Development of a multi-enzymatic cascade reaction for the synthesis of trans-3-hydroxy-L-proline from L-arginine
Development of a multi-enzymatic cascade reaction for the synthesis of trans-3-hydroxy-L-proline from L-arginine
复制标题
开发从 L-精氨酸合成反式-3-羟基-L-脯氨酸的多酶级联反应
DOI：
10.1007/s00253-015-6992-4
复制
发表时间：
2016
影响因子：
5
通讯作者：
K. Kino
中科院分区：
文献类型：
--
作者：
R. Hara;S. Kitatsuji;K. Yamagata;K. Kino
天然存在的四种区域和立体异构体的1-羟基脯氨酸已被探索作为药物前体的可能性，但尚未实现反式-3-羟基-L-脯氨酸的选择性合成。我们的目的是开发一种新的生物催化不对称合成反式-3-羟基-L-脯氨酸的方法。到目前为止，我们主要研究根瘤菌精氨酸分解代谢途径：精氨酸酶和鸟氨酸环脱氨酶参与L-精氨酸降解为L-脯氨酸-鸟氨酸。假设精氨酸酶和鸟氨酸环脱氨酶分别作用于(2S，3S)-3-羟基精氨酸和(2S，3S)-3-羟基鸟氨酸，即可合成反式-3-羟基-L-脯氨酸。为了验证这一假设，我们克隆了L-精氨酸3-羟基酶、精氨酸酶和鸟氨酸环脱氨酶基因，并在大肠杆菌中进行了高效表达，随后进行了酶纯化。在对每种酶进行了鉴定和优化后，以L-精氨酸为起始底物，进行了三步反应：1-精氨酸3-羟基酶、精氨酸酶和鸟氨酸环脱氨酶。在此过程的第二步，(2S，3S)-3-羟基精氨酸通过精氨酸酶定量合成了可能的羟基鸟氨酸。核磁共振和手性高效液相色谱分析表明，该化合物的绝对构型为(2S，3S)-3-羟基鸟氨酸。最后以(2S，3S)-3-羟基鸟氨酸为原料，经鸟氨酸环脱氨酶选择性合成反式-3-羟基-L-脯氨酸。因此，我们成功地开发了一条新的合成路线，由三个反应组成，将L-精氨酸转化为反式-3-羟基-L-脯氨酸。良好的选择性使这一过程比传统的化学合成更简单、更有效。
Naturally occurringl-hydroxyproline in its four regio- and stereoisomeric forms has been explored as a possible precursor for pharmaceutical agents, yet the selective synthesis oftrans-3-hydroxy-l-proline has not been achieved. Our aim was to develop a novel biocatalytic asymmetric method for the synthesis oftrans-3-hydroxy-l-proline. So far, we focused on the rhizobial arginine catabolic pathway: arginase and ornithine cyclodeaminase are involved inl-arginine degradation tol-proline vial-ornithine. We hypothesized thattrans-3-hydroxy-l-proline should be synthesized if arginase and ornithine cyclodeaminase act on (2S,3S)-3-hydroxyarginine and (2S,3S)-3-hydroxyornithine, respectively. To test this hypothesis, we cloned the genes ofl-arginine 3-hydroxylase, arginase, and ornithine cyclodeaminase and overexpressed them inEscherichia coli, with subsequent enzyme purification. After characterization and optimization of each enzyme, a three-step procedure involvingl-arginine 3-hydroxylase, arginase, and ornithine cyclodeaminase (in this order) was performed usingl-arginine as a starting substrate. At the second step of the procedure, putative hydroxyornithine was formed quantitatively by arginase from (2S,3S)-3-hydroxyarginine. Nuclear magnetic resonance and chiral high-performance liquid chromatography analyses revealed that the absolute configuration of this compound was (2S,3S)-3-hydroxyornithine. In the last step of the procedure,trans-3-hydroxy-l-proline was synthesized selectively by ornithine cyclodeaminase from (2S,3S)-3-hydroxyornithine. Thus, we successfully developed a novel synthetic route, comprised of three reactions, to convertl-arginine totrans-3-hydroxy-l-proline. The excellent selectivity makes this procedure simpler and more efficient than conventional chemical synthesis.