Dereplicative Combination of HPLC/DAD/MS and 2D NMR to Identify Lichexanthone Isomers in Lichen Extracts

Lichexanthones are the major xanthones found in lichens. They present a high degree of isomerism, which makes their identification tedious. Xanthones are known to occur in lichens according to chemosyndromes, and these compounds act as chemotaxonomic markers. Many lichens that produce xanthones are crustose lichens from Lecanora or Pertusaria genera, which often leads to small amounts of extracts being analyzed.

We aimed to set up a method able to identify the right isomers of lichexanthones contained in the extract of a xanthone-producing lichen. This method has to produce reliable results even without all the possible isomers at hand, as they are numerous and difficult to obtain.

The 16 norlichexanthones were obtained by a strategy combining isolation and synthesis. All of them were characterized by a full set of NMR experiments, highlighting key features, and an HPLC/DAD/MS method was developed. To exemplify the method, selected lichens were submitted to acetone microextraction and the extracts were analyzed by HPLC/DAD/MS and NMR.

All norlichexanthones were well separated by HPLC/DAD/MS, which enables their identification in the lichen extracts, provided that the retention time of all the isomers is known; ¹³C NMR is very informative about the position of chlorine in norlichexanthones but lacks sensitivity. 2D NMR provides a high level of structural information even on complex extracts.

In addition to HPLC/DAD/MS, NMR can be used directly on a lichen extract to confirm the positions of the chlorine atoms on the lichexanthone scaffold, thanks to the HSQC experiment. Furthermore, the NOESY experiment gives the position of methylations for a comprehensive overview of the substitution patterns involved in the extract of a xanthone-producing lichen, without requiring the entire series of the 64 derivatives.