title: Chiral separation of a diketopiperazine pheromone from marine diatoms using supercritical fluid chromatography.
abstract: The proline derived diketopiperazine has been identified in plants, insects and fungi with unknown function and was recently also reported as the first pheromone from a diatom. Nevertheless the stereochemistry and enantiomeric excess of this natural product remained inaccessible using direct analytical methods. Here we introduce a chiral separation of this metabolite using supercritical fluid chromatography/mass spectrometry. Several chromatographic methods for chiral analysis of the diketopiperazine from the diatom Seminavis robusta and synthetic enantiomers have been evaluated but neither gas chromatography nor high performance liquid chromatography on different chiral cyclodextrin phases were successful in separating the enantiomers. In contrast, supercritical fluid chromatography achieved baseline separation within four minutes of run time using amylose tris(3,5-dimethylphenylcarbamate) as stationary phase and 2-propanol/CO2 as mobile phase. This very rapid chromatographic method in combination with ESI mass spectrometry allowed the direct analysis of the cyclic dipeptide out of the complex sea water matrix after SPE enrichment. The method could be used to determine the enantiomeric excess of freshly released pheromone and to follow the rapid degradation observed in diatom cultures. Initially only trace amounts of c(d-Pro-d-Pro) were found besides the dominant c(l-Pro-l-Pro) in the medium. However the enantiomeric excess decreased upon pheromone degradation within few hours indicating that a preferential conversion and thus inactivation of the l-proline derived natural product takes place.
authors: Institute for Inorganic and Analytical Chemistry-Friedrich Schiller University Jena, Lessingstrasse 8, 07743 Jena, Germany. Electronic address: johannes.frenkel@uni-jena.de.
authors: Johannes
authors: J
authors: Frenkel
authors: Waters GmbH, Helfmann-Park 10, 65760 Eschborn, Germany. Electronic address: Carsten_Wess@waters.com.
authors: Carsten
authors: C
authors: Wess
authors: Laboratory of Protistology and Aquatic Ecology, Department of Biology, University Gent, Krijgslaan 281 S8 , 9000 Gent, Belgium. Electronic address: Wim.Vyverman@ugent.be.
authors: Wim
authors: W
authors: Vyverman
authors: Institute for Inorganic and Analytical Chemistry-Friedrich Schiller University Jena, Lessingstrasse 8, 07743 Jena, Germany. Electronic address: georg.pohnert@uni-jena.de.
authors: Georg
authors: G
authors: Pohnert
mesh_terms: D025924:Chromatography, Supercritical Fluid / Q000379:methods*; D003505:Cyclodextrins; D017377:Diatoms / Q000737:chemistry*; D054659:Diketopiperazines / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification*; D008401:Gas Chromatography-Mass Spectrometry; D010675:Pheromones / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification*; D013237:Stereoisomerism
chemical_list: D003505:Cyclodextrins; D054659:Diketopiperazines; D010675:Pheromones
keywords: Chiral separation; Cyclodextrine; Diketopiperazine; Pheromone; Supercritical fluid chromatography