Untargeted identification of alkyne containing natural products using ruthenium catalyzed azide alkyne cycloaddition reactions coupled to LC-MS/MS.

20 July 2021, Version 1
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

Alkyne containing natural products have identified from plants, insects, algae, fungi, and bacteria. This class of natural products have been characterized as having a variety of biological activities. Polyynes are a sub-class of acetylenic natural products that contain conjugated alkynes and are underrepresented in natural product databases due to the fact that they decompose during purification. Here we report a workflow that utilizes alkyne azide cycloaddition (AAC) reactions, using ruthenium or copper catalysts, followed by LC-MS/MS analysis to identify acetylenic natural products. In this report we demonstrate that AAC reactions with p-bromobenzyl azide resulted in p-bromobenzyl substituted triazole products that fragment to a common brominated tropylium ion. We were able to identify a synthetic alkyne spiked into the extract of Anabaena sp. PCC 7120 at a concentration of 10 μg/ml after optimization of MS/MS conditions. We then successfully identified the known natural product fischerellin A in the extract of Fischerella muscicola PCC 9339. Lastly, we identified the recently identified natural products protegenin A and C from P. protegens through a combination of genome mining and RuAAC reactions. We also compare CuAAC and RuAAC reactions and find that CuAAC reactions produce less by-products compared to RuAAC, but is limited to terminal alkyne containing compounds. We believe that both CuAAC and RuAAC reactions coupled to LC-MS/MS represent a method for the untargeted identification of acetylenic natural products.

Keywords

LC-MS/MS
RuAAC
CuAAC
alkyne
secondary metabolites

Supplementary materials

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Title
Supplementary Information for Untargeted identification of alkyne containing natural products using ruthenium catalyzed azide alkyne cycloaddition reactions coupled to LC-MS/MS.
Description
Supplementary Figures, Tables, NMR spectra and mass spectra
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