University of Zagreb, Faculty of Chemical Engineering and Technology, Savska c. 16, HR-10000 Zagreb, Croatia.
Institute of Pharmaceutical Sciences, Albert-Ludwigs-Universität Freiburg, Albertstrasse 25, 79104 Freiburg, Germany.
J Biotechnol. 2018 Feb 20;268:71-80. doi: 10.1016/j.jbiotec.2018.01.013.
The Stetter reaction, a conjugate umpolung reaction, is well known for cyanide-catalyzed transformations of mostly aromatic aldehydes. Enzymatic Stetter reactions, however, have been largely unexplored, especially with respect to preparative transformations. We have investigated the kinetics of the MenD-catalyzed 1,4-addition of α-ketoglutaric acid to acrylonitrile which has shown that acrylonitrile, while an interesting candidate, is a poor substrate for MenD due to low affinity of the enzyme for this substrate. The kinetic model of the reaction was simplified to double substrate Michaelis-Menten kinetics where the reaction rate linearly depends on acrylonitrile concentration. Experiments at different initial concentrations of acrylonitrile under batch, repetitive batch, and fed-batch reactor conditions were carried out to validate the developed mathematical model. Thiamine diphosphate dependent MenD proved to be quite a robust enzyme; nevertheless, enzyme operational stability decay occurs in the reactor. The spontaneous reactivity of acrylonitrile towards polymerization was also taken into account during mathematical modeling. Almost quantitative conversion of acrylonitrile was achieved in all batch reactor experiments, while the yield of the desired product was dependent on initial acrylonitrile concentration (i.e., the concentration of the stabilizer additive). Using the optimized reactor parameters, it was possible to synthesize the product, 6-cyano-4-oxohexanoic acid, in a concentration of 250 mM. The highest concentration of product was achieved in a repetitive batch reactor experiment. A fed-batch reactor experiment also delivered promising results, especially regarding the short reaction time needed to achieve a 200 mM concentration of product. Hence, the enzymatic Stetter reaction with a highly reactive acceptor substrate can be performed on a preparative scale, which should enable similar transformations with acrylate, methacrylate, and methyl vinyl ketone.
施蒂特反应（Stetter reaction）是一种共轭反转反应，以氰化物催化的大多数芳醛转化而闻名。然而，酶促施蒂特反应在很大程度上尚未得到探索，特别是在制备转化方面。我们研究了 MenD 催化的α-酮戊二酸对丙烯腈的 1,4-加成的动力学，结果表明丙烯腈虽然是一个有趣的候选物，但由于酶对该底物的亲和力低，因此对 MenD 来说是一种较差的底物。该反应的动力学模型简化为双底物米氏动力学，其中反应速率与丙烯腈浓度呈线性关系。在分批、重复分批和补料分批反应器条件下进行了不同初始丙烯腈浓度下的实验，以验证所开发的数学模型。依赖硫胺素二磷酸的 MenD 被证明是一种相当稳健的酶；然而，酶的操作稳定性在反应器中会衰减。在数学建模过程中，还考虑了丙烯腈的自发聚合反应性。在所有分批反应器实验中，丙烯腈几乎都实现了定量转化，而所需产物的产率取决于初始丙烯腈浓度（即稳定剂添加剂的浓度）。使用优化的反应器参数，可以在浓度为 250 mM 的情况下合成产物 6-氰基-4-氧己酸。在重复分批反应器实验中达到了最高的产物浓度。补料分批反应器实验也取得了有希望的结果，特别是在达到 200 mM 产物浓度所需的短反应时间方面。因此，具有高反应性受体底物的酶促施蒂特反应可以在制备规模上进行，这应该可以实现与丙烯酯、甲基丙烯酯和甲基乙烯基酮类似的转化。
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