RESUMEN

Cembranoids constitute a large family of 14-membered oxygenated macrocyclic diterpenoids with potential as therapeutic agents. Selective late-stage oxidations of cembranoid scaffolds remain a challenge for chemical catalysts but can be accomplished by enzymes. Here, a new chemoenzymatic route to oxyfunctionalized 14-membered macrocycles including cembranoids is described. This route combines a metal-catalyzed ring-closing metathesis with a subsequent P450 BM3-catalyzed hydroxylation and delivers cembranoid-like analogues. Systematic substrate probing with a set of synthetic 14-membered macrocycles revealed that the regioselectivity of a P450 BM3-based biocatalyst increased with increasing ring rigidity as well as size and polarity of the exocyclic substituents. Enzyme regioselectivity could further be improved by first-sphere active site mutagenesis. The V78A/F87A variant catalyzed hydroxylation of cembranoid-ol (9S/R)-3 d with 90 % regioselectivity for C5 position. Extensive NMR analysis of Mosher esters and single crystal X-ray structure determination revealed a remarkable diastereoselectivity of this P450 BM3 mutant depending on substrate stereochemistry, which led exclusively to the syn-cembranoid-diols (5S,9S)-4 and (5R,9R)-4.

Asunto(s)

Sistema Enzimático del Citocromo P-450/química , Ingeniería de Proteínas , Dominio Catalítico , Hidroxilación , Espectroscopía de Resonancia Magnética , Mutagénesis , Oxidación-Reducción , Especificidad por Sustrato

RESUMEN

A systematic study on ring-closing metathesis with Grubbs II catalyst to cembranoid macrocycles is described. Acyclic terpenoids with a functional group X in the homoallylic position relative to an RCM active terminus and substituents R, R(1) directly attached to the other terminal double bond were prepared from geraniol derived trienes and fragments that are based on bromoalkenes and dimethyl malonate. Such terpenoids were suitable precursors, despite the presence of competing double bonds in their framework. The size of R and R(1) is crucial for successful macrocyclization. Whereas small alkyl substituents at the double bond directed the RCM towards six-membered ring formation, cross metathesis leading to dimers dominated for bulkier alkyl groups. A similar result was obtained for precursors without functional group X. In the case of unsymmetrically substituted terpenoid precursor (R = Et, R(1) = Me) with homoallylic OTBS or OMe group, the RCM could be controlled towards formation of macrocyclic cembranoids, which were isolated with excellent E-selectivity. The role of the substituents was further studied by quantum chemical calculations of simplified model substrates. Based on these results a mechanistic rationale is proposed.