an unusual (r)-selective epoxide hydrolase with high activity for facile preparation of enantiopure...

DOI: 10.1002/adsc.201100031

An Unusual (R)-Selective Epoxide Hydrolase with High Activityfor Facile Preparation of Enantiopure Glycidyl Ethers

Jing Zhao,a Yan-Yan Chu,b Ai-Tao Li,a Xin Ju,a Xu-Dong Kong,a Jiang Pan,a

Yun Tang,b,* and Jian-He Xua,*a Laboratory of Biocatalysis and Synthetic Biotechnology, State Key Laboratory of Bioreactor Engineering, East China

University of Science and Technology, 130 Meilong Road, Shanghai 200237, People�s Republic of ChinaFax: (+86)-21-6425-0840; e-mail: [email protected]

b Department of Pharmaceutical Sciences, School of Pharmacy, East China University of Science and Technology, 130Meilong Road, Shanghai 200237, People�s Republic of ChinaFax: (+86)-21-6425-3651; e-mail: [email protected]

Received: January 14, 2011; Revised: March 21, 2011; Published online: June 16, 2011

Abstract: A novel epoxide hydrolase (BMEH) withunusual (R)-enantioselectivity and very high activitywas cloned from Bacillus megaterium ECU1001.Highest enantioselectivities (E>200) were achievedin the bioresolution of ortho-substituted phenyl gly-cidyl ethers and para-nitrostyrene oxide. Worthy ofnote is that the substrate structure remarkably affect-ed the enantioselectivities of the enzyme, as a re-versed (S)-enantiopreference was unexpectedly ob-served for the ortho-nitrophenyl glycidyl ether. As a

proof-of-concept, five enantiopure epoxides (>99%ee) were obtained in high yields, and a gram-scalepreparation of (S)-ortho-methylphenyl glycidyl etherwas then successfully performed within a few hours,indicating that BMEH is an attractive biocatalyst forthe efficient preparation of optically active epoxides.

Keywords: enantioselectivity; epoxide hydrolase;glycidyl ethers; kinetic resolution; molecular model-ing

Introduction

The enantioselective hydrolysis of racemic epoxidescatalyzed by epoxide hydrolases (EHs) is one of thepromising approaches to obtain enantiopure aryl gly-cidyl ethers,[1] which are important building blocks forthe production of bioactive compounds such as chiralamino alcohols[2] and b-blockers.[3] However, only fewpreparative-scale examples using EHs have been re-ported due to the quite low activities of the wild-typewhole cells.[4] Thus the task of searching for novel bio-catalysts still remains a challenge.

EHs are ubiquitously found in nature and havebeen identified in many organisms including mam-mals, plants, insects and various micro-organisms.[5]

Some of them exhibit modest enantioseletivitiestoward phenyl glycidyl ether (PGE), e.g., Aspergillusniger EH[4c] and Agrobacterium radiobacter EH.[6]

Thus protein engineering has been further applied toenhance the enantioselectivity of existing EHs.[7]

However, most of them are (S)-selective, and only afew could preferentially hydrolyze (R)-PGE, retainingthe useful (S)-epoxide for the synthesis of b-block-ers.[8] Unfortunately, the activities of the current (R)-selective EHs are generally low, e.g., recombinant Ba-

cillus subtilis EH (Bsueh)[8] with a specific activity ofmerely 0.01 mmol min�1 mg�1, which could not meetthe requirement for practical application. In our labo-ratory, a strain of B. megaterium ECU1001, which ishighly selective towards (R)-PGE, was successfullyisolated from soil samples.[9] It was the distinguishedproperty of EHs in B. megaterium ECU1001 that at-tracted our interest in cloning them for further inves-tigation and application.

Herein, we report a novel EH with unusual enan-tioselectivity and high activity toward (R)-glycidylethers, which has the potential to be used as an indus-trial biocatalyst for the production of enantiopure ep-oxides and diols.

Results and Discussion

The whole genome sequence of B. megaterium QMB1551 was recently released in the GenBank database(entry number CP001983.1). However, no putativeEH gene was available to date. Therefore, we predict-ed the EH gene based on the conserved regionsshared in the a/b-hydrolase fold EHs. As a result, anopen reading frame designated as bmeh was identified

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as a putative EH. As shown in Figure 1, BMEH con-tains all the conserved motifs and shares similar sec-ondary structures with other known EHs. This genewas then successfully cloned from the strain B. mega-terium ECU1001, and the sequencing result indicatesthat it consists of 864 bp, encoding 287 amino acidswith a molecular weight of 33,580 Da (GenBank ac-cession number HQ436037). Noteworthy, a BLASTsearch against the Protein Data Bank reveals thathuman sEH shares the closest structural homologywith a sequence identity of only 24%. The gene wasoverexpressed in Escherichia coli followed by a seriesof optimizations to obtain soluble expression. TheBMEH was then purified to an apparent homogeneitywith a specific activity of 83 U/mg protein by His-tagaffinity chromatography (Figure 2).

To investigate whether the BMEH had the sameenantiopreference as the wild-type strain, the model

substrate 1 was submitted to enzymatic hydrolysis. Asexpected, the BMEH is highly (R)-selective (E=58)and exhibited a pretty high activity (Table 1), indicat-ing a good potential for industrial applications.

To the best of our knowledge, the enantioselectivityis the highest among all the known native EHs for thebioresolution of rac-1 (Table 1). Hence, this encourag-ing result prompted us to expand the substrate scopefor further understanding of the interaction betweenthe enzyme and substrate. A range of racemic phenylglycidyl ethers, substituted with electron-donating andelectron-withdrawing groups, were employed for bio-hydrolysis using BMEH (Scheme 1, substrates 2–10).[12]

As shown in Table 2, the position and electronicproperties of the substituents were of great influenceon the activity and enantioselectivity. The resolutionof epoxides 1–8 proceeded with varying degrees of

Figure 1. Sequence alignment of EHs. The protein accession numbers are: B. megaterium ECU1001 (BMEH, this paper); B.subtilis (yfhM, O31581); Glycine max (GmEH, Q39856); Solanum tuberosum (StEH, Q41413); human sEH (EPHX2,P34913). Dark and light colours indicate sequence identity and similarity, respectively. The secondary structures are markedunder the sequences. Regions of putative motif are boxed, and the functionally essential residues are marked by stars. Thedashed line boxes represent the NC- and cap-loops, respectively.

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An Unusual (R)-Selective Epoxide Hydrolase with High Activity

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enantioselectivity (E=4 to >200), and a generaltrend can be found that the E value increases as thesubstituent on the phenyl ring is shifted from thepara- to the ortho-position. We also noticed that alonger (9) or a larger side-chain (10) could decreasethe E value to some extent, which is in agreementwith reported results.[4a] Interestingly, the substitutionpattern of the nitro group remarkably affected notonly the level of selectivity but also the configurationof the unreacted epoxide. As in the case of rac-5, theremained enantiomer was (R)-configuration whichwas opposite to all the other tested glycidyl ethers.Surprisingly, the unexpected excellent E values (>200) were observed in the cases of 5 and 6, although

the enantiopreference was opposite. These resultswere also confirmed in the following preparative-scale experiments. To the best of our knowledge, suchan interesting phenomenon has been observed for thefirst time. In contrast, the EH from B. alcalophilus[4a]

exhibited no inversed enantioselectivity for rac-5, andno activity towards rac-6 even after 24 h reactiontime.

To determine which epoxide carbon was preferen-tially attacked for substrates 2–5, the regioselectivitycoefficients aS and aR were calculated using Eq.(1).[13] aS and aR are regioselectivity coefficients relat-ed to the attack at the more substituted carbon atom(C-2) of the S and R enantiomers, respectively. Nor-mally, EH attack occurs preferentially at the less sub-stituted carbon atom (C-1), as demonstrated in thecases of compounds 2–4 (Table 3). However, for com-pound 5, it was noticed with surprise that the oxiranering was preferentially attacked at the C-2 for both

Figure 2. Purification of BMEH. Lane 1: the protein sizestandard; Lane 2 : soluble fraction; Lane 3 : insoluble frac-tion; Lane 4 : the purified BMEH.

Table 1. Comparison of PGE resolution between BMEH and other EHs.

Epoxide hydrolasesource

Catalystform

Catalyst concentra-tion [gL�1]

PGE concentra-tion [mM]

Time[min]

ee [%]/config-uration

Yield[%]

E Ref.

Aspergillus niger wet cells 75 20 240 100/(R) 26 n.a.[a] [4c]

Agrobacterium ra-diobacter

purifiedenzyme

0.025-0.125 1 n.a. >99/(R) 28 12 [6]

Trichosporon lou-bierii

wet cells 50 67 270 >99/(R) 35 20 [10]

Rhodobacteralesbacterium

purifiedenzyme

0.02 29.2 20 100/(R) 38.4 38.4 [11]

Bacillus alcalophi-lus

growingcells

n.a. 6.6 1440 >99/(S) 27 27 [4a]

Bacillus megateri-um

crudeenzyme

1.28 20 2 >99/(S) 44 58 thisstudy

[a] n.a.: not available.

Scheme 1. Epoxide substrates used in this study.

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FULL PAPERS Jing Zhao et al.


enantiomers, resulting in configuration inversion ofthe corresponding diol. This is consistent with the factthat at total conversion, the formed diol exhibited anee of about 10%. This phenomenon might be due tothe change of electronic properties caused by ortho-nitro substitution.[13] Interestingly, it was observedthat ortho-nitro substitution on the phenyl ring affectsthe enantiopreference as well as the regioselectivity

of the enzyme. A similar result has been described byFurstoss et al.[14] for Beauveria sulfurescens EH.During the biohydrolysis of para-substituted styreneoxide derivatives, they found that a p-NO2 substituentin the substrates caused a switch of EH enantioprefer-ence.

Homology modeling and molecular docking wereperformed to gain insights into the binding mode ofthe substrates and the origin of inverted enantioselec-tivity.[15] As a target template structure, a human solu-ble epoxide hydrolase with an inhibitor bound (PDBentry code: 3I28), showing 24% sequence identity and52% similarity to the BMEH, was selected after aPSI-BLAST search. Systematic analysis of the se-quence and structure of EHs shows that although thesequence identity of EHs is rather low, the cap regionand the conserved a/b-hydrolase fold of EHs could bemodeled reliably.[16] Two loops called NC-loop (resi-dues 121–127) and cap-loop (residues 358–369)(Figure 1), whose length determines the feasibility ofmodeling, should be taken into consideration.[16] Thecap-loop of BMEH is much shorter than that ofhuman EH, which was refined sufficently. Procheckand Verify-3D were used to evaluate the modeledstructure. The results indicate that the BMEH modelis reliable (see Experimental Section).

The binding modes of the substrates in the activesite from molecular docking are shown in Figure 3,and the related parameters including energies, distan-ces and angles are summarized in Table 4. As shownin Figure 3, all the substrates form hydrogen bonds

Table 2. Results for enzymatic resolution of epoxides 1–16 on an analytical scale.

Substrate Concentration [mM] Enzyme used [U][a] Time [min] eeS [%][b]/configuration Conversion [%][c] E value[d]

1 20 8 2 >99/(S) 56 582 10 9 30 >99/(S) 50 >2003 10 9 30 77/(S) 50 194 10 9 50 73/(S) 52 115 10 18 150 >99/(R) 50 >2006 10 18 30 >99/(S)[e] 50 >2007 10 108 120 67/(S) 54 78 10 18 30 87/(S)[e] 52 259 10 18 120 77/(S) 69 410 10 108 120 98/(S) 58 2511 5 400 5 >99/(R) 51 >20012 5 400 30 53/(R) 57 413 5 4 2 39/n.d.[f] 51 314 5 400 10 31/ ACHTUNGTRENNUNG(R,R) 54 215 20 8 5 20/ ACHTUNGTRENNUNG(2S,3R) 67 1.416 10 400 8 60/ ACHTUNGTRENNUNG(2S,3R) 70 3

[a] Lyophilized enzyme powders or cell-free extracts were used for substrates 1–10 and 11–16, respectively.[b] Determined by chiral HPLC or GC as described in the Experimental Section.[c] Conversion was calculated from the remaining substrate concentration and the initial substrate concentration.[d] E values were calculated by the following equation: E= lnACHTUNGTRENNUNG[(1�c)ACHTUNGTRENNUNG(1�ees)]/lnACHTUNGTRENNUNG[(1�c) ACHTUNGTRENNUNG(1+ees)] .[e] The absolute configurations were confirmed by comparing the optical rotations reported in the literature.[f] n.d.: not determined.

Table 3. Regioselectivity of BMEH for biohydrolysis of rac-emic epoxides 2–5.

Substrate aS[a]

[%]aR

[a]

[%]Absolute configuration[b]

residual epox-ide

diolformed

2 2.1 2.9 S R3 6.2 0 S R4 0 0 S R5 95 85 R R

[a] aS and aR were calculated from three sets of data (con-version, ees, eep) at seven different time points during thebiohydrolysis of racemic epoxides using the non-linearcurve fitting function of Origin software according to Eq.(1).

[b] The absolute configurations were established by chiralHPLC analyses or the optical rotations, for details seeExperimental Section.




with Y144 and Y203 by the epoxide oxygen. The cata-lytic efficacy of the enzyme is dependent on howoften the nucleophile and electrophile are present innear attack conformations (NACs).[15a] As Table 4shows, the favored enantiomers have relatively lowerbinding free energies (DG), shorter distance (d value)and larger angles (a1 and a2), corresponding to NACsor more generally to productive positions,[15d] as ex-pected. It is also observed that (S)-5 is more likely toproduce reactive conformations, which might be thereason for the inverted enantioselectivity. This analy-

sis can be related to NACs as postulated by Bruice[15a]

in a molecular dynamics (MD) study of another EH.We noticed that, for both enantiomers of compound5, the C-2 position is much more favorable to be at-tacked than the C-1 position considering both the dis-tances and the angles (data not shown), which is alsoin agreement with the measured regioselectivity coef-ficients.

What about other types of epoxides using BMEH?Thus, we further tested some other typical compounds11–16 (Scheme 1).[17] Unfortunately, low E values

Figure 3. Docking of compounds 2–5 (A–D) to the binding pocket of BMEH, respectively. The BMEH protein was shown inwhite cartoon, and the residues of the active pocket were shown by sticks in light grey carbon. The dark and light grey car-bons shown in ball and stick represent the (R) and (S) enantiomers, respectively. And hydrogen bonds were labeled bydashed lines, (R) in black and (S) in grey.




were observed for most of them, while in the case of11, an unexpected excellent E value (>200) was ob-served in favor of (S)-11. Encouraged by this result,we decided to further investigate various styreneoxide derivatives,[12,18] and this work is in progress.

In order to confirm the results from the analyticalscale experiments, semi-preparative 200-mg scale res-olutions of epoxides 1, 2, 5, 6 and 11 were carried outusing lyophilized BMEH powder (crude enzyme). Asa consequence, enantiopure epoxides (ee>99%) wereobtained with good yields (Table 5). To the best ofour knowledge, this is the first report of preparing(S)-6 by bioresolution which is the solely useful anti-pode for b-blocker synthesis.[19] Subsequently, withoutany optimization, a gram-scale preparation of (S)-2was achieved within 6 h at an elevated substrate con-centration of 30 g/L. This afforded (S)-2 in a nearlyenantiopure form (98% ee) in 32% isolated yield andthe antipodal (R)-diol (87% ee) in 40% isolated yield.In contrast, the biohydrolysis of 2 with growing cellsof B. alcalophilus afforded (S)-2 with 99% ee after24 h at a low concentration of 1 g/L,[4a] indicating thatBMEH is a more efficient catalyst. Further studies onoptimizing the reaction conditions are in progress, inorder to improve the utility of BMEH for large-scaleapplication.

Conclusions

In summary, the newly cloned BMEH from B. mega-terium ECU1001 was demonstrated to be a very effi-cient biocatalyst for the kinetic resolution of rac-gly-cidyl ethers. It was observed that the o-NO2 substitu-ent causes a dramatic switch in both the enantioselec-tivity and regioselectivity of the enzyme. Moleculardocking was then performed to uncover the origin ofthe inverted enantioselectivity. Given the low identityto other currently known EH structures (<25%), wehope that the crystal structure of BMEH might pro-vide deeper insights into the mechanism of EHs, andBMEH might become an attractive catalyst for facilepreparation of enantiopure epoxides.

Experimental Section

General Remarks

The racemic epoxides 3–7, 10–11 and 15–16 were synthe-sized according to the methods previously reported withsome modifications. All other chemicals were obtained com-mercially and used without further purification. The1H NMR spectra were recorded on a Brucker 500 MHzspectrometer, using the d scale (ppm) for chemical shifts.GC was performed on a Shimadzu GC-14C, and HPLC wasperformed on a Shimadzu LC-10AT. Thin layer chromatog-raphy (TLC) was carried out on RSG F254 silica gel sheetsusing a mobile phase of petroleum ether and ethyl acetate(EtOAc). Flash chromatography was performed on silica gel(300–400 mesh). Optical rotations were measured in a 10-cm cell on a Rudolph Research Autopol I automatic polar-imeter.

Identification and Cloning of the BMEH Gene

To clone EHs from B. megaterium ECU1001, sequencesearches (H-G-X-P and Sm-X-d-X-Sm-Sm motif) againstORFs of B. megaterium QM B1551 were performed usingthe GLIMMER program of NCBI and the basic local align-ment search tool (BLAST) program. The pairwise compari-sons of candidate EHs and reported EHs were performed

Table 4. Results of docking experiments.[a]

Substrate �DG [kcal mol�1][b] d [�ngstroms][c] a1 [deg][c] a2 [deg][c]

R S R S R S R S

2 25.3 22.8 3.1 3.7 136.8 106.8 85.1 55.03 23.4 22.6 3.2 3.8 136.9 110.1 84.2 61.74 23.0 21.4 3.2 3.6 128.3 105.1 74.6 51.85 27.5 29.0 3.2 2.9 121.9 153.7 73.4 99.9

[a] The data shown are the average values of three independent docking simulations.[b] The binding free energy was calculated by MM-GBSA of the compounds.[c] d: the distance between the Asp97 oxygen and the attacked epoxide carbon; a1: the angle from the Asp97 oxygen via the

attacked epoxide carbon to the epoxide oxygen; a2: the angle from the Asp97 oxygen via the attacked epoxide carbon tothe other epoxide carbon.

Table 5. Semi-preparative scale resolution of epoxides withBMEH.

Substrate Time[min]

Conversion[%][a]

eeS [%]/config-uration

Yield[%][b]

1 5 58 >99/(S) 312 15 52 >99/(S) 405 90 52 >99/(R) 416 30 51 >99/(S) 4511 150 50 >99/(R) 47

[a] Determined by HPLC analysis.[b] Isolated yield.




with the CLUSTAL W program. The resulting candidateswere manually confirmed for the presence of the putativeEHs active-site residues. Sequences that contained the cata-lytic triad, HGXP motif, Sm-X-D-X-Sm-Sm (Sm = small res-idue and X= any residue) motif and at least one ring-open-ing tyrosine were selected and aligned together with theknown EHs sequences, and the distribution of the secondarystructures was also labeled.

Genomic DNA of B. megaterium ECU1001 was isolatedusing the Genomic DNA extraction kit (TIANGEN, Bei-jing, China) following the manufacturer�s instructions. Thefull-length of bmeh gene flanked by BamHI and SalI siteswas amplified by a PCR with the forward primer(bmehF: 5’-CACGGATCCATGAGTAAA CAGTATATAAACGT-3’)and reverse primer (bmehR:5’-GGCGTCGACTTACTTATTTAAAAAATTCCACAT-3’). Theitalic sequences indicate the BamHI site in the forwardprimer and the SalI site in the reverse primer. The amplifiedDNA fragment was digested with BamHI and SalI, the frag-ment was ligated to BamHI/SalI-digested plasmid pET28a(+) and then the recombinant plasmid was transformed intoE. coli DH5a cells. The recombinant plasmid was intro-duced into E. coli BL21 ACHTUNGTRENNUNG(DE3) for expression after sequenceconfirmation.

Preparation and Purification of BMEH

Preparative-scale production of proteins was achieved bycultivating the recombinant E. coli in a 5-L fermentor(Baoxing Bioengineering Equipment Co. Ltd, Shanghai,China) as described previously.[20] The cells were harvestedby centrifugation and used directly for the enzyme lyophili-zation or protein purification. The harvested cells were re-suspended in potassium phosphate buffer (100 mM, pH 7.0)and disrupted twice by a high-pressure homogenizer(AH110B, ATS Engineering Inc.). Cell debris was removedby centrifugation (10,000 g, 4 8C, 15 min). To the supernatanta lactose solution of 1% (w/v) was added for enzyme protec-tion before lyophilization. As a result, 40 g of powder with aspecific activity of 12,500 U/g powder were obtained andstored at 4 8C for the following reactions unless otherwisestated. The purification of BMEH was performed with Ni2+-NTA beads according to the standard protocol. The purityof the protein was examined by SDS-PAGE using a 15%separating and 4% stacking gel.

General Procedure for Synthesis of 3–7 and 10

rac-Glycidyl ethers were synthesized from correspondingphenols and rac-epichlorohydrin according to the methodpreviously reported with some modifications.[21] Phenol(50 mmol) was dissolved in 75 mL of 0.8 M sodium hydrox-ide and the mixture was stirred for 30 min. Epichlorohydrin(5.85 mL, 75 mmol) was added and the mixture was stirredvigorously at ambient temperature for about 5 h. The homo-genous solution was then extracted three times with 15 mLdichloromethane and the organic fractions were combinedand washed with 5% sodium hydroxide followed by brine.The dichloromethane was dried over anhydrous sodium sul-fate and removed under reduced pressure, giving a solid oroil. The crude products were recrystallized from EtOAc/pe-troleum ether or purified by flash chromatography.

ACHTUNGTRENNUNG(R,S)-1,2-Epoxy-3-(3-methylphenoxy)propane (3): Ob-tained as a colourless oil after column chromatography;yield: 68%. 1H NMR (500 MHz, CDCl3, TMS): d=2.33 (s,3 H, CH3), 2.74–2.76 (m, 1 H, CH2), 2.89–2.91 (m, 1 H, CH2),3.33–3.36 (m, 1 H, CH), 3.95 (dd, J=11.0, 5.6 Hz, 1 H, CH2),4.19 (dd, J=11.0, 3.2 Hz, 1 H, CH2), 6.72–6.79 (m, 3 H,ArH), 7.15–7.18 (m, 1 H, ArH).ACHTUNGTRENNUNG(R,S)-1,2-Epoxy-3-(4-methylphenoxy)propane (4): Ob-tained as a colourless oil after column chromatography;yield: 71%. 1H NMR (500 MHz, CDCl3, TMS): d=2.28 (s,3 H, CH3), 2.74–2.75 (m, 1 H, CH2), 2.88–2.90 (m, 1 H, CH2),3.33–3.35 (m, 1 H, CH), 3.93 (dd, J=11.0, 5.6 Hz, 1 H, CH2),4.18 (dd, J=11.0, 3.1 Hz, 1 H, CH2), 6.75–6.83 (m, 2 H,ArH), 7.07–7.09 (m, 2 H, ArH).ACHTUNGTRENNUNG(R,S)-1,2-Epoxy-3-(2-nitrophenoxy)propane (5): Obtainedas a light yellow solid after recrystallization; yield: 31%.1H NMR (500 MHz, CDCl3, TMS): d=2.87–2.88 (m, 1 H,CH2), 2.92–2.94 (m, 1 H, CH2), 3.40 (s, 1 H, CH), 4.14–4.17(m, 1 H, CH2), 4.41 (dd, J=11.2, 2.5 Hz, 1 H, CH2), 7.06–7.14 (m, 2 H, ArH), 7.52–7.55 (m, 1 H, ArH), 7.85–7.86 (m,1 H, ArH).ACHTUNGTRENNUNG(R,S)-1,2-Epoxy-3-(3-nitrophenoxy)propane (6): Obtainedas a light yellow solid after recrystallization; yield: 56%.1H NMR (500 MHz, CDCl3, TMS): d=2.79–2.81 (m, 1 H,CH2), 2.94–2.96 (m, 1 H, CH2), 3.39–3.40 (m, 1 H, CH), 4.00(dd, J=11.0, 6.0 Hz, 1 H, CH2), 4.37–4.39 (m, 1 H, CH2),7.27–7.29 (m, 1 H, ArH), 7.43–7.47 (m, 1 H, ArH), 7.76–7.80(m, 1 H, ArH), 7.85–7.89 (m, 1 H, ArH).ACHTUNGTRENNUNG(R,S)-1,2-Epoxy-3-(4-nitrophenoxy)propane (7): Obtainedas a light yellow solid after recrystallization; yield: 24%.1H NMR (500 MHz, CDCl3, TMS): d=2.78–2.80 (m, 1 H,CH2), 2.95–2.96 (m, 1 H, CH2), 3.37–3.40 (m, 1 H, CH), 4.01(dd, J=11.1, 6.0 Hz, 1 H, CH2), 4.38 (dd, J=11.1, 2.6 Hz,1 H, CH2), 7.00 (d, J=9.2 Hz, 2 H, ArH), 8.22 (d, J= 9.2 Hz,2 H, ArH).ACHTUNGTRENNUNG(R,S)-1,2-Epoxy (1-napthoxy)propane (10): Obtained as alight yellow oil after column chromatography; yield: 34%.1H NMR (500 MHz, CDCl3, TMS): d=2.84–2.85 (m, 1 H,CH2), 2.95–2.97 (m, 1 H, CH2), 3.49–3.50 (m, 1 H), 4.14 (dd,J=10.9, 5.6 Hz, 1 H, CH2), 4.39 (dd, J=11.0, 3.0 Hz, 1 H,CH2), 6.80 (d, J= 7.6 Hz, 1 H, ring naphthyl), 7.34–7.37 (m,1 H, ring naphthyl), 7.44–7.51 (m, 3 H, ring naphthyl), 7.79–7.80 (m, 1 H, ring naphthyl), 8.29–8.31 (m, 1 H, ring naph-thyl).

Synthesis of Substrate 16

rac-16 was prepared from benzaldehyde and methyl chloro-ACHTUNGTRENNUNGacetate in the presence of sodium methoxide, and purifiedby flash chromatography according to the method previous-ly reported.[22] The product was obtained as a colourless oilafter column chromatography. 1H NMR (500 MHz, CDCl3,TMS): d=3.51–3.52 (m, 1 H, CH), 3.82 (s, 3 H, CH3), 4.10–4.10 (m, 1 H, CH), 7.28–7.31 (m, 2 H, ArH), 7.33–7.38 (m,3 H, ArH).

General Procedure for Enzymatic Reactions on anAnalytical Scale

To a stirred solution of BMEH powder (amount added asindicated in Table 2) in potassium phosphate buffer(100 mM, pH 7.0), the appropriate amount of epoxide (finalconcentration 5–20 mM) was added from a stock solution in




DMSO to a total volume of 0.5 mL. The final concentrationof DMSO was 5–10%. The reaction mixture was stirred in athermoshaker (Eppendorf, Germany) at 30 8C for the appro-priate time. The reaction mixture was extracted twice with0.5 mL of EtOAc, the combined organic layers were driedover anhydrous magnesium sulfate, filtered, and analyzed bychiral HPLC or GC to determine enantioselectivity.

General Procedure for Semi-Preparative ScaleBiohydrolysis of Epoxides 1–2, 5–6 and 11

BMEH powder (285 mg for 1 and 2, 570 mg for 5, 6 and 11)was rehydrated in potassium phosphate buffer (90 mL,100 mM, pH 7.0) for 30 min on a shaker (180 rpm, 30 8C).Then 10 mL DMSO containing 2 mmol of the substratewere added and the mixture was agitated at 30 8C. The reac-tion progress was monitored by TLC or HPLC, and termi-nated when the ee value of the residual epoxide reached99% by adding 150 mL of EtOAc. Then the reaction mix-ture was extracted twice again with EtOAc (150 mL) aftersaturation with NaCl. After drying over anhydrous sodiumsulfate, solvents were removed under vacuum. Purificationwas performed by flash chromatography.

(S)-1,2-Epoxy-3-phenoxypropane (1): Yield: 31%(93.1 mg); >99% ee ; [a]30

D : + 3.0 (c 1.0, CHCl3), Lit.[19] [a]20D :

+3.16 (c 0.95, CHCl3).(S)-1,2-Epoxy-3-(2-methylphenoxy)propane (2): Yield:

40% (130.4 mg); >99% ee ; [a]30D : + 13.0 (c 1.0, EtOH),

Lit.[23] [a]D: +14.8 (c 0.5, EtOH).(R)-1,2-Epoxy-3-(2-nitrophenoxy)propane (5): Yield: 41%

(160.0 mg); >99% ee ; [a]30D : �9.7 (c 1.0, CHCl3).

(S)-1,2-Epoxy-3-(3-nitrophenoxy)propane (6): Yield: 45%(175.6 mg); >99% ee ; [a]30

D : + 2.0 (c 1.0, CHCl3), Lit.[19]

[a]20D : +1.22 (c 1.02, CHCl3).

(R)-2-(4-nitrophenyl)oxirane (11): Yield: 47% (154.5 mg);>99% ee ; [a]30

D : �41.0 (c 1.0, CHCl3), Lit.[24] [a]D: �34.7 (c1.0, CHCl3) for 91% ee.

General Procedure for Preparative ScaleBiohydrolysis of Epoxide 2

In a 250-mL reactor, to rac-2 (18.3 mmol) dissolved inDMSO (10 mL) was added a solution of BMEH powder(1.0 g) in demineralized water (90 mL) then the mixture wasstirred at 500 rpm with a mechanical engine, and maintainedat 30 8C. The reaction was stopped when the ee of the resid-ual epoxide reached 98% by adding 150 mL of dichlorome-thane. The aqueous layer was saturated with NaCl then ep-oxide and diol were extracted twice again with dichlorome-thane (150 mL). After drying over anhydrous sodium sul-fate, the solvents were removed under vacuum. Purificationwas performed by flash chromatography. The diol was ob-tained as a white solid; yield: 40%; 87% ee ; [a]25

D : +18.0 (c1.0, hexane/i-PrOH, 4:1), Lit.[25] [a]20

D : +19.3 (c 1.2, hexane/i-PrOH, 4:1). 1H NMR (500 MHz, CDCl3, TMS): d= 2.00–2.03 (m, 1 H, OH), 2.21–2.24 (m, 3 H, CH3), 2.56 (d, J=5.0 Hz, 1 H, OH), 3.77–3.82 (m, 1 H, CH2), 3.85–3.90 (m,1 H, CH2), 4.04–4.09 (m, 2 H, CH2), 4.12–4.17 (m, 1 H, CH),6.83 (d, J=7.9 Hz, 1 H, ArH), 6.88–6.91 (m, 1 H, ArH),7.15–7.18 (m, 2 H, ArH).

Molecular Modeling

The crystal structure of human soluble epoxide hydrolasewith an inhibitor bound was retrieved from the ProteinData Bank (PDB, entry code: 3I28) and chosen as the tem-plate. The EH models were constructed using Modeller 9v8with loops refined sufficiently. Several models were obtainedand validated by Procheck and Profile-3D.[26] The bestmodel was shown in the Ramachandran plot, where 90.6%residues were located in the most favoured regions, 8.6% inthe additional allowed regions, 0.8% in the generously al-lowed regions and no residues located in disallowed regions.The RMSD between the model and 3I28 crystal structurewas 1.24 �. The alignment score was 0.06 calculated by pro-tein structure alignment panel of Schrçdinger software pack-age. The results from Profile-3D showed that overall com-patibility score for this model was 107.8 in the scale of theexpected value 58.75–130.55. All these data indicated thatthe model was reasonable and reliable.

Before carrying out molecular docking, all the tested sub-strates were treated using the program LigPrep of Maestro9.0 (Schrodinger LLC) to generate lowest energy conforma-tions for all enantiomers. And the molecular docking was re-peated three times using Glide (version 5.5) in standard pre-cision (SP) mode. The Schrçdinger�s proprietary GlideScorewas utilized to score the binding mode. One best bindingpose for each enantiomer was written out and analyzed.[27]

Assignment of Absolute Configuration

Absolute configurations were determined by chiral HPLCor GC analysis by comparison of retention times with refer-ence materials except for (S)-6 and (S)-8. In the case of ep-oxide 6, the absolute configuration of (S)-6 was establishedby comparisons of the specific rotation with the literaturevalues. The absolute configuration of (S)-8 was determinedfrom the sign of its optical rotation. Particularly, the abso-lute configuration of (R)-5 was confirmed by the followingtwo methods: (i) chiral HPLC analysis by comparison of re-tention time with reference values;[4a] (ii) deduced from thesign of optical rotation based on the fact that all the (R)-ep-oxides from various substituted phenyl glycidyl ether deriva-tives (of known absolute configuration) always exhibit nega-tive rotations.

Enzyme Assay

To a stirred solution of purified BMEH in potassium phos-phate buffer (100 mM, pH 7.0), the appropriate amount ofPGE (final concentration 2 mM) was added from a stock so-lution in DMSO to a total volume of 1 mL and then incu-bated at 30 8C. During incubation, the samples were with-drawn periodically, and the harvested mixtures were extract-ed with ethyl acetate. The resulting extracts were analyzedby chiral HPLC analysis. One EH unit (U) was defined asthe amount of enzyme required for the hydrolysis of 1 mmolof PGE (1) per minute under the assay conditions.

Chiral Analysis of Epoxides and Diols

The enantiomeric excess of the epoxides and diols was de-termined using the following columns: Chiralcel OD-H(Daicel, Japan): epoxides 1–4 and 9–10 ; Chiralpak AD-H




(Daicel, Japan): epoxides 5, 7, 11 and 15–16 ; BETA DEXTM

120 (Sulpeco Inc, USA): epoxides 12 and 13 ; Chiralcel OJ-H (Daicel, Japan): epoxides 6, 8 and 14. The antipodal diolsof 2–4 and 5 were analyzed using the Chiralcel OD-H andChiralpak AD-H column, respectively.

Acknowledgements

This work was financially supported by the National NaturalScience Foundation of China (Nos. 20902023 and 31071604),Ministry of Science and Technology, P.R. China(2011CB710800 and 2009ZX09501-016), and China NationalSpecial Fund for State Key Laboratory of Bioreactor Engi-neering (No. 2060204). Cordial thanks are also given to Prof.Dr. Manfred T. Reetz at Max-Planck-Institut f�r Kohlenfor-schung for his constructive discussion, and to Dr. Jie Sun atECUST for her valuable suggestions in the manuscript prepa-ration.

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an unusual (r)-selective epoxide hydrolase with high activity for facile preparation of enantiopure...

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