Enzymatic hydroxylation of L-pipecolic acid by L-proline cis -4-hydroxylases and isomers separation
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ORIGINAL RESEARCH PAPER
Enzymatic hydroxylation of L-pipecolic acid by L-proline cis-4-hydroxylases and isomers separation Fan Lu . Jiao Chen . Hai Ye . Hongli Wu . Feng Sha . Fujun Huang . Fei Cao . Ping Wei
Received: 26 May 2020 / Accepted: 6 September 2020 Springer Nature B.V. 2020
Abstract Objectives Establish a complete and efficient method for the preparation of cis-5-hydroxy-Lpipecolic acids (cis-5HPA), including biotransformation and isomers separation and purification. Results For non-heme Fe(II)/a-KG-dependent dioxygenases, a-ketoglutarate (a-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
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10529-020-03002-z) contains supplementary material, which is available to authorized users. F. Lu J. Chen H. Wu (&) F. Huang F. Cao (&) P. Wei College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, 30 South Puzhu Road, Nanjing 211816, People’s Republic of China e-mail: [email protected] F. Cao e-mail: [email protected] H. Ye Nanjing Heron Pharmaceutical Science and Technology Co., Ltd, Life Science and Technology Town, Jiangning District, Nanjing 211000, People’s Republic of China F. Sha School of Chemical Biology & Biotechnology, Shenzhen Graduate School, Peking University, Nanshan District, Shenzhen 518055, People’s Republic of China
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/FeSO47H2O and a-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. Keywords Cis-5-hydroxy-L-pipecolic acids Fe(II) ions oxidation Isomer separation L-proline cis-4hydroxylases
Introduction Inactively asymmetric hydroxylation of carbon atoms is an important way to industrially synthesize chiral compounds (Shul’pin 2016). In biosynthesis, the mononuclear non-heme Fe(II)/a-KG-dependent dioxygenases have the catalytic properties for hydroxylation at the substrate specific carbon positions and
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have extremely chiral selectivity (Hausinger 2015; Martinez and Hausinger 2015). Proline hydroxylase and isoleucine hydroxylase are most universal which have relatively broad substrate specificity to hydroxylate a set of compounds structurally similar to their physiological product (Pet
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