OBJECTIVES Establish a complete and efficient method for the preparation of cis-5-hydroxy-L-pipecolic acids (cis-5HPA), including biotransformation and isomers separation and purification. RESULTS For non-heme Fe(II)/α-KG-dependent dioxygenases, α-ketoglutarate (α-KG) has great influence on the stability of Fe(II) ions, which is also the basic of the hydroxylation reaction to the substrate. L-pipecolic acids (L-Pip) was converted to cis-5HPA by whole-cell catalysis in water, which can reduce the loss of Fe(II) ions. 120 mM L-Pip can be transformed to 93% via cell and Fe(II) ions continuous supplementation under the reaction system optimization (the molar ratio of ascorbic acid/FeSO4·7H2O and α-KG/L-Pip were 8:1 and 1:1, respectively). After the catalytic reaction, the amino protection strategy was adopted to improve the resolution of isomer products on silica gel chromatography, and the amino protected cis-5HPA was obtained with a yield of 86.7%. CONCLUSIONS We established a method which is promising to be used for cis-5HPA largescale preparation. It also provides a suitable reference for this type of enzyme-catalyzed reaction and the hydroxy pipecolic acid isomers separation.

中文翻译：

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L-脯氨酸顺式4-羟化酶和异构体分离对L-哌啶酸进行酶促羟基化

目的建立一种完整、高效的制备顺式5-羟基-L-哌啶酸(cis-5HPA)的方法，包括生物转化和异构体分离纯化。结果 对于非血红素 Fe(II)/α-KG 依赖性双加氧酶，α-酮戊二酸 (α-KG) 对 Fe(II) 离子的稳定性影响很大，它也是底物羟基化反应的基础. L-哌啶酸 (L-Pip) 在水中通过全细胞催化转化为顺式 5HPA，可以减少 Fe(II) 离子的损失。在反应体系优化（抗坏血酸/FeSO4·7H2O和α-KG/L-Pip的摩尔比为8:1和1:1，分别）。催化反应后，采用氨基保护策略提高异构体产物在硅胶色谱上的分离度，得到氨基保护的cis-5HPA，收率为86.7%。结论我们建立了一种有望用于cis-5HPA大规模制备的方法。也为此类酶催化反应和羟基哌啶酸异构体的分离提供了合适的参考。