Regio-and Stereoselective Hydroxylation Of Two Sesquiterpenes By P450 BM3

The selective hydroxylation of unactivated C-H bonds in complex natural products represents a difficult yet rewarding goal in synthetic organic chemistry.Curdione and its isomers are germacrane-type sesquiterpenoids with ten-membered ring,which possess multiple bioactive properties,such as anti-inflammatory and anticancer activities.Their transannular cyclization product,curcumalactones,upon acid treatment,possessing a spirolactone skeleton and have the potential to be used as lead compounds and scaffolds in drug discovery.Thus,an approach that can preparation of these mirror-image hydroxylated curdione and its three isomers derivatives and the corresponding curcumalactones with highly selectivity and activity is valuable.Here,we chosen P450 BM3 as catalyst for the selective hydroxylation of curdione and its isomer due to its high activity and wide substrate spectrum.For the rapidly engineering of P450 BM3,we showed that the exploitation of information derived from slightly reshaping the binding pocket and plotting noncovalent interaction of the substrate-enzyme complex for rationally designing iterative saturation mutagenesis constitutes a viable directed evolution strategy.In this strategy,the screening data for a minimal enriched P450 BM3 mutant library constructed by slightly reshaping of the binding pocket was collected,which can give effective structural characteristics for the computational simulations with the balance between speed and accuracy.Then,the following round of mutagenesis was performed to obtain the highest selective P450-based catalysts based on the computational data that gained from the noncovalent interaction of the substrate-enzyme complex which plotted by reduced density gradient versus the electron density multiplied.In this work,the combined approach was illustrated by the evolution of P450 BM3 mutants which enable nearly perfect regio-and diastereoselective hydroxylation of four sesquiterpenoid curdione isomers specifically at the neighboring positions C2 and C3 with unusually high activity,while the screening effort relatively low.And the P450 BM3 mutants were also used to prepare the desired hydroxylated products at a preparative scale.Cyperenoic acid,a sesquiterpenoid isolated from Croton crassifolius,possesses a patchoulane skeleton and have antiangiogenic-promoting effect.Its C7-and C9α-hydroxylated products can significantly inhibit the release of vascular endothelial growth factor(VEGF).An approach that can preparation of these hydroxylated cyperenoic acid derivatives with highly selectivity and activity is valuable.To prepare these hydroxylated products,cytochrome P450 BM3 monooxygenase was chosen as a catalyst for the hydroxylation of cyperenoic acid.A simple strategy,slightly reshaping the binding pocket of P450 BM3,is described to expedite the development of highly regio-and stereoselective P450 catalysts.In this work,P450 BM3 was evolved through one or two generations of mutations,and a highly enriched mutant library that contained fewer than 30 variants was then constructed.The obtained P450 BM3 variants achieved selective hydroxylation at positions C7(94%selectivity in the case of the F87A/A330W/F331L mutant)and C9α(90%regioselectivity and 100%stereoselectivity in the case of the L75V/F87A/T88F/A330W mutant)of cyperenoic acid and were also used to prepare the desired hydroxylated products at a preparative scale with high isolated yields.