Highly enantioselective hydrolysis of alicyclic meso-epoxides with a bacterial epoxide hydrolase from Sphingomonas sp. HXN-200: Simple syntheses of alicyclic vicinal trans-diols

Article abstract of DOI:10.1039/b300435j

Hydrolysis of N-benzyloxycarbonyl-3,4-epoxy-pyrrolidine and cyclohexene oxide with the epoxide hydrolase of Sphingomonas sp. HXN-200, respectively, gave the corresponding vicinal trans-diols in high ee and yield, representing the first example of enantioselective hydrolysis of a meso-epoxide with a bacterial epoxide hydrolase.

Full text of DOI:10.1039/b300435j

Highly enantioselective hydrolysis of alicyclic meso-epoxides with a
bacterial epoxide hydrolase from Sphingomonas sp. HXN-200: simple
syntheses of alicyclic vicinal trans-diols
Dongliang Chang, Zunsheng Wang, Maarten F. Heringa, Renato Wirthner, Bernard Witholt and Zhi
Li*
Institute of Biotechnology, Swiss Federal Institute of Technology Zurich, ETH-Hoenggerberg, CH-8093
Zurich, Switzerland. E-mail: zhi@biotech.biol.ethz.ch; Fax: +41 1 633 1051; Tel: +41 1 633 3811
Received (in Cambridge, UK) 14th January 2003, Accepted 5th March 2003
First published as an Advance Article on the web 18th March 2003
Hydrolysis of N-benzyloxycarbonyl-3,4-epoxy-pyrrolidine
and cyclohexene oxide with the epoxide hydrolase of
Sphingomonas sp. HXN-200, respectively, gave the corre-
sponding vicinal trans-diols in high ee and yield, represent-
ing the first example of enantioselective hydrolysis of a
meso-epoxide with a bacterial epoxide hydrolase.
The activity reached 17–18 U g cdw21. The ee of diol 2 was
determined by HPLC analysis with a chiral column. It remained
unchanged during the biotransformation at 95%. To our
knowledge, this is the first example of highly enantioselective
hydrolysis of N-containing meso-epoxide catalysed by either a
chemical or a biological catalyst.
Preparative hydrolysis was performed with 15 mM of 1 in
100 ml cell suspension (10 g L21) of Sphingomonas sp. HXN-
200 (Fig. 1).† 97% conversion was reached after 5 h, and 289.1
mg (81.3%) of 2 was isolated with 98.9% purity, 95% ee and an
Epoxide hydrolase (EH), an enzyme that catalyses the enantio-
selective addition of water to an epoxide forming the corre-
sponding vicinal diol, has been extensively investigated,1 and
several microbial EHs have been applied to prepare enantiopure
epoxide via kinetic resolution.2 In principle, EH could also
catalyse the enantioselective hydrolysis of a meso-epoxide to
give the corresponding trans-diol in high ee and 100%
theoretical yield, providing a simple and green synthesis in
addition to the other known chemical methods.3,4 Mammalian
epoxide hydrolase (mEH) is known to hydrolyse meso-epoxides
such as cycloalkene oxides5,6 and acyclic 1,2-disubstituted
epoxides6,7 with high enantioselectivity. However, synthetic
applications are limited due to the poor availability of mEH.
Microbial epoxide hydrolases can easily be produced in large
amounts, but examples of hydrolysis of meso-epoxides with
such EHs are rare: only a membrane-associated yeast EH from
Rhodotorula glutinis was found to enantioselectively hydrolyse
cyclohexene- and cyclopentene oxide;8 low enantioselectivity
was observed in hydrolysis of cyclohexene oxide with a fungal
EH;9 and thus far no bacterial EH was found to accept a meso-
epoxide as substrate.10 Here we report the first example of
highly enantioselective hydrolysis of a meso-epoxide catalysed
by a bacterial EH from Sphingomonas sp. HXN-200 and the
high yield preparation of the corresponding vicinal trans-
diols.
25
[a]D +7.6 (c = 1.80, CHCl3). The bio-product 2 is identical
with the authentic sample synthesized chemically, with respect
to MS, 1H- and 13C-NMR, UV, and IR spectra.
The configuration of bioproduct (+)22 was established by
chemical correlation: deprotection of bioproduct (+)22 by
hydrogenation with 20% Pd(OH)2/C gave 94% of 3,4-dihydrox-
25
ypyrrolidine with an [a]D of 218.6 (c = 0.80, in MeOH).
Since (3S, 4S)-3,4-dihydroxypyrrolidine has an [a]D26 of +20.7
(c = 0.30, in MeOH),15 the configuration of bioproduct (+)22
can be deduced to be (3R, 4R).
Table 1 Hydrolysis of N-benzyloxycarbonyl-3,4-epoxy-pyrrolidine 1 with
frozen/thawed cells of Sphingomonas sp. HXN-200
Activitya/U g
Con. of 1/mM cdw21
Conversion to 2 (%)b
Sphingomonas sp. HXN-200 is an alkane-degrading strain
and known to regio- and stereoselectively catalyse the hydrox-
ylation of a series of aliphatic heterocycles.11 Cells of
Sphingomonas sp. HXN-200 were produced by growth on n-
octane in 2 L E2 medium,11 the harvested cell pellets were
stored at 280 °C, and the frozen/thawed cells were used for
hydrolysis. 3,4-Epoxypyrrolidine 1 was chosen as substrate
since the trans-diol product is a useful synthetic intermediate
for the preparation of antibiotics,12 Sialyl Lewis X mimetics13
and aza-sugars.14 Substrate 1 was prepared by epoxidation of N-
benzyloxy-carbonyl-3-pyrroline with mCPBA,14 and small-
scale hydrolysis of 1 (10–20 mM) were performed with frozen/
thawed cells (10 g L21) of Sphingomonas sp. HXN-200 in 10 ml
50 mM K-phosphate buffer (pH = 8.0) containing glucose (2%)
at 30 °C. Aliquots (0.1–0.2 ml) were taken from the bioconver-
sion mixture at predetermined time points, diluted in MeOH,
and the cells were removed by centrifugation. The samples were
analyzed by reverse phase HPLC to follow the reaction directly
in the aqueous phase. The conversion was quantitated by
comparing the integrated peak areas at 210 nm of the samples
with the product standard which was prepared by hydrolysis of
1 with TFA. In all the cases, the desired diol 2 was generated
without formation of any byproduct. The conversion was
98–99% for the hydrolysis of 10–15 mM of substrate (Table 1).
0.5 h 1 h
2 h
3 h
4 h
5 h
10.0
15.0
20.0
17
18
18
52
37
27
72
53
43
88
70
56
98
86
70
99
96
79
99
98
85
a Activity was determined over the first 30 min. b Conversion was
determined by HPLC analysis; error limit: 2% of the stated values.
Fig. 1 Preparation of 2 by hydrolysis of 1 (15 mM) with frozen/thawed cells
(10 g L21) of Sphingomonas sp. HXN-200.
960
CHEM. COMMUN., 2003, 960–961
This journal is © The Royal Society of Chemistry 2003

Table 2 Hydrolysis of cyclohexene oxide 3 with frozen/thawed cells and
soluble cell-free extracts of Sphingomonas sp. HXN-200
containing glucose (2%) in a 500 ml shaking flask at 200 rpm and 30 °C for
5 h. The reaction was stopped by removing the cells via centrifugation, and
the product was extracted into ethyl acetate. The organic phase was
separated, dried over Na2SO4, and the solvent was removed by evaporation.
Purification by column chromatography on silica gel (Rf 0.27, ethyl acetate)
25
afforded 289.1 mg (81.3%) of 2: 95% ee; 98.9% purity; [a]D +7.6 (c =
1
1.80, CHCl3); H NMR (CDCl3, 400 MHz) d 7.34–7.26 (m, 5 H, Ph–H),
Cells/g CFEa/g
Scale/ Activityb/
Conv.c eed of 4
5.06 (s, 2 H, PhCH2); 4.07 (s, br, 2 H, CHOH 3 2); 3.86 (s, br, 1 H, OH),
3.63 (d, 2 H, J = 10.4 Hz, CH(H)N 3 2), 3.35 (t, 2 H, J = 10.4 Hz, CH(H)N
3 2), 2.63 (s, br, 1 H, OH); 13C NMR (CDCl3, 100 MHz) d 156.71 (s, CNO),
137.43 (s), 129.58 (d), 129.18 (d), 128.89 (d) (aromatic C), 76.27 (d), 75.63
(d) (CHOH 3 2), 68.28 (t, PhCH2), 52.85 (t), 52.51 (t) (CHN, 3 2); APCI-
MS(40eV) m/z 238 (30%, M + 1), 194 (100%); IR (CHCl3) n 3406 (br, OH),
1693 (C = O) cm21. The conversion was analysed by HPLC. Column:
Hypersil BDS-C18 (5 µm, 125 mm 3 4 mm); eluent: 10 mM K-phosphate
buffer (pH = 7.0)–acetonitrile (70+30); flow: 1.0 ml min21; detection: UV
at 210, 225, and 254 nm; tR of 2: 2.0 min, tR of 1: 4.2 min. The ee of 2 was
determined by HPLC analysis with a chiral column (Chiralpak AS, 250 mm
3/mM cdw L21 prot. L21 ml
U g21
Time/h (%)
(%)
10
20
10
20
13
13
20
12
5
1.8
3.1
1.0
1.4
7
7
7
99
96
95
91
87
86
86
85
20
20
5
14
a Cell free extract. b Average activity for the whole reaction period. U g
cdw21 and U g protein21 for whole cell and cell-free transformation,
respectively. c Conversion was determined by GC analysis; error limit: 2%
of the stated values. d Ee was determined by GC analysis with a chiral
column; error limit: 2% of the stated values.
3 4.6 mm). eluent: n-hexane–isopropanol (97+3); flow rate: 1.0 ml min21
UV detection at 210 nm; tR: 114.1 and 130.4 min.
;
‡ The biohydrolysis of cyclohexene oxide 3 to 4 was followed by GC
analysis. Column: Optima-5–0.25 µm (25 m 3 0.32 mm); temperature
program: 60 °C for 1 min, then to 140 °C at 15 °C min21, and finally to 260
°C at 49 °C min21; tR of 3: 2.46 min, tR of 4: 4.54 min. The ee of 4 was
determined by GC analysis with a chiral column (lipodex-A, 25 m 3 0.25
To further explore the hydrolysis potential, cyclohexene
oxide 3 was selected as the second substrate, since it represents
a carbocyclic meso-epoxide and the product 4 is an useful
synthon. The enzymatic reaction was performed at 25 °C
instead of 30 °C to reduce the non-enzymatic hydrolysis rate.
Hydrolysis of 3 (10–20 mM) was examined with frozen/thawed
cells (13 g L21) of HXN-200 in 50 mM Tris-HCl (pH = 7.5)
and followed by GC analysis of samples that were prepared by
taking aliquots (0.2 ml) at predetermined time points, removing
the cells, and extracting with ethyl acetate (1+2).‡ The
conversion was quantitated by comparison of the integrated
peak areas of the samples and the product standard and
correction with the extraction efficiency. Here again, only the
desired trans-diol 4 was formed, and the conversion reached
> 95% at 7 h (Table 2). Hydrolysis of 1 (20 mM) under the same
conditions with cells which were boiled for 20 min revealed that
the non-enzymatic hydrolysis was only about 1.2% at 7 h. The
ee of the product 4 was determined as 86–87% by GC analysis
with a chiral column, and the configuration was established as
(1R,2R) by comparison of the retention time with those of
(1R,2R)- and (1S,2S)-4. This stereochemistry outcome is similar
to the hydrolysis with mEH5,6 and the membrane-associated
yeast EH from R. glutinis.8
Different from the R. glutinis EH, the HXN-200 EH was
found to be a soluble enzyme. The preparation of the soluble
cell-free extracts of HXN-200 involved suspending the frozen/
thawed cells in 50 mM Tris-HCl (pH 7.5) to a cell density of 31
g L21, passing them through the French press to open the cells
and removing the cell wall fragments, membranes, and
membrane-associated proteins by ultracentrifugation at 244 000
g at 4 °C for 45 min. Hydrolysis of 3 (10–20 mM) with these
soluble cell-free extracts (20 g protein L21) gave the corre-
sponding diol (1R,2R)-4 in 85–86% ee and > 90% conver-
sion.
mm). Temperature program: 60 °C for 1 min, then to 120 °C at 2 °C min21
,
120 °C for 1 min, and finally to 160 °C at 40 °C min21; tR of (1S,2S)-4:
26.13 min, tR of (1R,2R)-4: 26.45 min.
1 I. V. J. Archer, Tetrahedron, 1997, 53, 15617; A. Archelas and R.
Furstoss, Curr. Opin. Chem. Biol., 2001, 5, 112; A. Steinreiber and K.
Faber, Curr. Opin. Biotech., 2001, 12, 552; M. Nardini, I. S. Ridder, H.
J. Rozeboom, K. H. Kalk, R. Rink, D. B. Janssen and B. W. Dijkstra, J.
Biol. Chem., 1999, 274, 14579.
2 I. Osprian, W. Kroutil, M. Mischitz and K. Faber, Tetrahedron:
Asymmetry, 1997, 18, 65; A. Steinreiber, I. Osprian, S. F. Mayer, R. V.
A. Orru and K. Faber, Eur. J. Org. Chem., 2000, 22, 3703; Y. Genzel,
A. Archelas, Q. B. Broxterman, B. Schulze and R. Furstoss, J. Org.
Chem., 2001, 66, 538; K. M. Manoj, A. Archelas, J. Baratti and R.
Furstoss, Tetrahedron, 2001, 57, 695; W. J. Choi, C. Y. Choi, J. A. M.
De Bont and C. A. G. M. Weijers, Appl. Microbiol. Biotechnol., 2000,
54, 641; H. Baldascini, K. J. Ganzeveld, D. B. Janssen and A. A. C. M.
Beenackers, Biotechnol. Bioeng., 2001, 73, 44.
3 R. A. Johnson and K. B. Sharpless, in Catalytic Asymmetric Synthesis,
ed. I. Ojima, VCH, New York, 2000, 357.
4 J. M. Ready and E. N. Jacobsen, J. Am. Chem. Soc., 2001, 123, 2687.
5 D. M. Jerina, H. Ziffer and J. W. Daly, J. Am. Chem. Soc., 1970, 92,
1056; G. Bellucci, C. Chiappe and F. Marioni, J. Chem. Soc., Perkin
Trans. 1, 1989, 2369.
6 B. Bellucci, I. Capitani, C. Chiappe and F. Mariono, J. Chem. Soc.,
Chem. Commun., 1989, 1170.
7 T. Watabe and K. Akamatsu, Biochim. Biophys. Acta, 1972, 279, 297;
G. Bellucci, C. Chiappe and G. Ingrosso, Chirality, 1994, 6, 577; D.
Wistuba, O. Trager and V. Schurig, Chirality, 1992, 4, 185; G. Bellucci,
C. Chiappe, A. Cordoni and G. Ingrosso, Tetrahedron Lett., 1996, 37,
9089.
8 C. A. G. M. Weijers, Tetrahedron: Asymmetry, 1997, 8, 639.
9 J. R. Cagnon, A. L. M. Porto and A. J. Marsaioli, Chemosphere, 1999,
38, 2237.
10 M. Mischitz, W. Kroutil, U. Wandel and K. Faber, Tetrahedron:
Asymmetry, 1995, 6, 1261; W. Kroutil and K. Faber, in Stereoselective
Biocatalysis, ed. R. N. Patel, Marcel Dekker, Inc., New York, 2000, pp.
205.
11 Z. Li, H.-J. Feiten, J. B. van Beilen, W. Duetz and B. Witholt,
Tetrahedron: Asymmetry, 1999, 10, 1323; D. Chang, B. Witholt and Z.
Li, Org. Lett., 2000, 2, 3949; Z. Li, H.-J. Feiten, D. Chang, W. A. Duetz,
J. B. van Beilen and B. Witholt, J. Org. Chem., 2001, 66, 8424; D.
Chang, H.-J Feiten, B. Witholt and Z. Li, Tetrahedron:Asymmetry,
2002, 13, 2141; D. Chang, H.-J. Feiten, K.-H. Engesser, J. B. van
Beilen, B. Witholt and Z. Li, Org. Lett., 2002, 4, 1859.
12 Y. S. Lee, J. Y. Lee, S. H. Jung, E.-R. Woo, D. H. Suk, S. H. Seo and
H. Park, J. Antibiot., 1994, 47, 609; Y. K. Kang, K. J. Shin, K. H. Yoo,
K. J. Seo, S. Y. Park, D. J. Kim and S. W. Park, Bioorg. Med. Chem.
Lett., 1999, 9, 2385.
Preparative hydrolysis was performed with 10 mM of 3 in
150 ml cell suspension (10 g L21) of Sphingomonas sp. HXN-
200 at 25 °C. Higher than 99% conversion was reached after 22
h, and 159.3 mg (91.4%) of 4 was isolated in 99.6% purity and
87% ee.
In summary, the soluble epoxide hydrolase of Sphingomonas
sp. HXN-200 has been found to catalyse the hydrolysis of N-
benzyloxycarbonyl-3,4-epoxy-pyrrolidine and cyclohexene
oxide with high enantioselectivity. The high yield preparation
of the corresponding vicinal trans-diols in high ee has been
demonstrated by simple enzymatic hydrolysis. This provides us
with the first efficient bacterial EH for hydrolysis of a meso-
epoxide.
13 H. Huang and C.-H. Wong, J. Org. Chem., 1995, 60, 3100.
14 B. G. Davis, M. A. T. Maughan, T. M. Chapman, R. Villard and S.
Courtney, Org. Lett., 2002, 4, 103.
15 F. Cardona, A. Goti, S. Picasso, P. Vogel and A. Brandi, J.
Carbohydrate Chem., 2000, 19, 585.
Notes and references
† (3R,4R)-N-Benzyloxycarbonyl-3,4-dihydroxy-pyrrolidine 2 was prepared
by hydrolysis of 1 (329 mg, 1.5 mmol) in a 100 ml cell suspension (10.0 g
L21) of Sphingomonas sp. HXN-200 in 50 mM K-phosphate buffer (pH 8.0)
CHEM. COMMUN., 2003, 960–961
961