Untargeted Identification of Alkyne-Containing Natural Products Using Ruthenium-Catalyzed Azide Alkyne Cycloaddition Reactions Coupled to LC-MS/MS

Alkyne-containing natural products
have been identified from plants,
insects, algae, fungi, and bacteria. This class of natural products
has been characterized as having a variety of biological activities.
Polyynes are a subclass 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
followed by LC-MS/MS analysis to identify acetylenic natural products.
In this report, we demonstrate that alkyne azide cycloaddition reactions
with p-bromobenzyl azide result 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 protegenins
A and C from Pseudomonas protegens Pf-5 through a
combination of genome mining and RuAAC reactions. This is the first
report of RuAAC reactions to detect acetylenic natural products. We
also compare CuAAC and RuAAC reactions and find that CuAAC reactions
produce fewer byproducts compared to RuAAC but is limited to terminal-alkyne-containing
compounds. In contrast, RuAAC is capable of identification of both
terminal and internal acetylenic natural products, but byproducts
need to be eliminated from analysis by creation of an exclusion list.
We believe that both CuAAC and RuAAC reactions coupled to LC-MS/MS
represent a method for the untargeted identification of acetylenic
natural products, but each method has strengths and weaknesses.