One-step eantioselective bioresolution for (S)-2-chlorophenylglycine methyl ester catalyzed by the immobilized Protease 6SD on multi-walled carbon nanotubes in a triphasic system

Highlights

• A novel triphasic system was constructed for bioresolution of (S)-2-chlorophenylglycine methyl ester ((S)-1).

• Optically pure (S)-1 was prepared via one-step direct bioresolution by the triphasic system.

• The triphasic system consisted of the immobilized Protease 6SD on NH₂-MWCNT, aqueous and MTBE phases.

Abstract

(S)-2-chlorophenylglycine methyl ester ((S)-1) is a key chiral building block of clopidogrel, which is a widely administered antiaggregatory and antithrombotic drug. Herein, Protease 6SD was covalently immobilized on multi-walled carbon nanotubes (MWCNT), and the as-prepared immobilizate P-6SD@NH₂-MWCNT was applied in the enantioselective resolution of (R,S)-1 to yield (S)-1. In order to overcome the poor solubility of (R,S)-1 in aqueous solution, a novel triphasic reaction system constituting P-6SD@NH₂-MWCNT, aqueous phase and methyl tert-butyl ether (MTBE) as the organic phase was constructed, which simultaneously improved the substrate solubility and the immobilizate recyclability. Under the optimized reaction conditions, P-6SD@NH₂-MWCNT catalyzed 10 mM (R,S)-1 for 2 h, yielding optically pure (S)-1 (>99.0 % ee_s) with 70.74 % conversion of the (R,S)-1. Moreover, P-6SD@NH₂-MWCNT can be reused for 15 batches, displaying an exquisite recycling performance. It is for the first time that enantiomerically pure (S)-1 was successfully synthesized by protease-catalyzed one-step resolution.

Introduction

Enantiomerically pure compounds are important building blocks of pharmaceuticals, agrochemicals, and food ingredients (Ahmed et al., 2012; Su et al., 2014). (S)-2-chlorophenylglycine methyl ester ((S)-1) is a key chiral precursor of clopidogrel, an antiaggregatory and antithrombotic drug widely administered for the reduction of atherosclerotic events, including myocardial infarction, ischemic stroke, and peripheral vascular disease (Xue et al., 2013; Bluet et al., 2014).

Industrial synthesis of (S)-1 is currently achieved through chemical resolution by forming a diastereomeric salt. (Qiao et al., 2016; Lőrincz et al., 2016). However, the chemical resolution of (R,S)-1 demands expensive chiral resolving agents, such as tartaric acid or camphor sulfonic acid (Uhm et al., 2007), bringing about low yield (Fadnavis et al., 2008) and abundant solid waste (Chou, 2010), which are unfavorable for clean commercial production (Xue et al., 2013). In contrast, bioresolution has unique advantages of high enantioselectivity, high substrate scope extension capability, low energy consumption, and low environmental pollution (Zheng and Xu, 2011; Belafriekh et al., 2017; Yu et al., 2019). Furthermore, using immobilized enzyme instead of free enzyme overcomes some drawbacks, such as poor operational stability, and difficulty in separation and recycling (Salis et al., 2009; Lin et al., 2018; Qiu et al., 2019). It was reported the penicillin G acylase (PGA) immobilized on Eupergit C mediated enantioselective hydrolysis racemic N-phenylacetyl-2-chlorophenylglycine (N-phenylacetyl-2) gave 99 % ee of (S)-2-chlorophenylglycine ((S)-2) within 5 h (Fadnavis et al., 2008). Immobilized subtilisin, alcalase-CLEA resoluted (R,S)-N-Boc-2-chlorophenylglycine methyl ester afforded (S)-2 in 32 % yield and 98 % ee (Ferraboschi et al., 2010). Xue et al. developed the kinetic resolution of racemic N-phenylacetyl-2 using immobilized PGA in a recirculating packed bed reactor, producing (S)-2 in 97 % ee within 5 h (Xue et al., 2013). Despite of acceptable enantioselectivity, these methods demand environmentally unfriendly protection and deprotection procedures (Fadnavis et al., 2008), resulting in cost and environmental problems (Miyazawa et al., 2005). More importantly, in terms of atom economy, the routine starting from (S)-1 is efficient and economical. Unfortunately, no research on biosynthesis of (S)-1 directly from racemic (R,S)-1 is available up to now, thus making it of great significance to develop direct bioresolution of (R,S)-1.

Recently, nanomaterials present a broad range of applications in enzyme immobilization (Min and Yoo, 2014; Ranjan et al., 2018) due to their small size, large surface area, mechanical strength, thermal stability and other unique properties (Szelwicka et al., 2019; Xue et al., 2019). Amongst various nanomaterials, multi-walled carbon nanotubes (MWCNT) have attracted much attention. The surface-modified MWCNT was used to immobilize Candida rugosa lipase (CRL) via covalent binding with glutaraldehyde, and the immobilized CRL had 2-folds higher activity than the free CRL (Asmat et al., 2019). Alkaline lipase from Pseudomonas fluorescen was non-covalently immobilized on oxidized MWCNT using an adsorption technique (Boncel et al., 2013). Immobilization of Candida antarctica lipase B on MWCNT produced a high enzyme loading and increased enzyme stability (Szelwicka et al., 2019).

Currently, proteases and lipases are widely applied in enantioselective bioresolution of esters (Gadler and Faber, 2007; Wiggers et al., 2009; Li et al., 2010). In the present study, Protease 6SD was screened out for its capacity and enantioselectivity towards (R,S)-1, and was immobilized onto amino-functionalized multi-walled carbon nanotubes (NH₂-MWCNT). Furthermore, a triphasic system constituting the immobilizate P-6SD@NH₂-MWCNT, aqueous phase and methyl tert-butyl ether (MTBE) as the organic phase was developed for the biocatalysis of (S)-1 (Fig. 1A).