Enantioselectivities of yeast epoxide hydrolases for 1,2-epoxides

Article abstract of DOI:10.1016/S0957-4166(99)00355-9

Kinetic resolution of homologous series of unbranched 1,2-epoxyalkanes (C-4 to C-12), 1,2-epoxyalkenes (C-4, C-6 and C-8), a 2,2-dialkylsubstituted epoxide (2-methyl-1,2-epoxyheptane) and a benzyloxy-substituted epoxide (benzyl glycidyl ether) was investigated using resting cells of 10 different yeast strains. Biocatalysts with excellent enantioselectivity (E>100) and high initial reaction rates (>300 nmol/min/mg dry weight) were found for the 2-monosubstituted aliphatic epoxides C-6 to C-8. Yeast strains belonging to the genera Rhodotorula, Rhodosporidium and Trichosporon all preferentially hydrolyzed (R)-1,2-epoxides with retention of configuration. The epoxide hydrolases of all the yeast strains are membrane-associated.

Full text of DOI:10.1016/S0957-4166(99)00355-9

Pergamon
Tetrahedron: Asymmetry 10 (1999) 3327–3336
Enantioselectivities of yeast epoxide hydrolases for 1,2-epoxides
Adriana L. Botes,a Carel A. G. M. Weijers,b Piet J. Botes a and Martie S. van Dyk a,∗
aDepartment of Microbiology and Biochemistry, University of the Orange Free State, PO Box 339, Bloemfontein, South Africa
bDivision of Industrial Microbiology, Department of Food Technology and Nutritional Sciences, Wageningen Agricultural
University, PO Box 8129, 6700 EV Wageningen, The Netherlands
Received 22 June 1999; accepted 11 August 1999
Abstract
Kinetic resolution of homologous series of unbranched 1,2-epoxyalkanes (C-4 to C-12), 1,2-epoxyalkenes (C-4,
C-6 and C-8), a 2,2-dialkylsubstituted epoxide (2-methyl-1,2-epoxyheptane) and a benzyloxy-substituted epoxide
(benzyl glycidyl ether) was investigated using resting cells of 10 different yeast strains. Biocatalysts with excellent
enantioselectivity (E>100) and high initial reaction rates (>300 nmol/min/mg dry weight) were found for the 2-
monosubstituted aliphatic epoxides C-6 to C-8. Yeast strains belonging to the genera Rhodotorula, Rhodosporidium
and Trichosporon all preferentially hydrolyzed (R)-1,2-epoxides with retention of configuration. The epoxide
hydrolases of all the yeast strains are membrane-associated. © 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction
Hydrolytic kinetic resolution of racemic epoxides offers a convenient route to obtain single enantio-
mer synthons for enantiopure fine chemicals. Since both the remaining epoxide and the diol product
(employed as cyclic sulfates or sulfites) are useful as reactive intermediates, high product recovery
can be achieved. Terminal epoxides are arguably the most important subclass of epoxides that serve as
building blocks for organic chemistry. Asymmetric hydrolytic catalysts able to resolve these inexpensive
racemates into optically active epoxides and vicinal diols have thus become an important focus of
research. An efficient (salen)Co(III)(OAc) catalyst for the hydrolytic kinetic resolution of terminal C-
3 to C-8 epoxides was recently developed.1 Resolution of long-chain alkyl epoxides (C-10 to C-20) was
also achieved (ees>95% and isolated yields >43%) with this catalyst.2 Biocatalytic hydrolytic resolution
of epoxides as an alternative to heavy metal catalysts has also been explored in recent years. The synthetic
potential of bacterial3,4 and filamentous fungal5 epoxide hydrolases as chiral biocatalysts was extensively
investigated. Epoxide hydrolase activity was found to be ubiquitous in these microorganisms, and the
structural requirements of their epoxide substrates for chiral recognition are now well established.6
∗
Corresponding author. E-mail: botesal@micro.nw.uovs.ac.za
0957-4166/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(99)00355-9

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In contrast, the occurrence of these enzymes in yeasts and their enantioselectivities for epoxides
of different structural classes had not been investigated until very recently. Pioneering work done by
Weijers on the yeast strain Rhodotorula glutinis CIMW 1477 and subsequent screening of yeasts from
many different genera for epoxide hydrolase activity8 revealed unusual enantioselectivities,9 which
differed significantly from those found in bacteria and filamentous fungi. The following results obtained
with yeast epoxide hydrolases are particularly noteworthy: (1) Rhodotorula glutinis displayed epoxide
hydrolase activity and enantioselectivity with an exceptionally broad range of structurally different
substrates, which included aryl, alicyclic, meso and aliphatic epoxides with terminal, subterminal,
trans and cis location of the epoxide moiety. In contrast, epoxide hydrolases of individual bacterial
and filamentous fungal strains have stringent structural requirements for enantioselective hydrolysis of
substrates, which severely limits the range of substrates that can be resolved with single strains; (2) kinetic
resolution of 1,2-epoxyhexane and 1,2-epoxyoctane was achieved with exquisite enantioselectivity using
yeast epoxide hydrolases. No similar enantioselectivity for unbranched 1,2-epoxyalkanes was achieved
with bacterial epoxide hydrolases.10 However, the results obtained with Chryseomonas luteola11 (safety
class II, which is risky to work with) suggest that this unusual enantioselectivity for 2-monosubstituted
1,2-epoxyalkanes may not be restricted to yeasts; (3) hydrolysis of most of the aliphatic substrates
proceeds with unusually high initial reaction rates. Direct comparison of specific activities obtained with
bacterial and fungal biocatalysts cannot be made from the available literature, but careful examination of
experimental conditions published12–15 indicates that hydrolysis of epoxides with bacterial or filamentous
fungal epoxide hydrolases using similar substrate to catalyst ratios would require significantly longer
reaction times; (4) high substrate concentrations (500 mM) of 1,2-epoxyhexane8 and 1,2-epoxyoctane9
can be used without any adverse effects on the reaction rate or enantioselectivity of yeast epoxide
hydrolases. This should facilitate efficient synthesis of enantiopure epoxides on a preparative scale.
From the limited data available on yeast epoxide hydrolases, it is clear that these enzymes have
promising synthetic potential. In the present study, the biocatalytic potential of yeast epoxide hydrolases
is further elucidated.
2. Results and discussion
Asymmetric hydrolysis of the terminal epoxides (ꢀ)-1 to (ꢀ)-4 was achieved by employing resting
cells of the different yeast strains in buffer at pH 7.5. All the strains preferentially hydrolyzed the (R)-
1,2-epoxide to the corresponding (R)-vic diol. From the fact that the formed diol and the remaining
non-hydrolyzed epoxide represent opposite enantiomeric forms, it can be assumed that the hydrolysis
is proceeding with retention of configuration to a large extent. However, more detailed studies (18O-
labelling) have to be done to elucidate the reaction mechanism. Although all the substrates were
hydrolyzed, both the enantioselectivities and the reaction rates were strongly influenced by both the
chain length and substitution pattern of the substrates. Enantiomeric ratios given in Tables 2 and 3
were calculated based on the enantiomeric excesses of the substrates (ees), since epoxide hydrolysis
does not necessarily proceed with complete regioselectivity,16 in which case Sih’s equation17 involving
the enantiomeric purity of the product (eep) is not applicable. E-values that were calculated using eep
indicated that hydrolysis did not proceed with complete regioselectivity.

A. L. Botes et al. / Tetrahedron: Asymmetry 10 (1999) 3327–3336
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2.1. Hydrolysis of 1,2-epoxyalkanes (ꢀ)-1 (Scheme 1)
Scheme 1. Hydrolytic kinetic resolution of a homologous range (C-4 to C-12) of 1,2-epoxyalkanes
Excellent enantioselectivities were displayed by several strains for C-6 to C-8 1,2-epoxyalkanes. The
efficiency of these biocatalysts, expressed as specific activity and catalytic activity (turnover frequency),
is given in Table 1. A decrease in enantioselectivity and reaction rate was displayed with both an
increase and decrease in the chain length (Table 2, Fig. 1). Strains (1), (3) and (5) retained useful (E>20)
enantioselectivity for the C-5 epoxide. Only strain (6) retained enantioselectivity for the C-10 epoxide,
while almost no enantioselectivity was displayed by any of the catalysts for the C-12 epoxide. It should be
noted, however, that the C-10 and C-12 epoxides are highly hydrophobic, and that the low reaction rates
obtained were probably due to low availability of the substrate to the catalyst in the aqueous buffer, since
no co-solvents were used.18 Secondly, low effective substrate concentration has been noted to decrease
the enantioselectivity of hydrolysis by epoxide hydrolases.8,19
Table 1
Efficiency of purified yeast epoxide hydrolase enzymes
2.2. Hydrolysis of 1,2-epoxyalkenes (ꢀ)-2 (Scheme 2)
Scheme 2. Hydrolytic kinetic resolution of a homologous range of 1,2-epoxyalkenes
A decrease in enantioselectivity (Table 3) was observed for most strains with the introduction of an
ω-double bond. As an exception, strain (1) displayed similar excellent enantioselectivity and reaction
rates (Fig. 2) for 1,2-epoxyoctene as for 1,2-epoxyoctane. From the results for the 1,2-epoxyalkenes
it can be observed that the enantioselectivity pattern has moved to longer chain lengths in relation to
the results for 1,2-epoxyalkanes. This might be caused by the small decrease in actual chain length of
epoxyalkenes in comparison with the corresponding epoxyalkanes. Of possible commercial interest will

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Table 2
Enantiomeric ratios (E)a and initial reaction rates (V0)b of yeast strains towards a homologous range
of 1,2-epoxyalkanes (20 mM)
be the hydrolysis of the simplest allylic epoxide, 1,2-epoxy-3-butene. Enantiopure forms of this epoxide
and its corresponding diol are small polyfunctional building blocks for the synthesis of a number of
natural products.22,23
2.3. Hydrolysis of 2-methyl-1,2-epoxyheptane (ꢀ)-3 (Scheme 3)
Scheme 3. Hydrolytic kinetic resolution of a 2,2-disubstituted 1,2-epoxyalkane, 2-methyl-1,2-epoxyheptane
A 2-alkyl-substituent resulted in a dramatic decrease in the enantioselectivity (Table 3) of all strains
except for strain (8), for which the methyl-substituent improved enantioselectivity. These results contrast
sharply with the results obtained for several bacterial epoxide hydrolases,10 which require a directing me-
thyl group in the 2-position of terminal aliphatic epoxides for enantioselectivity. The effect of the methyl
group on C-2 was less pronounced for strains (6) and (8), for which moderate enantioselectivity (E=10
and 18) (Fig. 2A) was found. Reaction rates (Fig. 2B) were comparable to those of the unsubstituted C-6
to C-8 epoxides.

A. L. Botes et al. / Tetrahedron: Asymmetry 10 (1999) 3327–3336
3331
Figure 1. Enantiomeric ratio (A) and initial reaction rate (B) of the five most enantioselective yeasts towards a homologous
range of 1,2-epoxyalkanes
2.4. Hydrolysis of benzyl glycidyl ether (ꢀ)-4 (Scheme 4)
Scheme 4. Hydrolytic kinetic resolution of a benzyloxy-substituted 1,2-epoxide, benzyl glycidyl ether. The absolute configura-
tion of the formed diol is (S) due to a switch in CIP-nomenclature
Asymmetric hydrolysis of benzyl glycidyl ether was achieved using strains (6) and (7). Although the
enantioselectivity (Table 3, Fig. 2) was too low (E<10) to be of practical use, this is the first report
of kinetic resolution of this important building block of β-blocker drugs by epoxide hydrolases. All
the strains, which were moderate (E>10) to excellent (E>30) biocatalysts for straight chain epoxides,
displayed a decrease in enantioselectivity and activity upon the incorporation of a benzyloxy-substituent.
It is noteworthy that the effect of both a 2-methyl- and a benzyloxy-substituent on strain (6) was less
pronounced than for the other strains.

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Table 3
Enantiomeric ratios (E)a and initial reaction rates (V0)b of yeasts towards a homologous range of
1,2-epoxyalkenes, 2-methyl-1,2-epoxyheptane and benzyl glycidyl ether (20 mM)
2.5. Cellular localization of yeast epoxide hydrolases
The epoxide hydrolases of strains (1)20 and (3)21 were previously shown to be membrane-associated.
No epoxide hydrolase activity could be detected in the 13 000×g supernatants of any of the yeast lysates
after extraction of soluble enzymes with Y-PER™ yeast protein extraction reagent. Total enzyme activity
was retained in the cell debris. The epoxide hydrolases of all the tested yeast strains are thus membrane-
associated.
3. Conclusion
Kinetic resolution of a homologous range of C-4 to C-12 epoxides by various yeast epoxide hydrolases,
albeit with different enantioselectivity and reaction rates, has been demonstrated. Excellent E-values
and extremely high reaction rates were displayed for unbranched C-6 to C-8 epoxides by several yeast
strains belonging to the genera Rhodotorula and Rhodosporidium. Hydrolytic kinetic resolution of these
substrates using yeast biocatalysts may be a viable alternative to the (salen)Co(III)(OAc) catalysts
developed by Jacobsen. In contrast to bacterial epoxide hydrolases, the presence of a directing group
on C-2 destroys chiral recognition in most cases. A benzyloxy group resulted in loss of enantioselectivity

A. L. Botes et al. / Tetrahedron: Asymmetry 10 (1999) 3327–3336
3333
Figure 2. Enantiomeric ratio (A) and initial reaction rate (B) of the five most enantioselective yeasts towards a homologous
range of 1,2-epoxyalkenes, 2-methyl-1,2-epoxyheptane and benzyl glycidyl ether
by the strains displaying high enantiopreference for straight-chain epoxides. Asymmetric hydrolysis of
benzyl glycidyl ether was, however, achieved with yeast strains that displayed lower enantiopreference
for straight-chain epoxides. Studies using various other aryl, alicyclic and meso-epoxides are in progress
to assess whether these strains share with Rhodotorula glutinis CIMW 147 the unusually broad range of
substrates7 that can be hydrolyzed enantioselectively.
4. Experimental
4.1. General
All yeasts were obtained from the Yeast Culture Collection of the University of the Orange Free State,
except Rhodotorula glutinis CIMW 147, which was from the laboratory culture collection of Industrial
Microbiology, AU Wageningen. Reactions were monitored and enantiomeric purities were analyzed by
GLC (Hewlett–Packard 4890 equipped with FID) on fused silica cyclodextrin capillary columns (β-DEX

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A. L. Botes et al. / Tetrahedron: Asymmetry 10 (1999) 3327–3336
225 and β-DEX 120, Supelco Inc., and Chiraldex A-TA, Astec, 30 m×0.25 mm, 0.25 µm film) using N2
as carrier gas. Concentrations of epoxides and diols were derived from calibration curves with heat-killed
cells. Optical rotation values were measured on a Perkin–Elmer 241 polarimeter at 589 nm.
4.2. Epoxides 1 and formed diols 1a
C-4, C-5, C-6, C-8 and C-12 racemic epoxides were obtained from Fluka. C-7 and C-10 epoxides
were synthesized as described previously.8,11 Chiral analysis of C-4 and C-5 epoxides was performed
on β-DEX 225 (40°C isotherm), C-6 and C-7 epoxides on β-DEX 120 (45°C and 50°C isotherm), C-8
on either β-DEX 225 or Chiraldex A-TA (55°C and 52°C) and C-10 and C-12 epoxides on Chiraldex
A-TA (90°C and 95°C isotherm). Absolute configurations of C-4 to C-8 epoxides were established from
the previously reported data.8 Determination of the absolute configurations of the resolved residual C-10
and C-12 epoxides was by co-injection on chiral GLC with the enantiopure (R) reference compounds
from Aldrich. C-4, C-5, C-6, C-8 and C-12 diols were obtained from Fluka. C-7 and C-10 diols were
synthesized as described previously.11 Chiral analysis of all the diols was performed on β-DEX 120.
Absolute configurations of the C-4 to C-8 diols were deduced from the previously reported data8 and
of C-10 and C-12 diols by co-injection on chiral GLC with the enantiopure reference compounds from
Aldrich.
4.3. Epoxides 2 and formed diols 2a
The C-4, C-6 and C-8 racemic 1,2-epoxyalkenes were obtained from Aldrich. Chiral analysis of C-4
and C-6 epoxyalkenes was performed on β-DEX 225 (50°C and 60°C isotherm) and C-8 on β-DEX
120 (60°C isotherm). The elution order of the C-6 and C-8 enantiomers was first (R) and second (S).
Surprisingly, however, a reversed elution order of the C-4 enantiomers on the β-DEX 225 column was
observed; first (S)- and second (R)-1,2-epoxy-3-butene was eluted from this column.
4.4. Absolute configurations of residual 1,2-epoxyalkenes 2
Absolute configurations of the residual 1,2-epoxyalkenes obtained after hydrolysis of 500 mg of the
appropriate epoxide by 1 g (dry weight) of Rhodotorula glutinis CIMW 147 cells were determined as
described before for corresponding (S)-1,2-epoxyalkanes.8 Chiral GLC was used for determination of
ees and concentrations were derived from calibration curves. Data of chiral GLC analysis and specific
22
optical rotation values of the residual epoxides are: epoxide (S)-2, C-4: [α]D +9.1 (c=0.11, pentane;
ee=37%) [lit.24 (R)-2, C-4: [α]D23 −0.68 (c=4.44, 2-propanol; ee=9%)] epoxide (S)-2, C-6: [α]D22 −12.4
22
(c=1.61, pentane; ee=95%) epoxide (S)-2, C-8: [α]D −14.0 (c=1.45, pentane; ee=90%) [lit.25 (R)-2,
C-8: [α]D23 +12.2 (neat; ee>80%)].
Determination of the absolute configurations was by comparison of our results with the data reported
in the literature.
4.5. Absolute configurations of formed alkene-1,2-diols 2a
Absolute configurations were determined for the diols formed from hydrolysis of 500 mg of the
appropriate epoxide by 1 g (dry weight) of Rhodotorula glutinis CIMW 147 cells as described before
for corresponding (R)-alkane-1,2-diols.8 Chiral GLC analysis was performed for determination of the ee
values. Data of chiral GLC analysis and specific optical rotation values of the formed diols are as follows.

A. L. Botes et al. / Tetrahedron: Asymmetry 10 (1999) 3327–3336
3335
Diol (R)-2a, C-4: [α]D +7.5 (c=4.8, methanol; ee=32%) [lit.24 (S)-2a, C-4: [α]D −43.6 (c=4.6,
22
20
2-propanol; ee>99%)] diol (R)-2a, C-6: [α]D22 +8.3 (c=8.8, methanol; ee=70%) diol (R)-2a, C-8: [α]D
+12.2 (c=8.3, methanol; ee=95%).
22
Determination of the absolute configurations was by comparison of our results with the data reported
in the literature.
4.6. Epoxide 3 and formed diol 3a
(ꢀ)-2-Methyl-1,2-epoxyheptane 3 was synthesized by direct epoxidation of the corresponding alkene
and the diol (ꢀ)-3a by acid hydrolysis of (ꢀ)-3 as described previously.11 Chiral analysis was performed
for the diol (ꢀ)-3a only, on β-DEX 120 (55°C). Absolute configuration of the diol was deduced from
reported data.22
4.7. Epoxide 4 and formed diol 4a
(ꢀ)-Benzyl glycidyl ether 4 was synthesized by BF3·Et2O-catalyzed addition of benzyl alcohol onto
epichlorohydrin followed by NaOH. Chiral analysis of epoxide (ꢀ)-4 and the acetonide derivative of diol
(ꢀ)-4a was performed on β-DEX 120 (110°C and 145°C). Absolute configurations were established by
co-injection of the commercially available enantiomerically pure epoxides and diols (Fluka).
4.8. Cultivation and preparation of yeast cells
Yeasts which displayed epoxide hydrolase activity were selected and grown at 30°C in 1 L shake-flask
cultures containing 200 ml YM medium supplemented with 1% glucose (w/v). At late exponential phase
(48–72 h) the cells were harvested by centrifugation (10 000×g, 10 min, 4°C), washed with phosphate
buffer (50 mM, pH 7.5), centrifuged again and frozen in glycerol (10%) at −20°C in micro centrifuge
tubes (1 ml cells per micro centrifuge tube). The cells could be stored for several months without
significant loss of activity.
4.9. General procedure for hydrolysis of epoxides
Frozen cells (1 ml) were thawed, washed with phosphate buffer (50 mM, pH 7.5) and resuspended in 4
ml buffer in 20 ml glass bottles with screw caps fitted with septa. The substrates were added neat to final
concentrations of 20 mM. The mixtures were agitated on a shaking water bath at 30°C. The course of
the bioconversions of epoxides was followed by withdrawing 50 µl headspace samples with a gas-tight
syringe and/or by withdrawing samples (200 µl) at appropriate time intervals for longer chain epoxides.
Samples were saturated with NaCl if necessary to facilitate improved extraction of the short chain diols
and extracted with an equal volume of ethyl acetate. After centrifugation (3000×g, 2 min), the remaining
epoxides and formed diols were analyzed by chiral GLC.
4.10. Determination of cellular localization of epoxide hydrolases
Pelleted cells (1 ml) were resuspended in 2 ml Y-PER™ Yeast Protein Extraction Reagent (Pierce), pH
7.4, gently vortexed until the mixture was homogeneous and allowed to stand at room temperature for 1
h. Cell debris and lysates were separated by centrifugation (13 000×g, 10 min). The cell debris pellets
were resuspended in 50 mM phosphate buffer, pH 7.4 (2 ml). 1,2-Epoxyoctane (20 µl) was added to

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A. L. Botes et al. / Tetrahedron: Asymmetry 10 (1999) 3327–3336
the cell pellets and cell lysates, and the reaction mixtures were incubated at 30°C for 6 h. The reactions
were terminated by extraction with 1 ml ethyl acetate, and epoxide hydrolase activities were assayed
by GC analysis of the formed diols. The effect of Y-PER™ on epoxide hydrolase activity was evaluated
by incubating 1 ml pelleted cells treated with 2 ml Y-PER™ as above with 20 µl 1,2-epoxyoctane as a
control.
Acknowledgements
The authors thank Elma Pretorius and Andrè van Wyk (Dept. of Microbiology and Biochemistry,
UOFS, SA) for maintenance of the yeast cultures and Mr. M. de Wit (Div. of Industrial Microbiology,
WAU, NL) and Mr. H. J. Swarts (Dept. of Organic Chemistry, WAU, NL) for assistance in the use
of analytical techniques. Financial support from Sasol Limited and the South African Foundation for
Research Development is also gratefully acknowledged.
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