A Systematic Approach to Discover New Natural Product Scaffolds Using Database-Derived Relative Mass Spectral Defects and Molecular Networking

Rapid advances in mass spectrometry (MS) data analysis
have accelerated
the identification of natural products from complex mixtures such
as natural product extracts. However, limitations in MS data in metabolite
libraries and dereplication strategies are still lacking for assigning
structures to known compounds and searching for unidentified compounds.
To overcome these limitations, we present an approach that combines
molecular networking with MS database-derived mass defect analysis
to preferentially discover new compounds with high structural novelty
in the initial stage of a discovery workflow. Specifically, unknown
metabolites or clusters generated from molecular networking are assigned
to a compound class based on their relative mass defects (RMDs) calculated
using open-source databases. If ancillary data such as ultraviolet
and MS/MS spectra of the unknown clusters are incongruent with the
RMD-assigned compound class, metabolites are considered to have a
new skeleton that exhibits a large difference in RMD value due to
structural changes. Here, we applied this RMD-assisted method to a
desert-derived bacterial strain library and validated it through the
discovery of brasiliencin A (1), a new 18-membered macrolide
from Nocardia brasiliensis. A putative
biosynthetic pathway of brasiliencin A was proposed through whole-genome
sequence analysis, and an additional 29 analogs were detected using
absolute mass defect filtering (AMDF) based on plausible biosynthetic
products. This led to the isolation of three additional macrolides,
brasiliencins B–D (2–4). The
structures of the brasiliencins (1–4) were fully elucidated through spectroscopic data analysis and quantum
chemical calculations including ROE distance and 13C NMR
chemical shift calculations, and experimental and theoretical electronic
circular dichroism (ECD). Brasiliencin A showed strong activity against Mycobacterium smegmatis and Streptococcus
australis (MIC = 31.3 nM and 7.81 μM, respectively)
compared to brasiliencin B (MIC = 1000 nM and 62.5 μM, respectively)
that differs at a single stereocenter.