title: Synthesis, configuration assignment, and simultaneous quantification by liquid chromatography coupled to tandem mass spectrometry, of dihydroanatoxin-a and dihydrohomoanatoxin-a together with the parent toxins, in axenic cyanobacterial strains and in environmental samples.
abstract: We have synthesized cis- and trans-dihydroanatoxin-a and cis- and trans-dihydrohomoanatoxin-a using a short synthetic route. The relative configuration of N-tert-butoxycarbonyl-cis-dihydroanatoxin-a was determined by X-ray crystallography, while that of N-tert-butoxycarbonyl-trans-dihydroanatoxin-a was confirmed by epimerization leading to the cis-diastereoisomer. The relative configuration of N-tert-butoxycarbonyl-trans- and cis-dihydrohomoanatoxin-a was inferred from their NMR spectra. Using an optimized LC-MS/MS analytical method and pure standards we have simultaneously determined anatoxin-a, homoanatoxin-a and their dihydroderivatives in axenic strains of cyanobacteria and in environmental samples from the Tarn River, France. However, in these analytical conditions, the cis- and trans-dihydroanatoxin-a and cis- and trans-dihydrohomoanatoxin-a could not be separated. In axenic strains, the dihydroderivatives represented less than 3% of the total toxin content, while in field samples dihydroanatoxin-a represented from 17% to 90% of the total toxin content. Thus, the reduction of anatoxin-a to dihydroanatoxin-a is predominant in the environment. The ratio of anatoxin-a concentration over that of homoanatoxin-a in axenic strains was variable, and among the eight strains studied we found three exclusive anatoxin-a producers and five producers of homoanatoxin-a and anatoxin-a, the latter representing from 0.5% to 2.0% of the total. In the strains studied, we have amplified by PCR, and sequenced the region of anaG coding for the methylation domain proposed to be responsible for the formation of homoanatoxin-a. The sequences showed at least 88% identity and we could not relate the toxin profile of the strains to the sequence of the methylation domain.
authors.affiliation: Chimie ParisTech, ENSCP, Laboratoire Charles Friedel, 11 rue Pierre et Marie Curie, 75231 Paris Cedex 05, France.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Mann
authors.forename: Mélanie
authors.initials: M
authors.lastname: Cohen
authors.forename: Florence
authors.initials: F
authors.lastname: Chapuis-Hugon
authors.forename: Valérie
authors.initials: V
authors.lastname: Pichon
authors.forename: Rabia
authors.initials: R
authors.lastname: Mazmouz
authors.forename: Annick
authors.initials: A
authors.lastname: Méjean
authors.forename: Olivier
authors.initials: O
authors.lastname: Ploux
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D018360:Crystallography, X-Ray; D000458:Cyanobacteria / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014326:Tropanes / Q000032:analysis*; D014871:Water Microbiology
chemical_list: D014326:Tropanes