Confirmation of Δ²-sterenes as precursors of 3-methylsteranes

Δ²-sterenes have long been postulated to represent the precursors of 3β-alkylsteranes, although this relationship has remained speculative in the absence of proven intermediates. The observed prevalence of C₅ and C₆ alkyl moieties has also prompted the suggestion that 3β-alkylsteranes may derive from precursors with a sugar substituent at C-3. Recognition of the presence of prominent Δ²-sterenes and 3-methyl-Δ²-sterenes, coupled with minor concentrations of 3β-methylsteranes in a Cenomanian-Turonian sediment sequence deposited during OAE2 provides convincing evidence that validates a pathway for formation of 3β-methylsteranes from sterols. The identity of both suites of compounds as Δ²-sterenes is confirmed by the presence of diagnostic ions formed by Retro-Diels Alder rearrangement of the A-ring, which are absent in co-occurring Δ⁴- and Δ⁵-sterenes. The carbon number distributions of 3-methyl-Δ²-sterenes are similar to the proportions of the corresponding Δ²-sterene homologs (C₂₈, C₂₉, and C₃₀ versus C₂₇, C₂₈, and C₂₉), which supports their assignment as a product/precursor relationship. The relative retention times of the 3-methyl-Δ²-sterenes are uniform among the three series and consistent with expectations for methylation at C-3, which was confirmed by augmented abundances of 3β-methylsteranes after hydrogenation. These data support the hypothesis that 3β-methylsteranes originate from 3-methyl-Δ²-sterenes precursors formed by microbial methylation of Δ²-sterenes. We speculate that the organisms capable of this process are probably anoxygenic bacteria possessing the genes required for ring-methylation. Methylation of Δ²-sterenes may also represent an alternative diagenetic pathway to formation of 5α- and 5β-steranes by hydrogenation.