Asymmetric synthesis of novel isoindolines: azasaccharide mimics as potential enzyme inhibitors.

2',3'-Dideoxy-2',3'-didehydronucleosides and azasaccharides are known to possess antiviral activity. The synthesized 1-methoxyisoindoline system (10), which is related to the above nucleosides, is potentially stable in-vivo. The 1-methoxyisoindoline was synthesized from the achiral phthalaldehyde in 10 steps via an enantiomerically pure diol obtained by Sharpless asymmetric dihydroxylation. The new heterocyclic compound is an azosaccharide mimic which provides an access to a new series of nucleoside analogues with potential as antiretroviral agents (anti-HIV) and as glycosidase inhibitors.

A dengue virus serotype-1 DNA vaccine induces virus neutralizing antibodies and provides protection from viral challenge in Aotus monkeys.

A DNA vaccine that expresses the premembrane/membrane (prM) and envelope (E) genes of dengue virus serotype-1 was tested for immunogenicity and protection against dengue-1 virus challenge in Aotus nancymae monkeys. The vaccine, in 1 mg doses, was administered intradermally (i.d.) to three monkeys and intramuscularly (i.m.) to three others. For controls, a 1 mg dose of vector DNA was administered i.d. to two monkeys and i.m. to one. All animals were primed and then boosted at one and five months post priming. Sera were collected monthly and analyzed for dengue-1 antibodies by enzyme linked immunosorbent assay (ELISA) and plaque reduction neutralization test (PRNT). Dengue-1 antibodies were detectable in the sera from i.d. and i.m. vaccine inoculated animals one month after the first boost and peaked one month after the second boost. The antibody levels from sera of animals that received the vaccine via the i.d. route were twice those from sera of animals that received the vaccine via the i.m. route. Six months after the second boost all inoculated and two naive monkeys were challenged with 1.25x10(4) plaque forming units (PFU) of dengue-1 virus. Two vaccine immunized animals were protected from viremia while the others showed a reduction in viremia. The mean days of viremia were 1 and 1.3 for the animals that were immunized with the vaccine via the i.d. or i.m. route, respectively vs 4 and 2 mean days of viremia in the animals inoculated with control DNA. Naive animals were viremic for an average of 4 days. All of the three control monkeys that received control DNA inoculum by either the i.d. or i.m. route had an intermittent viremia pattern with one or more negative days interspersed between the positive days. This pattern was not observed in any of the vaccine recipients or the naïve control monkeys. These results demonstrate that DNA immunization is a promising approach for the development of dengue vaccines and that A. nancymae monkeys are suitable for dengue vaccine trials.

Simple and stereoselective syntheses of nucleoside analogues with a benzo[c]furan glycone moiety.

A series of d4T analogues have been synthesised in which the 2',3'-didehydro-2',3'-dideoxyribose moiety is replaced by a benzo[c]furan core. A simple strategy has been developed to access a range of compounds for biological screening. In addition, a stereoselective approach has been achieved with view to permit more detailed studies.

Determination of the structure of exochelin MN, the extracellular siderophore from Mycobacterium neoaurum.

BACKGROUND: Siderophores are compounds produced by bacteria to acquire iron. Exochelin MN, the extracellular siderophore from Mycobacterium neoaurum, is of particular interest because it has been shown to transport iron into M. leprae, which is responsible for the disease leprosy. Exochelins from other species cannot mediate iron transport in M. leprae, suggesting a specific uptake mechanism involving exochelin MN. We set out to determine the structure of exochelin MN and identify the features of the molecule that may account for this specificity. RESULTS: The structure of exochelin MN was elucidated by a combination of techniques including nuclear magnetic resonance, mass spectrometry, derivatization and gas chromatography. Exochelin MN is a peptide, containing the unusual amino acid beta-hydroxyhistidine and an unusual N-methyl group. The peptide coordinates iron(III) octahedrally using its two cis-hydroxamate groups plus the hydroxyl and imidazole nitrogen of the beta-hydroxyhistidine. The three-dimensional structure of the hexadentate exochelin/gallium complex was deduced from NMR data. CONCLUSIONS: Exochelin MN has some structural features in common with other siderophores, but has a unique three-dimensional structure, which is presumably important for its specific activity in M. leprae. Exochelin MN may be a target for drug design in the fight against infection with this pathogen.

Isolation, purification and structure of exochelin MS, the extracellular siderophore from Mycobacterium smegmatis.

The extracellular siderophore from Mycobacterium smegmatis, exochelin MS, was isolated from iron-deficiently grown cultures and purified to > 98% by a combination of ion-exchange chromatography and h.p.l.c. The material is unextractable into organic solvents, is basic (pI = 9.3-9.5), has a lambda max at 420 nm and a probable Ks for Fe3+ of between 10(25) and 10(30). Its structure has been determined by examination of desferri- and ferri-exochelin and its gallium complex. The methods used were electrospray-m.s. and one- and two-dimensional (NOESY, DQF-COSY and TOCSY) 1H n.m.r. The constituent amino acids were examined by chiral g.l.c analysis of N-trifluoroacetyl isopropyl and N-pentafluoropropionyl methyl esters after hydrolysis, and reductive HI hydrolysis, of the siderophore. The exochelin is a formylated pentapeptide: N-(delta-N-formyl,delta N-hydroxy-R-ornithyl) -beta-alaninyl-delta N-hydroxy-R-ornithinyl-R-allo-threoninyl-delta N-hydroxy-S-ornithine. The linkages involving the three ornithine residues are via their delta N(OH) and alpha-CO groups leaving three free alpha-NH2 groups. Although there are two peptide bonds, these involve the three R (D)-amino acids. Thus the molecule has no conventional peptide bond, and this suggests that it will be resistant to peptidase hydrolysis. The co-ordination centre with Fe3+ is hexadenate in an octahedral structure involving the three hydroxamic acid groups. Molecular modelling shows it to have similar features to other ferric trihydroxamate siderophores whose three-dimensional structures have been established. The molecule is shown to have little flexibility around the iron chelation centre, although the terminal (Orn-3) residue, which is not involved in iron binding except at its delta N atom, has more motional freedom.

Synthesis of 4- and 5-amino-1-(2-deoxy-D-erythro-pentofuranosyl)imidazole nucleosides by chemical and biotransformation methods.

(2-Deoxy-D-erythro-pentofuranosyl)imidazole nucleosides have been synthesised by glycosylation of the sodium salt of ethyl 5-aminoimidazole-4-carboxylate with 2-deoxy-3,5-di-O-p-toluoyl-alpha-D-erythro-pentofuranosyl chloride. Glycosylation of ethyl 5-aminoimidazole-4-carboxylate with 2-deoxy-beta-D-erythro-pentofuranose was also achieved enzymically using E. coli, immobilised by ionotropic gelation in an alginate gel, with 2'-deoxyuridine as glycosyl donor. 5-Amino-1-(2-deoxy-beta-D-erythro-pentofuranosyl)imidazole-4-carboxylic acid 5'-phosphate and 4-amino-1-(2-deoxy-beta-D-erythro-pentofuranosyl)imidazole-5-carboxylic acid 5'-phosphate were synthesised by phosphorylation of the respective nucleosides and examined as inhibitors of phosphoribosylaminoimidazole carboxylase (EC 4.1.1.21) and phosphoribosylaminoimidazolesuccinocarboxamide synthetase (EC 6.3.2.6), which are involved in the de novo biosynthesis of purine nucleotides.

Accumulation of a poly(hydroxyalkanoate) copolymer containing primarily 3-hydroxyvalerate from simple carbohydrate substrates by Rhodococcus sp. NCIMB 40126.

A number of taxonomically-related bacteria have been identified which accumulate poly(hydroxyalkanoate) (PHA) copolymers containing primarily 3-hydroxyvalerate (3HV) monomer units from a range of unrelated single carbon sources. One of these, Rhodococcus sp. NCIMB 40126, was further investigated and shown to produce a copolymer containing 75 mol% 3HV and 25 mol% 3-hydroxybutyrate (3HB) from glucose as sole carbon source. Polyesters containing both 3HV and 3HB monomer units, together with 4-hydroxybutyrate (4HB), 5-hydroxyvalerate (5HV) or 3-hydroxyhexanoate (3HHx), were also produced by this organism from certain accumulation substrates. With valeric acid as substrate, almost pure (99 mol% 3HV) poly(3-hydroxyvalerate) was produced. N.m.r. analysis confirmed the composition of these polyesters. The thermal properties and molecular weight of the copolymer produced from glucose were comparable to those of PHB produced by Alcaligenes eutrophus.

Accumulation of a Polyhydroxyalkanoate Containing Primarily 3-Hydroxydecanoate from Simple Carbohydrate Substrates by Pseudomonas sp. Strain NCIMB 40135.

A number of Pseudomonas species have been identified which accumulate a polyhydroxyalkanoate containing mainly 3-hydroxydecanoate monomers from sodium gluconate as the sole carbon source. One of these, Pseudomonas sp. strain NCIMB 40135, was further investigated and shown to accumulate such a polyhydroxyalkanoate from a wide range of carbon sources (C(2) to C(6)); however, when supplied with octanoic acid it produced a polyhydroxyalkanoate containing mainly 3-hydroxyoctanoate monomers. Polymer synthesis occurred in batch culture after cessation of growth due to exhaustion of nitrogen. In continuous culture under nitrogen limitation up to 16.9% (wt/wt) polyhydroxyalkanoate was synthesized from glucose as the carbon source. The monomer units are mainly of the R-(-) configuration. Nuclear magnetic resonance studies confirmed the composition of the polymer. Differential scanning calorimetry suggested that the solvent-extracted polymer contained a significant proportion of crystalline material. The weight-average molecular weight of the polymer from glucose-grown cells was 143,000.

Isolation, identification, and structural analysis of the mycobactins of Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium scrofulaceum, and Mycobacterium paratuberculosis.

Methods were devised to purify the cell-associated, iron-binding compounds known as mycobactins from the closely related species Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum (i.e., the MAIS complex of organisms). The mycobactins from these three species showed a structure that is common to the mycobactins from all the mycobacteria examined to date. However, these mycobactins were unique in that they had more than one alkyl chain. The M. scrofulaceum mycobactins differed from other MAIS mycobactins by a shift in the position of the double bond in the R1 alkyl chain. Traces of other mycobactin types were observed in ethanol extracts of the three species, and examination of the chromatographic properties of these mycobactins showed that each species produced five mycobactin types. Each mycobactin could be subdivided further by the length of its R1 alkyl chain. No differences in the production of these novel mycobactin were observed among species. Mycobactins from three strains of Mycobacterium paratuberculosis and two wood pigeon strains of Mycobacterium avium which had lost their original growth requirements for mycobactin after repeated subculturing in laboratory growth media were examined by thin-layer chromatography and high-pressure liquid chromatography. Each organism produced a mycobactin with similar chromatographic properties to those synthesized by MAIS organisms. M. paratuberculosis NADC 18 produced at least two components in our laboratory, and nuclear magnetic resonance analysis of the major component showed this mycobactin to be identical to that produced by M. intracellulare M12. However, a sample of mycobactin J isolated by Merkal and McCullough (Curr. Microbiol. 7:333-335, 1982) from M. paratuberculosis NADC 18 was different from our isolates and appeared to correspond to a minor mycobactin component we had seen by thin-layer chromatography. No reason for this difference could be evinced. Our findings indicate that there is a close taxonomic relationship between M. paratuberculosis and the MAIS complex.

The separation of the mycobactins from Mycobacterium smegmatis by using high-pressure liquid chromatography.

The family of mycobactins from Mycobacterium smegmatis were resolved into seven fractions by high-pressure liquid chromatography. This separation was by virtue of the differences in length and character of the long acyl substituents as shown by g.l.c. of the methyl esters of the isolated fatty acids from the fractions. As t.l.c. could also resolve the individual mycobactin fractions, it too must rely on the same differences to effect separation. As the lengths of the acyl chains were modulated by the growth conditions, a specific range of acyl groups may not be needed for mycobactin to function. This technique provides a simple means of rapidly characterizing crude mycobactins from all mycobacteria.

Phospholipid phase transitions. Effects of n-alcohols, n-monocarboxylic acids, phenylalkyl alcohols and quaternary ammonium compounds.

The interactions of a series of alcohols, acids and quaternary ammonium salts with a phosphatidylcholine-water model biomembrane (dipalmitoyl phosphatidycholine) system have been studied using differential scanning calorimetry. In particular the effects of these molecules upon the lipid endothermic phase transitions were investigated over a range of concentrations. A variety of effects was observed. (a) Those molecules which shift or broaden the main lipid transition can also remove the pretransition endotherm. (b) n-Alcohols and n-monocarboxylic acids containing the same number of carbon atoms have very similar effects at molar concentrations up to 40%. Those molecules containing 12 or more carbon atoms raise the main lipid phase transition whilst those molecules containing 10 or less carbon atoms lower this transition temperature. (c) The phase diagram of stearoyl alcohol in the phosphatidylcholine-water system shows the formation of lipid-alcohol complexes. (d) Alkyl trimethyl ammonium bromides showed behaviour which differs considerably from n-alcohols and n-carboxylic acids of the same chain length. (e) Other alkyltrialkyl and tetraalkylammonium bromides show that a variety of effects on the lipid phase transition can be obtained. (f) With the homologous series of phenyl-alkyl alcohols from benzyl alcohol to 4-phenyl butanol increasing the number of methylenes between the terminal OH and the benzene ring leads to greater interaction between solute and bilayer. The range of different effects obtained with the compounds studied offers a means for introducing various degrees and types of perturbation into membrane systems.