Fig. 4: Stabilization of a high-energy radical intermediate via S–C bond formation.

a, Liquid chromatography–MS extracted-ion chromatogram of the GDGT–MAS reaction showing retention time of AG substrate (black trace) at 9.1 min and lipid IV (yellow trace) at 7.8 min. b, Structure of thiolated AG (lipid IV, S–AG). c, Structural characterization of S–AG by tandem MS/MS, with dashed lines representing the fragmented bond. Fragmentation of S–AG predominantly cleaves the ether bond, resulting in a 313.2929 m/z daughter ion, indicating the observation of a thiolated phytanyl chain. In addition, less favoured fragmentation patterns (present in the dashed box) reveal a similar fragmentation pattern to the AG substrate, in which the presence of the 527.3718 m/z daughter ion indicates the neutral loss of the thiolated phytanyl chain, whereas the 559.3439 m/z daughter ion indicates the neutral loss of the phytanyl chain. Yellow fragments indicate sulfur-containing daughter ions of S-AG; red fragments are nonsulfur-containing daughter ions of S-AG. d,e, Time-dependent production of the S–C bond intermediates, S–AG and S–GTGT, observed during in vitro activity assays with wild-type GDGT–MAS and limiting SAM, suggests that S–AG is the intermediate for mAG and GTGT synthesis and that S–GTGT is the intermediate for GDGT synthesis. Production of S–AG (teal trace) compared with the formation of mAG (red trace) (d), and production of S–GTGT (brown trace) from GTGT (blue trace) towards the formation of GDGT (green trace) (e) are shown. The error bars represent one standard deviation for reactions conducted in triplicate, with the centre representing the mean.