophoretically homogeneous form from guinea pig liver. The enzyme catalyzes the hydrolysis of the 2'- or 3'-phosphate from the appropriate nucleoside 2',5'- and 3',5'-bisphosphates and is active with 3'-phosphoadenosine 5'-phosphosulfate and with coenzyme A but not with ATP. The 40,000-dalton protein is a monomer that requires Mg2+ for activity.     

An end-healing enzyme from Clostridium thermocellum with 5' kinase, 2',3' phosphatase, and adenylyltransferase activities

We identify and characterize an end-healing enzyme, CthPnkp, from Clostridium thermocellum that catalyzes the phosphorylation of 5'-OH termini of DNA or RNA polynucleotides and the dephosphorylation of 2',3' cyclic phosphate, 2'-phosphate, and 3'-phosphate ribonucleotides. CthPnkp also catalyzes an autoadenylylation reaction via a polynucleotide ligase-type mechanism. These characteristics are consistent with a role in end-healing during RNA or DNA repair. CthPnkp is a homodimer of an 870-amino-acid polypeptide composed of three catalytic domains: an N-terminal module that resembles the polynucleotide kinase domain of bacteriophage T4 Pnkp, a central metal-dependent phosphoesterase module, and a C-terminal module that resembles the nucleotidyl transferase domain of polynucleotide ligases. The distinctive feature of CthPnkp vis-à-vis known RNA repair enzymes is that its 3' end modification component belongs to the calcineurin-type phosphatase superfamily. It contains putative counterparts of the amino acids that form the dinuclear metal-binding site and the phosphate-binding site of bacteriophage lambda phosphatase. As with lambda phosphatase, the 2',3' cAMP phosphatase activity of CthPnkp is specifically dependent on nickel or manganese. We identify homologs of CthPnkp in other bacterial proteomes.