Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Science and Technology, Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland.
Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Science and Technology, Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland.
Bioorg Chem. 2019 Dec;93:102751. doi: 10.1016/j.bioorg.2019.01.047. Epub 2019 Jan 30.
Presented work describes the first approach for the biocatalytic resolution of racemic mixtures of heterophosphonate derivative. Penicillium funiculosum and Rhodotorula mucilaginosa were successfully applied for the biological conversion of racemic mixture of 1-amino-1-(3'-pyridyl)methylphosphonic acid 3. Both microorganisms carried out the kinetically driven process leading to conversion of one from the substrate enantiomers, leaving the second one unreacted. Application of R. mucilaginosa allowed obtaining pure enantiomer of the substrate (yield 100%, e.e 100% - unreacted isomer) after 24 h of biotransformation of 3 in the laboratory scale process (Method E), applying biocatalyst pre-treatment step - 24 h of starvation. In case of other biocatalyst, application of whole cells of P. funiculosum in laboratory scale process, also resulted in conversion of the racemic mixture of substrate 3via oxidative deamination into ketone derivative, which was then bioreduced (second step of the process) into 1-hydroxy-1-(3'-pyridyl)methylphosphonic acid 4. This time two products were isolated: unreacted substrate and hydroxy compound 4. Conversion degree ranged from 30% (standard procedure, method A) to even 70% (with extra addition of sodium pyruvate - method B2). However, in this case, bioconversion was not enantioselective - products: amino- and hydroxyderivative were obtained as racemic mixtures. Both biocatalysts were also tested towards the scaling so other biocatalytic procedures were introduced - with immobilized fungal mycelium. In case of Rhodotorula mucilaginosa this approach failed (data not shown) but Penicillium funiculosum turned out to be active and also selective. Thus, application of this biocatalyst in the half-preparative scale, continuous-flow bioprocess (Method C2) resulted in the obtaining of pure S-3 (100% e.e.) isomer with the 100% of conversion degree, without any side products. Recorded NMR spectra allowed confirming the reaction progress and its selectivity and also postulating possible mechanism of conversion.
本文介绍了首例用于拆分外消旋杂磷酰衍生物的生物催化方法。青霉（Penicillium funiculosum）和粘红酵母（Rhodotorula mucilaginosa）被成功地应用于 1-氨基-1-（3′-吡啶基）甲基膦酸 3 的外消旋混合物的生物转化。两种微生物都进行了动力学驱动的过程，导致其中一个底物对映异构体转化，而另一个未反应。在实验室规模的过程（方法 E）中，应用粘红酵母，在生物催化剂预处理步骤-24 小时饥饿后，对 3 的外消旋混合物进行 24 小时的生物转化，可获得纯对映体的底物（产率 100%，ee 值 100%-未反应的异构体）。对于另一种生物催化剂，在实验室规模的过程中应用青霉完整细胞，也导致通过氧化脱氨将外消旋的 3 底物混合物转化为酮衍生物，然后将其生物还原（过程的第二步）成 1-羟基-1-（3′-吡啶基）甲基膦酸 4。此时，分离出两种产物：未反应的底物和羟基化合物 4。转化率范围从 30%（标准程序，方法 A）到甚至 70%（额外添加丙酮酸钠-方法 B2）。然而，在这种情况下，生物转化是非对映选择性的-产物为氨基和羟基衍生物，均为外消旋混合物。两种生物催化剂也进行了放大测试，引入了其他生物催化程序-固定化真菌菌丝体。对于粘红酵母，这种方法失败（未显示数据），但青霉表现出活性和选择性。因此，在半制备规模、连续流生物过程（方法 C2）中应用该生物催化剂，可获得纯 S-3（100%ee）异构体，转化率为 100%，无任何副产物。记录的 NMR 谱允许确认反应进展及其选择性，并推测转化的可能机制。
