Institute of Biochemistry, Heinrich-Heine University Düsseldorf, Universitätsstrasse 1, 40225, Düsseldorf, Germany.
Institute of Organic Chemistry, University Stuttgart, Pfaffenwaldring 55, 70569, Stuttgart, Germany.
Chemistry. 2018 Aug 14;24(46):12010-12021. doi: 10.1002/chem.201802250. Epub 2018 Jul 19.
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.
cembranoids 是一类具有潜在治疗作用的 14 元含氧大环二萜化合物。cembranoid 支架的选择性晚期氧化仍然是化学催化剂的一个挑战，但可以通过酶来完成。本文描述了一种新的化学酶法途径，用于合成含氧 14 元大环化合物，包括 cembranoids。该途径结合了金属催化的环 closing metathesis 与随后的 P450 BM3 催化的羟化反应，得到了类似 cembranoids 的化合物。用一组合成的 14 元大环化合物进行系统的底物探测表明，P450 BM3 基生物催化剂的区域选择性随着环刚性的增加以及外环取代基的大小和极性而增加。通过首次球活性位点突变可以进一步提高酶的区域选择性。V78A/F87A 变体催化 cembranoid-ol (9S/R)-3 的羟化，C5 位的区域选择性达到 90%。对莫舍酯的广泛 NMR 分析和单晶 X 射线结构测定表明，这种 P450 BM3 突变体具有显著的立体选择性，这主要归因于底物立体化学，导致仅生成顺式 cembranoid-二醇（5S,9S）-4 和（5R,9R）-4。
