Title: Untargeted identification of alkyne containing natural products using ruthenium catalyzed azide

Alkyne containing natural products have been 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 followed by LCMS/MS analysis to identify acetylenic natural products. In this report, we demonstrate that AAC 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 protegenin 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 less by-products 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 by-products 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.

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