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Chen, Haibin

Ji, Yaoyao

Zhang, Chengxiao

Zhang, Yingxin

English

Engineering Conferences International

IMMOBILIZED BIOCATALYTIC PROCESS TO PREPARE ENANTIOPURE PREGABALIN INTERMEDIATE USING ENGINEERED HYDANTOINASE  Haibin Chen, Enzymaster (Ningbo) Bio-Engineering Co., Ltd. haibin.chen@enzymaster.com  Yingxin Zhang, Enzymaster (Ningbo) Bio-Engineering Co., Ltd. Yaoyao Ji, Enzymaster (Ningbo) Bio-Engineering Co., Ltd. Chengxiao Zhang, Enzymaster (Ningbo) Bio-Engineering Co., Ltd.  Key Words: Enantioselective Desymmetrization, Enzyme Immobilization, Process Development and Intensification, Hydantoinase Engineering  Advances in enzyme discovery and protein engineering have paved the way for the development of new efficient routes for drug synthesis that outcompete purely chemical approaches. Pregabalin is a generic drug for the treatment of epilepsy and neuropathic pain, and the annual demand of Pregabalin API is about 1,000 mt with steady growth. The conventional synthesis of enantiopure (S)-3-isobutyl-γ-aminobutyric acid (S-Pregabalin) entails iterative cycles of chemical or enzymatic kinetic resolution and separate chemical re-racemization under harsh conditions to reach a high atom economy, which is inefficient and not eco-friendly. [1]  An attractive alternative is the application of a hydantoinase for enantioselective desymmetrization of a prochiral cyclic imine via hydrolytic ring-opening. However, wildtype hydantoinases show only moderate activity and enantioselectivity towards the desired Pregabalin Intermediate (IM), and also suffer from poor stability and/or product inhibition in biocatalytic processes with high substrate loading. Computer-aided directed enzyme evolution was carried out to improve the overall performance of a selected D-hydantoinase.[2] NHOOCONH2CO2H3-Isobutylglutarimid (R)-3-Carbamoymethyl- 5-methylhexanoic acid(S)-PregabalinNH2CO2HHofmannrearrangementD-hydantoinase  An immobilized biocatalytic process is desired to both allow process intensification and to meet regulatory requirements. The engineered hydantoinase variant is covalently bound on a carrier, and this immobilized enzyme can be reused for at least 23 times in consecutive batch reactions. In each batch reaction, 250 g/L substrate are fully converted into Pregabalin IM with excellent enantiomeric excess (ee) of 99.5%.     References:  [1] P. J. Dunn, The importance of Green Chemistry in Process Research and Development. Chem. Soc. Rev., 2012, 41, 1452–1461. [2] Y. Zhang et al., Biocatalysts and Methods for the Synthesis of Pregabalin Intermediates. PCT/CN2022/128468, 2022.  048121620242 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 19 20 21 22 23Time (hours) needed to reach >99% conversion# of consecutive batch reactions

Immobilized biocatalytic process to prepare enantiopure pregabalin intermediate using engineered hydantoinase

https://dc.engconfintl.org/cgi/viewcontent.cgi?filename=0&article=1023&context=enzyme_xxvii&type=additional

Immobilized biocatalytic process to prepare enantiopure pregabalin intermediate using engineered hydantoinase

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Engineering Conferences International