Biewenga Lieuwe, Crotti Michele, Saifuddin Mohammad, Poelarends Gerrit J
Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, Groningen 9713 AV, The Netherlands.
ACS Omega. 2020 Jan 28;5(5):2397-2405. doi: 10.1021/acsomega.9b03849. eCollection 2020 Feb 11.
The efficient engineering of iminium biocatalysis has drawn considerable attention, with many applications in pharmaceutical synthesis. Here, we report a tailor-made iminium-activated colorimetric "turn-on" probe, specifically designed as a prescreening tool to facilitate engineering of iminium biocatalysis. Upon complexation of the probe with the catalytic Pro-1 residue of the model enzyme 4-oxalocrotonate tautomerase (4-OT), a brightly colored merocyanine-dye-type structure is formed. 4-OT mutants that formed this brightly colored species upon incubation with the probe proved to have a substantial activity for the iminium-based Michael-type addition of nitromethane to cinnamaldehyde, whereas mutants that showed no staining by the probe exhibited no or very low-level "Michaelase" activity. This system was exploited in a solid-phase prescreening assay termed as activated iminium colony staining (AICS) to enrich libraries for active mutants. AICS prescreening reduced the screening effort up to 20-fold. After two rounds of directed evolution, two artificial Michaelases were identified with up to 39-fold improvement in the activity for the addition of nitromethane to cinnamaldehyde, yielding the target γ-nitroaldehyde product with excellent isolated yield (up to 95%) and enantiopurity (up to >99% ee). The colorimetric activation of the turn-on probe could be extended to the class I aldolase 2-deoxy-d-ribose 5-phosphate aldolase, implicating a broader application of AICS in engineering iminium biocatalysis.
亚胺生物催化的高效工程化已引起广泛关注，在药物合成中有许多应用。在此，我们报告了一种量身定制的亚胺激活比色“开启”探针，专门设计为一种预筛选工具，以促进亚胺生物催化的工程化。当探针与模型酶4-草酰巴豆酸互变异构酶（4-OT）的催化性Pro-1残基络合时，会形成一种颜色鲜艳的部花青染料型结构。与探针孵育后形成这种颜色鲜艳物种的4-OT突变体，对基于亚胺的硝基甲烷与肉桂醛的迈克尔型加成反应具有显著活性，而未被探针染色的突变体则没有或仅有极低水平的“迈克尔酶”活性。该系统被用于一种称为活化亚胺菌落染色（AICS）的固相预筛选试验，以富集活性突变体文库。AICS预筛选将筛选工作量减少了多达20倍。经过两轮定向进化，鉴定出两种人工迈克尔酶，其对硝基甲烷与肉桂醛加成反应的活性提高了多达39倍，以优异的分离产率（高达95%）和对映体纯度（高达>99% ee）得到目标γ-硝基醛产物。开启探针的比色激活可扩展到I类醛缩酶2-脱氧-d-核糖5-磷酸醛缩酶，这意味着AICS在亚胺生物催化工程中有更广泛的应用。
