Abstract: | Saxitoxin (STX) and its analogues are potent voltage‐gated sodium channel blockers biosynthesized by freshwater cyanobacteria and marine dinoflagellates. We previously identified genetically predicted biosynthetic intermediates of STX at early stages, Int‐A′ and Int‐C′2, in these microorganisms. However, the mechanism to form the tricyclic skeleton of STX was unknown. To solve this problem, we screened for unidentified intermediates by analyzing the results from previous incorporation experiments with 15N‐labeled Int‐C′2. The presence of monohydroxy‐Int‐C′2 and possibly Int‐E′ was suggested, and 11‐hydroxy‐Int‐C′2 and Int‐E′ were identified from synthesized standards and LC‐MS. Furthermore, we observed that the hydroxy group at C11 of 11‐hydroxy‐Int‐C′2 was slowly replaced by CD3O in CD3OD. Based on this characteristic reactivity, we propose a possible mechanism to form the tricyclic skeleton of STX via a bicyclic intermediate from 11‐hydroxy‐Int‐C′2. |