NMR spectroscopic search module for Spektraris, an online resource for plant natural product identification – Taxane diterpenoids from Taxus×media cell suspension cultures as a case study

Abstract

Development and testing of Spektraris-NMR an online spectral resource, is reported for the NMR-based structural identification of plant natural products (PNPs). Spektraris-NMRallows users to search with multiple spectra at once and returns a table with alist of hits arranged according to the goodness of fit between query data and database entries. For each hit, a link to a tabulated alignment of 1H-NMR and 13C-NMR spectroscopic peaks (query versus database entry) is provided. Furthermore, full spectroscopic records and experimental meta information about each database entry can be accessed online. To test the utility of Spektraris-NMR for PNP identification, the database was populated with NMR data (total of 466 spectra) for ∼250 taxanes, which are structurally complex diterpenoids (including the anticancer drug taxol) commonly found in the genus Taxus. NMR data generated used was then generated with metabolites purified from Taxus cell suspension cultures to search Spektraris-NMR, and were able to identify eight taxanes with high confidence. A ninth isolated metabolite could be assigned, based on spectral searches, to a taxane skeletal class, but no high confidence hit was produced. Using various spectroscopic methods, this metabolite was characterized as the taxane 2-deacetylbaccatin IV, a novel taxane. These results indicate that Spektraris-NMR is a valuable resource for rapid and reliable identification of known metabolites and has the potential to contribute to de-replication efforts in the search for novel PNPs.

Biosynthesis of Diterpenoids in Tripterygium Adventitious Root Cultures

“…Following HPLC fractionation of medium extracts of T. regelii adventitious root cultures, fractions containing metabolites that corresponded to prominent peaks in HPLC-QTOF-MS chromatograms were characterized by mass spectrometry (MS), tandem mass spectrometry (MS/ MS), and 1 H-NMR spectroscopy (Supplemental Fig. S2; Supplemental Methods and Data File S1), and metabolites were identified by searches against the Spektraris database (Fischedick et al, 2015). We ascertained the identities of five diterpenoids, were able to annotate three additional metabolites with high confidence, and acquired tentative identifications for an additional two metabolites (Table I and Fig.…”

Integrative Approaches for the Identification and Localization of Specialized Metabolites in Tripterygium Roots

“…Homogenates from root samples were extracted with acetone, and metabolites were separated and detected, based on previously published protocols, using HPLC-quadrupole time of flight (QTOF)-mass spectrometry (MS; Cuthbertson et al, 2013;Liu et al, 2013;Fischedick et al, 2015). Authentic standards of triptolide (Fig.…”

“…As a first step to remedy this situation we assembled the currently available information on the physicochemical properties of Tripterygium metabolites and integrated these data sets into the Spektraris online database (Cuthbertson et al, 2013;Fischedick et al, 2015). Based on our literature searches (as of June 2016), 415 specialized metabolites had been isolated and characterized from Tripterygium.…”

“…We have been working toward developing database resources that allow orthogonal searches with HPLC-MS, MS/MS, and NMR data sets. For example, our Spektraris database enabled the rapid identification of the major taxanes (diverse class of diterpenoids) in Taxus 3 media var Hicksii cell cultures (Cuthbertson et al, 2013;Fischedick et al, 2015). We have now expanded these efforts to include other highly diverse classes of specialized metabolites (those occurring in Tripterygium spp.)…”

“…While several online mass spectral databases now allow spectral comparisons for thousands of metabolites, including those occurring uniquely in plants, the coverage of open-source search tools for NMR spectra is much narrower (for review, see Lange, 2016). We developed Spektraris as a one-stop source for the integrative analysis of both accurate mass and NMR data (Cuthbertson et al, 2013;Fischedick et al, 2015). As part of this study, we updated the Spektraris database with multiple spectral data types for metabolites occurring in the genus Tripterygium.…”