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

TL;DR

This paper presents a method combining HPLC/DAD/MS and 2D NMR to identify lichexanthone isomers in lichen extracts, which are important for chemotaxonomy.

Contribution

A dereplicative method using HPLC/DAD/MS and 2D NMR to identify lichexanthone isomers without requiring all isomers to be available.

Findings

HPLC/DAD/MS effectively separates norlichexanthones when retention times are known.

2D NMR provides detailed structural information on lichen extracts without needing all isomers.

HSQC and NOESY experiments confirm chlorine and methylation positions in lichexanthones.

Abstract

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.

Xanthones are secondary metabolites deriving from the 9H‐xanthen‐9‐one scaffold. In lichens, lichexanthones are important chemotaxonomic markers to distinguish between morphologically related species. They can be chlorinated at Positions 2, 4, 5 and 7, and methylated at Positions 3 and 6, resulting in a high number of isomers. We created a library of the 16 non‐methylated derivatives (called norlichexanthones), developed an HPLC/DAD/MS method capable of separating them and elaborated a 2D NMR strategy to identify the isomers.

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