Stereoselectivity and substrate specificity in the kinetic resolution of methyl-substituted 1-oxaspiro[2.5]octanes by Rhodotorula glutinis epoxide hydrolase

Article abstract of DOI:10.1021/jo050533w

The kinetic resolution of a range of methyl-substituted 1-oxaspiro[2.5]octanes by yeast epoxide hydrolase (YEH) from Rhodotorula glutinis has been investigated. The structural determinants of substrate specificity and stereoselectivity of YEH toward these

Full text of DOI:10.1021/jo050533w

Stereoselectivity and Substrate Specificity in the Kinetic
Resolution of Methyl-Substituted 1-Oxaspiro[2.5]octanes by
Rhodotorula glutinis Epoxide Hydrolase
Carel A. G. M. Weijers, Petra Meeuwse, Robert L. J. M. Herpers,
Maurice C. R. Franssen,* and Ernst J. R. Sudho¨lter
Laboratory of Organic Chemistry, Wageningen University, Dreijenplein 8,
6703HB Wageningen, The Netherlands
maurice.franssen@wur.nl
Received March 16, 2005
The kinetic resolution of a range of methyl-substituted 1-oxaspiro[2.5]octanes by yeast epoxide
hydrolase (YEH) from Rhodotorula glutinis has been investigated. The structural determinants of
substrate specificity and stereoselectivity of YEH toward these substrates appeared to be the
configuration of the epoxide ring and the substitution pattern of the cyclohexane ring. For all
compounds tested, O-axial epoxides were hydrolyzed faster than the corresponding O-equatorial
compounds. In concern of the ring substituents, YEH preferred methyl groups on the Re side of
the ring. Placement of substituents close to the spiroepoxide carbon decreased the reaction rate
but increased enantioselectivity. YEH-catalyzed kinetic resolutions of 4-methyl 1-oxaspiro[2.5]octane
epimers were most enantioselective (E > 100).
Introduction
activity of mycotoxins. Many studies were initiated on
the preparation of effective 1-oxaspiro[2.5]octane deriva-
tives since the first observation that spiroepoxides such
as fumagillin show significant antitumor activity. Several
fumagillin analogues were synthesized which possess
angiogenesis inhibiting activity (Figure 1).4
Apart from their potential value as end products with
biological activity, 1-oxaspiro[2.5]octanes can serve as
chiral starting materials in asymmetric synthesis. As an
1-Oxaspiro[2.n]alkanes are compounds in which an
epoxide moiety is joined to an alicyclic fragment by a
common carbon atom. The spiro attachment makes this
type of epoxides easy accessible and thus highly sensitive
for nucleophilic ring opening. This will result in an en-
hanced reactivity when compared to their epoxycyclo-
alkane counterparts.1 In nature, this high reactivity is
translated to the high toxicity of spiroepoxide-containing
mycotoxins. Typical examples thereof are trichothecenes
and fumagillin-related toxins.2,3
(3) (a) Todd Lowther, W.; McMillen, D. A.; Orville, A. M.; Matthews,
B. W. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 12153-12157. (b) Hala´sz,
J.; Poda´nyi, B.; Vasva´ri-Debrecczy, L.; Szabo´, A.; Hajdu´, F.; Bo¨cskei,
Z.; Hegedu´s-Vajda, J.; Gyo´rb´ıro´, A.; Hermecz, I. Tetrahedron 2000, 56,
10081-10085. (c) Zhou, G.; Tsai, C. W.; Liu, J. O. J. Med. Chem. 2003,
46, 3452-3454.
(4) (a) Griffith, E. C.; Su, Z.; Niwayama, S.; Ramsay, C. A.; Chang,
Y.-H.; Liu, J. O. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 15183-15188.
(b) Baldwin, J. E.; Bulger, P. G.; Marquez, R. Tetrahedron 2002, 58,
5441-5452. (c) Vosburg, D. A.; Weiler, S.; Sorensen, E. J. Chirality
2003, 15, 156-166. (d) Pyun, H.-J.; Fardis, M.; Tario, J.; Yang, C.
Y.; Ruckman, J.; Henninger, D.; Jin, H.; Kim, C. U. Bioorg. Med.
Chem. Lett. 2004, 14, 91-94. (e) Rodeschini, V.; Boiteau, J.-G.; Van
de Weghe, P.; Tarnus, C.; Eustache, J. J. Org. Chem. 2004, 69, 357-
373.
Preparation of synthetic spiroepoxides has focused to
a great extent on compounds mimicking the biological
* To whom correspondence should be addressed: Tel: +31-317-
482976. Fax: +31-317-484914.
(1) Kas’yan, L. I.; Kas’yan, A. O.; Tarabara, I. N. Russ. J. Org. Chem.
2001, 37, 1361-1404.
(2) (a) Bennet, J. W.; Klich, M. Clin. Microbiol. Rev. 2003, 16, 497-
516. (b) Amagata, T.; Rath, C.; Rigot, J. F.; Tarlov, N.; Tenney, K.;
Valeriote, F. A.; Crews, P. J. Med. Chem. 2003, 46, 4342-4350. (c)
Eriksen, G. S.; Pettersson, H.; Lundh, T. Food Chem. Toxicol. 2004,
42, 619-624.
10.1021/jo050533w CCC: $30.25 © 2005 American Chemical Society
Published on Web 07/16/2005
J. Org. Chem. 2005, 70, 6639-6646
6639

Weijers et al.
SCHEME 1. Sulfur Ylide Epoxidation of
Substituted Cyclohexanones
FIGURE 1. Antiangiogenic agents based on the fumagillin
structure.
example, preparation of enantiopure 4,4,5,8-tetramethyl-
1-oxaspiro[2.5]octane as a key intermediate for highly
active fragrances has been reported.5
BL21(DE3)(pEph1), was used for the enantioselective
hydrolysis of racemic 2-isocyano-1-oxaspiro[2.5]octane.12
In most synthetic approaches, enantiopure spiroep-
oxides are prepared by sulfur ylide epoxidation of the
corresponding cyclic ketones. Initially, the desired ster-
eochemistry was simply defined by the starting carbonyl
compound, but subsequent development of chiral sulfur
ylide catalysts has rendered this method into a catalytic
process for asymmetric epoxidation.6 Nevertheless, the
novel methodology is still limited in terms of substrate
scope and has been applied only recently for the epoxi-
dation of a symmetric cyclic ketone in the preparation of
enantiopure 2-phenyl-1-oxaspiro[2.5]octane.7
The alternative approach to obtain enantiopure ep-
oxides is kinetic resolution of racemic epoxides by ste-
reoselective ring opening.8,9 Although this method has
clearly proven its practical utility, it has been very rarely
tested for the resolution of spiroepoxides.1 Stereoselective
hydrolytic ring opening of a spiroepoxide has been
initially demonstrated in the enzymatic hydrolysis of the
(3R)- and (3S)-epimers of 3-spiro[5R-androstane-3,2′ox-
irane]-17â-ol by whole cells of Mycobacterium aurum.10
In two other studies, enzymatic hydrolytic kinetic resolu-
tion of 1-oxaspiro[2.5]octanes has been described. Mam-
malian microsomal epoxide hydrolase (mEH) has been
used in the kinetic resolution of several methyl-substi-
tuted 1-oxaspiro[2.5]octanes.11 Bellucci and co-workers
showed in their study that spiroepoxides in general were
excellent substrates for the mammalian enzyme and that
enantioselectivity was high in case of the presence of a
geminal dimethyl group on the cyclohexane ring. More
recently, epoxide hydrolase of Rhodotorula glutinis,
which was overexpressed in recombinant Escherichia coli
In the present study, yeast epoxide hydrolase (YEH)
containing cells of Rhodotorula glutinis ATCC 201718
were tested for kinetic resolution of a range of methyl-
substituted 1-oxaspiro[2.5]octanes (see structure block).
Discrimination between epimeric structures 1 and 2 was
investigated, as well as effects of methyl substituents on
varying positions of the cyclohexane ring, to gain insight
into the structural requirements of spiroepoxides to serve
as substrates for YEH. In this way, our study aims to
extend the scope of enzymatic kinetic resolution of
epoxides to the structurally and chemically divergent
spiroepoxides.
Results and Discussion
Preparation of Racemic Substrates.
Racemic 1-oxaspiro[2.5]octanes are easy accessible by
sulfur ylide epoxidation of the corresponding cyclohex-
anones (Scheme 1).13 Dimethyl sulfonium ylide was used
for the preparation of spiroepoxides 2a-2c, which were
obtained as mixtures of O-axial and O-equatorial epimers.
Our results, as summarized in Table 1, are in agreement
with findings reported in the literature.13,14
(5) (a) Chapuis, C.; Brauchli, R. Helv. Chim. Acta 1993, 76, 2070-
2088. (b) Liebens, A.; Laufer, W.; Ho¨lderich, W. Catal. Lett. 1999, 60,
71-75.
(6) (a) Aggarwal, V. K.; Richardson, J. Chem. Commun. 2003, 2644-
2651. (b) Aggarwal, V. K.; Winn, C. L. Acc. Chem. Res. 2004, 37, 611-
620.
(7) Aggarwal, V. K.; Bae, I.; Lee, H.-Y.; Richardson, J.; Williams,
D. T. Angew. Chem., Int. Ed. 2003, 42, 3274-3278.
(8) (a) Jacobsen, E. N. Acc. Chem. Res. 2000, 33, 421-431. (b)
Schaus, S. E.; Brandes, B. D.; Larrow, J. F.; Tokunaga, M.; Hansen,
K. B.; Gould, A. E.; Furrow, M. E.; Jacobsen, E. N. J. Am. Chem. Soc.
2002, 124, 1307-1315. (c) Nielsen, L. P. C.; Stevenson, C. P.; Black-
mond, D. G.; Jacobsen, E. N. J. Am. Chem. Soc. 2004, 126, 1360-
1362.
(9) (a) Weijers, C. A. G. M.; de Bont, J. A. M. J. Mol. Catal., B 1999,
6, 199-214. (b) Orru, R. V. A.; Faber, K. Curr. Opin. Chem. Biol. 1999,
3, 16-21. (c) Archelas, A.; Furstoss, R. Curr. Opin. Chem. Biol. 2001,
5, 112-119. (d) Smit, M. S. TIBTECH 2004, 22, 123-129.
(10) Escoffier, B.; Prome, J.-C. Bioorg. Chem. 1989, 17, 53-63.
(11) Bellucci, G.; Chiappe, C.; Ingrosso, G.; Rosini, C. Tetrahedron:
Asymmetry 1995, 6, 1911-1918.
The stereochemical outcomes of sulfur ylide epoxida-
tions are known to be controlled by the extent of revers-
ibility in the formation of intermediate betaines: high
reversibility leads to high stereoselectivity.6,15 Epoxida-
(12) Visser, H.; de Oliveira Villela Filho, M.; Liese, A.; Weijers, C.
A. G. M.; Verdoes, J. Biocatal. Biotransform. 2003, 21, 33-40.
(13) (a) Corey, E. J.; Chaykovsky, M. J. Am. Chem. Soc. 1965, 87,
1353-1364. (b) Gololobov, Y. G.; Nesmeyanov, A. N.; Lysenko, V. P.;
Boldeskul, I. E. Tetrahedron 1987, 43, 2609-2651.
(14) (a) Toda, F.; Imai, N. J. Chem. Soc., Perkin Trans. 1 1994,
2673-2674. (b) Migneco, L. M.; Vecchi, E. Gazz. Chim. Ital. 1997, 127,
19-24.
(15) Johnson, C. R.; Schroeck, C. W.; Shanklin, J. R. J. Am. Chem.
Soc. 1973, 95, 7424-7431.
6640 J. Org. Chem., Vol. 70, No. 17, 2005

Kinetic Resolution of Spiroepoxides
TABLE 1. Preparation of Racemic Spiroepoxide
Substrates by Sulfonium Ylide Epoxidation of
Methyl-Substituted Cyclohexanones
prepared
diMe-sulfonium ylide diMe-oxo-sulfonium ylide
spiroepoxide
substrate
yield 1 + 2 ratio 1:2
yield 1 + 2
ratio 1:2
1a, 2a
1b, 2b
1c, 2c
1d
77%
69%
72%
80:20
49:51
40:60
81%
84%
83%
80%
85%
97:3
97:3
91:9
95:5
99:1
1e
SCHEME 2. YEH-Catalyzed Hydrolysis of 1c and
2c
FIGURE 2. Stereoselective hydrolysis of 6-methyl-1-oxaspiro-
[2.5]octane (400 µmol) by YEH-containing cells of Rhodotorula
glutinis (132 mg dw). The substrate was prepared as a 4:6
mixture of O-axial epimer 1c (b) and O-equatorial epimer 2c
(O).
nucleophilic attack on the easily accessible exocyclic
epoxide carbon. The observed reaction rate (vi ) 85 nmol/
min, mg dw) was by far above values determined for
YEH-catalyzed hydrolysis of any other alicyclic epoxide.17
Further investigation on biohydrolysis of epimeric mix-
ture 1c/ 2c showed however a much lower activity for
spiroepoxide 2c (Figure 2).
In solution, compounds 1c and 2c occur in two different
chair conformations, which are in equilibrium. Equatorial
methyl conformations are reported to predominate for
both epimers (1c, 96% eq Me; 2c, 93% eq Me, respec-
tively).18 Stereochemical discrimination by YEH will thus
primarily concentrate on the configuration of the epoxide
moiety. From the results shown in Figure 2, we can
conclude that the YEH enzyme has a clear preference
for substrate 1c, having the epoxide in the O-axial
position.
Docking of substrates in the enzyme active site has
been studied in detail for various R/â hydrolase fold
epoxide hydrolases (EHs).19 It is known that initial
binding of the substrate occurs by hydrogen bonding of
the epoxide oxygen to two Tyr residues from the covering
lid. Further modulation of the position of the epoxide
carbon takes place by hydrophobic interactions with other
residues in the active-site cavity. Since the X-ray struc-
ture of YEH is still under investigation, substrate docking
in the YEH active site has not been studied in much
detail yet. It is likely that binding of substrates by YEH
is comparable with that of other eukaryotic EHs. The
reason is that YEH has been characterized as an R/â
hydrolase fold enzyme, and the Eph1 gene sequence
revealed high similarity to mammalian mEHs and other
eukaryotic EHs, including the two conserved Tyr resi-
dues.9,20 YEH is assumed to be present as the single EH
tion by dimethylsulfonium ylide is more or less irrevers-
ible due to the low stability of the sulfonium ylide. As a
consequence, reactions with substituted cyclohexanones
will initially generate a mixture of betaine intermediates,
which are irreversibly converted to their respective
spiroepoxides. Increased steric hindrance around the
ketone carbonyl changed the preference of the direction
of methylene addition from axial toward equatorial, thus
reducing the amount of O-equatorial epimers in 2a
compared to 2b and 2c. Dimethyl sulfonium ylide was
not used for epoxidation of di- and trisubstituted cyclo-
hexanones because the O-equatorial epimers of 1d and
1e were expected to be obtained in even less amounts,
due to enhanced steric effects.14
Epoxidation with the sulfoxide-stabilized dimethyloxo-
sulfonium ylide facilitated the reversion of unfavored
betaines to their more stable starting materials. Diaste-
reoselectivity was thereby enhanced to such an extent
that spiroepoxides 1a-1e were obtained almost exclu-
sively as O-axial epimers.
Configurational assignments of the epimers 1 and 2
were performed using NMR analysis. Particularly 13C
chemical shift values for epimeric 1-oxaspiro[2.5]octanes
are reported to be very consistent.16 Characteristic
deshielding by an equatorial epoxide oxygen results in
higher values of 13C chemical shifts for the cyclohexane
ring carbons C3-C8 of O-equatorial epimers. 1H and 13
C
NMR spectra of the synthesized epimers 1 and 2 are
given in the Supporting Information.
Stereoselective Hydrolysis of Epimeric Spiroep-
oxides 1c and 2c.
In preliminary experiments, spiroepoxide 1c was tested
for bioconversion by YEH-containing cells of Rhodotorula
glutinis ATCC 201718. We noticed a very rapid hydroly-
sis of 1c and a concomitant formation of the diol cis-3c
(Scheme 2). Apparently, there is a facile enzymatic
(17) Weijers, C. A. G. M. Tetrahedron: Asymmetry 1997, 8, 639-
647.
(18) Uebel, J. J. Tetrahedron Lett. 1967, 47, 4751-4754.
(19) (a) Schiøtt, B.; Bruice, T. C. J. Am. Chem. Soc. 2002, 124,
14558-14570. (b) Arand, M.; Cronin, A.; Oesch, F.; Mowbray, S. L.;
Jones, T. A. Drug Metab. Rev. 2003, 35, 365-383.
(20) (a) Visser, H.; Vreugdenhil, S.; de Bont, J. A. M.; Verdoes, J.
C. Appl. Microbiol. Biotechnol. 2000, 53, 415-419. (b) Visser, H.;
Weijers, C. A. G. M.; van Ooyen, A. J. J.; Verdoes, J. C. Biotechnol.
Lett. 2002, 24, 1687-1694. (c) Visser, H. Ph.D. Thesis, Wageningen
University, The Netherlands, 2002.
(16) (a) Bottin-Strzalko, T.; Roux-Schmitt, M. C. Org. Magn. Reson.
1980, 14, 145-146. (b) Davis, R.; Kluge, A. F.; Maddox, M. L.;
Sparacino, M. L. J. Org. Chem. 1983, 48, 255-259. (c) Bellucci, G.;
Chiappe, C.; Lo Moro, G. J. Org. Chem. 1995, 60, 6214-6217. (d)
Vedejs, E.; Dent, W. H.; Kendall, J. T.; Oliver, P. A. J. Am. Chem. Soc.
1996, 118, 3556-3567.
J. Org. Chem, Vol. 70, No. 17, 2005 6641

Weijers et al.
FIGURE 4. Time course of the kinetic resolution of spiroep-
oxides 1b/2b (200 µmol) by YEH-containing cells of Rhodot-
orula glutinis (106 mg dw). The solution contained an epimeric
1:1 mixture of 1b (O-ax) and 2b (O-eq), consisting of (3R,5R)-
1b (b), (3S,5S)-1b (O), (3R,5S)-2b ([), and (3S,5R)-2b (]).
FIGURE 3. Schematic presentation of proposed bindings of
substrates 1c and 2c in the YEH active site. Orientation of
the epoxide moiety of 1c and 2c represents initial noncovalent
binding of the preferred O-axial epimer 1c and is based on
key interactions with Asp190, Tyr262, and Tyr331 residues.
O-Equatorial epimer 2c is oriented with epoxide oxygen
upward for most optimum binding to the two tyrosines, just
like in 1c. Residues of Rhodotorula glutinis YEH have been
determined in a previous study.20
tiomers remained unaffected in resolution experiments
(see Figure S1 Supporting Information).
The kinetic resolutions of substrates 1a and 2a and
1b and 2b are presented in Scheme 3. Substrates 2a and
2b are depicted in their proposed binding conformation
with the oxygen atoms pointing upward, according to the
orientation of 2c in Figure 3. In Scheme 3, it is shown
that enantiomers of 1a, 2a, 1b, and 2b with methyl
substituents on the Re side of the cyclohexane ring were
preferably hydrolyzed by YEH of Rhodotorula glutinis.
Our results were most striking since it had been reported
that the mammalian mEH did not show a significant
discrimination between the O-axial and O-equatorial
epimers of substrates 1a and 2a and 1b and 2b.11 In
addition, enantioselectivities of mEH were only moderate
in hydrolysis of the individual substrates. Because of the
close similarity of YEH and mammalian mEH, no such
large differences in substrate specificity were expected.20
A possible explanation for the much better stereoselective
performance of YEH might be a difference in protein con-
formation, caused by the difference in environment where
both membrane associated enzymes were situated during
the biocatalysis experiments. In the present study, YEH
was still embedded in its most optimal surroundings, the
parent yeast cell, whereas the mammalian mEH has been
used as an isolated enzyme. However, preferential con-
version of O-axial spiroepoxide epimers is not restricted
to the yeast enzyme, since an O-axial epimeric preference
has been described for the hydrolysis of steroidal spiro-
epoxides by cells of Mycobacterium aurum.10
enzyme in Rhodotorula glutinis ATCC 201718 since there
is only one EH-encoding gene found in this yeast.20c
Proposed binding of epimers 1c and 2c in the YEH
active site is schematically depicted in Figure 3. The
proposed binding indicates that the two epimers will
require a different spatial orientation in the active site,
from which the stereochemical discrimination by YEH
can be understood.
Diastereoselective and Enantioselective Hydro-
lysis of Spiroepoxides 1b and 2b.
To investigate the diastereoselectivity and enantiose-
lectivity of YEH toward 1-oxaspiro[2.5]octanes, we de-
cided to study to the nonsymmetrical 5-methyl-substi-
tuted substrates 1b and 2b. YEH-catalyzed hydrolyses
of racemic 1b and the epimeric mixture 1b/2b were
investigated. In the latter reaction, the O-axial epimer
1b was preferably hydrolyzed, thus confirming the results
obtained with mixture 1c/ 2c. Furthermore, significant
enantioselectivity was found in the hydrolysis of indi-
vidual racemic substrates 1b and 2b, respectively (Figure
4). This enabled the isolation of the enantiopure epoxides
(3S,5S)-1b and (3S,5R)-2b by sequential kinetic resolu-
tion of the epimeric mixture 1b/2b, showing the potential
of YEH for the resolution of these compounds.
The effect of methyl substituents on varying positions
of the cyclohexane ring of substrates 1a-1c and 2a-2c
on the activity of YEH is summarized in Figure 5. The
results demonstrate that the effect of the substitution
pattern of the cyclohexane ring on the YEH activity is
similar for both the O-axial and O-equatorial spiroep-
oxides. The substrate specificity, as presented in Figure
5, clearly shows that YEH has a preference for (i)
spiroepoxides with substituents remote from the epoxide
moiety and (ii) spiroepoxide isomers bearing substituents
on the Re side of the cyclohexane ring.
Diastereoselective and Enantioselective Hydro-
lysis of Spiroepoxides 1a and 2a.
Increased diastereoselectivity and enantioselectivity,
when compared to hydrolysis of 1b and 2b, were observed
in kinetic resolutions of the more sterically hindered
4-methyl-1-oxaspiro[2.5]octanes 1a and 2a. Discrimina-
tion between 1a and 2a was very high, with preferential
hydrolysis of the O-axial substrate 1a. Enantioselectivity
toward enantiomers of the individual epimers 1a and 2a
was in both cases nearly absolute (E > 100). Only the
(3R,4S)-isomer of 1a and (3R,4R)-isomer of 2a were
accepted by the YEH enzyme, whereas the other enan-
The determination of the absolute configurations of
residual spiroepoxides and formed diols was performed
6642 J. Org. Chem., Vol. 70, No. 17, 2005

Kinetic Resolution of Spiroepoxides
SCHEME 3. YEH-Catalyzed Kinetic Resolution of 1a, 2a, 1b, and 2b and Conversion of Products to the
Corresponding Cyclohexanones for Stereochemical Assignment
as follows: (i) the relative configuration of the quaternary
carbon was assigned directly by 13C NMR spectroscopy
or by enantioselective gas chromatography (GC) by co-
injection with a prepared reference compound with
known configuration; (ii) the configuration of the methyl-
substituted carbons was determined by oxidation to the
corresponding cyclohexanones, followed by analysis of
their optical rotation. Specific optical rotations of enan-
tiopure methyl-substituted cyclohexanones were avail-
able as reference data.23a,25,27
selective extraction, with pentane and ethyl acetate,
respectively. Sodium metaperiodate-mediated oxidative
cleavage was used for the subsequent conversion of diols
to their respective cyclohexanones.21 Base-catalyzed hy-
drolysis to the corresponding diols preceded metaperio-
date oxidation in case of residual spiroepoxides (Scheme
3). The results of the kinetic resolutions are summarized
in Table 2.
Di- and Trimethyl-Substituted Spiroepoxides 1d
and 1e.
More specifically, residual spiroepoxides and formed
diols were isolated from the final reaction mixture by
Investigations were further extended toward sub-
strates bearing methyl substituents on the Si side of the
cyclohexane ring. Dimethyl-substituted spiroepoxide 1d
was not converted by YEH-containing cells. This result
is in accordance with the observation that compounds
bearing a methyl substituent adjacent to the spirocarbon
on the Si side of the cyclohexane ring were not hydrolyzed
(21) (a) Zhong Y.-L.; Shing, T. K. M. J. Org. Chem. 1997, 62, 2622-
2624. (b) Doderer, K.; Lutz-Wahl, S.; Hauer, B.; Schmid, R. D. Anal.
Biochem. 2003, 321, 131-134. (c) Mateo, C.; Archelas, A.; Furstoss,
R. Anal. Biochem. 2003, 314, 135-141.
(22) (a) Prelog, V. Pure Appl. Chem. 1964, 9, 119-130. (b) Tanikaga,
R.; Obata, Y.; Kawamoto, K.-I. Tetrahedron: Asymmetry 1997, 8,
3101-3106.
(23) (a) Allinger, N. L.; Riew, C. K. J. Org. Chem. 1975, 40, 1316-
1321. (b) Laruelle, C.; Lepant, M. U.S. Patent, 1992, WO 92/22529.
(24) Morisseau, C.; Nellaiah, H.; Archelas, A.; Furstoss, R.; Baratti,
J. C. Enzyme Microb. Technol. 1997, 20, 446-452.
(25) Meyers, A. I.; Williams, D. R.; Erickson, G. W.; White, S.;
Druelinger, M. J. Am. Chem. Soc. 1981, 103, 3081-3087.
(26) Park, J.; Pedersen, S. Tetrahedron 1992, 48, 2069-2080.
(27) Thurkauf, A.; Hillery, P.; Jacobson, A. E.; Rice, K. C. J. Org.
Chem. 1987, 52, 5466-5467.
FIGURE 5. Substrate specificity in YEH-catalyzed kinetic
resolution of monomethyl-substituted 1-oxaspiro[2.5]octanes.
J. Org. Chem, Vol. 70, No. 17, 2005 6643

Weijers et al.
TABLE 2. YEH-Catalyzed Kinetic Resolution of Methyl-Substituted 1-Oxaspiro[2.5]octanes
residual spiroepoxide
diol producta
substrate
E value
eeb
yieldb
abs. conf.
[R]20
eeb
abs. conf.
[R]20
D
D
(rac)-1a
(rac)-2a
(rac)-1b
(rac)-2b
1c/2ce
>100
>100
28
>98%
76%
49%
11%
42%
19%
48%
(3S,4R)-1a
(3S,4S)-2a
(3S,5S)-1b
(3S,5R)-2 b
trans-2c
no reaction
(3R,7S)-1e
+6.2 (c 1.1, hexane)
ndc
+39.1 (c 1.2, hexane)
+24.0 (c 0.1, hexane)
91%
nd
(1R,2S)-3a
(1R,2R)-3a
(1R,3R)-3b
(1R,3S)-3b
cis-3c
no reaction
(1S,5R)-3e
-6.5 (c 1.1, CHCl3)
nd
>98%
70%
62%
nd
-1.2 (c 1.1, CHCl3)
nd
4
>98%d
74%d
1d
(rac)-1e
12
>98%
25%
f
48%
g
a 1-Hydroxy-x-methyl-cyclohexanemethanol. b ee and yield of single epimer. c Not determined. d Value of de. e Mixture of epimers 1c
(cis) and 2c (trans). f Specific rotation reference compound (3R,7S)-1e (ee ) 70%) [R]20 ) +5.2. (c 0.7, hexane). g Specific rotation diol
D
product (1S,5R)-3e (ee ) 73%) [R]20 ) -2.8. (c 0.6, CHCl3).
D
SCHEME 4. Preparation of (3R,7S)-1e by Baker’s
Yeast Mediated Reduction
(Fermipan Red) was selected for this reaction. Baker’s
yeast reduction of (rac)-4e proceeded according to Prelog’s
rule (Re side hydride attack on the ketone carbonyl),
resulting in predominant formation of trans-(1S,5R)-5.22
Alcohol (1S,5R)-5 and residual ketone (5S)-4e were
isolated from the reaction mixture and subsequently
purified by column chromatography. Determination of
the absolute configurations of (1S,5R)-5 and (5S)-4e was
done by determination of the specific rotations and
comparison with reference data.23 Epoxidation of (5S)-
4e by dimethyloxosulfonium ylide yielded reference
compound (3R,7S)-1e, enantiomeric excess (ee) ) 70%;
[R]20 ) +5.2 (c 0.7, hexane).
D
By comparative GC analysis, we determined the con-
figuration of the residual spiroepoxide 1e, obtained by
YEH-catalyzed resolution, as (3R,7S)-1e. Consequently,
the enantiomer bearing the geminal dimethyl group on
the Si side of the cyclohexane ring was preferably
hydrolyzed by YEH. This result was not in accordance
with the observed preferences for other tested spiroep-
oxides, but it has to be considered that substrate 1e in
any case was a poor substrate for YEH. The axial methyl
of the geminal dimethyl group will possibly hamper
optimal orientation of either enantiomer of 1e in the YEH
active site. For a better understanding of this observa-
tion, more detailed information about the crystal struc-
ture of the YEH enzyme will be necessary.
(enantiomers (3S,4R)-1a and (3S,4S)-2a, respectively,
Figure 5).
Acceptance of substituents on the carbon atom â to the
spirocarbon on the Si side was expected to be less tight,
considering the lower enantioselectivity observed in the
hydrolysis of 1b and 2b (Figure 4). Trimethyl-substituted
spiroepoxide 1e was used to study the effect of substitu-
tion on this â-position. Although the reaction rate has
dropped dramatically (vi ) 0.52 nmol/min, mg dw)
compared to the rate for 1b (Figure 5, C5), it was still
possible to perform a complete kinetic resolution of
substrate 1e with cells of Rhodotorula glutinis (see
Supporting Information).
Determination of the absolute configurations of re-
sidual spiroepoxide 1e and diol 3e was initially set up
according to the procedure shown in Scheme 3. However,
due to the greatly increased hydrophobic character of diol
3e, selective extraction of residual spiroepoxide 1e was
hampered by co-extraction of amounts of diol 3e. We
therefore turned to an alternative approach for determi-
nation of the absolute configuration by preparing an
enantiomerically enriched spiroepoxide reference com-
pound 1e. This was achieved by yeast-mediated enanti-
oselective reduction of ketone (rac)-4e, followed by sulfur
ylide epoxidation to give the desired epoxide (Scheme 4).
For enantioselective reduction of ketone (rac)-4e, bak-
er’s yeasts from several commercial sources were screened
and a highly enantioselective baker’s yeast preparation
Conclusions
Kinetic resolution of 1-oxaspiro[2.5]octanes by YEH-
containing cells of Rhodotorula glutinis ATCC 201718
has been demonstrated. Several methyl-substituted spiro-
epoxides have been prepared in enantiopure form by this
methodology. Substrate recognition of spiroepoxides by
YEH was found to be primarily dependent on the con-
figuration of the epoxide moiety: O-axial spiroepoxides
were preferably hydrolyzed.
Methyl substituents on varying positions of the cyclo-
hexane ring were found to have a large effect on both
activity and enantioselectivity of substrate hydrolysis.
Placement of substituents closer to the epoxide moiety
resulted in a decrease of reaction rate and an increase
of enantioselectivity. Substitution on the Re side of the
cyclohexane ring was strongly preferred above Si side
substitution. Preferential conversion of Re substituted
substrates actually defined the enantiomeric discrimina-
tion in spiroepoxide hydrolysis.
Throughout this study, O-axial-preferred spiroepoxide
hydrolysis has been consistently observed in YEH-
6644 J. Org. Chem., Vol. 70, No. 17, 2005

Kinetic Resolution of Spiroepoxides
selective GC. Initial reaction rates were determined from
substrate disappearance. In general, reactions were termi-
nated when the residual spiroepoxides reached ee > 98%.
Subsequently, yeast cells were removed from the reaction
mixture by centrifugation. Residual spiroepoxides in formed
diols were isolated from the supernatants by selective extrac-
tion with pentane and ethyl acetate, respectively.
catalyzed conversions. It is hence assumed that epimeric
discrimination is an intrinsic feature of the YEH enzyme.
Although O-equatorial epimers were gradually converted
as well, it cannot be fully excluded that some apparent
activity for O-equatorial epimers was (partly) due to
hydrolysis of their O-axial conformers, concomitantly
present in the reaction mixture by conformational equi-
libria. The latter phenomenon could in this way possibly
mask an even more definite intrinsic preference of YEH
for O-axial epimers. We have therefore continued our
study with investigations on the behavior of conforma-
tionally locked spiroepoxide substrates in YEH-catalyzed
reactions. The complete results of the subsequent studies
will be presented elsewhere. Initial results of these
studies confirm the preferential hydrolysis of O-axial
spiroepoxide epimers.
General Procedure for the Determination of the
Enantiomeric Ratio (E value).
For determination of the enantiomeric ratio E, ln[fast]0/[fast]
vs ln[slow]0/ [slow] was plotted. The slope of this curve
represents the E value.24 Concentrations of the fast-reacting
enantiomer at times 0 and t are represented by [fast]0 and
[fast], while [slow]0 and [slow] are the concentrations of the
slow-reacting enantiomer at times 0 and t, respectively.
General Procedure for the Determination of the
Absolute Configuration of Residual Spiroepoxides and
Formed Diols.
Kinetic resolutions were scaled-up for conversion of 0.5 g of
substrate. For hydrolysis of substrates 1a, 1b, and 2a-2c,
0.3-1.3 g (dw) of cells of Rhodotorula glutinis were used and
incubations were continued for complete resolution within 6
h. Resolution of substrate 1e (0.5 g) was performed by 2.0 g
(dw) of yeast cells and incubation for 24 h. The reactions were
monitored by analysis with enantioselective GC. Residual
spiroepoxides and formed diols were isolated by selective
extraction, dried, and concentrated. Determination of the
absolute configuration of residual spiroepoxides and formed
diols was as follows: (i) the relative configuration of the
quaternary carbon was assigned directly by 13C NMR spec-
trometry or by enantioselective GC by co-injection with a
prepared reference compound with known configuration; (ii)
configuration of the methyl-substituted carbons was deter-
mined by subsequent oxidative cleavage with periodate to the
corresponding cyclohexanones, followed by analysis of their
optical rotation. Specific optical rotations of enantiopure
methyl-substituted cyclohexanones were available as reference
data.23a,25,27
Oxidative cleavage of diols to cyclohexanones was performed
by adding 0.10 g of diol to a stirred solution of 0.15 g of NaIO4
in 5 mL of water. The mixture was stirred at room temperature
for 1 h and extracted with pentane (3 × 5 mL). The combined
organic layers were dried and concentrated under reduced
pressure. The resulting cyclohexanones were redissolved in
appropriate solvents for the measurement of ee by enantiose-
lective GC and specific optical rotation.
Experimental Section
General Procedure for the Synthesis of 1, Exemplified
by O-ax 4-Methyl-1-oxaspiro[2.5]octane (1a).
To a clear-yellow solution of trimethylsulfoxonium iodide
(7.75 g, 35 mmol) in 75 mL of dry DMSO was added 2.52 g
(22.5 mmol) of 2-methylcyclohexanone with stirring. The
mixture was brought under a N2 atmosphere, and a solution
of potassium tert-butoxide (3.95 g, 35 mmol) in 50 mL dry
DMSO was slowly added. The resulting solution was stirred
at room temperature for 16 h under N2. The reaction was
quenched by addition of water (150 mL) and extracted with
diethyl ether (3 × 50 mL). Combined organic layers were
washed with water (50 mL), dried over Na2SO4, and concen-
trated under reduced pressure. Bulb-to-bulb distillation gave
(rac)-1a (2.04 g, 81%) as a colorless liquid. 1H NMR (200 MHz,
CDCl3): δ 0.80 (3H, d), 1.39-1.77 (9H, m), 2.46 (1H, d), 2.73
(1H, d). 13C NMR (50 MHz, CDCl3): δ 14.3 (CH3), 24.0 (CH2),
24.6 (CH2), 32.6 (CH2), 32.9 (CH2), 34.5 (CH), 52.6 (CH2), 60.9
(C). MS m/z 126 [M]+; HRMS calcd for C8H14O 126.1045, found
126.1042.
General Procedure for the Synthesis of 2, Exemplified
by O-eq 4-Methyl-1-oxaspiro[2.5]octane (2a).
Substrate 2a was prepared from 2-methylcyclohexanone
(2.52 g) according to the method for compound 1a, using
trimethylsulfonium iodide (7.75 g, 38 mmol) for epoxidation.
Compound 2a was obtained as a mixture of 1a/2a (80:20) in
1
77% yield (1.94 g) as a colorless liquid. H NMR (200 MHz,
The residual spiroepoxides were hydrolyzed to their corre-
sponding diols, preceding oxidative cleavage. For base-
catalyzed hydrolysis, 0.10 g of spiroepoxide was added to a
solution of 10 mL of 1 M NaOH and stirred at room temper-
ature for 24 h. The mixture was neutralized with H3PO4,
saturated with NaCl, and extracted with ethyl acetate. The
combined organic layers were dried and the solvent removed
under reduced pressure. Acid-catalyzed hydrolysis was ac-
cordingly performed in 1 N H3PO4 solution instead of NaOH.
The obtained diols were subsequently used for reaction with
NaIO4.
CDCl3): δ 0.70 (3H, d, CH3 1a/2a), 1.22-1.72 (9H, m, 1a/2a),
2.33 (1H, d, CH2 of C2 2a), 2.35 (1H, d, CH2 of C2 1a), 2.57
(1H, d, CH2 of C2 2a), 2.63 (1H, d, CH2 of C2 1a). 13C NMR
(50 MHz, CDCl3): δ 14.1 (CH3 1a), 14.7 (CH3 2a), 23.7 (CH2
2a), 23.9 (CH2 1a), 24.5 (CH2 1a), 25.3 (CH2 2a), 32.4 (CH2
1a), 32.7 (CH2 1a), 33.1 (2*CH2 2a), 34.3 (CH 1a), 35.4 (CH
2a), 50.3 (CH2 2a), 52.3 (CH2 1a), 60.6 (C 1a), 65.6 (C 2a). MS
m/z 126 [M]+; HRMS calcd for C8H14O 126.1045, found
126.1045.
General Procedure for YEH-Catalyzed Kinetic Reso-
lution with cells of Rhodotorula glutinis.
(3S,4R)-4-Methyl-1-oxaspiro[2.5]octane (1a) and (1R,2S)-
1-Hydroxy-2-methyl-cyclohexanemethanol (3a) by YEH-
Catalyzed Resolution.
Rhodotorula glutinis ATCC 201718 was cultivated for 48 h
in a mineral medium supplemented with 2% glucose and 0.2%
yeast extract, at 30 °C in a shaking incubator. Cells were
harvested by centrifugation at 10 000 g, washed with 50 mM
potassium phosphate buffer pH 8.0, concentrated, and stored
at -20 °C for future experiments.
Hydrolysis of spiroepoxides was routinely performed in 100-
mL screw-capped bottles sealed with rubber septa. The bottles
contained cells of Rhodotorula glutinis (0.1-1.0 g dw) and 50
mM potassium phosphate buffer pH 8.0 to a total volume of
10 mL. The bottles were placed into a shaking water bath at
35 °C, and the reaction was started by addition of 0.20 mmol
of the appropriate (neat) substrate. The course of the reaction
was followed by monitoring headspace samples with enantio-
Residual spiroepoxide (3S,4R)-1a was obtained in 49% yield
as a colorless liquid: ee > 98%; [R]20 +6.2 (c 1.1, hexane).
D
Base-catalyzed hydrolysis, followed by oxidative cleavage of
enantiopure (3S,4R)-1a, yielded ketone (2R)-4a as a colorless
1
liquid: ee ) 91%; [R]20 -21.5 (c 0.4, methanol). H and 13C
D
spectra of (2R)-4a matched the spectra of commercially avail-
able (rac)-4a. The absolute configuration of (2R)-4a was
determined by comparison of the specific optical rotation with
literature data (2S)-4a: [R]23 +12.2 (c 4.0, methanol); ee )
D
87%. The absolute configuration of (3S,4R)-1a was derived
from the configuration of (2R)-4a and the O-axial configuration
of (rac)-1a (Scheme 3).
J. Org. Chem, Vol. 70, No. 17, 2005 6645

Weijers et al.
Diol (1R,2S)-3a was obtained in 44% yield as a yellow oil:
7.0. The reaction was started by addition of (rac)-4e (2.2 g),
and incubation was continued for 2 days in an orbital shaker
at 35 °C. Yeast cells were discarded from the reaction mixture
by centrifugation (10 000 g, 10 min, 4 °C), and the superna-
tants extracted with diethyl ether. The combined organic
layers were dried and the solvent removed under reduced
pressure. Alcohol 5 and residual ketone 4e were obtained as
a mixture. Purification was done by flash chromatography on
silica gel with petroleum ether (bp 40-60 °C)/diethyl ether (10:
1), followed by concentration under reduced pressure.
ee ) 91%; [R]20 -6.5 (c 1.1, CHCl3). 1H NMR (200 MHz,
D
CDCl3): δ 0.86 (3H, d), 1.20-1.64 (8H, m), 1.72-1.79 (1H, m),
2.11 (2H, OH, broad), 3.35 (1H, d), 3.60 (1H, d). 13C NMR (50
MHz, CDCl3): δ 15.1 (CH3), 21.5 (CH2), 25.1 (CH2), 30.5 (CH2),
33.9 (CH2), 36.3 (CH), 69.0 (CH2), 73.2 (C). Comparison of 13
C
NMR values with data from literature revealed that the
relative stereochemistry of diol (1R,2S)-3a was cis.26 Oxidative
cleavage of (1R,2S)-3a yielded ketone (2S)-4a as a colorless
liquid: ee ) 83%; [R]20D +19.2 (c 0.73, methanol). The absolute
configuration of (2S)-4a was determined by comparison of the
Residual ketone (5S)-4e was isolated in 36% yield (0.8 g)
specific optical rotation with literature data (2S)-4a: [R]25
as a colorless liquid: ee ) 70%; [R]20 +20.8 (c 0.25, CHCl3).
D
D
+12.2 (c 4.0, methanol); ee ) 87%.25 The absolute configuration
of (1R,2S)-3a was derived from the configuration of (2S)-4a
and the assigned cis stereochemistry of (1R,2S)-3a (Scheme
3).
1H and 13C NMR spectra of (5S)-4e matched the spectra of
commercially available (rac)-4e. The absolute configuration of
(5S)-4e was determined by comparison of the specific optical
rotation with literature data (5S)-4e: [R]24D +20.3 (c 1, CHCl3);
ee ) 75%.23a
trans-6-Methyl-1-oxaspiro[2.5]octane (2c) and cis-1-
Hydroxy-4-methyl-cyclohexanemethanol (3c) by YEH-
Catalyzed Resolution.
Alcohol (1S,5R)-5 was isolated in 10% yield (0.2 g) as a
yellow oil: ee ) 77%; [R]20D -9.2 (c 1.1, CHCl3). The absolute
configuration of trans-(1S,5R)-5 was determined by comparison
of the specific optical rotation with literature data of trans-
Spiroepoxide trans-2c was prepared by resolution of sub-
strate 2c and obtained as a colorless liquid: diasteromeric
excess (de) > 98%; yield 48% (0.24 g). Purification was done
by flash chromatography on silica gel with petroleum ether
(bp 40-60 °C)/diethyl ether (4:1), followed by concentration
(1S,5R)-5: [R]24 -15.5 (c 1, CHCl3).23b
D
Reference compound (3R,7S)-1e was prepared by epoxida-
tion of 0.3 g of ketone (5S)-4e with dimethyloxosulfonium ylide,
according to the method for 1a. Compound (3R,7S)-1e was
obtained in 81% yield (0.24 g) as a colorless liquid: ee ) 70%;
1
under reduced pressure. H NMR (300 MHz, CDCl3): δ 0.87
(3H, d), 1.03-1.26 (4H, m), 1.42 (1H, m), 1.71-1.82 (4H, m),
2.51 (2H, s). 13C NMR (75 MHz, CDCl3): δ 21.6 (CH3), 31.5
(CH), 33.2 (2*CH2), 33.9 (2*CH2), 54.8 (CH2), 59.5 (C).
Diol cis-3c was prepared by a duplicate experiment and
isolated after termination of the reaction at lower conversion
of substrate 2c. Diol cis-3c was obtained as a yellow solid: de
> 98%; yield 28% (0.14 g). Purification was done by flash
chromatography on silica gel with petroleum ether (bp 40-60
°C)/diethyl ether (4:1), followed by removal of the solvent under
reduced pressure. 1H NMR (200 MHz, CDCl3): δ 0.90 (3H, d),
1.16-1.36 (5H, m), 1.42-1.72 (4H, m), 2.27 (2H, OH, broad),
3.38 (2H, s). 13C NMR (50 MHz, CDCl3): δ 22.3 (CH3), 29.0
(2*CH2), 32.4 (CH), 33.5 (2*CH2), 71.1 (C), 71.7 (CH2). The
stereochemistry of biohydrolysis product diol cis-3c was de-
termined by enantioselective GC analysis by co injection with
a reference compound cis-3c, prepared by known base-
catalyzed hydrolysis of cis-1c.
[R]20 ) +5.2 (c 0.7, hexane). 1H and 13C NMR spectra of
D
(3R,7S)-1e matched the spectra of substrate (rac)-1e.
Acknowledgment. The authors are indebted to Dr.
M. A. Posthumus for performing mass spectral analyses,
to Mr. B. van Lagen for recording the NMR spectra, and
to Mr. E. J. C. van der Klift for support in the use of
other analytical techniques.
Supporting Information Available: Graphs of YEH-
catalyzed kinetic resolution of substrates 1a, 2a, and 1e.
General experimental procedures, descriptions of the synthe-
sis, and characterization of substrates 1b-1e, 2b, and 2c.
Product characterization data of compounds obtained by YEH-
catalyzed kinetic resolutions of 1b, 2a, 2b, and 1e. 1H and 13
C
NMR spectra of substrates and reaction products, mass
spectroscopy peak listings of substrates, and conditions of GC
analysis. This material is available free of charge via the
Internet at http://pubs.acs.org.
Reference Compound (3R,7S)-5,5,7-Trimethyl-1-oxa-
spiro[2.5]octane (1e), Prepared by Baker’s Yeast Medi-
ated Reduction of 3,3,5-Trimethylcyclohexanone.
For reduction of ketone 4e, 300 g of lyophilized baker’s yeast
(Fermipan Red) was suspended in 1 L of phosphate buffer pH
JO050533W
6646 J. Org. Chem., Vol. 70, No. 17, 2005