Department of Chemical and Pharmaceutical Biology, University of Groningen, Antonius Deusignlaan 1, 9713 AV, Groningen, The Netherlands.
Chemistry. 2022 Oct 21;28(59):e202201651. doi: 10.1002/chem.202201651. Epub 2022 Aug 26.
Peroxygenases selectively incorporate oxygen into organic molecules making use of the environmentally friendly oxidant H O with water being the sole by-product. These biocatalysts can provide 'green' routes for the synthesis of enantioenriched epoxides, which are fundamental intermediates in the production of pharmaceuticals. The peroxyzyme 4-oxalocrotonate tautomerase (4-OT), catalysing the epoxidation of a variety of α,β-unsaturated aldehydes with H O , is outstanding because of its independence from any cost-intensive cofactor. However, its low-level peroxygenase activity and the decrease in the enantiomeric excess of the corresponding α,β-epoxy-aldehydes under preparative-scale conditions is limiting the potential of 4-OT. Herein we report the directed evolution of a tandem-fused 4-OT variant, which showed an ∼150-fold enhanced peroxygenase activity compared to 4-OT wild type, enabling the synthesis of α,β-epoxy-aldehydes in milligram- and gram-scale with high enantiopurity (up to 98 % ee) and excellent conversions. This engineered cofactor-independent peroxyzyme can provide new opportunities for the eco-friendly and practical synthesis of enantioenriched epoxides at large scale.
过氧化物酶选择性地将氧掺入有机分子中，利用环境友好的氧化剂 H 2 O 2 ，水是唯一的副产物。这些生物催化剂可以为手性富集的环氧化物的合成提供“绿色”途径，环氧化物是制药生产中的基本中间体。过氧化物酶 4-草酰基丁烯酸互变异构酶（4-OT）能够催化各种α,β-不饱和醛的环氧化，与 H 2 O 2 反应，由于其不需要任何昂贵的辅因子，因此非常出色。然而，其低水平的过氧化物酶活性和在制备规模条件下相应的α,β-环氧醛对映过量的降低限制了 4-OT 的潜力。在此，我们报告了串联融合的 4-OT 变体的定向进化，与 4-OT 野生型相比，该变体的过氧化物酶活性提高了约 150 倍，能够以毫克和克级规模合成高对映纯度（高达 98%ee）和优异转化率的α,β-环氧醛。这种工程化的无需辅因子的过氧化物酶可以为大规模环保和实用的手性富集环氧化物合成提供新的机会。
