Regulation of P450 TleB catalytic flow for the synthesis of sulfur-containing indole alkaloids by substrate structure-directed strategy and protein engineering

摘要

Indole alkaloids have attracted considerable attention from synthetic chemists and biochemists for their structural diversity and important biological activities.Compared with traditional organic synthesis methods,the strategy of using cytochrome P450s'extraordinary abilities to selectively activate carbon-hydrogen bonds to assist in the synthesis of various indole alkaloids has the characteristics of short synthetic route,mild conditions and high atomic economy.Here,we utilized P450 monooxygenases HinD and TleB to synthesize a novel 6/5/8 tricyclic product from(S)-N-((S)-1-(4-fluoro-1H-indol-3-yl)-3-hydroxypropan-2-yl)-2-mercapto-3-methylbutanamide through the substrate structure-directed strategy.TleB was more effective in catalyzing C–S coupling,and was used to synthesize a series of 6/5/8 tricyclic indololactam derivatives to provide drug candidates.Interestingly,the S–S coupling product was observed in HinD catalysis,which was a minor product in the wild-type TleB catalysis.With the help of protein engineering,we accurately regulated the catalytic flow and reversed the selectivity of TleB to obtain the S–S coupling product.At the same time,the reaction mechanism was reasonably speculated by means of site blocking and protein-substrate complex analysis.