Epoxide hydrolase-catalyzed enantioselective synthesis of chiral 1,2-diols via desymmetrization of meso-epoxides

Article abstract of DOI:10.1021/ja0466210

The discovery, from nature, of a diverse set of microbial epoxide hydrolases is reported. The utility of a library of epoxide hydrolases in the synthesis of chiral 1,2-diols via desymmetrization of a wide range of meso-epoxides, including cyclic as well as acyclic alkyl- and aryl-substituted substrates, is demonstrated. The chiral (R,R)-diols were furnished with high ee's and yields. The discovery of the first microbial epoxide hydrolases providing access to complementary (S,S)-diols is also described. Copyright

Full text of DOI:10.1021/ja0466210

Published on Web 08/20/2004
Epoxide Hydrolase-Catalyzed Enantioselective Synthesis of Chiral 1,2-Diols
via Desymmetrization of meso-Epoxides
Lishan Zhao,* Bin Han, Zilin Huang, Mark Miller, Hongjun Huang, Dan S. Malashock, Zuolin Zhu,
Aileen Milan, Dan E. Robertson, David P. Weiner,* and Mark J. Burk*
DiVersa Corporation, 4955 Directors Place, San Diego, California 92121
Received June 8, 2004; E-mail: lzhao@diversa.com; dweiner@diversa.com; mburk@diversa.com
Table 1. EH-Catalyzed Desymmetrization of Cyclic
Enantioselective construction of chiral 1,2-diols has been the
subject of extensive studies because of their importance in asym-
metric synthesis.1 Catalytic production of chiral diol derivatives
via desymmetrization of meso-epoxides is an attractive route which
simultaneously “constructs” two contiguous stereogenic centers and
can furnish 100% theoretical yield of product.2 Several asymmetric
catalyst systems have been reported that promote this reaction using
oxygen nucleophiles to generate 1,2-diol derivatives.3 However,
the lack of broad substrate scope, moderate enantioselectivity, and
the need for expensive and sensitive metal catalysts have limited
synthetic application of these systems.4 Epoxide hydrolases (EHs)
(EC 3.3.2.x), which catalyze epoxide ring opening by water, could
offer a promising alternative strategy.5 There have been reports of
the discovery and application of microbial EHs, but examples of
desymmetrization of meso-epoxides are scarce.6,7 Here we report
the discovery of a large and diverse set of microbial EHs and their
utilization in the desymmetrization of meso-epoxides to produce
chiral 1,2-diols.
meso-Epoxidesa
Since no single enzyme is likely to function optimally on all
types of epoxides, the full potential of EHs as a synthetic tool will
be realized only through creation of a library or collection of
enzymes with differing substrate scopes. Traditional approaches
to enzyme discovery by microbial cultivation have resulted in fewer
than 20 reported microbial EHs, and fewer still that have been
carefully studied. The generation of DNA libraries directly from
environmental samples has been described previously.8 To access
the most diverse range of enzymes in nature, we screened our
environmental libraries, which were created from numerous global
habitats. To date, over 50 novel microbial EHs have been discovered
using sequence-based and activity-based high-throughput assays.9
All new EHs were found to be unique at the sequence level and
were shown to possess the conserved aspartate residue that has been
postulated as the active-site nucleophile attacking the epoxide ring
in the first step of the catalysis.10 Phylogenetic analysis showed
that the newly discovered EH sequences are highly diverse and
represent a wide range of sequence space.
Each EH in our library was overexpressed and stored as a
lyophilized cell lysate. We first screened these enzymes for the
desymmetrization of cyclic meso-epoxides. Under standard condi-
tions, 2 mg/mL of substrate and 1 mg/mL of cell lysate were
incubated in 20 mM phosphate buffer (pH 7.5) with 5% acetonitrile
(v/v) at 22 °C. Five representative epoxides were examined, and
for each substrate, multiple enzymes were found that catalyzed the
formation of (R,R)-diol products. Several enzymes were character-
ized further, revealing that our EH library contained enzymes
capable of hydrolyzing heterocyclic and alicyclic meso-epoxides
of different ring sizes with both high rates and high enantioselec-
tivities (Table 1). To demonstrate synthetic utility, entries 3, 4, and
5 (using BD9833) were carried out successfully on 0.1 g scale.9
a Reactions were conducted under our standard conditions (see text).
Determination of absolute configuration is described in the Supporting
Information. b Specific activities were measured at 30 min time points and
are expressed as µmol mg-1 min-1 c TOF ) turnover frequency, mol
.
product/mol catalyst/sec. d Enantioselectivities were determined by chiral
GC analysis. e Enantioselectivities were determined by chiral HPLC analysis.
To our knowledge, there are no reports of microbial EHs that
are able to desymmetrize bulky internal epoxides such as cis-stilbene
oxide and its derivatives.11 We therefore were pleased to find that
11 of our novel EHs were active on this substrate, and four afforded
(R,R)-1,2-diphenyl-1,2-ethanediol with excellent selectivity (>96%
ee). A significant difference in the specific activities of these
enzymes toward cis-stilbene oxide was observed, with turnover
frequencies ranging from 0.04 to nearly 5 per second (Table 2,
entry 1). Interestingly, these four EHs share less than 35% sequence
identity. To confirm synthetic utility and simplicity, 1.0 g of cis-
stilbene oxide (250 mM) was treated with 109 mg of cell lysate
containing 4.6 mg of BD8877, and after 48 h, (R,R)-1,2-diphenyl-
1,2-ethanediol was isolated in 83% yield and 99% ee.9
Preliminary analysis of substrate scope was performed using one
of our lead enzymes, BD8877, along with several substituted cis-
stilbene oxides and dipyridyl analogues (Table 2, entries 2-8).9
Substituents at the meta and para positions of the phenyl ring were
well tolerated, as the enzymes catalyzed the hydrolysis of the 3-
and 4-chloro analogues with high ee and at rates comparable to
the parent substrate. Ortho substitutions, however, resulted in slower
rates of hydrolysis. For the 2-chloro analogue, enantioselectivity
remained high, although the specific activity was 300-fold lower
than with cis-stilbene oxide (Table 2, entry 2). A 5-fold rate
9
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C O M M U N I C A T I O N S
Table 2. EH-Catalyzed Desymmetrization of Aryl meso-Epoxidesa
Enantioselective ring opening of meso-epoxides has proven to
be challenging to accomplish through conventional chemical
methods. We have demonstrated that a viable biocatalytic solution
can be developed using a diverse set of microbial epoxide
hydrolases that were discovered from nature. Enzymes were
identified that are capable of selectively hydrolyzing a wide range
of meso-epoxides and the corresponding chiral (R,R)-diols were
furnished with high ee’s and yields. Moreover, the first EHs
providing access to complementary (S,S)-diols also were found.
Given the high activity and selectivity, as well as broad sequence
divergence and substrate scope, our expanding EH library is
expected to find wide utility in the synthesis of a range of chiral
1,2-diols.
Acknowledgment. We thank P. Kretz, D. Wyborski, W. Callen,
T. Richardson, M. Podar, T. Todaro, M. Lafferty, J. McElhinney,
L. Bibbs, W. Yu, X. Tan, P. Chen, A. Flordeliza, C. Cowden, F.
Bartnek, S. Wells, and E. Mathur for their assistance. J. M. Short
is gratefully acknowledged for guidance. This work was partially
supported by NIH SBIR grant 1R43GM65669-01A1.
a Reaction conditions as in Table 1. b,c See Table 1. d Enantioselectivities
were determined by chiral HPLC analysis.
Table 3. (S,S)-Diols via Desymmetrization of meso-Epoxidesa
Supporting Information Available: Materials and methods and
amino acid sequences. This material is available free of charge via the
Internet at http://pubs.acs.org.
References
(1) Hannessian, S. Total Synthesis of Natural Products. The Chiron Approach;
Pergamon Press: New York, 1983; Chapter 2.
(2) For an alternative approach to the synthesis of optically active 1,2-diols,
see: Kolb, H. C.; VanNieuwenhze, M. S.; Sharpless, K. B. Chem. ReV.
1994, 94, 2483-2547.
(3) (a) Jacobsen, E. N.; Kakiuchi, F.; Konsler, R. G.; Larrow, J. F.; Tokunaga,
M. Tetrahedron Lett. 1997, 38, 773-776. (b) Matsunaga, S.; Das, J.; Roels,
J.; Vogl, E. M.; Yamamoto, N.; Iida, T.; Yamaguchi, K.; Shibasaki, M.
J. Am. Chem. Soc. 2000, 122, 2252-2260.
(4) For example, only two meso-epoxides were ring-opened at high ee and
others were at <80% ee in the (salen)-Co-catalyzed addition of benzoic
acid.3a
(5) (a) Archelas, A.; Furstoss, R. Annu. ReV. Microbiol. 1997, 51, 491-525.
(b) Armstrong, R. N. In ComprehensiVe Natural Products Chemistry;
Barton, D., Nakanishi, K., Meth-Cohn, O., Eds.; Pergamon Press: New
York, 1999; Vol. 5, Chapter 4, pp 51-70.
(6) Hydrolysis of several meso-epoxides by liver microsomes containing
mammalian EH has been reported: (a) Bellucci, G.; Capitani, I.; Chiappe,
C.; Marioni, F. J. Chem. Soc., Chem. Commun. 1989, 1170-1171. (b)
Bellucci, G.; Chiappe, C.; Marioni, F. J. Chem. Soc., Perkin Trans. 1
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(7) (a) Faber, K.; Orru, R. V. A. In Enzyme Catalysis in Organic Synthesis:
A ComprehensiVe Handbook; Drauz, K., Waldmann, H., Eds.; Wiley-
VCH: Weinheim, 2002; Vol. II, p 592, and references therein. (b) The
only reported example using a bacterial epoxide hydrolase was the
hydrolysis of cyclohexene oxide and N-benezyloxycarbonyl-3,4-epoxy-
pyrrolidine: Chang, D.; Wang, Z.; Heringa, R. W.; Witholt, B.; Li, Z.
Chem. Commun. 2003, 960-961. Whole cells were used, and the sequence
and identity of the responsible EH were unknown.
a Reaction conditions as in Table 1. b,c See Table 1. Enantioselectivities
were determined by chiral HPLC d or GC e analysis.
reduction also was observed for the 2-fluoro analogue, which
suggests that steric hindrance may be primarily responsible for the
rate decrease. BD8877 performed particularly well on dipyridyl
substrates; the highest specific activity and ee were observed with
bis(2-pyridyl) epoxide (entry 6). Reactions with substituted cis-
stilbene oxide and dipyridyl epoxides were successfully performed
at the 0.1 g scale with BD8877.9
To our knowledge, all previously reported microbial EHs have
been observed to predominantly form (R,R)-diols from meso-
epoxides.7,12 We now have found in our EH library the first
examples of (S,S)-selective enzymes for desymmetrization of meso-
epoxides. As summarized in Table 3, these EH-catalyzed reactions
exhibited moderate to excellent ee (56-99%), although reaction
rates were generally ∼50-200-fold lower relative to those of (R,R)-
diol-producing enzymes.
(8) (a) Robertson, D. E.; Mathur, M. J.; Swanson, R. V.; Marrs, B. L.; Short,
J. M. SIM News 1996, 46, 3-8. (b) Short, J. M. Nature Biotech. 1997,
15, 1322-1323. (c) Henne, A.; Daniel, R.; Schmitz, R. A.; Gottschalk,
G. Appl. EnViron. Microbiol. 1999, 65, 3901-3907. (d) Short, J. M. U.S.
Patent 5,958,672, 1999.
(9) For further details, see the Supporting Information.
(10) The formed acyloxy intermediate is subsequently hydrolyzed by water to
afford 1,2-diol product. Lacourciere, G. M.; Armstrong, R. N. J. Am.
Chem. Soc. 1993, 115, 10466-10467.
(11) (a) Rink, R.; Fennema, M.; Smids, M.; Dehmel, U.; Janssen, D. B. J.
Biol. Chem. 1997, 272, 14650-14657. (b) Morisseau, C.; Archelas, A.;
Guitton, C.; Faucher, D.; Furstoss, R.; Baratti, J. C. Eur. J. Biochem. 1999,
263, 386-395.
(12) Mammalian EH showed opposite enantioselectivity on several substrates
with rigid tricyclic structures: Bellucci, G.; Berti, G.; Chiappe, C.; Fabri,
F.; Marioni, F. J. Org. Chem. 1989, 54, 968-970.
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