John Mann

Metabolic pools associated with monoterpene biosynthesis in higher plants

Abstract Partial degradations of (+)-isothujone biosynthesised in Tanacetum vulgare after feeding IPP-[4- 14 C], DMAPP-[4- 14 C] or 3,3-dimethylacrylate-[Me- 14 C], and of geraniol and (+)-pulegone formed in Pelargonium graveolens and Mentha pulegium respectively after uptake of 3,3-dimethylacrylate-[Me- 14 C], indicated that none of these metabolites was a direct source of the part of the monoterpene skeleton derived hypothetically from DMAPP. Uptake of glucose-[U 14 C] into P. graveolens led, in contrast, to both IPP and DMAPP-derived moieties of geraniol being extensively labelled. Feeding of l -valine-[U- 14 C] and l -leucine-[U- 14 C] to all three plants resulted in negligible incorporation of tracer into monoterpenes. A soluble enzyme system prepared from foliage of T. vulgare that had been exposed to CO 2 -[ 14 C] for 20 days converted isotopically-normal IPP into GPP with the DMAPP-derived portion containing essentially all (>98%) of the radioactivity present. These observations and those previously obtained from feeding experiments with other [ 14 C]-labelled precursors on the same plant species are consistent with the occurrence of two metabolic pools of intermediates for monoterpene biosynthesis, one of which is probably protein-bonded.

Biosynthesis of monoterpenes in plants from 14C-labelled acetate and CO2☆

Abstract Degradation of (+)-isothujone biosynthesized by Tanacetum vulgare or Thuja plicata from acetate-[1- 14 C], -[2- 14 C] and -[2- 3 H 3 ] or from CO 2 -[14C] at physiological concentration revealed a pattern of asymmetric labelling whereby tracer predominantly (72–98% resided in that part of the skeleton derived from IPP. This is similar to the patterns previously obtained for uptake of MVA-[2- 14 C] but differed from those reported in other species with acetate-[ 14 C] as precursor. Within the IPP-derived moiety the 3 parts derived from acetate units were not equivalently labelled. Partial degradations of geraniol and (+)-pulegone formed in Pelargonium graveolens and Mentha pulegium after uptake of 14 C-labelled acetate or CO 2 showed that the C-2 units of the skeletons of these monoterpenes were also labelled to widely differing extents and these patterns persisted over a range of feeding and seasonal conditions. These results suggest that metabolic pools of acetyl-CoA and/or acetoacetyl-CoA exist in these plants. The general occurrence of such pools and the consequent nonequivalent labelling patterns in secondary metabolism could invalidate biosynthetic conclusions drawn from partial degradations of labelled natural products.

Symmetric bis-benzimidazoles are potent anti-staphylococcal agents with dual inhibitory mechanisms against DNA gyrase.

Various bis-benzimidazole derivatives have been reported to possess activity against Gram-positive pathogens. No mechanism of action has been elucidated to fully account for the antibacterial activity of this class of compounds. A group of symmetric bis-benzimidazoles (BBZ) designed as anticancer agents have previously been shown to possess moderate antiproliferative activity. We sought to assess the antibacterial activity and mechanism of action of BBZ compounds against Staphylococcus aureus. Antibacterial activities were assessed by determination of minimal inhibitory concentrations (MICs), time-kill curves, and scanning electron microscopy. Transcriptional responses to BBZ treatment were determined using whole genome microarrays. Activities against bacterial type II topoisomerases were investigated using in vitro supercoiling, decatenation, DNA binding, and DNA cleavage inhibition assays. MICs for EMRSA-16 were between 0.03 and 0.5 μg/mL. The compounds showed concentration-dependent bactericidal activity and induced cell swelling and lysis. Transcriptional responses to BBZ were consistent with topoisomerase inhibition and DNA damage. A subset of BBZ compounds inhibited S. aureus DNA gyrase supercoiling activity with IC(50) values in the range of 5-10 μM. This inhibition was subsequently shown to operate through both inhibition of binding of DNA gyrase to DNA and accumulation of single-stranded DNA breaks. We conclude that BBZ compounds are potent anti-staphylococcal agents and operate at least in part through DNA gyrase inhibition, leading to the accumulation of single-stranded DNA breaks, and by preventing the binding of gyrase to DNA.

Symmetric bis-benzimidazoles: new sequence-selective DNA-binding molecules

A series of bis-benzimidazole compounds with a head-to-head orientation have been designed as sequence-specific DNA binders; crystallographic analysis of oligonucleotide complexes has been combined with DNase I footprinting to confirm that the predicted optimal site for the core bis-benzimidazole motif is the four-base-pair sequence 5′-AATT; this sequence specificity results in inhibition of transcription at A/T sites and may be responsible for the cytotoxic and antitumour effects shown by these head-to-head bis-benzimidazoles.

1,2 Hydrogen-shifts in the biosynthesis of the thujane skeleton

Abstract Degradation of (+)-isothujone ( trans -thujan-3-one) biosynthesized in Tanacetum vulgare from (3 RS )-mevalonic acid (MVA)-[2- 14 C, 2- 3 H 2 ] showed that one hydrogen from C-2 of the precursor was specifically incorporated at C-4 of product whereas the other was lost. Feeding of α-terpineol-[9- 14 C, 4- 3 H 1 , 10- 3 H 3 ] ( p -menth-1-en-8-ol) yielded isothujone with the same isotope ratios as in precursor. These results indicate 1,2 hydrogen-shifts at two locations in the construction of the the thujane skeleton from α-terpineol or its biogenetic equivalent, and are consistent with a mechanism involving direct cyclization of the latter to a product that by-passes the formation of the biogenetic equivalent of terpinen-4-ol ( p -menth-1-en-4-ol) as an intermediate. (3 R )-MVA-[ 14 C, 3 H] was more effectively incorporated (up to 1.5 %) into (+)-isothujone in vivo during autumn or winter than in summer (up to 0.02%).

Antibacterial activity of head-to-head bis-benzimidazoles.

Symmetric bis-benzimidazole (BBZ) conjugates were profiled for activity against a range of Gram-positive and Gram-negative bacteria. para-Substituted ethoxy, amino and methoxy derivatives displayed potent bacteriostatic activity against meticillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, streptococci and Listeria monocytogenes. Moderate to good activity was also found against mycobacteria; two compounds were strongly active against logarithmic phase and hypoxia-induced latent Mycobacterium tuberculosis. No compound displayed significant activity towards Gram-negative bacteria. Only high concentrations of antibacterial BBZs showed cytotoxic effects towards fibroblasts, and the most active compound was well tolerated by zebrafish embryos.

The synthesis of 4-cyanoprogesterone: a potent inhibitor of the enzyme 5-α-reductase

We describe a number of synthetic approaches to 4-cyanoprogresterone. This compound is a potent inhibitor, both in vitro and in vivo of the enzyme 5-alpha-reductase

The discovery of a novel antibiotic for the treatment of Clostridium difficile infections: a story of an effective academic–industrial partnership

The story of the discovery of the bis-benzimidazole derivative SMT19969, which is currently in clinical trials against the pathogen Clostridium difficile.

Synthesis of novel N- and S-mustards as potential pro-drugs activated by bioreductive processes

We describe the synthesis of 4-{p-[bis-(2-chloroethyl)amino]phenyl}butanoic acid N-oxide, which is the N-oxide of the anti-cancer drug chlorambucil, and 4-[p-(2(chloroethylsulphinyl)phenyl]butanoic acid. The 3-nitro and 3,6-dinitro derivatives of chlorambucil have also been prepared.

Two synthetic routes to 2′,3′-dideoxy-3′-C-(hydroxymethyl)-4′-thionucleosides

A number of 2′,3′-dideoxy-3′-C-(hydroxymethyl)-4′-thionucleosides have been prepared from either (4R, 5S)-4,5-bis(tert-butyldimethylsiloxymethyl)tetrahydrofuran-2-one or from (2R, 3R)-1-benzyloxy-3-(benzyloxymethyl)hex-5-en-2-ol. The former was converted into methyl (3R, 4S)-4-acetylsulfanyl-5-(tert-butyldimethylsiloxy)-3-(tert-butyldimethylsiloxymethyl)pentanoate and then into 1-O-acetyl-2,3-dideoxy-3-C-(tert-butyldimethylsiloxymethyl)-5-O-(tert-butyldimethylsilyl)-4-thio-D-erythro-pentofuranose prior to coupling with silylated pyrimidines. The second key intermediate was converted into (2R, 3R)-1-benzyloxy-3-benzyloxymethyl-5,5-bis(benzylsulfanyl)pentan-2-ol and thence via the mesyl ester into 1-S-benzyl-5-O-benzyl-3-C-(benzyloxymethyl)-2,3-dideoxy-1,4-dithio-D-erythro-pentofuranose prior to coupling with silylated pyrimidines.