Now showing 1 - 10 of 40
  • Publication
    Production of the Novel Lipopeptide Antibiotic Trifluorosurfactin via Precursor-Directed Biosynthesis
    Incorporation of fluorine into antibiotics can moderate their biological activity, lipophilicity and metabolic stability. The introduction of fluorine into an antimicrobial lipopeptide produced by Bacillus sp. CS93 via precursor-directed biosynthesis is described. The lipopeptide surfactin is synthesised non-ribosomally by various Bacillus species and is known for its biological activity. Administering 4,4,4-trifluoro-dl-valine to cultures of Bacillus sp. CS93 results in the formation of trifluorosurfactin in quantities sufficient for detection by LC–MS/MS. 19F NMR analysis of the culture supernatant revealed that the bulk of the fluorinated amino acid was transformed and thus was unavailable for incorporation into surfactin. Detection of ammonia, and MS analysis indicated that the transformation proceeds with deamination and reduction of the keto acid, yielding 4,4,4-trifluoro-2-hydroxy-3-methylbutanoic acid.
    Scopus© Citations 9  551
  • Publication
    The synthesis and biological testing of bacilysin analogues
    A series of compounds based on the structure of bacilysin were synthesised and tested for antibacterial activity. The key steps in the syntheses are the coupling of an iodide to a diketopiperazine (DKP) and mono-lactim ether scaffold, respectively. The diastereoselectivity of the coupling reactions was dependant on the scaffold, with selectivity for DKP of about 4:1 and mono-lactim ether exceeding 98:2. Subsequent elaboration of the compounds to give open chain dipeptides and DKPs that mimic the structure of bacilysin but substitute the epoxy ketone for a saturated or unsaturated ketone is described. Overall yield from coupling to final product was between 5 and 21 %, with the yield of the saturated products notably higher. The open chain dipeptides demonstrated moderate antibacterial and antifungal activity.
      302Scopus© Citations 3
  • Publication
    Biosynthesis of pyrrolylpolyenes in Auxarthron umbrinum
    (Royal Society of Chemistry, 2008-10-31) ;
    The biosynthesis of the pyrrolylpolyene rumbrin (1) in the fungus Auxarthron umbrinum was elucidated using feeding studies with labelled precursors. Incorporation of stable isotopes from [15N]-proline, [13C]-methionine and [13C]-acetate confirmed that these were the precursors of the pyrrole moiety, methyl groups, and backbone of rumbrin, respectively. Label-dilution experiments with pyrrole-2-carboxylate confirmed it was a direct precursor in the biosynthesis of rumbrin. Both 3- and 4-chloropyrrolecarboxylates were also accepted as precursors in polyene production.
    Scopus© Citations 18  607
  • Publication
    Bacterial defluorination of 4-fluoroglutamic acid
    Fluorinated amino acids are used as enzyme inhibitors, mechanistic probes and in the production of pharmacologically active peptides. Because enantiomerically pure 4-fluoroglutamate is difficult to prepare, the selective degradation of the l-isomer is a potentially convenient method of obtaining d-4-fluoroglutamate from the racemate. In this paper, we describe our investigations on the degradation of 4-fluoroglutamate by bacteria. Fluoride ion was detected in resting-cell cultures of a number of bacteria that were incubated with racemic 4-fluoroglutamate. Analysis of the culture supernatants by chiral gas chromatography–mass spectrometry revealed that only the l-isomer was degraded. The degradation of 4-fluoroglutamate was also examined in cell-free extracts of Streptomyces cattleya and Proteus mirabilis, and it was observed that equimolar concentrations of fluoride ion and ammonia were generated. The activity was located in the soluble fraction of cell extracts, thus is not related to the l-2-amino-4-chloro-4-pentenoic acid dehydrochlorinase previously identified in membrane fractions of P. mirabilis.
      1417Scopus© Citations 6
  • Publication
    Biodegradation of pentafluorosulfanyl-substituted aminophenol in Pseudomonas spp.
    The pentafluorosulfanyl (SF5–) substituent conveys properties that are beneficial to drugs and agrochemicals. As synthetic methodologies improve the number of compounds containing this group will expand and these chemicals may be viewed as emerging pollutants. As many microorganisms can degrade aromatic xenobiotics, we investigated the catabolism of SF5-substituted aminophenols by bacteria and found that some Pseudomonas spp. can utilise these compounds as sole carbon and energy sources. GC–MS analysis of the culture supernatants from cultures grown in 5-(pentafluorosulfanyl) 2-aminophenol demonstrated the presence of the N-acetylated derivative of the starting substrate and 4-(pentafluorosulfanyl)catechol. Biotransformation experiments with re-suspended cells were also conducted and fluorine-19 NMR analyses of the organic extract and aqueous fraction from suspended cell experiments revealed new resonances of SF5-substituted intermediates. Supplementation of suspended cell cultures with yeast extract dramatically improved the degradation of the substrate as well as the release of fluoride ion. 4-(Pentafluorosulfanyl)catechol was shown to be a shunt metabolite and toxic to some of the bacteria. This is the first study to demonstrate that microorganisms can biodegrade SF5-substituted aromatic compounds releasing fluoride ion, and biotransform them generating a toxic metabolite.
    Scopus© Citations 16  325
  • Publication
    Metabolism of fluoroorganic compounds in microorganisms: Impacts for the environment and the production of fine chemicals
    Incorporation of fluorine into an organic compound can favourably alter its physicochemical properties with respect to biological activity, stability and lipophilicity. Accordingly, this element is found in many pharmaceutical and industrial chemicals. Organofluorine compounds are accepted as substrates by many enzymes, and the interactions of microorganisms with these compounds are of relevance to the environment and the fine chemicals industry. One the one hand the microbial transformation of fluorinated compounds can lead to the generation of toxic compounds that are of environmental concern, yet similar biotransformations can yield difficult-to-synthesise products and intermediates, in particular derivatives of biologically active secondary metabolites. In this paper we review the historical and recent developments of organofluorine biotransformation in microorganisms, and highlight the possibility of using microbes as models of fluorinated drug metabolism in mammals.
    Scopus© Citations 49  1022
  • Publication
    Microbial biotransformation of aryl sulfanylpentafluorides
    We report, for the first time, the biotransformation of potential pollutants bearing the pentafluorosulfanyl (SF5-) functional group in a fungus and bacteria. Cunninghamella elegans transformed p-methoxy phenyl SF5 via demethylation; Pseudomonas knackmussii and P. pseudoalcaligenes KF707 transformed amino-, hydroxyamino- and diamino- substituted phenyl SF5, forming the N-acetylated derivatives as the main product. Cell-free extract of Streptomyces griseus transformed 4-amino-3-hydroxy-phenyl SF5 to the N-acetylated derivative in the presence of acetyl CoA, confirming that an N-acetyltransferase is responsible for the bacterial biotransformations. Approximately 25 % of drugs and 30 % of agrochemicals contain fluorine, and the trifluoromethyl group is a prominent feature of many of these since it improves lipophilicity and stability. The pentafluorosulfanyl substituent is seen as an improvement on the trifluoromethyl group and research efforts are underway to develop synthetic methods to incorporate this moiety into biologically active compounds. It is important to determine the potential environmental impact of these compounds, including the potential biotransformation reactions that may occur when they are exposed to microorganisms.
    Scopus© Citations 9  373
  • Publication
    Production of human metabolites of the anti-cancer drug flutamide via biotransformation in Cunninghamella species
    (Springer Netherlands, 2011-02-01) ;
    Fungi belonging to the genus Cunninghamella have enzymes similar to those employed by mammals for the detoxification of xenobiotics, thus they are useful as models of mammalian drug metabolism, and as a source for drug metabolites. We report the transformation of the anti-cancer drug flutamide in Cunninghamella sp. The most predominant phase I metabolites present in the plasma of humans, 2-hydroxyflutamide and 4-nitro-3-(trifluoromethyl)aniline, were also produced in Cunninghamella cultures. Other phase I and phase II metabolites were also detected using a combination of HPLC, GC–MS and 19F-NMR.
      510Scopus© Citations 18
  • Publication
    Purification and properties of fluoroacetate dehalogenase from Pseudomonas fluorescens DSM 8341
    The degradation of fluoroacetate by microorganisms has been established for some time, although only a handful of dehalogenases capable of hydrolyzing the stable C-F bond have been studied. The bacterium Pseudomonas fluorescens DSM 8341 was originally isolated from soil and very readily degraded fluoroacetate, thus it was thought that its dehalogenase might have some desirable properties. The enzyme was purified from cell free extracts and characterised: it is a monomer of 32,500 Da, with a pH optimum of 8 and is stable between pH 4 and 10; its activity is stimulated by some metal ions (Mg2+, Mn2+ and Fe3+), but inhibited by others (Hg2+, Ag2+). The enzyme is specific for fluoroacetate, and the Km for this substrate (0.68 mM) is the lowest determined for enzymes of this type that have been investigated to date.
      1291Scopus© Citations 34
  • Publication
    Filamentous fungal biofilm for production of human drug metabolites
    In drug development, access to drug metabolites is essential for assessment of toxicity and pharmacokinetic studies. Metabolites are usually acquired via chemical synthesis, although biological production is potentially more efficient with fewer waste management issues. A significant problem with the biological approach is the effective half-life of the biocatalyst, which can be resolved by immobilisation. The fungus Cunninghamella elegans is well established as a model of mammalian metabolism, although it has not yet been used to produce metabolites on a large scale. Here, we describe immobilisation of C. elegans as a biofilm, which can transform drugs to important human metabolites. The biofilm was cultivated on hydrophilic microtiter plates and in shake flasks containing a steel spring in contact with the glass. Fluorescence and confocal scanning laser microscopy revealed that the biofilm was composed of a dense network of hyphae, and biochemical analysis demonstrated that the matrix was predominantly polysaccharide. The medium composition was crucial for both biofilm formation and biotransformation of flurbiprofen. In shake flasks, the biofilm transformed 86% of the flurbiprofen added to hydroxylated metabolites within 24 h, which was slightly more than planktonic cultures (76%). The biofilm had a longer effective lifetime than the planktonic cells, which underwent lysis after 2×72 h cycles, and diluting the Sabouraud dextrose broth enabled the thickness of the biofilm to be controlled while retaining transformation efficiency. Thus, C. elegans biofilm has the potential to be applied as a robust biocatalyst for the production of human drug metabolites required for drug development.
    Scopus© Citations 25  672