Enantioselective synthesis of a chiral intermediate of himbacine analogs by Burkholderia cepacia lipase A
Yanming Chen . Fei Gao . Guojun Zheng . Shuaihua Gao
Abstract
The enantiomers of (4R/S)-4-hydroxy-N, N-diphenyl-2-pentynamide are key chiral synthons for the synthesis of thrombin receptor antagonists such as vorapaxar. In this paper, we report the enzymatic preparation of enantiomerically enriched (4R)-4-hydroxy-N, N-diphenyl-2-pentynamide using lipase A from Burkholderia cepacia ATCC 25416 as the catalyst. First, the lipase gene (lipA) and its chaperone gene (lipB) was cloned and expressed in Escherichia coli system. After purification, lipase A activation was performed with the assistance of foldase lipase B. Enzyme assay revealed that the activated lipase A showed the optimal catalytic activity at 60 8C and pH 7. The effects of various metals on the activity were investigated and results demonstrated that most of the metals inhibited the activity. To further improve the catalytic outcome, two-phase reaction was studied, and n-hexane proved to be a good organic solvent for the combination system. Using the optimize conditions, (4R)-4-hydroxy-N, N-diphenyl-2-pentynamide with 94.5% ee value and 48.93% conversion ratio was achieved. Our investigation on this lipase reveals lipase A as a promising biocatalyst for producing chiral propargyl alcohol for preparation of novel himbacine analogs.
Keywords Enantioselective hydrolysis Klebsiella oxytoca Lamivudine Oxathiolane Stereoselectivity
Introduction
Vorapaxar (brand name Zontivity, formerly known as SCH 530348) is a thrombin receptor antagonist which was developed by Merck & Co based on the natural product himbacine to reduce the rate of combined endpoint cardiovascular death, stroke, and urgent coronary revascularization (Bailey and Campbell 2011; Chackalamannil 2006; Olivier et al. 2013; Tello-Montoliu et al. 2011). It is the first proteaseactivated receptor (PAR-1) inhibitor approved by the U.S. Food and Drug Administration (FDA) in 2014 (Chelliah et al. 2014). Vorapaxar can potently inhibit thrombin-induced platelet aggregation via a selective antagonism of the PAR-1, thus serving as a potent antiplatelet agent (Faraji et al. 2020; Goto and Goto 2015). The chemical synthesis of himbacine-derived thrombin receptor antagonists has been reported (Knight et al. 2016). The chiral compound of (4R)-4hydroxy-N, N-diphenyl-2-pentynamide has been considered as a significantly important synthons in the synthetic process (Scheme 1) and efforts have been made from researchers to obtain this optical enantiomer (Li et al. 2006; Zaks et al. 2009). In this study, we first report the preparation of the functional lipase A from Burkholderia cepacian then an enzymatic approach to preparing this optically pure enantiomer through enantioselective hydrolysis of racemic 4-hydroxy-N, N-diphenyl-2-pentynamide using lipase A (Sasso et al. 2016; Theil and Bjo¨rkling 1993).
Though the major applications of Burkholderia cepacia lipase A (E.C. 3.1.1.3) are in transesterification reactions, studies have shown its broad substrates scope for other reactions (Kahveci and Xu 2011; Padilha and Osorio 2019; Sanchez et al. 2018; Schmid and Verger 1998; Tasna´di et al. 2009). Herein, we investigate its hydrolytic activity and enantioselectivity towards the racemic (4R/S)-4-acetyloxy-N, Ndiphenyl-2-pentynamide. Most lipases from gramnegative bacteria require a lipase-specific foldase in order to fold in the periplasm into their active, protease-resistant conformation prior to their secretion (El Khattabi et al. 2000). To obtain functional lipase A, the assistance of the lipase-specific chaperone lipase B encoded in the same operon with the structural gene of lipase A is mandatory. Lipasespecific chaperone is either required during or after translation (Kranen et al. 2014; Madan and Mishra 2010) Thus, the functional expression of lipase A can either be achieved through co-expression of lipA and lipB (whether both genes are positioned in the same plasmid or in separate plasmids) or by in vitro refolding of lipase using various approaches (Omori et al. 2005; Oshima-Hirayama et al. 1993).
In this study, lipase A and lipase B were first expressed in E. coli system and purified to homogeneity separately, then in vitro refolding of lipase A was performed in the presence of lipase B to activate lipase A. After activity activation, lipase A was used to test the hydrolytic activity toward (4R/S)-4-acetyloxy-N, N-diphenyl-2-pentynamide. Resolution result was detected by high performance liquid chromatography (HPLC). Results demonstrated that activated lipase A could enantioselectively hydrolyze the (4S)-configuration enantiomer from 4-hydroxy-N, N-diphenyl-2pentynamide with high conversion ratio (48.93%) and ee value (94.5%), thus achieving (4R)-4-hydroxy-N, N-diphenyl-2-pentynamid with high purity.
Materials and methods
Chemicals, bacterial strains, plasmids, and molecular biology tools
Unless stated otherwise, all analytical grade chemical reagents were purchased from Beijing Beihua Fine Chemicals Co., Ltd. (Beijing, China). Burkholderia cepacia ATCC 25416 was from the cell stock stored in our lab. The strain was cultured in lysogeny broth (LB) medium at 37 C for genome extraction. E. coli DH5a competent cells were used as a general cloning host for expression plasmid propagation. E. coli Rosetta (DE3) competent cells were utilized for the heterologous expression of the recombinant proteins. Q5 Highfidelity DNA polymerase, T4 DNA ligase and restriction enzymes from New England Biolab (Beverly, MA, USA) were used for the gene manipulation. DNA sequencing was used to confirm the accuracy of the construction of the recombinant plasmids. The genomic DNA extraction kit, gel extraction kit, and plasmid Miniprep kit were purchased from Qiagen (Germany). Vectors pMD18-T and pET28a(?) (Invitrogen) were used for TA cloning and the production of recombinant protein, respectively. In order to select E. coli carrying recombinant plasmids, kanamycin was added to the medium at a final concentration of 50 lg/mL.
Preparation of the racemic substrate of (4R/S)-4acetyloxy-N, N-diphenyl-2-pentynamide N-diphenyl-2-pentynamide, (2R/S)-3-butyn-2-ol, and 3,4-dihydro-2H-pyran (DHP) were used as the starting materials. The materials were dissolved in methylene chloride with the catalyst of p-toluenesulfonic acid (TsOH) (Clasby et al. 2007; Floyd et al. 1980; Wissner 1977). The resulting mixture was slowly charged in nhexyllithium at – 40 C. Subsequently, N, N-Diphenylcarbamic chloride which was dissolved in a mixed solvent of THF and toluene was added to the reaction system followed by distillation (Bailey and Campbell 2011). The product of (4R/S)-4-hydroxy-N, N-diphenyl-2-pentynamide was obtained and used for next reaction to produce the final product by reacting with TEA, DMAP and acetic anhydride in MTBE at room temperature.
Construction of the recombinant plasmids
Polymerase chain reaction (PCR) was conducted to amplify the genes of lipA (NCBI: AIO27730.1) and lipB (NCBI: AIO27817.1) using primers listed in Table 1. The genome DNA extracted from Burkholderia cepacia ATCC 25416 was used as the PCR template. PCR product for each was then purified and ligated to the pMD18-T simple vector (Novagen) to construct the recombinant plasmid of pMD18-TlipA(lipB). Double digestions of the recombinant plasmid and vector pET-28a by the restriction endonucleases of Nde I and Hind III resulted in fragments with identical sticky blunt ends. Ligating the fragments using T4 ligase yielded the final expression plasmid pET-28a-lipA(lipB). The recombinant plasmid was transferred in E. coli DH5a cells for DNA amplification followed by DNA sequencing to confirm the accuracy of the sequence.
Overexpression of lipase A and foldase B
Sequenced plasmid harboring the gene of lipA was then transformed to E. coli Rosetta (DE3) cells for protein expression using lysogeny broth (LB) medium containing kanamycin (50 lg/mL) and chloramphenicol (34 lg/mL). To initialize the expression, isopropyl-b-D-1- thiogalactopyranoside (IPTG, final concentration of 0.1 mM) was added when the OD600 of the medium reached 0.8–1.0. The induction process was carried out at 30 C for 5 h with shaking. Cells after expression were harvested by centrifugation (6000 rpm for 20 min at 4 C and cell pellets were stored for future purification. Foldase B was expressed with a 6 9 His tag at the N-terminus under the same condition as lipase A and it mainly formed soluble protein. It was purified by affinity column chromatography using Ni–NTA resins as stated before (Gao et al. 2016, 2017, 2018). SDS-PAGE was performed to assess the protein purity.
As lipase A was expressed without the presence of its foldase, it mainly formed inclusion bodies which were not soluble in aqueous solution. For purification, first lipase A cell pellet was resuspended in buffer A (50 mM Tris–HCl, 0.2 mM DTT, 1 mM EDTA, 5% glycerol, pH 8.0). Resuspended cells were sonicated on an ice bath to release the proteins. The soluble proteins were removed from the precipitant by centrifuging at 10,000 rpm/min at 4 C for 20 min. Collected inclusion bodies were resuspended with buffer A with 2 mg sodium deoxycholate and equilibrated for 30 min at 4 C. The sediment was collected by centrifuging at 4 C. To future reduce the impure but soluble proteins, the sediment was washed three times with 5 mL of buffer A. Prior to dissolving the precipitant, 1 mg Triton X-100 was added and the system was equilibrated for 20 min at room temperature. After the removal of the supernatant, 4 mL of buffer B (8 M urea, 50 mM Tris–HCl, 0.2 mM DTT, 0.1 M sodium phosphate, pH 8.0) was added to the sediment. The dissolving process was conducted via incubation at room temperature for 60 min with slight stirring. After the incubation, 6 mL of buffer A was added to the system dropwise to precipitate impurities. Final supernatant containing the solubilized and denatured lipase A was collected after centrifuge (Gao et al. 2016).
After obtaining the denatured lipase A and purified foldase B, the refolding process of lipase A was carried out. First, 400 lL of supernatant fraction containing the solubilized and denatured lipase A was added to 40 mL renaturation buffer (3 mM CaCl2, 50 mM Tris–HCl, 0.5 mM oxidized glutathione, 40% glycerol, pH 8.0). The renaturation process was equilibrated at 4 C for 1 h with slow shaking. To refold lipase A, 2 mL foldase B (0.825 mg/mL) was added to the soluble lipase A (0.083 mg/mL, in renaturation buffer) and unfolded lipase A refolded to active form after ca. 2 h. SDS-PAGE (10%) was run to confirm the molecular weight of the purified protein to determine if it matched the theoretical molecular weight value of lipase A. Protein concentration was determined at 280 nm using Thermo NanoDrop 2000 and further calibrated based on the theoretical extinction coefficient of the protein.
Enzyme assay
The reaction system contained 500 lL lipase A and 1 mg/mL (4R/S)-4-acetyloxy-N, N-diphenyl-2-pentynamide. The duration for each reaction was 2 h with shaking. The control was set as the spontaneous hydrolysis of the substrate at different temperatures in the absence of lipase A. After reaction, 500 lL ethyl acetate was used to extract the remaining substrate from the solution for testing. The content of both of the enantiomers of the substrate was measured by HPLC. A chiral column of Chiralcel OJ-H (Daicel, Japan) was used and substrate was detected at the UV wavelength of 260 nm, with the mobile phase of 40% iPrOH in Hexane. A volume of 10 lL extract was applied to the column. Relative activity was calculated relative to the maximum activity in the corresponding assay. All the experiments were run in triplicate. One unit (U) of enzyme activity was defined as the amount of enzyme that catalyzes the conversion of 1 nmol of substrate per min. The ee, was calculated as follows: Enantiomeric excess (ee) =|LS – LR|/(LS ? LR) 9 100%, where LS and LR stand for the concentration of (4R)-4acetyloxy-N, N-diphenyl-2-pentynamide and (4S)-4acetyloxy-N, N-diphenyl-2-pentynamide, respectively.
Physical characteristics of lipase A towards the target substrate
The effects of temperature, pH, metallic ions, and organic solvent on the enantioselective resolution of the racemic substrate by lipase A were investigated. All reactions were carried out according to the standard enzyme activity assay unless stated otherwise. All experiments were conducted in triplicates.
To determine the optimum temperature for lipase A, reactions were performed at a range of temperatures (30–90 C). Then, the samples were analyzed accordingly based on the standard assay method.
The optimum pH for the enzyme was studied at pH values ranging from pH 4.0 to 12.0 (citrate buffer (pH 4.0–5.0), phosphate buffer (pH 6.0–8.0), and glycinesodium hydroxide buffer (pH 9.0–12.0) at 60 C. All the pH-dependent assays were carried out at 60 C. The following reaction system was used: 250 lL lipase A, 250 lL buffer at corresponding pH, and 1 mg/mL (4R/S)-4-acetyloxy-N, N-diphenyl-2-pentynamide. The system was shaking at 60 C for 2 h. To quench the reaction, 500 lL ethyl acetate was added to the solution and the remaining substrate was extracted. Enzyme activity and ee value of the reactions were measured by HPLC.
The effect of metal ions on enzyme activity was investigated by adding metal ions (Ca2?, Co2?, Cu2?, Fe2?, Li?, K?, Mg2?, Mn2?, Na?, Ni?, Zn2?) to the reaction solution (pH 7.0) at a final concentration of 1 mM. The reaction was incubated at 60 C with shaking, and then the enzyme activity was assayed as described in standard enzyme activity assay. Two-phase system reactions were tested: 250 lL lipase A, 150 lL buffer at pH 7.0, 100 lL organic solvent, and 1 mg/mL (4R/S)-4-acetyloxy-N, N-diphenyl-2-pentynamide. The control was the sample without organic solvent under the same experimental condition.
Results and discussions
Racemic substrate preparation
The final racemic substrate of (4R/S)-4-acetyloxy-N, N-diphenyl-2-pentynamide for the enzyme assay in this study was produced with the starting materials of N-diphenyl-2-pentynamide, (2R/S)-3-butyn-2-ol and 3,4-dihydro-2H-pyran (DHP). Nuclear magnetic resonance (NMR) was used to confirm the compound information of (4R/S)-4-acetyloxy-N, N-diphenyl-2pentynamide. The NMR measurement on the product showed properties as follows: 1H NMR (400 MHz, CDCl3, d in ppm) 1.29(s, 3H), 1.95(d, 3H), 5.3(q, 1H), 7.20 (m, 1H), 7.25–7.45 (m, 9H). It matched the theoretical characteristics of the compound and was further used for the enzyme assays performed by refolded lipase A.
Refolding of lipase A
In the absence of foldase B, lipase A mainly formed inclusion bodies which were not soluble in aqueous solution. To obtain the active form lipase A, the inclusion bodies were first unfolded using the denaturant urea. Subsequently, the denatured lipase A was properly refolded at the presence of its foldase. It was convinced that the efficiency of the correct refolding of lipase A was high ([ 90% recovery ratio). SDSPAGE gel revealed a single protein band with a molecular weight of ca 37 kDa which matched the theoretical molecular weight value of lipase A(Fig. 1).
Temperature dependence of lipase A catalyzed reaction
To determine the optimal temperature for the lipase A catalyzed bio-resolution process, reactions at temperatures ranging from 30 to 90 C were conducted. Relative activity was calculated relative to the maximum activity in the temperature-dependent assay. As shown in Fig. 2, the highest activity was observed at 80 C above which the activity declined rapidly with increasing temperature. As for ee value, it reached 92% at the temperature of 80 C, which was a good result for an enantioselective resolution. The decreased ee at higher temperatures may indicate that the enzyme could catalyze both enantiomers. It was also noted that, the spontaneous hydrolysis of the substrate became dramatic at higher temperatures (data not shown), which may partially account for the decrease in ee. The loss of enzyme activity at high temperature may also be considered as another factor for the loss of the enantioselectivity for this case due to the thermostability of lipase A. Taking all the parameters into consideration, we decided that the optimal temperature to perform the reaction was 60 C. pH dependence of lipase A catalyzed reaction
The impact of reaction pH on the resolution outcome was then tested. A series of buffers with pH values ranging from 4.0 to 12.0 were prepared. The control was set as the spontaneous hydrolysis of the substrate at different pHs without lipase A. Enzyme activity and ee value of the reactions were calculated by HPLC. As demonstrated in Fig. 3, lipase A exhibited the optimal activity and highest ee at pH 7.0 and no spontaneous hydrolysis were detected. The activity started to slightly reduce when the pH value increased to 10.0. When pH was higher than 10, both the activity and ee values decreased drastically.
The impact of metal ions on the bio-resolution reaction
Studies have shown that some of the metals can bind in the enzyme active site to form a complex that is beneficial for enzyme to achieve higher activity or exacting enantioselectivity (Gao et al. 2015, 2016). For metalloenzyme systems, tightly bound metal ions might be a necessity for the reaction to proceed. For non-metal involved enzymatic systems, addition of metals will slow down the reaction systems in some cases but facilitate the reaction in other cases. To figure out whether the metals could be a boosting agent for the bio-resolution, we tested the effects of different metal ions (Ca2?, Co2?, Cu2?, Fe2?, Li?, K?, Mg2?, Mn2?, Na?, Ni?, Zn2?) on the enzyme activity and resolution outcome. The reaction system was similar to that for the pH-dependent study, except for that the pH for all resections was set to pH 7.0 and metals were added at a final concentration of 1 mM. Metaldependent results were detected via HPLC. Relative activity was calculated compared to the control activity which was set as the reaction without addition of metals. (Fig. 4). Unfortunately, we found that none of the metal ions tested in the experiment could enhance the enzymatic activity. In fact, Li? inhibited the activity significantly and only * 50% of the activity remained after the addition of this metal. The reason for the metal inhibition can be rationalized in the way that metal ions can bind to the side chains of some polar or charged residues and perturb the exacting electrostatics of the active site. That in turn changes the conformation of the active site to reach the transition state which will result in affecting the enzyme activity in a negative way. Thus, adding metal ions to the biotransformation process was not recommended.
The effect of organic solvent on the enantioselective hydrolysis
As industrial biocatalysts, lipases are active in both aqueous medium as well as non-aqueous medium, (Kumar et al. 2016). Here we tested two-phase reaction with the goal of reducing the degree of spontaneous hydrolysis since the hydrolysis of waterinsoluble substrates must occur at an interface (Santos et al. 1991). Prior to testing the enantioselective reaction, the stability of the enzyme in different organic solvents (isopropyl ether, MTBE, ethyl acetate, n-hexane, n-heptane, toluene, and dichloromethane) were investigated to confirm the enzyme was stable at organic solvent (Fig. 5). The control was the sample without organic solvent under the same experimental condition. HPLC measurement revealed that the introduction of the organic solvent of n-hexane would lead to an increase in the activity by 4.78% compare to the control activity. However, considering the fact that the substrate would evaporate with n-hexane at 60 C, the two-phase reaction system was not adopted.
Reaction catalyzed by refolded lipase A at optimal conditions
At last, the optimized reaction was carried out in the following system: 400 lL lipase A, 100 lL buffer of pH 7.0 and 0.5 mg (4R/S)-4-acetyloxy-N, N-diphenyl2-pentynamide, no metal ions and organic solvents were added. The reaction was performed at 60 C for 4 h with shaking. HPLC (Fig. 6) measurement demonstrated that (4R)-4-hydroxy-N, N-diphenyl-2pentynamide was obtained with 48.93% conversion ratio and 94.5% ee. This result demonstrates the potential of lipase A as a promising biocatalyst for preparing a medically important chiral compound in a scalable manner.
Conclusion
Due to the irreplaceable place of green catalysts such as whole cells, purified enzymes, and immobilized enzymes in the field of bio-synthesis of important pharmaceuticals, especially the preparation of chiral building blocks for the chiral drugs on the market. Many chemical catalysts that facilitate enantioselective bond formation are made from rare occurring and non-renewable materials, such as expensive transition metals or toxic chemical reagents. Substituting them with efficient and sustainable alternatives would provide us the chance to obtain key chemicals in a more environmentally friendly manner. Biocatalysts has proven to be the solution for us to access the chirality of lots of medical related compounds (Shin et al. 2019; Yamazaki and Hosono 1989). In this study, we sought to first correctly refold the lipase A with its assisting foldase B and then investigate its incorporation into the bio-resolution of racemic substrate that provided us chiral intermediate for the downstream production of himbacine-derived thrombin receptor antagonists.
In conclusion, we report an approach to obtaining the functional lipase A from Burkholderia cepacia ATCC 25416 with the assistance of its foldase B. Preliminary enzyme assay showed that lipase A displayed high enantioselectivity towards the substrate(4R/S)-4-acetyloxy-N, N-diphenyl-2-pentynamide. To optimize the reaction, temperature-, pH, metal, and organic solvent-dependent experiments were screened. The best performance was achieved with the conversion of 48.93% and ee value of 94.5%. As a comparation to previously reported chemical approaches to synthesizing the chiral precursor of himbacine analogs, herein, we proposed an efficient and cost-effective biocatalytic process to obtain optically pure (4R)-4-hydroxy-N, N-diphenyl-2-pentynamide, which can be used for the synthesis of multiple himbacine analogs such as vorapaxar.
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