Epoxide Hydrolysis: An Important Transformation in Organic Synthesis

529
2.6.3 Epoxide Hydrolysis
R. Wohlgemuth
General Introduction
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In synthetic organic chemistry, the strained three-membered cyclic ethers, which are
named as epoxides or oxiranes, as well as the diol products of epoxide hydrolysis, repre-
sent versatile elements in functional-group-oriented synthesis planning for a variety of
reactions and are therefore of great interest as intermediates.[1] The asymmetric epoxida-
tion (AE) and asymmetric dihydroxylation (AD) reactions, which have been developed by
Sharpless and co-workers for a wide range of alkenes with various substitution patterns,
enable direct and simple access to epoxides and diols by the oxidation of C=C bonds using
highly enantioselective small-molecule catalysts.[2] As boundary conditions for synthetic
route selection, such as raw materials, selectivities, and safety, health, and environment
issues, may change, it is valuable to have complementary access routes to epoxides and
diols. The hydrolytic ring opening of epoxides by nucleophiles is an important transfor-
mation in laboratory-scale and industrial synthesis, and is also utilized by nature in vari-
ous biosynthetic transformations. For direct and highly selective epoxide-to-product
transformations, mild and environmentally benign reaction conditions, as well as direct
one-step reactions using catalytic rather than stoichiometric systems, are required to
meet boundary conditions and sustainability criteria. The hydrolysis of epoxides to vici-
nal diol products, which are of synthetic use as well, has not only been catalyzed by acids,
but also by milder reagents such as solid or solid-supported Lewis acids, one-electron-
transfer reagents, or even just hot water as a modest catalyst, reactant, and solvent.[3]
The chemo-, regio-, and enantioselective ring opening of epoxides by nucleophiles pro-
vides opportunities for building new compounds by creating a variety of new chemical
bonds. As epoxides have been synthesized at large, industrial scale, the safety, health,
and environmental effects on biological systems required special attention and investiga-
tions.
From early work on detoxification metabolism and the fact that trans-diol metabo-
lites were obtained from naphthalene, anthracene, and phenanthrene, Boyland and co-
workers suggested that such products may arise by hydration of an epoxide.[4] Pioneering
work on insecticides by Brooks and co-workers[5] as well as on detoxification by Uden-
friend and co-workers[6,7] and on human metabolism by Oesch,[8] led to the discovery of
epoxide-hydrolyzing enzymes, initially termed epoxide hydratases, and their importance
in foreign-compound metabolism.[9] The excellent selectivity of epoxide-hydrolyzing en-
zymes from pig liver microsomes toward racemic cyclodiene epoxide insecticides has
been demonstrated by the discovery that the products formed by the enzymatic hydroly-
sis are the corresponding trans-diol enantiomers.[5] These enzymes, which have finally
been classified as epoxide hydrolases (EC 3.3.2.X), are capable of hydrolyzing epoxides to
vicinal diols with increased water solubility, altered biological activity, or reduced chem-
ical reactivity, and can catalyze the nucleophilic attack on a particular epoxide enantio-
mer and carbon position with high selectivity. Depending on which epoxide carbon and
enantiomer is attacked by the nucleophile, the stereochemical configuration of the diol
products can be retained or inverted. Although biocatalytic epoxide hydrolysis is not the
only route to detoxification by biological cells, epoxide-hydrolyzing enzymes occur ubiq-
uitously in nature and have been discovered in microbial, plant, animal, and human
for references see p 553

530 Biocatalysis 2.6 Epoxide Conversions
cells.[10–12] The epoxide biodegradation and biotransformation in living cells is not only of
key importance for toxic epoxides, but also for epoxides with high biological activities in
a variety of metabolic pathways.
The highly energetic epoxide or oxirane functional group occurs naturally in a num-
ber of endogenous metabolites with widely different physiological functions in various
biochemical pathways, ranging from isoprenoid, cofactor, and fatty acid metabolism
(Scheme 1) to dicarboxylic acid, steroid, and terpene metabolism (Scheme 2).
Scheme 1 Naturally Occurring Epoxides Involved in Isoprenoid, Cofactor, and Fatty Acid
Metabolism
(S)-2,3-oxidosqualene
O
vitamin K1 2,3-epoxide
O
O
n
CO2H
O
O
menaquinone 2,3-epoxides (n = 1−13) leukotriene A4
O
HN
3-(10,11-epoxygeranylgeranyl)indole
CO2H CO2H
HO O
O
HO
hepoxilin A3 hepoxilin B3
Scheme 2 Naturally Occurring Epoxides Involved in Dicarboxylic Acid, Steroid, and Terpene
Metabolism
O
CO2Me
HO2C CO2H
O
(R,R)-epoxysuccinate juvenile hormone III

2.6.3 Epoxide Hydrolysis 531
O O
CO2H
(13S,14S)-epoxymaresin (4S)-limonene-1,2-epoxide (4R)-limonene-1,2-epoxide
H H
H H
H H H H
HO HO
O
(24R,28R)-fucosterol epoxide cholesterol-5β,6β-epoxide
The epoxide functional group is also important for the biosynthesis as well as for the bio-
logical functions of many complex natural products such as the epothilones A, B, E, and F,
penicillitone, epoxomicin, neocarzinostatin chromophore, and (+)-scyphostatin (Scheme
3), with the exact stereochemical configuration being decisive. The biodegradation and
biotransformation of epoxides with high biological activities or with high toxicity to bio-
logical cells is also of key importance in a variety of catabolic pathways.
Scheme 3 Complex Epoxide Natural Products
R1 H
O HO
S H
R2 OH
N
O
O
HO O
O OH O O
epothilone A (R1 = H; R2 = Me) penicillitone
epothilone B (R1 = R2 = Me)
epothilone E (R1 = H; R2 = CH2OH)
epothilone F (R1 = Me; R2 = CH2OH)
HO
O
OH
NH
O
(+)-scyphostatin

OMe
OH O O
O O
H H O
Ac N N OH O O
N N O
H
Me O O Me
Pri O
HN O
HO
O
HO
epoxomicin neocarzinostatin chromophore
As the focus of this chapter is on the selective biocatalytic ring opening of epoxides by
water, leading to vicinal diols or other reaction products, it is worth noting that this strat-
egy is also used by nature to prepare a range of important metabolites and natural prod-
ucts by epoxide hydrolase catalyzed ring-opening reactions (Scheme 4). The hydrolysis of
easily accessible racemic epoxides to give enantiomerically pure epoxides or vicinal diols
has become of increasing interest as a route toward a great variety of chiral intermediates
for the synthesis of pharmacologically active compounds, agrochemicals, flavors and fra-
grances, and metabolites.
Scheme 4 Diol Natural Products Prepared by Enzymatic Ring Opening of Epoxides
O OH O
OH OH
HO ONa
OH ONa
OH O
O OH OH
meso-tartaric acid sodium (S)-2,3-dihydroxyisovalerate sodium (R)-2,3-dihydroxyisovalerate
OH OH
CO2H
CO2H
OH OH
OH OH
lipoxin A4 lipoxin B4
OH OH
OH OH OH
HO
CO2H
leukotriene B4 (1S,2S,4R)-limonene-1,2-diol (1R,2R,4S)-limonene-1,2-diol

OH OH
H OH H OH
H H
HO HO
(24R)-24,25-dihydroxyvitamin D3 (24S)-24,25-dihydroxyvitamin D3
The hydrolytic kinetic resolution of racemic terminal epoxides using cobalt(III)(salen)
complexes has shown an extraordinarily high stereoselectivity and a broad substrate
scope.[13] The rate- and stereoselectivity-determining epoxide-ring-opening step has been
explained by a cooperative bimetallic mechanism where one cobalt(III) complex acts as a
Lewis acid and another delivers the hydroxide nucleophile.[14] With the requirements for
sustainability in industrial processes becoming more pronounced, applications may suf-
fer from the use of potentially toxic catalysts, high catalyst-to-substrate ratios, low to mod-
erate turnover frequencies, and varying selectivities. The regio- and enantioselective ring
opening of epoxides as carbon electrophiles with a variety of nucleophiles to provide
unique 1,2-difunctionalized products is a highly important general reaction type. A varie-
ty of biocatalysts involved in epoxide-ring-opening reactions have also shown extraordi-
narily high stereoselectivity, and in addition score well in safety, health, and environmen-
tal criteria for the production of chiral epoxides and diols. As more natural biocatalysts, as
well as an increasing number of engineered enzymes optimized for the intended reaction
conditions of the epoxide ring opening, have become available, the synthetic interest in
using biocatalytic epoxide hydrolysis for the synthesis of chiral epoxides and diols has in-
creased tremendously.[15–21] The chemical versus the biocatalytic approaches to epoxide
hydrolysis have been reviewed.[22–24]
2.6.3.1 Monosubstituted Epoxides Containing an Aromatic Group and

Their Vicinal Diols
Monosubstituted epoxides containing aromatic groups can be easily prepared in racemic

form and have therefore been investigated for hydrolytic kinetic resolutions over many
years in order to prepare residual epoxides or the corresponding vicinal diol products in
enantiomerically pure form. A variety of pyridyl- and phenyloxiranes, many ring-substi-
tuted phenyloxiranes, and the corresponding vicinal diols have been produced in enantio-
merically pure form by biocatalytic hydrolytic kinetic resolution using enantiocomple-
mentary epoxide hydrolases, and the success of these preparations have made these chi-
ral epoxides and vicinal diols ideal model systems for analyzing epoxide hydrolases with
more complex natural substrates.[19–30]
2.6.3.1.1 Monosubstituted Epoxides Containing a Phenyl Group and Their Vicinal Diols
Enantiomerically pure styrene oxides and their derivatives, as well as the corresponding
vicinal diol products, are valuable and versatile chiral building blocks for the synthesis of
more complex target compounds for applications in the pharmaceutical, agrochemical,
chemical, and polymer industries. Various (R)- and (S)-selective epoxide hydrolases have
been used for the resolution of racemic epoxides rac-1, as illustrated in Scheme 5 for the
case of retaining epoxide hydrolases.

Scheme 5 Enantioselective Enzymatic Hydrolysis of Styrene Oxides
(S)-selective
OH
epoxide hydrolase O
HO +
Ar1
Ar1
O O (R)-2 (S)-1
+
Ar1 Ar1
(R)-selective OH
(R)-1 (S)-1
epoxide hydrolase O
+ HO
Ar1
Ar1
(R)-1 (S)-2
Among the various chemical and biocatalytic routes to these monosubstituted epoxides
and their vicinal diols 2, the hydrolytic kinetic resolution of racemic epoxides is attrac-
tive, because the racemic epoxides are easily accessible starting materials.
2.6.3.1.1.1 Chiral Phenyloxiranes
The widely investigated synthesis of chiral phenyloxiranes has involved various routes,
from purely chemical routes, lipase-catalyzed resolutions of suitably protected racemic
alcohols, and monooxygenase-catalyzed epoxidations of the corresponding styrenes, to
epoxide hydrolase catalyzed ring opening of racemic epoxides. Enantiocomplementary
epoxide hydrolases have been used to prepare both R- and S-enantiomers of styrene
oxide, 4-chlorostyrene oxide, and 4-nitrostyrene oxide as well as a number of other (S)-sty-
rene oxides with chloro-, fluoro-, and bromo-substitutions on the phenyl ring.[31,32] The ep-
oxide hydrolase catalyzed synthesis of (S)-styrene oxide [(S)-3, Ar1 = Ph] compares favor-
ably with the cobalt–salen complex catalyzed hydrolytic kinetic resolution because the
catalyst-based specific productivity of the biocatalytic method is more than 30 times high-
er and the biocatalytic method is also less expensive and more environmentally benign.[32]
A selection of the results of these studies is shown in Scheme 6.[31–42] In addition to their
synthetic utility, chiral phenyloxiranes have also been useful for the characterization of
the regio- and enantioselectivities of new retaining and inverting epoxide hydrolases in
the biosynthesis of nine-membered enediyne antitumor antibiotics.[30]
Scheme 6 Epoxide Hydrolase Catalyzed Resolution of Phenyloxiranes[31–42]
O epoxide hydrolase O OH
∗ + OH
Ar1 Ar1 Ar1 ∗
rac-3 (S)- or (R)-3
Ar1 Source of Epoxide Config of ee (%) Yield (%) Ref

Hydrolase Residual Epoxide 3 of 3 of 3
[31]
Ph Beauveria sulfurescens ATCC 7159 R >98 34
[32]
Ph Sphingomonas sp. HXN-200 S >99 36.4–41.6
expressed in E. coli
[33]
Ph Aspergillus niger S 99 50
[32]
3-BrC6H4 Sphingomonas sp. HXN-200 S 98.0 46.5
[34]
2-ClC6H4 Agrobacterium radiobacter S >99 35
AD1 expressed in E. coli
Ar1 Source of Epoxide Config of ee (%) Yield (%) Ref

Hydrolase Residual Epoxide 3 of 3 of 3
[35]
2-ClC6H4 Sphingopyxis sp. BSNA05 S 99.9 39.3
[32]
3-ClC6H4 Sphingomonas sp. HXN-200 S >99 37.9–44.3
[34]
4-ClC6H4 Agrobacterium radiobacter S >99 34
[36]
4-ClC6H4 Aspergillus niger LCP 521 S 99 47
[32]
4-FC6H4 Sphingomonas sp. HXN-200 S >99 31.3–42.3
[34]
4-Tol Agrobacterium radiobacter S >99 36
[37,42]
4-F3CC6H4 Aspergillus niger S 98 37
[42]
4-F3COC6H4 Aspergillus niger S 99 43
[38]
4-O2NC6H4 Aspergillus niger LCP 521 S >99 49
[39]
4-O2NC6H4 Yarrowia lipolytica Oxy-10 R 99.3 43
[40]
2-O2NC6H4 Aspergillus niger CGMCC 0496 S 98 34
[41]
Rhodotorula glutinis SC 16293 S >99 48
O
(S)-2-Phenyloxirane [(S)-3, Ar1 = Ph]; Typical Procedure:[32]
CAUTION: 2-Phenyloxirane (styrene oxide) is a possible human carcinogen, a skin and eye irri-
tant, and may cause skin sensitization.
Racemic styrene oxide (rac-3, Ar1 = Ph; 120 g per L organic solvent) was hydrolyzed in a
simple two-phase system consisting of hexane/aqueous buffer (1:1) at a density of 5.0 g
of E. coli (SpEH) cells (dry weight) per liter of aqueous phase. After a linear decrease in
the concentration of the R-substrate during the first 40 min, the reaction was finished
within 1 h to give the title enantiomer (S)-3 (Ar1 = Ph); yield: 43%; >99% ee. The E value at
this high substrate concentration was 39, which is the highest among all epoxide hydro-
lases in the form of free enzyme, cell extracts, or whole cells. The product concentration
reached 0.43 M in the organic phase (51 g per L organic phase), and the overall space-time
yield amounted to 26 g • L–1 • h–1. The cell-based specific productivity reached 10.3 g • h–1
per g cell dry weight. This value is 542 times higher than that (0.019 g • h–1 per g dry weight
of cell-free extracts) with Sphingomonas sp. HXN-200.28. It is also 264 times higher than
that (0.039 g • h–1 per g cell dry weight) achieved in recombinant R. glutinis epoxide hydro-
lase catalyzed kinetic resolution of 1.8 M racemic styrene oxide at a cell density of 92 g cell
dry weight per L for 24 h. Thus, E. coli (SpEH) cells catalyze the resolution of racemic sty-
rene oxide in a highly productive way to afford enantiomer (S)-3 (Ar1 = Ph), which was ob-
tained after column chromatography as a colorless liquid; yield: 36.4%; >99% ee.
(S)-2-(3-Chlorophenyl)oxirane [(S)-3, Ar1 = 3-ClC6H4]; Typical Procedure:[32]

Gram-scale resolution of racemic 2-(3-chlorophenyl)oxirane (rac-3, Ar1 = 3-ClC6H4; 200
mM, 30 g per L organic phase) was achieved with the resting cells of E. coli (SpEH) under
non-optimized conditions within 80–90 min. In addition to the high product concentra-
tions, the product/cells ratios (7.9–11.7 g per g cell dry weight) as well as cell-based specific
productivities (5.7–8.8 g • h–1 per g cell dry weight) were also high. The product (S)-2-
(3-chlorophenyl)oxirane [(S)-3, Ar1 = 3-ClC6H4] was obtained as a light-yellow liquid after
column chromatography; yield: 37.9%; >99% ee.

(S)-2-(4-Fluorophenyl)oxirane [(S)-3, Ar1 = 4-FC6H4]; Typical Procedure:[32]

Freshly prepared E. coli (SpEH) cells were diluted with 50 mM Tris buffer (pH 7.5) to a 20–
110 mL system with the required cell density in a 250–1000-mL flask. A second phase with
hexane (20–100 mL) containing an appropriate amount of the racemic epoxide was then
added to the flask. The mixture was shaken at 250 rpm in an incubator at 30 8C for the
appropriate time. The reaction was monitored by HPLC, and terminated by cooling on
ice once the ee of residual epoxide reached 99%. The reaction system was then immediate-
ly extracted with hexane (3 20–100 mL), and the organic phases were combined. After
drying (Na2SO4), the solvents were removed by evaporation. The crude product was then
purified by flash chromatography (silica gel, hexane/EtOAc 50:1; Rf = 0.3).
(S)-4-(Oxiran-2-yl)-2,3-dihydrobenzo[b]furan {(S)-3, Ar1 = 2,3-Dihydrobenzo[b]furan-4-yl};
Typical Procedure:[41]
Rhodotorula glutinis SC 16293 cells (150 g) were suspended in 0.1 M phosphate buffer
(pH 8.0; 500 mL) kept at 28 8C in a jacketed three-necked flask with condenser and stop-
per, and the suspension was stirred with an air-driven stirrer. A soln of racemic 4-(oxi-
ran-2-yl)-2,3-dihydrobenzo[b]furan (2.5 g) in t-BuOMe (50 mL) was added to the stirred cell
suspension and the progress of the hydrolysis was monitored by withdrawing samples
and determining the ee. A portion (1.2 g) of the epoxide remained after a reaction time
of 5 h, and the reaction was stopped after 5.5 h. NaCl (180 g) was added, and the mixture
was extracted with EtOAc (2 1 L). The two EtOAc layers were combined, dried (Na2SO4),
and filtered, and the solvent was removed on a rotary evaporator. HPLC analysis showed
that the reaction product contained only the S-epoxide and the R-diol. No peak for the
R-epoxide was detected and the total amount of remaining S-epoxide was calculated to
be 1.2 g (48% yield). After a portion of the reaction product was dissolved in heptane/
EtOAc (1:1) and repeatedly extracted with 0.10 M aq Na2B4O7, the organic layer provided
0.95 g of S-epoxide (S)-3 (Ar1 = 2,3-dihydrobenzo[b]furan-4-yl); >99% ee.
2.6.3.1.1.2 Vicinal Diols Derived from Monosubstituted Epoxides Containing

a Phenyl Group
The enantiomeric purity of the residual epoxide in an epoxide hydrolase catalyzed kinetic
resolution just depends on the enantioselectivity of the enzyme; however, the enantio-
meric purity of the formed chiral 1,2-diol depends on both the enantioselectivity and
the regioselectivity of the epoxide hydrolase, because the racemic epoxide substrate can
be attacked at the C2 or C3 position of the oxirane ring, and this regioselectivity can be
different for the R- and the S-epoxide (Scheme 7).[43–45] The hydrolysis of racemic epoxides
monosubstituted with a phenyl group to a single enantiomer of the corresponding chiral
1,2-diol can be achieved in an enantioconvergent mono- or bienzymatic way, with one or
two epoxide hydrolases, using the right combination of the enantioselectivities of retain-
ing and inverting epoxide hydrolases; for example, the hydrolysis of the R-epoxide to the
R-1,2-diol is catalyzed by a retaining epoxide hydrolase and the hydrolysis of the S-epoxide
to the R-1,2-diol is catalyzed by an inverting epoxide hydrolase.[24]
Scheme 7 Regioselective Hydrolysis of Phenyloxiranes
inverting retaining
epoxide epoxide
hydrolase OH hydrolase
OH
R1
O O
1 1
R retaining inverting R
epoxide epoxide
hydrolase OH hydrolase
OH
R1
Scheme 8 presents some examples of the synthesis of nonracemic vicinal diols from race-
mic phenyloxiranes.[25,31,35,46–51]
Scheme 8 Hydrolysis of Phenyloxiranes to Vicinal Diols[25,31,35,46–51]
O epoxide hydrolase OH
OH
Ar1 Ar1 ∗
rac-4 (S)- or (R)-5
Ar1 Source of Epoxide Hydrolase Config of Diol 5 ee (%) Yield (%) Ref
[46]
3-ClC6H4 Solanum tuberosum expressed in E. coli R 97 88
[47]
4-ClC6H4 Caulobacter crescentus R 95 78
[48]
4-FC6H4 Aspergillus niger LCP 521 R 81 47
a [25]
4-O2NC6H4 Phaseolus radiatus L. R >99 68.7
[31]
Ph Aspergillus niger LCP 521 and R 89 92
Beauveria sulfurescens ATCC 7159
[49]
Ph Aspergillus niger SQ-6 R >99 58
[50]
Ph Solanum tuberosum expressed in E. coli and R 98 100
Agrobacterium radiobacter AD1, EchA-I219F
[51]
Ph Sphingomonas sp. HXN-200 S >99.9 40.2
[35]
4-ClC6H4 Aspergillus niger and Solanum tuberosum R 96 93
a
After recrystallization.
(R)-(3-Chlorophenyl)ethane-1,2-diol [(R)-5, Ar1 = 3-ClC6H4]; Typical Procedure:[46]

Racemic (3-chlorophenyl)oxirane (4, Ar1 = 3-ClC6H4; 340 mg, 2.2 mmol) dissolved in a mix-
ture of DMSO (0.3 mL) and H2O (22 mL) was placed in a 100-mL reactor and epoxide hydro-
lase from Solanum tuberosum (StEH; 960 U) was added as an aqueous soln with 10% glycer-
ol. The mixture, containing an overall substrate concentration of about 10 g • L–1, was then
vigorously stirred. In order to further minimize any spontaneous hydrolysis of the sub-
strate and favor enzyme stability, the enzymatic hydrolysis was carried out at 20 8C. The
conversion was complete after 1.5 h, as shown by HPLC analysis. The enzyme was recov-
ered by ultrafiltration as a concentrated soln and reused to perform the next run on an-
other batch of substrate (340 mg) using the same conditions as before. The aqueous solns
of nine successive runs with the same enzyme, corresponding to the hydrolysis of 3 g of
racemic (3-chlorophenyl)oxirane, were pooled and extracted with EtOAc. (R)-(3-Chloro-
phenyl)ethane-1,2-diol [(R)-5, Ar1 = 3-ClC6H4; 3.5 g (100% yield)] was isolated and purified
by bulb-to-bulb distillation to give pure product as a waxy solid; yield: 3 g (88%); 97% ee.

2.6.3.1.2 Chiral Pyridyloxiranes
The preparation of chiral building blocks, such as pyridyloxiranes, is important for key
steps in the synthesis of biologically active compounds. The limitations of various metal-
catalyzed oxidation procedures[52–54] and hydrolytic kinetic resolutions[13,54,55] are shown
for 2-pyridyloxirane in Scheme 9. As neither of the two enantiomers of 2-pyridyloxirane
are accessible in good enantiomeric purity by conventional chemical procedures based on
the use of heavy metal catalysts, the biocatalytic hydrolytic kinetic resolution of 2-pyridyl-
oxirane (rac-6, Ar1 = 2-pyridyl) was explored using 14 different epoxide hydrolases.[56] The
fungal epoxide hydrolase from Aspergillus niger GBCF 79[54,57] and the bacterial epoxide hy-
drolase from Agrobacterium radiobacter AD1[58] were found to be very efficient biocatalysts
for this hydrolytic kinetic resolution, making this simple procedure using water as a reac-
tant and solvent the preferred and most direct way to prepare enantiomerically pure (S)-2-
pyridyloxirane [(S)-6, Ar1 = 2-pyridyl] (Scheme 10).
Scheme 9 Metal-Mediated Asymmetric Synthesis and Resolution of 2-Pyridyloxi-

rane and Its Diol Derivative[52–57]
O
Mn(salen) (cat.)
N N
9% ee
O O OH
Co(salen) (cat.)
OH
+
N N N
rac 4% ee
OH
AD-mix-β
OH
N N
79% ee
Scheme 10 Epoxide Hydrolase Mediated Resolution of Pyridyloxiranes[54,56–58]
+ OH
Ar1 Ar1 Ar1
rac-6 (S)-6
Ar1 Source of Epoxide Hydrolase ee (%) of 6 Yield (%) of 6 Ref

[57,58]
2-pyridyl Agrobacterium radiobacter >98 36
[54]
2-pyridyl Aspergillus niger GBCF 79 99 27
[54]
[54]
(S)-2-Pyridyloxirane [(S)-6, Ar1 = 2-Pyridyl]; Typical Procedure:[57]

A soln of racemic 2-pyridyloxirane (rac-6, Ar1 = 2-pyridyl; 0.61 g, 5 mmol) in pure H2O (1 L)
was treated with crude enzyme extract of epoxide hydrolase from Aspergillus niger (0.99 g).
After 97 min, the mixture was extracted with CHCl3 (this extraction solvent could be re-
placed for safety and health reasons by CH2Cl2 or a more environmentally friendly alter-
native). The title enantiomeric oxirane was obtained after normal workup; yield: 260 mg
(43%); >99% ee.
2.6.3.2 Glycidyl Ethers and Their Vicinal Diols
Chiral glycidyl (oxiran-2-ylmethyl) ethers and the related vicinal diols are versatile build-
ing blocks for the synthesis of a variety of industrially useful intermediates and products
such as pharmaceuticals, agrochemicals, cross-linkers for surface coatings, and polymers.
Microbial epoxide hydrolases of bacterial, yeast, and fungal origins have been used for the
resolution of racemic glycidyl ethers with good enantioselectivity, preparing enantioen-
riched glycidyl ethers and the related vicinal diols (Scheme 11).
Scheme 11 Enantioselective Hydrolysis of Aryl Glycidyl Ethers
Ar1O Ar1O + Ar1O OH
rac
2.6.3.2.1 Enantioenriched Glycidyl Ethers
Many important pharmaceuticals used in the treatment of neurological disorders and car-
diovascular diseases are derived from chiral glycidyl ethers 7. The need to prepare these
active pharmaceutical ingredients in enantiomerically pure form has stimulated the de-
velopment of safer, simpler, and more efficient synthetic routes. The chromogenic sub-
strate glycidyl 4-nitrophenyl ether has been used for screening Agrobacterium radiobacter
epoxide hydrolase mutant enzymes with enhanced enantioselectivity and activity.[59]
The search for epoxide hydrolases with high enantioselectivity toward glycidyl phen-
yl ether [2-(phenoxymethyl)oxirane], useful for the synthesis of -blockers and chiral
amino alcohols, led to the discovery of the (R)-selective epoxide hydrolase from Bacillus
megaterium ECU1001, which leaves behind in the reaction mixture the S-epoxide in
>99.5% ee (Scheme 12).[60] For this hydrolytic kinetic resolution of racemic glycidyl phenyl
ether, a highly enantioselective mutant of the epoxide hydrolase from Aspergillus niger has
been evolved, which showed that the basis of the enhanced enantioselectivity was the
change in the active site, which enables a better positioning of the preferred (S)-glycidyl
phenyl ether, but not the R-enantiomer.[20] Excellent enantioselectivities have been
achieved in the resolution of racemic ortho-substituted glycidyl phenyl ethers[61] by a
novel epoxide hydrolase with very high activity, which was cloned from Bacillus megateri-
um ECU1001. The substrate spectrum of the Bacillus megaterium epoxide hydrolase has
been expanded by redesigning the active site, and the activities toward a series of bulkier
substrates as typical -blocker precursors have been significantly improved.[62]

Scheme 12 Epoxide Hydrolase Catalyzed Kinetic Resolution of Glycidyl Ethers[20,34,60,62–66]
R1O R1O + R1 O OH
∗ ∗
rac-7 (S)- or (R)-7
R1 Source of Epoxide Hydrolase Config of eea (%) Yielda (%) Ref

Residual Epoxide 7 of 7 of 7
[60]
Ph Bacillus megaterium ECU1001 S >99.5 25.6
[67]
Ph Bacillus megaterium ECU1001 S 100 44.5
[34]
Ph Agrobacterium radiobacter AD1 R >99 28
[63]
2-Tol Bacillus alcalophilus MTCC 10234 S >99 38
[62]
3-Tol Bacillus megaterium ECU1001, M145I, S 99.5 38.7
[63]
3-Tol Bacillus alcalophilus MTCC 10234 S 98.4 37
[63]
4-Tol Bacillus alcalophilus MTCC 10234 S >99 38
[63]
3-ClC6H4 Bacillus alcalophilus MTCC 10234 S >99 33
[63]
3-BrC6H4 Bacillus alcalophilus MTCC 10234 S >99 33
[62]
1-naphthyl Bacillus megaterium ECU1001, F128S, S 99.5 44.6
[66]
2-O2NC6H4 Trichosporon loubierii ECU 1040 R 97 41
[64]
Bn Rhodotorula glutinis CIMW 147 R >98 14
[65]
t-Bu Aspergillus niger M200 R >99 n.r.
[20]
4-MeOC6H4 Aspergillus niger LCP521, variant LW202, evolved R n.r. n.r.
a
n.r. = not reported.
(S)-2-(Phenoxymethyl)oxirane [(S)-7, R1 = Ph]; Typical Procedure:[60]

Preparative hydrolysis was performed with lyophilized Bacillus megaterium ECU1001 cells
(30 g • L–1) in 100 mM potassium phosphate buffer (pH 8.0; 200 mL) containing 5% (v/v)
DMSO and 60 mM 2-(phenoxymethyl)oxirane (rac-7, R1 = Ph). Following a 16 h reaction,
enantiopure (S)-7 (R1 = Ph) was obtained as a colorless oil; yield: 0.46 g (25.6%); >99.5% ee;
[Æ]D33 9.8 (c 1.0, EtOH)
(S)-2-[(1-Naphthyloxy)methyl]oxirane [(S)-7, R1 = 1-Naphthyl]; Typical Procedure:[62]

In a 2-L three-necked flask, crude epoxide hydrolase enzyme powder was added to 0.1 M
potassium phosphate buffer (pH 7.0; 800 mL) containing 0.02% Tween-80. The soln was
mechanically agitated at 180 rpm and the reaction was started by adding a soln of racemic
2-[(1-naphthyloxy)methyl]oxirane (rac-7, R1 = 1-naphthyl; 10 g, 50 mmol) in iPr2O (200 mL).
After 10–16 h of reaction at rt, the ee of the residual epoxide reached 99%. The mixture
was extracted with CH2Cl2 (3 500 mL) and the extracts were dried (Na2SO4). After solvent
evaporation, the resulting epoxide and diol were separated by column chromatography,
providing the title product; yield: 44.6%; 99.5% ee; [Æ]D25 37.4 (c 1.0, MeOH).
2.6.3.2.2 Vicinal Diols from Glycidyl Ethers
In contrast to the chiral epoxides, where high enantiomeric excesses have been achieved
(Section 2.6.3.2.1), the synthesis of vicinal diols 8 with high enantiomeric purity from gly-
cidyl ethers by epoxide hydrolase catalyzed resolution of the racemic epoxides is more
challenging (Scheme 13).[62,63,68,69]
Scheme 13 Vicinal Diols by Epoxide Hydrolase Catalyzed Hydrolysis of Glycidyl Ethers[62,63,68,69]
Ar1O Ar1O OH
rac (R)-8
Ar1 Source of Epoxide Hydrolase ee (%) Yield (%) Ref

[63]
3-Tol Bacillus alcalophilus MTCC 10234 93 46
[68]
Ph Aspergillus niger mutant LW202 95 48
[62]
2-(H2C=CHCH2)C6H4 Bacillus megaterium ECU1001, M145A, expressed in E. coli 92 46.0
[62]
2,3-Me2C6H3 Bacillus megaterium ECU1001, M145T, expressed in E. coli 96.6 39.9
[62]
1-naphthyl Bacillus megaterium ECU1001, F128S, expressed in E. coli 99.9 37.2
[69]
Ph Bacillus sp. Z018 96.3 45.8
2.6.3.3 Monosubstituted Epoxides Containing Alkyl or Alkenyl Substituents and

Their Vicinal Diols
Unbranched aliphatic terminal epoxides carrying highly flexible nonpolar alkyl or alken-
yl chains have been resolved by membrane-associated epoxide hydrolases from fungal
and yeast strains (Scheme 14).[70] A number of epoxide hydrolases from Rhodotorula, Rho-
dosporidium, and Trichosporon yeast strains have shown high enantioselectivity for the res-
olution of racemic 2-hexyloxirane, yielding (S)-2-hexyloxirane with >98% ee.[70,71]
Scheme 14 Enantiomeric Hydrolysis of Monosubstituted Epoxides
+ ∗ OH
R1 R1 ∗ R1
rac
R1 = alkyl, alkenyl
2.6.3.3.1 Monosubstituted Epoxides Containing Alkyl or Alkenyl Substituents
The application of epoxide hydrolases to non-aromatic epoxide substrates such as linear

aliphatic terminal epoxides 9 and functionalized epoxides has been extended by using
novel yeast epoxide hydrolases. A number of aliphatic terminal S-epoxides have been pre-
pared in high enantiomeric purities by epoxide hydrolase from yeasts such as Rhodotorula
glutinis[72,73] and Yarrowia lipolytica[39] as well as engineered epoxide hydrolases (Scheme
15).[59]

Scheme 15 Epoxide Hydrolase Catalyzed Kinetic Resolution of Alkyloxiranes[39,64,72–76]
+ OH
R1 R1 ∗ R1 ∗
rac-9 (S)- or (R)-9
R1 Source of Epoxide Hydrolase Config of ee (%) Yield (%) Ref

Residual Epoxide 9 of 9 of 9
[64]
Me Rhodotorula glutinis CIMW 147 S >98 15
[72]
Et Rhodotorula glutinis CIMW 147 S >98 21
[72]
Pr Rhodotorula glutinis CIMW 147 S >98 40
[72]
Bu Rhodotorula glutinis CIMW 147 S >98 48
[72]
(CH2)4Me Rhodotorula glutinis CIMW 147 S >98 44
[72]
(CH2)5Me Rhodotorula glutinis CIMW 147 S >98 38
[73]
(CH2)5Me Chryseomonas luteola S 98 38
[72]
CH2Cl Rhodotorula glutinis CIMW 147 R >98 10
[74]
CH2Cl Agrobacterium radiobacter expressed in E. coli R >99 42.7
[75]
CH2Cl Novosphingobium aromaticivorans ECU 1040 S 99.9 20.7
a [76]
CH2N3 Aspergillus niger ZJUTZQ208 R >95 –
[39]
(CH2)2Br Yarrowia lipolytica Oxy-9 S 98.6 26
[39]
(CH2)3Br Yarrowia lipolytica Oxy-1 S 98.7 26
[39]
(CH2)2OAc Yarrowia lipolytica Oxy-3 S 97.8 20
a
Yield not reported.
2.6.3.3.2 Diols from Monosubstituted Epoxides Containing Alkyl or Alkenyl

Substituents
While the resolution of racemic aliphatic terminal epoxides by epoxide hydrolases yields
enantiopure epoxides (see Section 2.6.3.3.1), it is still a challenge to obtain the corre-
sponding diols 10 in the same high enantiomeric purities (Scheme 16).[72]
Scheme 16 Vicinal Diols by Epoxide Hydrolase Catalyzed Hydroly-

sis of Alkyloxiranes[72]
epoxide hydrolase from

O Rhodotorula glutinis CIMW147 OH
OH
n n
rac (R)-10
n ee (%) Yield (%) Ref

[72]
2 66 54
[72]
3 83 47
[72]
4 73 52
[72]
5 55 60
2.6.3.4 Other Monosubstituted Epoxides and Related Diols
Chiral epoxides carrying additional functional groups such as epoxy alcohols, epoxyalde-
hydes, epoxy ketones, epoxy acids, and epoxy esters are particularly interesting building
blocks for the introduction of the 1,2-diol functionality, whereby mild hydrolysis meth-
ods using epoxide hydrolases do not affect the other functional groups (Scheme 17).
Scheme 17 Enantioselective Hydrolysis of Functionalized Monosubstituted

Epoxides
O O OH
epoxide hydrolase
OR1 OR1 + HO OR1
OR1 OR1 OR1
rac
2.6.3.4.1 Other Chiral Monosubstituted Epoxides
Enantiomerically pure acetal-protected glycidaldehyde derivatives (R)- or (S)-11 have been

prepared by hydrolytic kinetic resolution using recombinant Aspergillus niger epoxide hy-
drolases (Scheme 18).[77] Enantiomerically pure (S)-2-(2,2-diethoxyethyl)oxirane could also
be obtained in 30% yield by resolving racemic 2-(2,2-diethoxyethyl)oxirane using crude ep-
oxide hydrolase from Aspergillus niger.[78] The epoxide hydrolase from Acinetobacter bau-
mannii showed the highest enantioselectivity in the resolution of racemic ethyl 2-(oxi-
ran-2-yl)acetate, and ethyl (R)-2-(oxiran-2-yl)acetate could be obtained in 46% yield and
>99% ee.[79]
Scheme 18 Epoxide Hydrolase Catalyzed Kinetic Resolution of Functionalized

Alkyloxiranes[77–79]
epoxide hydrolase OH
O O
from Aspergillus niger
OR1 OR1 ∗ OR1
+ HO
OR2 OR2 OR2

rac-11 (R)-11
R1 R2 ee (%) of (R)-11 Yield (%) of (R)-11 Ref

[77]
Me Me >99 39.4
[77]
iPr iPr >99 46
[77]
CH2CMe2CH2 >99 43.5
epoxide hydrolase
O OEt from Aspergillus niger O OEt OH OEt
+ HO ∗
OEt OEt OEt
rac 30%; >98% ee
epoxide hydrolase
O from Acinetobacter baumannii O OH
CO2Et CO2Et + HO ∗ CO2Et
rac 46%; >99% ee

2.6.3.4.2 Other Chiral Monosubstituted Diols
Chiral monosubstituted diols (S)-12 have been obtained from the enzymatic hydrolysis of
racemic alkyloxirane acetals in moderate to excellent enantiomeric excess, depending on
the structure of the acetal moiety (Scheme 19). As the optimal acetal derivative can be
chosen and the obtained diols can be cyclized back into the corresponding epoxide with-
out loss of enantiomeric purity, this would allow the preparation of the S-diols in very
high enantiomeric purity by performing a second resolution cycle.[78]
Scheme 19 Vicinal Diols by Epoxide Hydrolase Catalyzed Hydrolysis of

Alkyloxirane Acetals[77,78]
epoxide hydrolase OH
O from Aspergillus niger
OR1 HO OR1
OR2
OR2
rac (S)-12
R1 R2 ee (%) Yield (%) Ref

[77]
Me Me 54 41
[77]
iPr iPr 97 43
[77]
CH2CMe2CH2 92 45.8
epoxide hydrolase
O OEt from Aspergillus niger OH OEt
54%; 47% ee
HO
OEt OEt
rac
2.6.3.5 Geminally Disubstituted Epoxides and Related Diols
A wide range of (R)- and (S)-selective epoxide hydrolases, illustrated in Scheme 20 for the
retaining epoxide hydrolases, have been used for the resolution of racemic 2,2-disubsti-
tuted epoxides. Optically active 2,2-disubstituted epoxides and the diols derived from
them by hydrolysis remain challenging and important building blocks for the synthesis
of biologically active compounds. Therefore, efficient, scalable, and sustainable synthetic
methods are highly desirable. The discovery of epoxide hydrolase activities in microor-
ganisms and in a commercial crude enzyme from Rhodococcus sp., and the availability of
stable biocatalyst preparations and their synthetic benefits, provided a great stimulus[31,80]
and promoted their increasing use in the synthesis of geminally disubstituted epoxides
and diols. Epoxide hydrolases are widely distributed in nature and are easy to use as a se-
lective catalyst without cofactors. A large group of microbial epoxide hydrolases have
been applied in sustainable preparations of synthetically useful enantiopure 2,2-disubsti-
tuted epoxides, enabling various subsequent asymmetric transformations such as epox-
ide-ring-opening reactions with anionic nucleophiles or chirality-conserving epoxide
ring enlargements to oxetanes and tetrahydrofurans, as well as in preparations of their
corresponding diols, which in this case are also of special interest as valuable optically
active tertiary alcohols. The resolution of a racemic 2,2-disubstituted oxirane using an ep-
oxide hydrolase and a halohydrin dehalogenase with opposite enantiopreferences has
been used in a route toward enantiomerically pure (R)- and (S)-chromanemethanol [2-(hy-
droxymethyl)-2,5,7,8-tetramethyl-2,3-dihydro-4H-benzopyran-6-ol], which can be applied
as key intermediates in the synthesis of pure Æ-tocopherol stereoisomers.[81]
Scheme 20 Enantioselective Hydrolysis of 2,2-Disubstituted Epoxides
O epoxide hydrolase type 1 O HO R1

R1 R1 + HO
R2
R2 R2
rac R-epoxide S-diol
O epoxide hydrolase type 2 O HO R1

R1 R1 + HO
R2
R2 R2
rac S-epoxide R-diol
2.6.3.5.1 Chiral 2,2-Disubstituted Epoxides
The preparation of (S)-2-(chloromethyl)-2-(2,4-difluorophenyl)oxirane [(S)-13, R1 = CH2Cl;

R2 = 2,4-F2C6H3] by hydrolytic kinetic resolution using recombinant Aspergillus niger epox-
ide hydrolase is remarkable from the point of view of high substrate concentration. The
epoxide product is obtained with 99.9% ee and 41.5% yield using a 2.5 M concentration of
the racemic epoxide substrate in water containing 5% dimethyl sulfoxide at 27 8C.[82] Bi-
phasic systems have also been useful for the hydrolytic kinetic resolution of spiro epox-
ides such as 1-oxaspiro[2.5]octane 14[83,84] and 1-oxaspiro[2.4]heptane 15[85] at high sub-
strate concentrations (Scheme 21). Epimeric discrimination has been demonstrated by
the highly selective resolution of methyl-substituted 1-oxaspiro[2.5]octanes using Rhodo-
torula glutinis epoxide hydrolase.[84] The enantiocomplementary epoxide hydrolases from
Rhodococcus erythropolis DCL 14 and Aspergillus niger have enabled the preparation of both
the R,R- and the S,S-enantiomers of 4,7-divinyl-1-oxaspiro[2.4]heptane (15) with excellent
enantiomeric purities using high substrate concentrations.[85] From resolutions of 2,2-di-
substituted functionalized epoxides it was found that reaction rates and enantioselectiv-
ities are decreased with increasing polarity of the functional group. Variation of the ether-
protecting group can improve selectivities, and substrates with short spacers give better
selectivities than substrates with a longer spacer.[86]
Scheme 21 Epoxide Hydrolase Catalyzed Kinetic Resolution of 2,2-Disubstituted Epoxides[34,42,82,83,85,87–90]
O epoxide hydrolase O HO R1
R1 R1 + HO
∗ R2
R2 R2
rac-13 (S)- or (R)-13
R1 R2 Source of Epoxide Hydrolase Config of ee Yield Ref

Residual Epoxide (%) (%)
[82]
CH2Cl 2,4-F2C6H3 Aspergillus niger S >99 42
[87]
Me (CH2)4Me Nocardia EH1 and TB1 R >99 50
[88]
Me 4-BrC6H4 Aspergillus niger LCP 521 S >99 39
[34]
Me Ph Agrobacterium radiobacter AD1 S >99 27
[89]
Me (CH2)3Br Nocardia H8 R >99 50
[87]
Me Bn Rhodococcus NCIMB 11216 R >98 11
[90]
Me CH2OBn Rhodococcus ruber SM 1789 R 98 43
[42]
Me 4-F3CC6H4 Aspergillus niger Gbcf79 S 99.7 42

OH
O OH
O
Rhodotorula glutinis ATCC 201718
+
rac-14 (3S,4R)-14 49%; >98% ee
epoxide hydrolase O HO OH
from Aspergillus niger
+
O
(R,R)-15 26%; 99% ee
Rhodococcus O HO
rac-trans-15 erythropolis DCL 14
OH
+
(S,S)-15 25%; 98% ee
(R)-2-Alkyl-2-methyloxiranes (R)-13 (R1 = Me); General Procedure:[87]

Lyophilized Nocardia EH1 or TB1 microbial cells (100 mg) were rehydrated in 0.05 M Tris
buffer (pH 8.0; 5 mL) for 30 min on a rotary shaker at 180 rpm and ambient temperature.
Racemic epoxide rac-13 (100 mg) was then added and the mixture was agitated at rt while
the reaction was monitored by TLC or GLC. After a reasonable degree of conversion was
reached, acetone (3 mL) was added and the cells were centrifuged. The formed diols and
remaining epoxides were extracted with EtOAc from the buffer medium and the pellet in
90% overall yield.
(S)-2-Methyl-2-[4-(trifluoromethyl)phenyl]oxirane [(S)-13, R1 = Me; R2 = 4-F3CC6H4];

Typical Procedure:[42]
In a standardized stirred-tank reactor (of 0.5 L total volume) racemic 2-methyl-2-[4-(trifluo-
romethyl)phenyl]oxirane (rac-13, R1 = Me; R2 = 4-F3CC6H4; 25 g, 123.8 mmol) was added to
isooctane (25 mL) and demineralized H2O (112.5 mL), and an emulsion was formed by ag-
itation at 600 rpm. Then, the reaction was started by adding a crude enzymatic epoxide
hydrolase soln (112.5 mL, 5600 U). The reaction was stopped when the ee of the residual
epoxide reached 99% (ca. 150 min) by adding EtOAc (60 mL) directly into the reactor. After
decanting, the aqueous phase was extracted with EtOAc (2 60 mL). The organic fractions
were collected, washed with brine, dried (MgSO4), and concentrated under reduced pres-
sure. Final purification by flash chromatography (pentane/Et2O 4:1 then 0:1) provided the
title product; yield: 10.5 g (42%); 99.7% ee.
(R,R)-4,7-Divinyl-1-oxaspiro[2.4]heptane [(R,R)-15]; Typical Procedure:[85]

To racemic trans-4,7-divinyl-1-oxaspiro[2.4]heptane (rac-trans-15; 1 g, 6.66 mmol) dis-
solved in isooctane (1 mL) in a 50-mL reactor, a soln of Aspergillus niger epoxide hydrolase
(0.5 g) in demineralized H2O (9 mL) was added, and the mixture was stirred mechanically
at 500 rpm and 27 8C. When the ee of the residual epoxide reached 99%, the reaction was
stopped by adding EtOAc (15 mL). The aqueous phase was saturated with NaCl, the epox-
ide and diol were extracted with EtOAc (2 25 mL), and the combined extract was dried
(MgSO4). The solvent was removed under reduced pressure and the residue was purified
by flash chromatography (pentane/EtOAc 1:1). The product (R,R)-15 obtained was further
purified by bulb-to-bulb distillation (65 8C/2 mbar); yield: 26%; 99% ee; [Æ]D25 –5.0 (c 0.875,
EtOH).
(S,S)-4,7-Divinyl-1-oxaspiro[2.4]heptane [(S,S)-15]; Typical Procedure:[85]

Racemic trans-4,7-divinyl-1-oxaspiro[2.4]heptane (rac-trans-15; 1 g, 6.66 mmol) was added
to a soln of Rhodococcus erythropolis DCL14 epoxide hydrolase powder (3 g) in 100 mM Tris-
HCl buffer (pH 7; 125 mL) in a 250-mL reactor, and the mixture was stirred at 27 8C. When
the ee of the residual epoxide reached 98%, the reaction was stopped, and the mixture was
extracted and purified as described in the procedure above; yield: 25%; 98% ee; [Æ]D25 6.4 (c
0.85, EtOH).
2.6.3.5.2 Chiral 2,2-Disubstituted Vicinal Diols
Chiral 2,2-disubstituted 1,2-diols 16, formed by enzymatic hydrolysis of 2,2-disubstituted
oxiranes (Scheme 22),[87,88,91–95] are valuable for a number of applications in asymmetric
synthesis and bioactive compound synthesis. Besides a tertiary alcohol moiety, which
cannot be oxidized to a carbonyl group, these compounds contain a second hydroxy
group, which is available to form hydrogen bonds.
Scheme 22 Vicinal Diols by Epoxide Hydrolase Catalyzed Hydrolysis of 2,2-Disubstituted

Oxiranes[87,88,91–95]
R1 R1 ∗ OH
R2 R2
rac (R)- or (S)-16
R1 R2 Source of Epoxide Hydrolase Config of 16 ee (%) Yield (%) Ref

[91]
Me (CH2)6Me Rhodococcus NCIMB 11216 S 98 20
[91]
Me (CH2)8Me Rhodococcus NCIMB 11216 S >99 36
[92]
Me (CH2)4Me Nocardia EH1 S 98 98
[92]
Me (CH2)3CH=CH2 Nocardia EH1 S 99 99
[93]
Me CH2OBn Bacillus subtilis R >99 82
[95]
Me CH2OBn Rhodococcus sp. SM 1789 R >98 75
[87]
Me Bn Rhodococcus NCIMB 11216 S >98 11
[94]
CH2Cl 2,4-F2C6H3 Aspergillus niger R 98.3 38
[88]
Me 4-BrC6H4 Aspergillus niger LCP 521 R 96 49
(R)-1-(Chloromethyl)-1-(2,4-difluorophenyl)ethane-1,2-diol [(R)-16, R1 = CH2Cl; R2 = 2,4-

F2C6H3]; Typical Procedure:[94]
Racemic 2-(chloromethyl)-2-(2,4-difluorophenyl)oxirane (400 mg, 1.96 mmol) and crude
Aspergillus niger epoxide hydrolase (13 mg) with a specific activity 8 U per mg of protein
were mixed in 0.1 M phosphate buffer (pH 7) containing 10% DMSO. When nearly 50% con-
version was reached after a reaction time of 4.75 h, the reaction was stopped. The unreact-
ed epoxide (S)-2-(chloromethyl)-2-(2,4-difluorophenyl)oxirane was obtained (yield: 41%;
98.3% ee) as well as the title diol (R)-16 (R1 = CH2Cl; R2 = 2,4-F2C6H3); yield: 38%; 98.3% ee.
2.6.3.6 Vicinally Disubstituted Epoxides and Related Diols
Although extensive work on the hydrolysis of vicinally disubstituted cis-, trans-, and meso-
epoxides has been carried out with mammalian epoxide hydrolases, it was only after over-
coming the bottleneck of the limited availability of these enzymes with the discovery of

highly enantioselective microbial epoxide hydrolases and their large-scale production, as

well as the development of molecular biological tools, that this methodology became at-
tractive for organic synthesis.[96]
2.6.3.6.1 Nonsymmetrical Vicinally Disubstituted Epoxides
Epoxide hydrolase catalyzed resolution of racemic cis- and trans-2,3-disubstituted epox-

ides 17 compares favorably with hydrolytic kinetic resolution catalyzed by heavy metal
complexes. An increasing number of epoxide hydrolases from a variety of microorgan-
isms have become available for highly enantioselective hydrolysis of cis- and trans-epox-
ides (Scheme 23).[23,24,26,32,44,64,97–100]
Scheme 23 Epoxide Hydrolase Catalyzed Kinetic Resolution of cis- and trans-Disubstituted
Epoxides[26,32,44,64,97–100]
O O epoxide hydrolase O HO OH
+ + ∗ ∗
R1 R2 R1 R2 R1 R2 R1 R2
rac-trans 17A
R1 R2 Source of Epoxide Hydrolase Config of ee (%) Yield (%) Ref

Residual Epoxide 17A of 17A of 17A
[64]
Me Me Rhodotorula glutinis CIMW 147 R,R >98 45
[64]
Et Me Rhodotorula glutinis CIMW 147 R,R >98 48
[64]
Me Ph Rhodotorula glutinis CIMW 147 R,R >98 45
[97]
Me Ph Kau2 (metagenome) R,R 99.3 48
a [98]
Ph CO2Et Galactomyces geotrichum ZJUTZQ 200 2R,3S 99.4 65
Ph CO2Me Galactomyces geotrichum ZJUTZQ 200 2R,3Sa 98.6 62.5 [98]
a [98]
2-Tol CO2Me Galactomyces geotrichum ZJUTZQ 200 2R,3S 97.2 68
a
The aryl group (R1) is at the C3 position.
O O epoxide hydrolase O HO OH
+ + ∗ ∗
R1 R2 R1 R2 R1 R2 R1 R2
rac-cis 17B

Residual Epoxide 17B of 17B of 17B
Et Me Rhodotorula glutinis CIMW 147 2R,3Sa >98 48 [64]
Me Ph Aspergillus terreus R,R >98 –b [44]
[97]
Me Ph Kau2 (metagenome) R,R 98.3 97
Rhodotorula glutinis ATCC 201718 2S,3Ra >98 22 [64]
Beauveria sulfurescens 2S,3Ra >98 20 [100]


Residual Epoxide 17B of 17B of 17B
Diplodia gossypina ATCC 16391 2R,3Sa 100 14 [99]
Beauveria sulfurescens ATCC 7159 2S,3Ra >98 38 [26]
Sphingomonas sp. HXN-200
–c 99.5 37.6 [32]
a
The R1 group is at the C3 position.
b
Yield not reported.
c
(–)-Config; absolute configuration not reported.
2.6.3.6.2 Diols from Nonsymmetrical Vicinally Disubstituted Epoxides
Biocatalytic ring opening of 2,3-disubstituted oxiranes to vicinal diols has been shown to
occur with excellent enantiomeric excess and complete conversion (Scheme
24),[21,64,100,101] e.g. by using mammalian microsomal and soluble epoxide hydrolases in
the enantioconvergent hydrolysis of racemic cis-2,3-dialkyloxiranes.[102,103] Screening mi-
croorganisms for enzymes capable of hydrolyzing cis- and trans-2,3-disubstituted oxiranes
provided an important step toward easily available epoxide hydrolases for preparative
work.[101]
Scheme 24 Vicinal Diols by Epoxide Hydrolase Catalyzed Hydrolysis of cis- and trans-Di-
substituted Oxiranes[21,64,100,101]
O O epoxide hydrolase HO OH
+ ∗ ∗
R1 R2 R1 R2 R1 R2
rac-cis
R1 R2 Source of Epoxide Hydrolase Config of Diol ee (%) Yield (%) Ref

[101]
Me Bu Nocardia EH1 R,R 97 79
[100]
Me Ph Beauveria sulfurescens ATCC 7159 R,R 99 42
[64]
Et Me Rhodotorula glutinis CIMW 147 R,R >98 52
O O epoxide hydrolase HO OH
+ ∗ ∗
R1 R2 R1 R2 R1 R2
rac-trans
R1 R2 Source of Epoxide Hydrolase Config of Diola ee (%) Yield (%) Ref

[64]
Ph Me Rhodotorula glutinis CIMW147 2R,3S >98 55
[100]
Ph Me Beauveria sulfurescens 2R,3S 90 38
[21]
Me Bn Aspergillus niger variant DK-17 2R,3S >99 92
a
The R1 group is at the C2 position.

2.6.3.6.3 Diols from Symmetrical Vicinally Disubstituted meso-Epoxides
The enantioselective desymmetrization of meso-epoxides is an attractive route from an

achiral starting material to a product with two neighboring stereocenters, and is of high
interest for obtaining the corresponding chiral R,R-diols or the complementary S,S-diols
because both enantiomeric excess and yield can theoretically reach 100%. A series of re-
combinant epoxide hydrolases have been found to catalyze the enantioselective synthesis
of a variety of chiral 1,2-diols with excellent enantiomeric excesses and yields by (R,R)- and
(S,S)-selective desymmetrizations of meso-epoxides (Scheme 25).[32,64,104–108]
Scheme 25 Vicinal Diols by Epoxide Hydrolase Catalyzed Hydrolysis of Disubstituted meso-

Epoxides[32,64,104,107,108]
(R,R)-selective
epoxide hydrolase HO OH
R1 R2
O (R,R)-diol
R1 R2
meso (S,S)-selective
epoxide hydrolase HO OH
R1 R2
(S,S)-diol
R1 R2 Source of Epoxide Hydrolase Config of Diol ee (%) Yield (%) Ref

[104]
Ph Ph Rhodococcus erythropolis DCL14 mutant 173 R,R >99 15
[64]
(CH2)3 Rhodotorula glutinis CIMW147 R,R >98 98
[32]
(CH2)3 Sphingomonas sp. HXN-200 expressed in E. coli R,R 88 99
[108]
(CH2)4 Rhodococcus erythropolis DCL14 mutant 178 S,S 97 90
[32]
(CH2)4 Sphingomonas sp. HXN-200 expressed in E. coli R,R 99 68.5
[107]
Ph Ph recombinant BD8877 R,R 99 83
[107]
3-pyridyl 3-pyridyl recombinant BD8877 R,R 97 80
[108]
(CH2)5 Rhodococcus erythropolis DCL14 mutant 178 S,S 98 86
[108]
CH2N(Cbz)CH2 Sphingomonas sp. HXN-200 expressed in E. coli R,R 93 90
Epoxide hydrolases from Rhodococcus sp., Solanum tuberosum, Aspergillus niger, and Myco-
bacterium tuberculosis have also been used in a hydrolytic ring-opening/cyclization cascade
of meso-bis(oxiranyl)methanes to give substituted chiral tetrahydrofurans with excellent
diastereo- and enantioselectivity.[109]
2.6.3.7 Trisubstituted Epoxides and Related Diols
Various metabolites and natural products contain trisubstituted epoxide groups, as

shown for selected examples in Schemes 1–3, either alone or even in combination with
mono- and disubstituted epoxide groups, and are involved in key steps in biosynthetic
pathways.[110,111]
2.6.3.7.1 Chiral Trisubstituted Epoxides
Enantiopure trisubstituted oxiranes are not only important motifs in nature, but are also
highly valuable intermediates for organic synthesis; in addition to being substrates for the
preparation of chiral tertiary alcohols via epoxide ring opening by water,[112] they have
also been used for epoxide-opening cascade reactions.[113] Some examples of biocatalytic
epoxide hydrolysis of trisubstituted oxiranes are shown in Scheme 26.[64,112,114]
Scheme 26 Epoxide Hydrolase Catalyzed Ring Opening of Trisubstituted Epoxides[64,112,114]
NHPh NHPh NHPh
O O O O O O
Aspergillus niger LCP 521
+
OH
O O
OH
rac 97% ee 47%; 40% ee
O O OH
OH
Rhodotorula glutinis CIMW
+
48%; >98% ee >98% ee
O O OH
OH
Rhodotorula glutinis CIMW
+
28%; >98% ee 30% ee
2.6.3.7.2 Chiral Diols from Trisubstituted Epoxides
Epoxide hydrolases have been used to prepare vicinal diols from trisubstituted oxiranes
(Scheme 27; see also Scheme 26 for related examples).[115–117] The key step in a short asym-
metric total synthesis of myrcenediol, a beer flavor, and the related compound 18, con-
sists of a biocatalytic hydrolysis of a racemic trisubstituted epoxide bearing an alkenyl
side chain.[115]
Scheme 27 Vicinal Diols by Epoxide Hydrolase Catalyzed Hydrolysis of Trisubstituted

Oxiranes[115–117]

O OH
Mycobacterium paraffinicum
HO
71% conversion; 81% ee
rac (R)-18

O O
O O

R. ruber DSM 43338 OH
O
O 15%; 95% ee HO O
rac-19 (R)-20
O OH
HO
Corynebacterium C12
42%; 95% ee
rac (S,S)
The epoxide hydrolase Lsd19, which was the first enzyme shown to catalyze epoxide-
opening cascades, has been demonstrated to accept various trisubstituted bis-epoxides
and to catalyze either the epoxide-opening cascade involving 5-exo cyclization to give
linked tetrahydrofuran–tetrahydropyran products or the epoxide-opening cascade involv-
ing 6-endo cyclization to give tetrahydrofuran–tetrahydrofuran products with excellent
regioselectivity.[113]
(R)-(+)-Marmin {(R,E)-7-[(6,7-Dihydroxy-3,7-dimethyloct-2-en-1-yl)oxy]-2H-benzopyran-2-
one, (R)-20}; Typical Procedure:[116]
Lyophilized cells of Rhodococcus ruber DSM 43338 (0.3 g) were rehydrated for 1 h in 0.05 M
Tris-HCl buffer (pH 8.0; 6 mL) by shaking at 130 rpm at 30 8C. Racemic substrate rac-19
(100 mg, 0.3 mmol) was added. After 24 h, products were extracted with CH2Cl2 and the
organic layer was dried (Na2SO4). After concentration, the crude product was purified by
flash chromatography (petroleum ether/EtOAc 1:1); yield: 20 mg (15%); 95% ee; mp 104–
109 8C; [Æ]D20 9.1 (c 2.5, MeOH).
References 553
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Epoxide Hydrolysis: An Important Transformation in Organic Synthesis

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Epoxide Hydrolysis: An Important Transformation in Organic Synthesis

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529

2.6.3 Epoxide Hydrolysis

for references see p 553

(R,R)-epoxysuccinate juvenile hormone III

(13S,14S)-epoxymaresin (4S)-limonene-1,2-epoxide (4R)-limonene-1,2-epoxide

Scheme 3 Complex Epoxide Natural Products

for references see p 553

Scheme 4 Diol Natural Products Prepared by Enzymatic Ring Opening of Epoxides

leukotriene B4 (1S,2S,4R)-limonene-1,2-diol (1R,2R,4S)-limonene-1,2-diol

2.6.3.1 Monosubstituted Epoxides Containing an Aromatic Group and

Monosubstituted epoxides containing aromatic groups can be easily prepared in racemic

for references see p 553

Scheme 5 Enantioselective Enzymatic Hydrolysis of Styrene Oxides

2.6.3.1.1.1 Chiral Phenyloxiranes

Scheme 6 Epoxide Hydrolase Catalyzed Resolution of Phenyloxiranes[31–42]

Ar1 Source of Epoxide Config of ee (%) Yield (%) Ref

Ar1 Source of Epoxide Config of ee (%) Yield (%) Ref

(S)-2-Phenyloxirane [(S)-3, Ar1 = Ph]; Typical Procedure:[32]

(S)-2-(3-Chlorophenyl)oxirane [(S)-3, Ar1 = 3-ClC6H4]; Typical Procedure:[32]

for references see p 553

(S)-2-(4-Fluorophenyl)oxirane [(S)-3, Ar1 = 4-FC6H4]; Typical Procedure:[32]

2.6.3.1.1.2 Vicinal Diols Derived from Monosubstituted Epoxides Containing

Scheme 7 Regioselective Hydrolysis of Phenyloxiranes

Scheme 8 Hydrolysis of Phenyloxiranes to Vicinal Diols[25,31,35,46–51]

(R)-(3-Chlorophenyl)ethane-1,2-diol [(R)-5, Ar1 = 3-ClC6H4]; Typical Procedure:[46]

for references see p 553

2.6.3.1.2 Chiral Pyridyloxiranes

Scheme 9 Metal-Mediated Asymmetric Synthesis and Resolution of 2-Pyridyloxi-

Scheme 10 Epoxide Hydrolase Mediated Resolution of Pyridyloxiranes[54,56–58]

Ar1 Source of Epoxide Hydrolase ee (%) of 6 Yield (%) of 6 Ref

(S)-2-Pyridyloxirane [(S)-6, Ar1 = 2-Pyridyl]; Typical Procedure:[57]

2.6.3.2 Glycidyl Ethers and Their Vicinal Diols

Scheme 11 Enantioselective Hydrolysis of Aryl Glycidyl Ethers

2.6.3.2.1 Enantioenriched Glycidyl Ethers

for references see p 553

Scheme 12 Epoxide Hydrolase Catalyzed Kinetic Resolution of Glycidyl Ethers[20,34,60,62–66]

R1 Source of Epoxide Hydrolase Config of eea (%) Yielda (%) Ref

(S)-2-(Phenoxymethyl)oxirane [(S)-7, R1 = Ph]; Typical Procedure:[60]

(S)-2-[(1-Naphthyloxy)methyl]oxirane [(S)-7, R1 = 1-Naphthyl]; Typical Procedure:[62]

2.6.3.2.2 Vicinal Diols from Glycidyl Ethers

Scheme 13 Vicinal Diols by Epoxide Hydrolase Catalyzed Hydrolysis of Glycidyl Ethers[62,63,68,69]

Ar1 Source of Epoxide Hydrolase ee (%) Yield (%) Ref

2.6.3.3 Monosubstituted Epoxides Containing Alkyl or Alkenyl Substituents and

Scheme 14 Enantiomeric Hydrolysis of Monosubstituted Epoxides

2.6.3.3.1 Monosubstituted Epoxides Containing Alkyl or Alkenyl Substituents

The application of epoxide hydrolases to non-aromatic epoxide substrates such as linear

for references see p 553

Scheme 15 Epoxide Hydrolase Catalyzed Kinetic Resolution of Alkyloxiranes[39,64,72–76]

R1 Source of Epoxide Hydrolase Config of ee (%) Yield (%) Ref

2.6.3.3.2 Diols from Monosubstituted Epoxides Containing Alkyl or Alkenyl

Scheme 16 Vicinal Diols by Epoxide Hydrolase Catalyzed Hydroly-

epoxide hydrolase from

n ee (%) Yield (%) Ref

2.6.3.4 Other Monosubstituted Epoxides and Related Diols

Scheme 17 Enantioselective Hydrolysis of Functionalized Monosubstituted

2.6.3.4.1 Other Chiral Monosubstituted Epoxides

Enantiomerically pure acetal-protected glycidaldehyde derivatives (R)- or (S)-11 have been

Scheme 18 Epoxide Hydrolase Catalyzed Kinetic Resolution of Functionalized

OR2 OR2 OR2

R1 R2 ee (%) of (R)-11 Yield (%) of (R)-11 Ref

rac 46%; >99% ee

for references see p 553

2.6.3.4.2 Other Chiral Monosubstituted Diols