Computational metabolomics reveals overlooked chemodiversity of alkaloid scaffolds in Piper fimbriulatum

Abstract

Plant specialized metabolites play key roles in diverse physiological processes and ecological interactions. Identifying structurally novel metabolites, as well as discovering known compounds in new species, is often crucial for answering broader biological questions. The Piper genus (Piperaceae family) is known for its special phytochemistry and has been extensively studied over the past decades. Here, we investigated the alkaloid diversity of Piper fimbriulatum, a myrmecophytic plant native to Central America, using a metabolomics workflow that combines untargeted LC-MS/MS analysis with a range of recently developed computational tools. Specifically, we leverage open MS/MS spectral libraries and metabolomics data repositories for metabolite annotation, guiding isolation efforts toward structurally new compounds (i.e., dereplication). As a result, we identified several alkaloids belonging to five different classes and isolated one novel seco-benzylisoquinoline alkaloid featuring a linear quaternary amine moiety which we named fimbriulatumine. Notably, many of the identified compounds were never reported in Piperaceae plants. Our findings expand the known alkaloid diversity of this family and demonstrate the value of revisiting well-studied plant families using state-of-the-art computational metabolomics workflows to uncover previously overlooked chemodiversity. To contextualize our findings within a broader biological context, we employed a workflow for automated mining of literature reports of the identified alkaloid scaffolds and mapped the results onto the angiosperm tree of life. By doing so, we highlight the remarkable alkaloid diversity within the Piper genus and provide a framework for generating hypotheses on the biosynthetic evolution of these specialized metabolites. Many of the computational tools and data resources used in this study remain underutilized within the plant science community. This manuscript demonstrates their potential through a practical application and aims to promote broader accessibility to untargeted metabolomics approaches.

Keywords: Piper fimbriulatum; Piperaceae; Wikidata; alkaloids; angiosperms; computational metabolomics; mass spectrometry; technical advance.

Figure 1

Analytical and computational workflow used in the present study. Different organs of the plant were sampled and analyzed by untargeted LC–MS/MS. Computational tools were used to aid the exploration of the detected chemical space, while publicly available spectral libraries and data repositories were used to focus isolation efforts toward structurally novel phytochemicals.

Figure 2

(a) A global molecular network of the chemical space detected in Piper fimbriulatum (leaf, stem, and root organs). Nodes predicted as alkaloids by CANOPUS are colored based on the alkaloid class. Alkaloid‐related molecular families are highlighted as MN1‐5. (b) Chemical scaffolds corresponding to the alkaloid classes highlighted in the global molecular network.

Figure 3

(a) Chemical structures of the Piper fimbriulatum alkaloids identified in the present study. Compounds confirmed by retention time match with commercial standard or NMR structural characterization are marked with asterisks. More information about the identified compounds are provided in Table S1. (b) Base peak LC–MS chromatogram of P. fimbriulatum leaf acquired in positive ionization mode. (c) Heatmap representing the abundance (shown as log10‐transformed LC–MS peak area) of the annotated alkaloids across the different plant organs (i.e., leaf, stem, root). It must be noted that the heatmap aims at highlighting the interorgan distribution of individual metabolites. Since absolute quantification for each compound was not performed, abundance comparisons between metabolites [e.g., compound (1) more abundant in the plant than compound (15)] cannot be made due to differences in ionization efficiency and other factors.

Figure 4

Angiosperm tree of life from Zuntini et al. (2024) mapped with literature reports, mined from Wikidata, of the alkaloid scaffolds considered in the present study (i.e., benzylisoquinoline, aporphine, piperolactam, piperidine, seco‐benzylisoquinoline). Each leaf in the tree corresponds to a representative species for each genus as described in the original publication. Different plant orders are separated by dashed black lines, and the Piperales order is highlighted in light blue. Reports for each scaffold are represented with different colored shapes (see legend in the figure). Reports for the Piper genus are highlighted in red. Colored arcs around the tree indicate the four main clades of angiosperms as described in the original publication: Magnoliids, Monocots, and Eudicots. The tree in the figure only shows the plant orders for which at least one scaffold was reported. A full version of the tree is provided in Figure S15. More details about the construction of the tree are provided in the Experimental procedures section. The figure was created using iTOL (Letunic & Bork, 2024). The original tree can be accessed at https://itol.embl.de/tree/14723112167224931731658296.