Efficient Chemoenzymatic Synthesis of Optically Active Pregabalin

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Cite This: Org. Process Res. Dev. XXXX, XXX, XXX−XXX

Efficient Chemoenzymatic Synthesis of Optically Active Pregabalin from Racemic Isobutylsuccinonitrile Qin Zhang,†,‡ Zhe-Ming Wu,†,‡ Shuang Liu,†,‡ Xiao-Ling Tang,†,‡ Ren-Chao Zheng,*,†,‡ and Yu-Guo Zheng†,‡ Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering and ‡Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou 310014, P. R. China

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S Supporting Information *

ABSTRACT: An efficient chemoenzymatic route has been developed for the synthesis of optically active pregabalin (PGB) from isobutylsuccinonitrile (IBSN). (S)-3-cyano-5-methylhexanoic acid ((S)-CMHA), a critical chiral intermediate of PGB, was synthesized using regio- and enantioselective hydrolysis of IBSN by immobilized Escherichia coli cells harboring nitrilase BrNIT from Brassica rapa. The catalytic performances of immobilized cells were investigated, and high enantioselectivity (E > 150) and substrate conversion (>41.1%) were obtained at a substrate loading of 100 g/L by immobilized cells after 12 batches of reaction. The unreacted (R)-IBSN was recycled by racemization with a high yield of 94.5%, and the resultant (S)-CMHA was hydrogenated directly to the desired PGB with a high purity of 99.6% and optical purity of 99.4%. The input of raw materials and E factor of this chemoenzymatic route were demonstrated to be much lower than those of the first- and second-generation routes for PGB synthesis. KEYWORDS: pregabalin, nitrilase, chemoenzymatic route, isobutylsuccinonitrile, immobilization cost effectiveness in comparison to the first-generation manufacturing process,22 the process flow and atom economy still need to be improved. The nitrilase-mediated chemoenzymatic route for PGB from isobutylsuccinonitrile (IBSN) displays significant advantages over the second-generation process, such as more straightforward process flow (Scheme 2, from four steps to two steps) and higher atom economy.23 Nitrilases, including Arabidopsis thaliana (A. thaliana) NIT1 (AtNIT1), NIT-101, NIT-102, and NIT-103, have been reported for kinetic resolution of IBSN to synthesize (S)-3-cyano-5-methylhexanoic acid ((S)CMHA).23 AtNIT1 displayed the highest enantioselectivity (E > 150), but the substrate loading was low (20 g/L). Previously, a nitrilase from Brassica rapa (B. rapa) (BrNIT) was screened in our laboratory, which exhibited good catalytic performances toward IBSN at high substrate concentration (100 g/L).24 However, utilization of recombinant Escherichia coli (E. coli) cells harboring BrNIT led to difficulties in downstream (S)CMHA separation, purification, and subsequent hydrogenation. Cell immobilization is often regarded as an appropriate strategy for efficient industrial production,25,26 since the utilization of immobilized cells not only enhances the reusability of biocatalysts but also simplifies the separation of products.27−30 In addition, immobilized cells have better catalytic performances (thermal stability, pH variation, or activity) than free cells31−33 and easily adapt to the difference between the cellular environment and the industrial setting.34,35

1. INTRODUCTION Optically active (S)-3-(aminomethyl)-5-methyl hexanoic acid (pregabalin, PGB) is a lipophilic γ-aminobutyric acid analogue.1 It is launched by Pfizer as Lyrica for the treatment of nervous system disorders, such as epilepsy, post-herpetic neuralgia, diabetic peripheral neuropath, anxiety and social phobia, and restless legs syndrome.2−4 PGB has already taken the lead in the global neuropathic pain drug with significant advantages of lower dosages, fewer side effects, and longer duration of action5,6 and became a blockbuster with global sales of $4.97 billion in 2018. The profit of enormous market demand for PGB arouses great interest and enables its synthesis to become a hotspot in the pharmaceutical industry. Although the chemical synthesis routes for PGB are well studied,1,7−10 they usually require expensive catalysts or chiral auxiliaries, excessive usage of organic solvents, and harsh reaction conditions.11 As an alternative, the chemoenzymatic route for PGB is attracting tremendous attentions due to its advantages of environmental friendliness and mild reaction conditions.12 Up to now, a number of chemoenzymatic routes have been reported for the synthesis of PGB with lipase and esterase,13−15 nitrilase,16−18 or ene-reductase19 as biocatalysts. However, the application of these enzymes suffers from poor substrate tolerance,17 enantioselectivity,20 and catalytic activity,21 restricting their practical applications. Significantly, a chemoenzymatic route using a commercially available lipase (Lipolase) as a biocatalyst has been successfully developed for large-scale production of PGB, which is referred to be the second-generation manufacturing route (Scheme 1).13 The process comprises four steps including enzymatic hydrolysis, decarboxylation, chemical hydrolysis, and hydrogenation. Although this route displays high environmental benefit and © XXXX American Chemical Society

Received: June 24, 2019

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DOI: 10.1021/acs.oprd.9b00285 Org. Process Res. Dev. XXXX, XXX, XXX−XXX

Organic Process Research & Development

Article

Scheme 1. Second-Generation Manufacturing Process for PGB

Scheme 2. Chemoenzymatic Synthesis of PGB Catalyzed by Immobilized Cells Harboring Nitrilase

China). Raney nickel catalyst (T-1) was provided by Dalian Tongyong Chemicals Co., Ltd. (Dalian, China). Diatomite and poly(ethyleneimine) (PEI) were bought from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Sodium ethoxide (ca. 20% in ethanol) was purchased from J&K Chemical Co., Ltd. (Beijing, China). All the other chemical reagents were of analytical grade as commercially available. 2.2. Fermentation of Recombinant E. coli Overexpressing BrNIT. A single colony of E. coli BL21 (DE3)/ pET28b(+)-BrNIT was inoculated in an LB medium and cultivated at 37 °C and 150 rpm. When the optical density of E. coli cells at 600 nm reached 0.6−0.8, the primary seed culture was sterilely transferred at an inoculation concentration of 5% (v/v) to a 50 L fermenter containing 30 L of sterilized LB medium. The fermentation process was carried out at 37 °C for 3 h with an aeration of 1.3 vvm (air volume/culture volume/minute) and agitation of 500 rpm. The pH of the fermentation medium was adjusted to 7.0 using ammonia and phosphoric acid. Subsequently, 15 L of secondary seed culture was sterilely transferred to a 500 L fermenter containing 300 L of sterilized LB medium at the same inoculation size. The fermentation parameters were the same as above. After incubation for 4 h, 150 L of third seed medium was then inoculated into a 5000 L fermenter containing 3000 L of sterilized fermentation medium (pH 7.0), which was consisted of 15 g/L peptone, 12 g/L yeast extract, 10 g/L NaCl, 15 g/L glycerol, 5 g/L (NH4)2SO4, 2.28 g/L K2HPO4·3H2O, 1.36 g/L KH2PO4, and 0.375 g/L MgSO4·7H2O. The fermentation medium was incubated at 37 °C for 4 h with an aeration of 1.3 vvm and agitation of 180 rpm. Subsequently, lactose (12 g/L) was added to induce the expression of BrNIT protein at 28 °C. After 10 h of induction, recombinant E. coli cells were harvested by centrifugation and stored at −20 °C until used for further study. 2.3. Biocatalytic Hydrolysis of IBSN in 5 L Reactor. Enzymatic hydrolysis of IBSN was performed in a 5 L reactor containing 3 L of Tris-HCl buffer (50 mM, pH 8.0), 300 g of IBSN, and 39.8 g of dry cell weight (DCW) whole cells at 30 °C with an agitation rate of 300 rpm for 6 h. The reaction

In the present study, a commercially feasible chemoenzymatic process for synthesis of PGB with immobilized E. coli cells harboring BrNIT was developed. The immobilized cells were employed for biosynthesis of (S)-3-cyano-5methylhexanoic acid ((S)-CMHA) with prominent biocatalytic performances. The resultant (S)-CMHA was subsequently converted to PGB by one-step hydrogenation, and the unreacted (R)-IBSN was recycled by racemization. The high atom economy, simple process flow, and low catalyst cost demonstrated the great potential of this process for industrial production of PGB.

2. EXPERIMENTAL SECTION 2.1. Strains, Culture Condition, and Chemicals. Gene sequences of nitrilase BrNIT from B. rapa (ABM55734.1) and nitrilase AtNIT1 from A. thaliana (NP_851011.1) were synthesized by Shanghai Xuguan Biotechnological Development Co., Ltd. (Shanghai, China) and inserted into the expression vector pET28b(+). E. coli BL21 (DE3) was used as the expression host for the recombinant plasmids. The recombinant E. coli BL21 (DE3)/pET28b(+)-BrNIT and recombinant E. coli BL21 (DE3)/pET28b(+)-AtNIT1 were stored in our laboratory. The recombinant E. coli BL21 (DE3) strains were cultured at 37 °C in a Luria-Bertani (LB) medium with following compositions: 5 g/L yeast extract, 10 g/L peptone, 10 g/L NaCl, and kanamycin at a final concentration of 50 μg/mL. The expression of recombinant enzymes was induced by 0.1 mM IPTG at 28 °C and 150 rpm for 10 h. The whole cells were harvested by centrifugation at 9000 rpm for 10 min at 4 °C and washed twice with saline solution (0.9%, w/v). The substrate IBSN was synthesized according to a previously described method with modifications.23 (S)CMHA was purchased from Hui Chem Co., Ltd. (Shanghai, China). PGB was purchased from J&K Chemical Co., Ltd. (Beijing, China). (Trimethylsilyl)diazomethane (TMSD) solution in hexane (2.0 M) was obtained from Macklin Biochemical Co., Ltd. (Shanghai, China). Glutaraldehyde (GA) was purchased from J&K Chemical Co., Ltd. (Beijing, B



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extracted with dichloromethane (2 × 300 mL). The combined organic layers (∼600 mL) were then mixed with 30% sodium hydroxide solution. After extraction separation, the aqueous solution (∼200 mL, containing 48.1 g of (S)-CMHA) was further decolorized with activated carbon. Finally, the purified solution of (S)-CMHA (∼185 mL, containing 45.6 g of (S)CMHA) was obtained in 85.1% isolated yield. The enantiomeric excess (ee) of (S)-CMHA was 98.1%. For spectral characterization, an ammonium salt of (S)-CMHA was prepared. The pH of the purified solution was adjusted to 1.8 with 4.0 M HCl, and the solution was extracted with dichloromethane. Liquid ammonia was injected into the organic layers under stirring conditions, affording a white solid under low temperatures that identified by NMR and MS (ESI). The spectral data of (S)-CMHA were in line with the previous report.18 2.9. Hydrogenation of (S)-CMHA to PGB. Hydrogenation reaction was performed at room temperature for 9 h under 2.0 MPa H2 with 180 g of concentrated aqueous solution of (S)-CMHA (24.6% w/w, 44.3 g), 5.4 g of NaOH, and 27 g of Raney nickel catalyst (T-1) in a 500 mL hydrogenation vessel (CJK-0.5, Weihai, China). The reaction process was monitored by HPLC analysis. When hydrogenation reaction of (S)-CMHA was completed, the T-1 catalyst was separated by filtration and used for the next batch. The pH of the aqueous solution of PGB (41.3 g) was adjusted to 6.0 using 6.0 M HCl, and the resulting solutions were stirred slowly at low temperature (0−5 °C) until no further white solid was precipitated (0.5−1.0 h). After filtration, the white solid was dried in a vacuum oven at 50 °C for 24 h to yield crude PGB (30.5 g, 86.7% purity, 99.4% ee). For the purpose of obtaining high-purity PGB, both crude PGB and activated carbon were added into a solution of 60% isopropyl alcohol (400 mL), and the resulting solution was refluxed at 70 °C for 1 h. After filtration and drying again, high-quantity PGB (21.1 g, 99.6% purity, 99.4% ee) was obtained with a yield of 62.1%. 1 [α]25 D + 10.7° (c = 1.06, H2O). H NMR (500 MHz, D2O, δ): 2.99 (ddd, J = 19.8, 13.0, 6.1 Hz), 2.29 (ddd, J = 22.1, 14.7, 6.6 Hz), 2.21−2.14 (m), 1.66 (td, J = 13.5, 6.7 Hz), 1.23 (dd, J = 10.9, 3.9 Hz), 0.90 (t, J = 6.3 Hz). 13C NMR (126 MHz, D2O, δ): 183.71 (s), 46.32 (s), 43.37 (s), 43.21 (s), 34.27 (s), 26.98 (s), 24.59 (s), 24.12 (s). MS (ESI) m/z: [M + H]+ 160.1. 2.10. Racemization of (R)-IBSN. The unreacted (R)IBSN (50 g) was separated from the reaction mixture by extraction with methylene dichloride. Subsequently, methylene dichloride was completely removed by vacuum rotary evaporation. The anhydrous oily liquid (R)-IBSN obtained was mixed with 50 mL of sodium ethylate solution (20% w/v in the ethanol), and the mixture was heated to reflux for 4 h. After the completion of racemization, the mixture was washed twice with 10% hydrochloric acid and then separated. Finally, 47.25 g of racemic IBSN (94.5% yield) was obtained by reduced pressure distillation heated by an oil bath. The ee of IBSN obtained was monitored by GC analysis. 2.11. Analytical Methods. The concentrations of (S)CMHA and PGB were determined by HPLC using a Welchrom C18 column (250 mm × 4.6 mm, 5 μm) and a UV detector at 210 nm, with 85% (v/v) buffer (containing 3.4 g/L KH2PO4, pH 6.3) and 15% (v/v) methanol at a flow of 1 mL/min at 30 °C. The retention times were 10.12 min for PGB and 29.41 min for (S)-CMHA. To determine the optical purity of IBSN and CMHA, a precolumn derivatization gas chromatography method was

samples (200 μL) were taken every 1 h and acidified by the addition of 50 μL of 2.0 M HCl. The resulting mixtures were extracted with 800 μL of ethyl acetate and centrifuged at 12,000 rpm for 2 min. The organic layer was dried over anhydrous Na2SO4 for gas chromatography (GC) analysis. The resultant (S)-CMHA was separated from the reaction mixture by centrifugation, filtration, and extraction. The concentration of (S)-CMHA was measured by HPLC analysis. 2.4. Preparation of Immobilized E. coli Cells. Harvested whole cells (26.5 g of DCW) harboring BrNIT were suspended in 1000 mL of distilled water. When the whole cells were completely blended, 6 g of diatomite was added into the cell suspension and thoroughly stirred for 1 h at room temperature. The cross-linking step was carried out by addition of 5% PEI solution (30 mL) to the resulting mixture of diatomite and whole cells, and the mixture was stirred for 1 h at room temperature. Subsequently, 25% GA solution (10 mL) was added to the suspension solution.27 After stirring for 1 h at room temperature, the reaction mixture was filtered to separate the immobilized E. coli cells. The immobilized E. coli cells were washed three times with distilled water to remove the residual reagents. Finally, 132.6 g of immobilized cells was obtained and employed for the following research. 2.5. Enzyme Assay. Biotransformation trials were carried out in 10 mL of reaction mixture consisting of 1 g of IBSN, 50 mM Tris-HCl buffer (pH 8.0), and 0.27 g of DCW whole cells or 1.33 g of immobilized cells (corresponding to 0.27 g of DCW whole cells). The mixtures were incubated at 30 °C with shaking at 200 rpm for 30 min. All assays were performed in triplicate. One unit of the enzyme activity was defined as the amount of enzyme that produced 1 μmol of CMHA per minute. 2.6. Optimization of Biotransformation Conditions. The biotransformation reactions mediated by immobilized cells were carried out in a 10 mL reaction system using different variable parameters. The effect of temperature on the catalytic property of immobilized cells was investigated at temperatures ranging from 25 to 45 °C. The effect of pH was examined by using different buffers including sodium phosphate buffer (50 mM, pH 6.0−8.5), Tris-HCl buffer (50 mM, pH 7.0−9.0), and glycine−NaOH buffer (50 mM, pH 8.5−10.5). The effect of different substrate loadings (100, 130, and 150 g/L) on the hydrolysis of IBSN via immobilized cells was also studied. 2.7. Reusability of Immobilized Cells. To assess the reusability of immobilized cells, the biotransformation reaction was performed in a repeated batch operation mode with 300 mL of reaction mixture (pH 8.0) containing 39.78 g of immobilized cells and 30 g of racemic IBSN. The reaction mixtures were incubated at 30 °C for 3 h with shaking at 300 rpm. After each batch of reaction, immobilized cells were recycled by filtration, washed three times with distilled water, and subsequently used in the next biotransformation. The reaction process was monitored by GC analysis. The filtrate was collected and combined for downstream separation of (S)CMHA. 2.8. Isolation of (S)-CMHA. Immobilized cells were filtered after the enzymatic reaction was completed. The combined filtrate (∼1 L, containing 53.6 g of (S)-CMHA) was directly extracted with dichloromethane (300 mL) to remove the unreacted IBSN. As the pKa value of (S)-CMHA was about 3.0, the pH of the aqueous solution (∼1 L) was subsequently adjusted to 1.8 with concentrated hydrochloric acid and then C



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volume of 3000 L) under the optimized culture conditions (data not shown). The fermentation process was monitored by measuring the biomass and nitrilase activity at regular intervals. As illustrated in Figure 1, the BrNIT activity and biomass were obtained as 3395 U/L and 11.4 g (DCW)/L after fermentation for 11 h, respectively, which were suitable for large-scale applications.

established. Samples were treated with an excess of TMSD to give their methyl ester derivatives and determined on an Agilent 6890 N gas chromatography equipped with an FID and a chiral capillary column BGB-174 (30 m × 0.25 mm, 0.25 μm film thickness). The column flow rate was 1.5 mL/min (helium). The temperatures of the injector and detector were set at 250 °C, and the oven temperature was programmed as follows: hold at 120 °C for 15 min, 10 °C/min to 170 °C, and hold at 170 °C for 10 min. One microliter sample was injected, and the retention times of (R)-CMHA, (S)-CMHA, (S)-IBSN, and (R)-IBSN were 12.76, 13.16, 24.61, and 25.02 min, respectively. The conversion and enantiomeric ratio (E) were calculated based on the enantiomeric excess of the residual substrate (ees) and that of the product (eep) as the method described.36 The biomass was calculated as dry cell weight. A 30 mL of fermentation liquor of recombinant E. coli was centrifuged at 8000 rpm for 10 min, which was dried to a constant weight at 80 °C. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance III (Bruker, Switzerland) operating at 500 MHz for 1H NMR and 126 MHz 13C NMR acquisitions. Highresolution mass (MS) spectra were recorded on a Bruker micrOTOF-QII.

Figure 1. Fermentation of recombinant E. coli cells in 5000 L fermenter with a working volume of 3000 L. Recombinant cells were cultivated at 37 °C for 4 h and then at 28 °C for 10 h with an aeration of 1.3 vvm and agitation of 180 rpm.

3. RESULTS AND DISCUSSION 3.1. Production of Recombinant Cells Harboring BrNIT in 5000 L Fermenter. AtNIT1 and BrNIT were identified as suitable biocatalysts for hydrolysis of IBSN in terms of enantioselectivity and activity.16,24 Here, the catalytic performances of recombinant cells harboring BrNIT and AtNIT1 were compared. The results showed that the whole cell activity of BrNIT reached 147.8 U/g DCW, which was 5.6fold higher than that of AtNIT1 (26.3 U/g DCW). Moreover, the recombinant cells harboring BrNIT exhibited higher substrate tolerance than recombinant cells harboring AtNIT1, and both of them exhibited high enantioselectivities (E > 150). Concretely, as listed in Table 1, after 10 h of

3.2. Biocatalytic Hydrolysis of IBSN with Free Whole Cells. Enzymatic hydrolysis of IBSN catalyzed by free whole cells was performed in a 3 L reaction system with a substrate loading of 100 g/L. As depicted in Figure 2, the conversion of

Table 1. Biocatalytic Hydrolysis of IBSN by Recombinant E. coli Harboring Different Nitrilases at Various Substrate Loadings nitrilase

substrate (g/L)

reaction time (h)

conversion (%)a

E

BrNIT

30 50 100 30 50 100

2 5 10 10 24 24

45.67 48.33 45.20 45.74 39.35 20.73

>150 >150 >150 >150 >150 >150

AtNIT1

Figure 2. Catalytic hydrolysis of IBSN via free whole cells in a 3 L reaction system. The reactions were performed in a 5 L three-necked flask containing 3 L of Tris-HCl buffer (50 mM, pH 8.0), 39.8 g of DCW whole cells, and 300 g of IBSN at 30 °C and 300 rpm for 6 h.

a

Biotransformation reactions were performed in 10 mL of Tris-HCl buffer (50 mM, pH 8.0) consisting of 13.3 g/L DCW whole cells and different substrate loadings (30, 50, and 100 g/L) at 30 °C and 200 rpm.

IBSN reached 45.62% with 98.2% eep within 9 h. Although the biotransformation of IBSN was performed successfully, proteins leaking from the damaged cells lowered oil−water interfacial tension and emulsified oil and water, leading to difficulty in downstream processing and the low yield of (S)CMHA. Given that immobilization could improve the mechanical strength of cells and simplify the separation process,37 immobilized cells were suggested as biocatalysts for practical application.

reaction at high substrate concentration (100 g/L), the conversion of IBSN catalyzed by BrNIT reached 45.20%, which was much higher than that catalyzed by AtNIT1 (20.73%). As such, recombinant cells harboring BrNIT were selected as a potential biocatalyst for large-scale hydrolysis of IBSN. To achieve large-scale production of nitrilase, recombinant E. coli cells were cultivated in a 5000 L fermenter (a working D



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3.3. Biocatalytic Performances of Immobilized Cells. In present work, recombinant E. coli cells expressing BrNIT were adsorbed on the surface of diatomite, followed by crosslinking with GA/PEI to obtain immobilized cells. The effects of temperature, pH, and substrate concentration on biocatalytic performances of the immobilized E. coli cells were investigated. 3.3.1. Effect of Temperature and pH. The influence of temperature on the catalytic property of immobilized cells was studied. As shown in Figure 3, the immobilized cells showed

immobilized E. coli cells were in accordance with those of free whole cells. 3.3.2. Effect of Substrate Loading. The effect of substrate concentration on the biotransformation was investigated at different substrate concentrations ranging from 100 to 150 g/ L. As illustrated in Figure 5, the conversion of IBSN reached

Figure 5. Effect of substrate loading on biocatalytic hydrolysis of IBSN. The reactions were performed in a 10 mL reaction system consisting of 10 mL of Tris-HCl buffer (50 mM, pH 8.0), 1.0 g of immobilized cells, and different substrate loadings at 30 °C and 200 rpm.

Figure 3. Effect of temperature on the catalytic property of immobilized cells harboring BrNIT. The reactions were performed in a 10 mL reaction system consisting of 10 mL of Tris-HCl buffer (50 mM, pH 8.0), 0.6 g of immobilized cells, and 0.5 g of IBSN at various reaction temperatures for 30 min.

47.5, 42.0, and 37.9% after 6 h reaction with substrate concentrations of 100, 130, and 150 g/L, respectively. When the substrate loading was up to 150 g/L, a conversion of 45.2% was obtained after 15 h. These data evidenced the good catalytic property of immobilized cells at high substrate concentration. However, 100 g/L substrate concentration was more suitable for reusability of immobilized cells from the viewpoint of reaction time. 3.4. Reusability of Immobilized Cells in Kinetic Resolution of IBSN. The repeated biotransformation reactions were carried out under optimized reaction conditions (30 °C, pH 8.0). As depicted in Figure 6, the conversion and eep were 47.5 and 98.0% in the first batch. As reaction went on, the conversion and eep kept at a high level (>41.1% conversion, >98.0% eep) for the first 12 batches. After then, the conversion of IBSN was decreased to 38.3%, while the immobilized E. coli cells remained 92.8% of its activity. It was found that the weight of immobilized cells decreased from 39.78 to 26.72 g in the process of biotransformation and filtration, leading to the decrease of conversion. More remarkably, immobilized cells could be easily separated from the reaction mixture by filtration, and emulsification was not observed during extraction of the remaining solution. In short, immobilized cells exhibited high enantioselectivity and conversion in batch reactions. Moreover, immobilized cells displayed good reusability compared with free whole cells, which was conducive to reducing costs and simplifying the downstream separation process. 3.5. Preparation of Optically Active PGB. After separation and extraction, the transparent aqueous solution of (S)-CMHA with 85.1% isolated yield was obtained, which could be converted to PGB by one-step hydrogenation. Raneynickel catalyst (T-1) was employed as a catalyst for hydrogenation of (S)-CMHA, and sodium hydroxide was

maximal activity at 30 °C, while the eep kept almost constant at various temperatures. The effect of pH on the catalytic property of immobilized cells was investigated at pH ranging from 6.0 to 10.5. The highest activity was achieved at pH 8.0 (Figure 4). When the reactions were performed at a pH range of 8.0−10.5, the enzyme retained 80% of its initial activity. The eep showed little variation and remained above 98.0% at different pH values. The optimal temperature and pH for

Figure 4. Effect of pH on the catalytic property of immobilized cells harboring BrNIT. The reactions were performed in a 10 mL reaction system consisting of 10 mL of different buffers, 0.6 g of immobilized cells, and 0.5 g of IBSN at 30 °C and 200 rpm for 30 min. E



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racemized IBSN as a substrate, the overall yield for PGB was 62.0%. It was obvious that the racemization and recycling of unreacted (R)-IBSN increased the total yield of PGB synthesis. 3.7. Green Features of the Chemoenzymatic Route. In this work, the utilization of immobilized cells, as well as racemization of unreacted (R)-IBSN, dramatically increased atom economy and reduced waste streams. To verify the process efficiency of this route, the E factor (kg total waste per kg PGB product)39,40 for the whole process was calculated to be 3.72, which was much lower than that of the first- (with E factor of 86) and second-generation routes (with E factor of 17).13 Moreover, as evidenced in Table 2, the total input of Table 2. Inputs for 1000 kg of PGB in This Process Figure 6. Reusability of immobilized cells in hydrolysis reaction of IBSN. The reactions were performed in a 300 mL reaction system consisting of 300 mL of Tris-HCl buffer (50 mM, pH 8.0), 39.78 g of immobilized cells, and 30 g of IBSN at 30 °C and 300 rpm for 3 h. The immobilized E. coli cells were recovered and used for the next batch reaction.

simultaneously added as a catalyst promoter to suppress the formation of secondary and tertiary amines.38 As illustrated in Figure 7, the conversion of (S)-CMHA reached 95.6% after 9 h

inputs

nitrilase-mediated chemoenzymatic route (kg)

IBSN immobilized cells Raney nickel solvents total

1319 82.9 82.5 2982.5 4466.9

raw materials for preparation of 1000 kg of PGB via this process was 4466.9 kg, which was 2.5 times less than that of the second-generation route and 13 times less than that of the first-generation route.13 It was obvious that the process described here was more in line with the requirements of green chemistry.

4. CONCLUSIONS In conclusion, we developed a green and efficient chemoenzymatic route for the production of PGB from IBSN. The biocatalytic hydrolysis of IBSN was conducted using immobilized cells harboring nitrilase BrNIT, which exhibited prominent catalytic activity and excellent enantioselectivity (E > 150). High-quality PGB (99.6% purity and 99.4% optical purity) was obtained by one-step hydrogenation of resultant (S)-CMHA, and the unreacted (R)-IBSN was easily racemized with a yield of 94.5%. Moreover, the E factor and input of raw materials of this chemoenzymatic route were calculated to be much lower than that of the previous processes. The promising results demonstrated the effectiveness of the chemoenzymatic approach for the large-scale preparation of PGB.

Figure 7. Catalytic hydrogenation of (S)-CMHA to PGB over Raneynickel (T-1) catalyst. The hydrogenation of (S)-CMHA was performed at room temperature for 9 h with 27 g of Raney-nickel (T-1) catalyst under conditions of a hydrogen of pressure 2.0 MPa.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.oprd.9b00285. 1 H and 13C NMR and MS and HPLC chromatograms for PGB and (S)-CMHA (PDF)

reaction, and the yield of PGB was 93.2%. Subsequently, Raney-nickel catalyst (T-1) was recovered by filtration and used in the next batch. The crude PGB was isolated with a yield of 84.6%. After recrystallization, the chemical purity (HPLC) and optical purity of PGB were up to 99.6 and 99.4% ee, respectively. 3.6. Recycling of Unreacted (R)-IBSN. To overcome the limitation of a theoretical yield of 50% in kinetic resolution of IBSN, racemization of unreacted (R)-IBSN was investigated. The unreacted (R)-IBSN was enriched by extraction and vacuum rotary evaporation. In the presence of sodium ethoxide, the intermediate carbanion was generated after deprotonation on the chiral center of (R)-IBSN and then racemized at high temperature. Finally, racemic IBSN was obtained with a yield of 94.5%. After one recycle of the

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +86-571-88320391. Fax: +86-571-88320884. ORCID

Ren-Chao Zheng: 0000-0001-9682-1759 Yu-Guo Zheng: 0000-0002-6358-6243 Notes

The authors declare no competing financial interest. F



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ACKNOWLEDGMENTS This work was supported by Zhejiang Provincial Natural Science Foundation (no. LR19B060001) and Program for Changjiang Scholars and Innovative Research Team in University (IRT13096).

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Efficient Chemoenzymatic Synthesis of Optically Active Pregabalin

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