CuAAC click chemistry for the enhanced detection of novel alkyne-based natural product toxins

In the context of discovering and quantifying terminal alkyne-based natural products, here we report the combination of CuAAC click chemistry with LC-MS for the detection of polyether toxins (prymnesins) associated with harmful algal blooms.


General experimental conditions
Reagents and anhydrous solvents were purchased from by Sigma Aldrich and were used without further purification. Analytical grade solvents were purchased from Fischer Scientific. Glassware was oven-dried and purged with nitrogen immediately before use, and reactions requiring inert atmosphere were run under N2.
Reactions were monitored by thin-layer chromatography (TLC) on aluminium-backed, pre-coated silica gel plates (Silica Gel 60 F254, E. Merk) with the indicated eluents, and the TLC plates were

Extraction of prymnesin toxins from P. parvum cell cultures
Prymnesin toxin extractions were performed using a modified protocol of Manning & La Claire 2 . In brief, cultures of Prymnesium (150 mL) were grown in 5PSU f/2 media at room temperature in a 14/10 h (light/dark) cycle as previously described 3 . After 3 weeks, the cells were harvested by centrifugation (4000 × g for 5 minutes) and the supernatant discarded. The cells were suspended in cold acetone (2 mL, -20 °C) and subject to vortex mixing for two minutes. The resulting suspensions were then centrifuged at 30,000 × g for 5 minutes. The supernatant was discarded, being careful not to disturb the cell debris, and the pellets were subject twice more to the same acetone wash. The cell pellets were then suspended in MeOH (2 mL) and vortex mixed for two minutes, after which time the cell debris was pelleted by centrifugation (30,000 × g for 5 minutes) and the supernatant was collected.
This methanol extraction was repeated twice more, followed by three rounds of analogous extraction using n-PrOH. The MeOH and n-PrOH extracts were combined, dried in vacuo, and stored at -20 o C until further use.

CuAAC coupling of prymnesin extracts
Dried Prymnesium cell extracts were resuspended in 1 mL of 50% aqueous EtOH. An excess of 3-azido-7-hydroxycoumarin (500 µg in 200 µL of 50% EtOH) was added to the prymnesin extracts. A freshly prepared solution of 0.1M aqueous copper sulfate and 0.2M aqueous sodium ascorbate (10 µL) was added to start the coupling reaction. The reaction was left at room temperature in the dark overnight.
To follow reaction progress, thin layer chromatography analysis was carried out following solvent conditions described by Manning and La Claire 2 . The TLC plate was allowed to dry in a fume hood for 30 minutes before being visualized by long wave UV (λ 365 nm) (Fig. S6). The clicked extracts were dried in vacuo and stored at -20 o C until further use.