Biomedical subjects
Publications and source records attributed to R T Walker.
The reaction of various nucleophiles with 5-vinyl-2'-deoxyuridine has been studied in an attempt to explain the in vitro toxicity of the compound. Attempts to synthesise pro-drugs of 5-vinyl- and 5-ethynyl-2'-deoxyuridine are described.
A fibre-type x-ray diffraction photograph from thymidylyl-3',5'-thymidine is interpreted in terms of a seven-residues per turn, left-handed helical structure in which the first thymine of one molecule is linked to the second thymine of the next molecule by 'wobble' type hydrogen bonds. The first thymidine is in the anti conformation, with a C2'endo - C3'exo sugar and the second is in the syn conformation with a C3'endo - C4'exo sugar. The C5' - 05' bond in the inter-nucleotide linkage is in the g- conformation and that of the P-05' is g+.
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By following the course of the reaction between a suitably-protected base and a chlorosugar in an NMR tube at 250 MHz, it has been shown that the products are consistent with those expected from an SN2 mechanism with inversion of configuration at the anomeric carbon of chlorosugar. In order to achieve high yields of beta-2'-deoxynucleoside, the crystalline alpha-chlorosugar used must react swiftly so that anomerization of the sugar moiety is kept to a minimum. If the base is sufficiently reactive (e.g. 5-methyluracil, uracil), then no catalyst is required and chloroform is the preferred solvent. Using equimolar quantities of the reactants, almost quantitative yields of nucleoside can be obtained in one hour with a beta:alpha ratio greater than 4. With an excess of base, the beta:alpha ratio can be increased even further. With less reactive bases (e.g. 5-nitrouracil, 5-acetyluracil), addition of catalyst can increase the rate of condensation more than the rate of anomerization or decomposition of the sugar. ZnCl2 (0.1 equivalents) has been found to give satisfactory results, although the slower the reaction, inevitably the more alpha-2'-deoxynucleoside is formed. Essentially pure alpha-2'-deoxynucleoside can be isolated in high yield by allowing chlorosugar to anomerize by letting it stand in a polar solvent (acetonitrile) before addition of the base.
5-Vinylpyrimidine nucleosides can be readily synthesized via organometallic intermediates from commercially available nucleosides. Highly potent and selective inhibitors of herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV) are (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and some related analogs such as (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU), 1-beta-D-arabinofuranosyl-(E)-5-(2-bromovinyl)uracil (BVaraU) and (E)-5-(2-bromovinyl)-2'-deoxycytidine (BVDC). The selective antiviral action of BVDU is based upon a specific phosphorylation by the virus-encoded deoxythymidine kinase (TK), inhibition of the viral DNA polymerase and/or incorporation into viral DNA. The efficacy of BVDU against HSV-1 and VZV infections has been demonstrated in animal models and phase I clinical trials. Possible limitations in the clinical usefulness of 5-vinylpyrimidine nucleosides in general and BVDU in particular are discussed.
The following derivatives of 2'-deoxy-5'-O-1",3",2"-dioxaphosphacyclohex-2" -yluridine 2"-oxide have been synthesised: 5-fluoro (4), 5"-(benzyloxy)-5-methyl (6), 5"-(benzyloxy)-5-fluoro (7), 5"-hydroxy-5-methyl (8), 5-fluoro-5"-hydroxy (9), 5",5"-difluoro-5-methyl (11), and 5,5",5"-trifluoro (12). Compounds 4, 9, and 12 have been evaluated for their inhibitory effects on the growth and metabolism of murine leukemia L1210 cells. Compound 12 was nearly as potent as 2'-deoxy-5-fluorouridine in its inhibitory effect on these cells (ID50 = 0.003 and 0.001 micrograms/mL, respectively). Compounds 4 and 9 were about 300 times less active than 12. None of the compounds was an inhibitor of the cell-free thymidylate synthetase, although their antiproliferative effects were achieved by the inhibition of this enzyme.
Most antiviral agents have been discovered by chance. These discoveries and the rationalization of their mode of action in terms of their selective action in the viral replicative cycle has demonstrated that viral targets exist. It is unlikely that in the near future that rational design will result in the synthesis of new classes of antiviral agents. Progress is more likely from a combination of the continuing identification of potential targets and the design of screens to identify prototype compounds, the properties of which may then be improved by chemical synthesis of analogues.
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Using molecular cloning techniques, a clone containing a 7-kb insert of Spiroplasma species BC3 DNA that hybridized strongly to total labeled Spiroplasma tRNA was identified. Sequence analysis of a portion of this recombinant plasmid identified a cluster of tRNA genes. The gene order was as follows: tRNACys-tRNAArg-tRNAPro-tRNAAla-tRNAMet-tRNAIle and a portion of tRNASer All the genes encode the 3'-terminal CCA and have very high A + T and relatively long intergenic regions. An RNA polymerase promoter site was found upstream of the tRNACys gene. The tRNA gene cluster found in Spiroplasma can be compared with a similar Bacillus subtilis gene cluster, which raises interesting questions concerning gene organization and transcription.
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Using in vitro labelling techniques, the complete nucleotide sequence of the 5S ribosomal RNAs isolated from the honeybee pathogen, Spiroplasma species BC3 and Mycoplasma mycoides sp. capri PG3, have been determined. The latter shows only 3 differences from the reported sequence of M. capricolum 5S rRNA, indicating that these two species are very closely related. The Spiroplasma sequence is also 107 nucleotides long and a comparative analysis of the sequence confirms that this Spiroplasma species is closely related to the Mycoplasma species and that they and the Gram-positive eubacteria have descended from a common ancestor and in the process the cell wall-less organisms have lost a large percentage of their genome.