Insights into the formation of 3-Methyl-2-butene-1-thiol in Cannabis sativa flowers through the identification of its glutathionylated and cysteinylated precursors

Odor emissions from cannabis cultivation have been described as “skunky”, a sensory characteristic recently associated with the compound 3-methylbut-2-ene-1-thiol (3MBT). However, the biosynthetic origin of this key odor-active compound in Cannabis sativa remains unknow. This study investigated the occurrence of the S-cysteine and S-glutathione conjugates of 3MBT (Cys-3MBT and GSH-3MBT) in CBD- and THC- dominant cannabis flowers and proposed them as possible precursor of the odor-thiol 3MBT. Both conjugates were identified for the first time in Cannabis sativa in CBD and THC cultivars by HPLC-QTOF-MS/MS and confirmed with reference standards. In vitro release experiments showed that GSH-3MBT generated 3MBT under moderate thermal conditions, whereas both conjugates released substantial amounts of 3MBT during fermentation with S.cerevisiae. Cys-3MBT and GSH-3MBT were quantified by HPLC-MS/MS at two stages of flower maturation in both cultivars, showing a decrease in their quantity during maturation. The THC-cultivar sample contained higher levels of both precursors at the earliest stage of maturation (Cys-3MBT: 13.01± 0.061 mg/kg ; GSH-3MBT 10.98 ± 0.14 mg/kg) than the CBD-cultivar (Cys-3MBT: 0.85 ± 0.057 mg/kg ; GSH-3MBT 2.96 ± 0.044 mg/kg) sample. Higher levels of the precursors in the THC-cultivar were also correlated with higher levels of free 3MBT (1.79 ± 0.19 mg/kg in THC and 0.075 mg/kg in CBD). These findings identify Cys-3MBT and GSH-3MBT as potential precursors of 3MBT and provide the basis for elucidating the biochemical origin and evolution of this odor-active thiol during Cannabis sativa maturation.