Enantioselective Bio-Hydrolysis of Geranyl-Derived rac-Epoxides: A Chemoenzymatic Route to trans-Furanoid Linalool Oxide

Article abstract of DOI:10.1002/adsc.201801094

In contrast to many chemical dihydroxylation methods, enzymatic epoxide hydrolysis provides an environmentally benign route to vicinal diols, which are important intermediates in the synthesis of fine chemicals and pharmaceuticals. Using epoxide hydrolases, enantiopure diols are accessible under mild conditions. In order to assess the selectivity of epoxide hydrolases on geraniol-derived oxiranes, a range of derivatives were screened against a large variety of enzyme preparations. For nearly all substrates, a matching hydrolase with excellent enantioselectivity (≥95% ee) could be found. In addition, a chemoenzymatic approach for the stereoselective synthesis of furanoid linalool oxide was developed. Combination of enzymatic enantioselective hydrolysis with stereoselective Tsuji-Trost reaction granted diastereoselective access to trans-(2R,5R)-configured linalool oxide with high diastereomeric and enantiomeric excess (97% de and 97% ee). (Figure presented.).

Full text of DOI:10.1002/adsc.201801094

Title: Enantioselective Bio-Hydrolysis of Geranyl-Derived rac-Epoxides:
A Chemoenzymatic Route to trans-Furanoid Linalool Oxide
Authors: Matthijs van Lint, Aysegül Gümüs, Eelco Ruijter, Kurt Faber,
Romano Orru, and Mélanie Hall
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To be cited as: Adv. Synth. Catal. 10.1002/adsc.201801094
Link to VoR: http://dx.doi.org/10.1002/adsc.201801094

10.1002/adsc.201801094
Advanced Synthesis & Catalysis
FULL PAPER
DOI: 10.1002/adsc.201((will be filled in by the editorial staff))
Enantioselective Bio-Hydrolysis of Geranyl-Derived rac-
Epoxides: A Chemoenzymatic Route to trans-Furanoid Linalool
Oxide
Matthijs J. van Linta, Aysegül Gümüsb,c, Eelco Ruijtera, Kurt Faberb, Romano V.A.
Orrua* and Mélanie Hallb*
a
Department of Chemistry & Pharmaceutical Sciences and Amsterdam Institute for Molecules, Medicines and Systems
(AIMMS), Vrije Universiteit Amsterdam, De Boelelaan 1108, 1081 HZ Amsterdam, The Netherlands.
e-mail: r.v.a.orru@vu.nl
Department of Chemistry, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria
b
e-mail: melanie.hall@uni-graz.at
Present address: Department of Chemistry, Yuzuncu Yil University, 65080 Van, Turkey
c
Received: ((will be filled in by the editorial staff))
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201
Abstract. In contrast to many chemical dihydroxylation chemoenzymatic approach for the stereoselective synthesis
methods, enzymatic epoxide hydrolysis provides an of furanoid linalool oxide was developed. Combination of
environmentally benign route to vicinal diols, which are enzymatic enantioselective hydrolysis with stereoselective
important intermediates in the synthesis of fine chemicals Tsuji-Trost reaction granted diastereoselective access to
and pharmaceuticals. Using epoxide hydrolases, enantiopure trans-(2R,5R)-configured linalool oxide with high
diols are accessible under mild conditions. In order to assess diastereomeric and enantiomeric excess (97% de and 97%
the selectivity of epoxide hydrolases on geraniol-derived ee).
oxiranes, a range of derivatives were screened against a large
variety of enzyme preparations.
For nearly all substrates, a matching hydrolase with excellent
enantioselectivity (≥95% ee) could be found. In addition, a
Keywords: Biocatalysis; Epoxide hydrolases; Furanoid
linalool oxide; Geraniol epoxide; Tsuji-Trost reaction
of EHs was further exploited in the total synthesis of
marmin,[7b] vitamin E-related chromane-systems[9]
and cis-gigantrionenin.[10]
Introduction
Biocatalytic
reactions
often
provide
an
In addition to high regio- and stereoselectivities,
easy access and straightforward implementation of
these biocatalysts is an appealing aspect. Epoxide
hydrolase activity of crude cell lyophilisates can often
be used directly without prior protein purification,
providing a cost-effective and practical catalytic
platform for the stereoselective generation of vic-
diols from corresponding epoxides.
Encouraged by promising results obtained in
previous studies,[7b] the potential of epoxide
hydrolases for the stereoselective hydrolysis of
isoprenoid-based oxiranes bearing various functional
groups, such as alcohols, ketones, esters and ethers,
was assessed. Several of these compounds constitute
juvenile hormones and are used in insect pest
control.[11]
environmentally benign alternative to heavy metal-
catalyzed reactions. Especially for dihydroxylation of
olefins, green alternatives to hazardous Os-based
functionalization are desirable.[1] Alternatively,
peroxide-based olefin epoxidation followed by
catalytic hydrolysis may be used to generate vic-
diols.[2] These compounds are versatile intermediates
in the synthesis of industrial products, such as chiral
drugs and pesticides, while chiral vicinal diols often
serve as intermediates in total syntheses.[3]
In recent years, stereoselective hydrolysis of
epoxides to yield enantiopure diol products has been
thoroughly
investigated.[4]
Besides
recently
developed organometallic and organocatalytic
methods, enzymes have also received substantial
interest[5] and a large number of microbial strains and
isolated enzymes have been classified according to
their substrate preference.[6] These epoxide
hydrolases (EHs) have been used on a variety of
substrates with significant success,[7] and excellent
regioselectivity, as well as notable stereoselectivity,
were typically observed.[8] The broad substrate scope
Results and Discussion
Our first goal was to assess the effect of substitution
of racemic geraniol oxide ethers on enzymatic
hydrolysis performance. Ethyl ether rac-1 (Scheme 1)
1
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10.1002/adsc.201801094
Advanced Synthesis & Catalysis
experimental section).[14] For determination of
absolute configuration of unreacted epoxide 1, the
latter was treated with aqueous acid to produce the
corresponding
diol
2
with
retention
of
configuration.[15] Next, the influence of the substrate
type on enzyme enantioselectivity was studied with
allyl ether rac-3 (Scheme 1 and Table 1). The results
generally indicated no significant influence of the
allyl moiety compared to the ethyl moiety of rac-1 on
enantioselectivity and activity, except in a few cases
(Table 1, entries 11 & 17). As with 2, nearly
enantiopure diol 4 (98% ee) could be generated using
isolated epoxide hydrolase E-23 from Synechocystis
sp. PCC 6803 (Table 1, entry 19). Notably, a
remarkable drop in activity was found using epoxide
hydrolase E-21 from Aspergillus niger, together with
a different selectivity pattern (i.e. overall preference
for retention of (S)-1 and inversion of (R)-3, Table 1,
entry 17). E-15 on the other hand displayed opposite
stereopreference on 3 but followed the same pathway
(apparent inversion) as with 1 (Table 1, entry 11).
Encouraged by selective hydrolysis of geranyl
oxide-based ethers, the epoxide of the pheromone
geranylacetone (rac-5a)[16] was subjected to the
enzyme library, in parallel with its Z-isomer,
nerylacetone (rac-5b). The results of this bioassay
revealed dual activity for the lyophilisates. As
anticipated with whole-cell preparations, the methyl
ketone moiety was readily reduced by endogenous
alcohol dehydrogenases (ADHs, Tables 2-3), while
some strains and isolated enzymes displayed
chemoselectivity for epoxide hydrolysis (Table 4).
This resulted in a range of products (Scheme 2) with
unknown stereochemistry, rendering elucidation of
pathways and absolute configurations challenging.
The latter were determined by independent synthesis
of enantioenriched 5b followed by stereoselective
reduction of the methyl ketone using Prelog (ADH-
A) and anti-Prelog (ADH200) ADHs to give 7b.[17]
Scheme 1. Epoxide hydrolase (EH) mediated hydrolysis of
racemic ethyl geraniol-6,7-oxide ether (1) and allyl
geraniol-6,7-oxide ether (3). Inversion: nucleophilic attack
at C2; retention: nucleophilic attack at C1.
was subjected to an array of epoxide hydrolases from
different sources and in various forms (lyophilized
cells and isolated enzymes, Table S1). Most enzyme
preparations showed moderate to good activity after
24 h with varying selectivity (see Table 1). In most
cases, the obtained enantiomeric excess of the
remaining epoxide (1) was higher than that of the
product (2), providing >99% ee for some substrates.
Depending on the type of enzyme and substitution
pattern of the substrate, epoxide hydrolases can
follow up to four hydrolysis pathways: Hydrolysis of
both enantiomers can proceed either via retention
(nucleophilic attack at ‘C1’) or inversion
(nucleophilic attack at ‘C2’) of absolute configuration
(Scheme 1). Provided the enzyme exhibits different
reaction rates for the two enantiomers (i.e.
enantiopreference), kinetic resolution may occur. In
some cases however, an enantioconvergent process
can take place, which is caused by hydrolysis of one
enantiomer via retention and of the other via
inversion.[12] Given the possible occurrence of all four
Comparison
with
independently
synthesized
reference material indicated that Rhodococcus strains
mainly showed Prelog-type alcohol dehydrogenase
activity, while opposite enantiopreference was only
observed for E-3 (Table 2, entry 2). In line with
previous results, the overall stereoselectivity of
epoxide hydrolases was moderate to good (substrate
(S)-5a obtained in 87% ee and product (R)-6a in up to
97% ee, Table 4, entries 3 and 12, respectively). Four
enzyme preparations showed complete hydrolysis
within 24 h (Table 2, entries 1 & 8 and Table 4,
entries 5 & 11), while E-3 and E-10, as with 1a and
3a, did not show any epoxide hydrolysis activity
(Tables 2-3, entries 2 & 9). This simplified
assignment of stereochemistry, as syn- and anti-
diastereoisomers of 7a (and 7b) were produced in
equal amounts as sole products. Since the ADH
enantiopreference of E-3 was found opposite of that
of E-10, both diastereoisomers were easily
recognized.
pathways
simultaneously,
calculation
of
enantioselectivities (E-values) is not possible.[13]
Detailed analysis of the data (conversion, eeS and
eeP) indeed revealed mixed stereochemical pathways,
which were highly strain-dependent. With 1, apparent
kinetic resolution was observed primarily among
Rhodococcus ruber strains (Table 1, entries 4-7),
through dominating inversion (i.e. remaining
substrate and product share same absolute
configuration), yielding (R)-1 in up to 99% ee. Partial
enantioconvergent hydrolysis [(R)-1 -> (R)-2 along
with (S)-1 -> (R)-2, Scheme 1] could be identified
with E-1 and E-9 as well as isolated epoxide
hydrolase E-22 from Deinococcus radiodurans
(Table 1, entries 1, 8 & 18, almost full conversion
along with moderate to good enantiopurity of product
2, eep up to 83%). The absolute configuration of vic-
diol 2 was determined by independent synthesis of
the respective (S)-enantiomer by asymmetric
Sharpless dihydroxylation using AD-mix-α (see
2
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10.1002/adsc.201801094
Advanced Synthesis & Catalysis
Table 1. Conversion and selectivity of epoxide hydrolase preparations in biocatalytic hydrolysis of rac-1 and rac-3.
Entry
Enzyme Conv. of 1
Conv. of 3
1
2
3
4
preparation
(%)
ees (%)
eep (%)
(%)
ees (%)
eep (%)
Lyophilized cells
1
2
3
4
5
6
7
8
9
10
11
12
13
14
E-1
E-2
E-4
E-5
E-6
E-7
E-8
E-9
E-12
E-14
E-15
E-16
E-17
E-18
87
17
n.d.
53
37
65
85 (R)
37 (R)
n.a.
96 (R)
84 (R)
68 (R)
>99 (R)
n.a.
n.a.
n.a.
>99 (R)
>99 (R)
>99 (R)
n.a.
67 (R)
80 (R)
n.a.
83 (R)
86 (R)
80 (R)
67 (R)
74 (R)
n.a.
>99
7
n.a.
5
74 (R)
(R) 89 (R)
43
65
55
76
75
>99
25
>99
85
80
61
5
58 (R) 90 (R)
89 (R) 86 (R)
80 (R) 88 (R)
38 (R) 82 (R)
88 (R) 62 (R)
n.a.
64 (S)
n.a.
69
>99
n.d.
>99
94
79
61
65 (R)
67 (S)
rac
10 (R)
27 (R)
rac
67 (S)
20 (S)
>99 (R) rac
>99 (R) 30 (S)
16 (S) rac
8
(S)
n.d.
n.a.
Isolated enzymes
15
16
17
18
19
E-19
E-20
E-21
E-22
E-23
17
>99
33
>99
18
>99 (R)
n.a.
20 (R)
n.a.
34 (S)
(R)
59 (S)
55 (R)
95 (R)
35
>99
8
>99
22
93 (R) 28 (S)
5
n.a.
rac
33 (S)
n.a.
58 (S)
57 (R)
77 (R)
40 (R) 98 (R)
Standard conditions: Incubation of 5 µL substrate and 50 mg epoxide hydrolase preparation in Tris-HCl buffer (1 mL, 50
mM, pH 8.0) at 30 °C for 24 h; Origin of all epoxide hydrolases can be found in the Supporting Information (Table S1);
n.d. not determined; n.a. not applicable; no conversion with E-3, E-10 and E-11.
Scheme 2. Enzymatic hydrolysis (red) and concomitant ADH-catalyzed carbonyl reduction (blue) of rac-geranylacetone
9,10-oxide (rac-5a) and rac-nerylacetone 9,10-oxide (rac-5b) by epoxide hydrolase preparations (numbering of C-atoms 3
and 10 only for labelling purpose, see Tables 2-3).
3
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10.1002/adsc.201801094
Advanced Synthesis & Catalysis
Table 2. Conversion of rac-5a with epoxide hydrolase preparations containing ADHa) activity and product distribution.
Entry Enzyme
Preparation Conv.
(%)
5a
6a
ee
7a
8a
5a/6a/7a/8a
ee
(%)
de ee (%)b) ee (%)c) de ee (%)b) ee (%)c) distribution
(%) (%) (3R,10S) (3S,10S) (%) (3R,10S) (3R,10R)
(%)
1
2
3
4
5
6
7
8
9
E-1
E-3
E-4
E-5
E-6
E-7
E-8
E-9
E-10
>99 n.a.
66 (R) n.a.
n.a.
n.a
66d)
83
94
94
81
98
n.a.
69
n.a.
60
63
61
n.a.
65
88
84
73
76
81
n.a.
0:20:0:80
46:0:54:0
11:13:39:37
7:21:26:46
11:21:29:39
22:52:10:16
7:17:31:45
0:23:0:76
61:0:39:0
54
89
93
89
78
93
rac
0
51e) n.a. n.a.
83 (R) 87 (R) 74
88 (R) 87 (R) 85
75 (R) 84 (R) 72
20 (R) 81 (R) 20
96 (R) 80 (R) 94
87
73
41
26
82
n.a.
77
89
61
79
88
56
58
n.a.
87
86
84
84
76
77
n.a.
>99 n.a.
39 rac
83 (R) n.a.
n.a.
3
Standard conditions: 5 µL substrate and 50 mg epoxide hydrolase were incubated in Tris-HCl buffer (1 mL, 50 mM, pH
8.0) at 30 °C for 24 h; a) alcohol dehydrogenase selectivity was determined by comparison with authentic material obtained
b)
c)
d)
with Prelog (S-selective) ADH-A and anti-Prelog (R-selective) ADH200; major enantiomer; minor enantiomer;
(3S,10R); e) (3R,10R); n.a.: not applicable.
Table 3. Conversion of rac-5b with epoxide hydrolase preparations containing ADHa) activity and product distribution.
Entry Enzyme
5b
6b
ee
7b
8b
6b/7b/8b
Preparation Conv.
ee
de ee (%)b) ee (%)c) de ee (%)b) ee (%)c) distribution
(%)
58
71
24
74
71
40
80
89
10
(%)
rac
(%) (%) (3R,10S) (3S,10S) (%) (3R,10S) (3R,10R)
(%)
1
2
3
4
5
6
7
8
9
E-1
E-3
E-4
E-5
E-6
E-7
E-8
E-9
E-10
45 (R)
n.a.
6
0
9
5
2
4
41
43d)
87
76
85
79
66
11
83
24
40
77
72
n.a.
94
42:11:41:10
29:0:71:0
76:7:11:6
26:4:55:15
29:3:57:11
60:10:23:7
20:40:24:16
11:8:49:32
90:0:10:0
rac
rac
4
rac
3
57e) n.a. n.a.
49 (R)
87
85
92
88
94
35
83
33
47
27
14
63
56
n.a.
73
63
79
70
44
71
n.a.
(R) 33 (R)
44 (R)
(R) 61 (R)
94
97
96
65
64
n.a.
93 (R) 70 (R) 83
5
rac
60 (R) 10
n.a.
0
Standard conditions: 5 µL substrate and 50 mg epoxide hydrolase were incubated in Tris-HCl buffer (1 mL, 50 mM, pH
8.0) at 30 °C for 24 h; a) alcohol dehydrogenase selectivity was determined by comparison with authentic material obtained
b)
c)
d)
with Prelog (S-selective) ADH-A and anti-Prelog (R-selective) ADH200; major enantiomer; minor enantiomer;
(3S,10R); e) (3R,10R); n.a.: not applicable.
Table 4. Conversion of rac-5a and 5b with epoxide hydrolase preparations displaying no ADH activity.
Entry
Enzyme
Conv. of
Conv. of
5a
6a
5b
6b
preparation
5a (%)
ee (%)
ee (%)
5b (%)
ee (%)
ee (%)
Lyophilized cells
1
2
3
4
5
6
7
8
E-2
36
23
38
17
>99
98
41 (R)
55 (S)
87 (S)
19 (S)
n.a.
n.a.
70 (R)
70 (R)
79 (R)
83 (R)
88 (S)
60 (S)
10 (S)
24 (S)
rac
18
46
26
27
>99
43
11 (R) 68 (R)
E-11
E-12
E-13
E-14
E-15
E-16
E-17
75 (S)
50 (S)
40 (S)
n.a.
84 (R)
20 (S)
10 (S)
rac
10 (S)
32 (R)
87
71
60
46
73 (R) 20 (S)
35 (R) 20 (S)
rac
Isolated enzymes
9
E-19
E-20
E-22
E-23
33
88
>99
23
86 (R)
n.d.
n.a.
21 (S)
rac
50 (R)
97 (R)
5
n.d.
n.a.
n.a.
6
n.d.
10
11
12
>99
>99
8
13 (R)
30 (R)
78 (R)
50 (R)
(S)
Standard conditions: 5 µL substrate and 50 mg epoxide hydrolase were incubated in Tris-HCl buffer (1 mL, 50 mM, pH
8.0) at 30 °C for 24 h; n.a.: not applicable; n.d.: not determined.
4
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10.1002/adsc.201801094
Advanced Synthesis & Catalysis
Table 5. Conversiona) and selectivity in biocatalytic
hydrolysis of rac-9 to 10.
A notable finding is the stereochemistry of the
minor diastereoisomer of 8a (Tables 2-3). Two
pathways are possible: 5a->6a->8a (EH->ADH) or
5a->7a->8a (ADH->EH). In the latter, the carbonyl is
first reduced to the (S)-alcohol (except with E-3),
further leading to formation of (3R,10S)-8a. Thus the
presence of (3R,10R)-8a in the final product mixture
indicates that the ADH selectivity in the first pathway
is influenced by the presence of hydrolyzed epoxide.
In general, lower activities and selectivities were
observed using nerylacetone (5b) (Tables 3-4). Diol
6b and epoxide 5b were obtained in 70% and 93% ee
(max.), respectively. In some cases, the total ADH
activity (formation of 7a and 8a) present in the
lyophilisates was much lower with 5b compared to
5a (Table 3, entries 1, 3, 7 & 9). Combination of
highest conversion and highest enantiopurity of both
substrate 5b and product was obtained through
overall retaining EH activity from E-11 (Table 4,
entry 2).
Entry Enzyme Conv. (%) eeS (%) eeP (%)
Ec)
Lyophilized cells
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
E-1
E-2
E-4
E-5
E-6
E-7
E-8
E-9
E-11
E-12
E-13
E-14
E-15
E-16
E-17
25
n.d. b)
34
34
35
24
42
34
36
24
3
>99
>99
>99
56
27 (R) 80 (R)
12
45 (R) 87 (R)
43 (R) 85 (R)
47 (R) 87 (R)
20 (R) 63 (R)
59 (R) 82 (R)
42 (R) 80 (R)
22
19
23
5
18
14
26
10
2
n.a.
n.a.
n.a.
3
50 (S)
24 (S)
88 (R)
77 (S)
1 (R) 34 (S)
n.a.
n.a.
n.a.
49 (S)
45 (R)
42 (R)
43 (R)
38 (R)
Isolated enzymes
In analogy to EH-mediated total synthesis of
marmin,[7b] a new route to furanoid natural product
linalool oxide (11) – found in many essential oils and
commercial perfumes – was proposed (Scheme 3).
This route is based on a 5-exo-trig cyclization of triol
10 to form the furan ring. Based on the work of
Carreira et al.,[18] an intramolecular enantioselective
iridium-catalyzed allylic etherification of the
secondary alcohol onto the allylic moiety was
envisioned. The required vicinal diol 10 is available
by enzymatic hydrolysis of the parent geraniol
epoxide 9, similarly to 1a, 3a and 5a.
16
17
18
19
E-19
E-20
E-22
E-23
61
>99
51
43 (R) 27 (R)
n.a. 24 (R)
>99 (R) 97 (R) >200
21 (R) 93 (R) 34
3
n.a.
18
Standard conditions: 5 µL substrate and 50 mg epoxide
hydrolase were incubated in Tris-HCl buffer (1 mL, 50
mM, pH 8.0) at 30 °C for 24 h; a) Conversion calculated by
[ees/(ees+eep)];[19] Instead of product 10, several other
b)
products were obtained, including all isomers of linalool
oxide; c) apparent E-value[19] for whole-cell preparations;
n.a.: not applicable; n.d.: not determined.
resulting in a fully enantioconvergent process.
Enantiopure (R)-10 could finally be used in the
enantioselective iridium-catalyzed ring closure
reaction towards 11. Unfortunately, the catalytic
complex had little stereocontrol on the
diastereoselectivity of the formation of cyclic ether
11 (data not shown). The lack of selectivity might be
explained by the ease of cyclization following acid-
mediated activation of the allylic alcohol in 10. With
this in mind, a short study on the effect of acid and
solvent was conducted in order to achieve
diastereoselective ring closure (Scheme 4). Results
from acid-mediated cycloetherification test reactions
(Table 6) indicate an intrinsic preference for the
trans-product, although both solvent polarity and
catalyst acidity influenced the trans/cis ratio.
Scheme 3. Envisaged epoxide hydrolase (EH)-mediated
hydrolysis of geraniol-6,7-oxide (rac-9)/iridium-catalyzed
cycloetherification sequence.
In order to identify the most enantioselective
enzymatic conversion, geraniol-6,7-oxide (rac-9) was
subjected to the library of EHs (Table 5). Most
lyophilized cell preparations catalyzed an apparent
inversion process (except for E-13 and E-17, entries
11&15) with moderate enantioselectivity (max. E =
26, Table 5, entry 9), while high enantioselectivity
was observed using EH of Deinococcus radiodurans
(E-22), which catalyzed kinetic resolution of rac-9
via inversion [(R)-9 in >99% ee and (R)-10 in 97% ee,
E >200]. Remaining (R)-9 was then subjected to acid-
catalyzed hydrolysis with retention of configuration,
Scheme 4. Acid-catalyzed cycloetherification of rac-10 to
trans- and cis-furanoid linalool oxide (11).
5
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10.1002/adsc.201801094
Advanced Synthesis & Catalysis
Table
6.
Diastereoselectivity
of
acid-mediated
yielding (R)-14c with ee up to 96% (Table 7, entries 2,
4 & 17). Remarkably, E-1 and E-9 led to almost full
conversion in combination with high eeP values,
cycloetherification of rac-10 a).
d.r.b)
Entry
Solvent
DMSO-d6
CCl4
Acetone-d6
CDCl3
Acid
TFA
TFA
TFA
TFA
pKa
indicating
a
stereoconvergent process. E-4
1
2
3
4
3.45[20] no conv.
(Rhodococcus ruber strain DSM44539) was selected
for further optimization, with regard to enzyme
loading, buffer type, and pH (Table 8). Reducing the
enzyme preparation loading by 50% (final 25
mg/mL) still afforded nearly full kinetic resolution in
24 h. Buffer type and pH had only moderate
influence on conversion and ee values.
3.45
3.45
3.45
1.2:1
2.2:1
2.8:1
5
CDCl3
4.0:1
2.42-
2.63[21]
6
Toluene-d8
6.1:1
Table 7. Conversion and selectivity in biocatalytic
hydrolysis of rac-13c to 14c.
a)
Entry Enzyme Conv. a) (%) eeS (%) eeP (%)
Lyophilized cells
All reactions performed in an NMR tube with excess of
acid. b) Based on trans-/cis-ratio as determined by 1H NMR
analysis.
1
2
3
4
5
E-1
E-4
E-5
E-6
E-7
97
51
64
50
50
>99 (R) 83 (R)
>99 (R) 96 (R)
>99 (R) 89 (R)
>99 (R) 96 (R)
87 (R) 93 (R)
88 (R) 78 (R)
6
E-8
68
7
8
9
10
11
12
13
14
E-9
>99
64
90
20
>99
92
n.a.
90 (R)
E-11
E-12
E-13
E-14
E-15
E-16
E-17
39 (S) 95 (R)
>99 (S) 63 (S)
7 (S) 85 (S)
n.a.
>99 (S)
17 (R)
(R)
7
89
72
>99 (S) 18 (S)
53 (S) 35 (S)
Isolated enzymes
15
16
17
18
E-19
E-20
E-22
E-23
89
65
57
52
92 (S) 35 (S)
>99 (R) 30 (S)
>99 (R) 95 (R)
62 (R) 96 (R)
Scheme 5. Epoxide hydrolase (EH)-mediated hydrolysis of
geraniol-6,7-oxide derivatives 13a-c with subsequent
Tsuji-Trost allylic substitution.
Standard conditions: 5 µL substrate and 50 mg epoxide
hydrolase were incubated in Tris-HCl buffer (1 mL, 50
mM, pH 8.0) at 30 °C for 24 h; a) based on GC integrals.
Due to unsatisfactory results with the iridium-
based catalyst, the strategy was altered to a Tsuji-
Trost based approach,[22] in which biocatalysis could
advantageously be combined with transition metal
catalysis to obtain 11 stereoselectively (Scheme 5).
In order to identify the right allylic substitution for
both the biocatalytic step and the cycloetherification,
three leaving groups that have been already used in
intramolecular Tsuji-Trost reactions[22-23] were
investigated (methyl carbonate, acetate, and pivalate).
First, the stability of the activation groups was tested
in aqueous buffer, required for epoxide hydrolysis.
Carbonate (13a) was largely hydrolyzed (pH 8) after
24 h at 30 °C. In contrast, acetate 13b and pivalate
13c derivatives were hardly affected by the buffer.
Owing to the likely presence of active endogeneous
lipases in whole cell preparations, 13c was selected
for further studies since pivalates are much poorer
substrates for lipases compared to acetates.[24] Based
on previous results with 1, 3, 5a-b and 9, a selection
of epoxide hydrolases was tested (Table 7).
Table 8. Conversion and selectivity of E-4 mediated
hydrolysis of rac-13c using various buffers and enzyme
loadings.
Buffer
type
Loading
(mg/mL)
Conv. eeS eeP
(%) (%) (%)
Entry
pH
a)
1
2
3
4
5
6
7
8
9
25
25
25
25
5
5
5
5
1
Tris·HCl 7.0
Tris·HCl 8.0
Phosphate 7.0
Phosphate 8.0
Tris·HCl 7.0
Tris·HCl 8.0
Phosphate 7.0
Phosphate 8.0
Tris·HCl 7.0
Tris·HCl 8.0
Phosphate 7.0
Phosphate 8.0
41
43
46
45
20
22
28
24
25
24
38
29
77 97
83 98
92 98
93 98
20 98
25 98
32 98
27 99
4
1
3
7
95
94
97
98
10
11
12
1
1
1
Standard conditions: 5 µL substrate and E-4 were
The most notable result was the high selectivity of
Rhodococcus lyophilisates (E-1 and E-4-9) on 13c
(Table 7, entries 1-7). In some cases, full kinetic
resolution (E >200) via inversion was achieved,
a)
incubated in 1 mL buffer at 30 °C for 24 h; 50 mM Tris
or 100 mM potassium phosphate.
6
This article is protected by copyright. All rights reserved.

10.1002/adsc.201801094
Advanced Synthesis & Catalysis
Preparative scale hydrolysis of 13c in a
aluminium) from Merck Serono KGaA (Darmstadt) and
compounds were visualized by UV detection (254 or 366
nm) and stained with basic aq. KMnO4 or cerium-
molybdate stain.
stereoconvergent fashion using E-4 followed by acid
hydrolysis was performed (Scheme 6). Once
enzymatic kinetic resolution was complete (high ee
values for both substrate and product), the product
mixture was extracted and treated with aqueous acid.
This treatment resulted in full hydrolysis to the diol
and yielded (R)-14c in high enantiopurity (97% ee).
Subsequent palladium-catalyzed ring closure using
(S)-C3-TunePhos (L2) as the ligand proceeded
smoothly to give the desired trans-configured linalool
oxide 11 with high diastereomeric and enantiomeric
excess (75% yield, 97% de and 97% ee) in 39%
overall yield starting from rac-13c.
1H and 13C NMR spectra were recorded on a Bruker
Avance 500 (500.23 MHz for 1H and 125.78 MHz for 13C),
1
a Bruker Avance 400 (400.13 MHz for H and 100.62
1
MHz for 13C) or a Bruker AMX 360 (360.17 MHz for H
and 90.56 MHz for 13C) in CDCl3 using the residual
1
solvent as internal standard (CDCl3: δ = 7.26 for H NMR
and δ = 77.16 for 13C NMR). Chemical shifts (δ) are given
in ppm and coupling constants (J) in Hz. Resonances are
described as s (singlet), d (doublet), t (triplet), q (quartet),
bs (broad singlet) and m (multiplet) or combinations
thereof. HSQC-NMR spectra were used for the assignment
of the proton and APT-NMR spectra for carbon signals.
NMR data were processed using MestReNova.
Chiral GC-FID measurements were carried out on a
Shimadzu GC-2010 Plus equipped with an FID detector
and a AOC-20i auto injector using a Chirasil Chiraldex
Dex-CB (25 m x 0.32 mm, 0.25 μm film) column. Chiral
HPLC analysis was performed using a JASCO-System
equipped with a multiwavelength-detector (MD-910) and a
temperature-chamber (AS-950).
Epoxide hydrolases were obtained from bacterial, yeast
and fungal strains, FCC stands for in-house ‘Fab-Crew-
Collection’ (see Supporting Information). ADH200 was
from Evocatal (evo-1.1.200).
Standard Procedure for Epoxidation of Olefins[12a,25]
Finely powdered Na2HPO4 (2.6 eq.) was added to a
vigorously stirred solution of alkene (5.8 mmol) in CH2Cl2
(50 mL). The mixture was stirred for 15 min at room
temperature and was then cooled to 0 °C. Then m-CPBA
(1 eq.) was added slowly. The mixture was allowed to
warm to room temperature and the reaction was monitored
with TLC. After the reaction was complete, the white
suspension was filtered. The resulting solution was treated
with Na2S2O5 (10% aq., 15 mL) to destroy excess of
peracid. The two-phase system was stirred for 30 min, the
layers were separated, and the organic phase was washed
twice with NaHCO3 (sat. aq.). The organic phase was dried
over Na2SO4 and evaporated. The crude product was
purified by flash column chromatography.
Scheme 6. E-4-mediated hydrolysis of geraniol-6,7-oxide
derivative 13c followed by acid-catalyzed hydrolysis and
subsequent Tsuji-Trost allylic substitution.
Conclusion
Selective hydrolysis of terpenoid geranyl-derived
epoxides was established using epoxide hydrolases,
allowing simple access to enantiopure vicinal diols
and oxiranes. Highest enantioselectivities were
obtained with epoxides derived from geranyl acetone
and geraniol esters. Furthermore, a novel route to
optically pure linalool oxide was developed by
combining enantioselective biocatalytic epoxide
hydrolysis with diastereoselective Tsuji-Trost
rac-(E)-3-(5-Ethoxy-3-methylpent-3-en-1-yl)-2,2-
dimethyloxirane (rac-1).
cycloetherification,
affording
diastereoselective
Flash column chromatography (c-Hex/EtOAc, 6:1)
provided rac-1 as a colorless liquid (0.69 g, 60%).
1H NMR (500 MHz, CDCl3) δ 5.39 (tq, J = 6.7, 1.3 Hz,
1H), 3.97 (d, J = 6.7 Hz, 2H), 3.47 (q, J = 7.0 Hz, 2H),
2.71 (t, J = 6.2 Hz, 1H), 2.25-2.08 (m, 2H), 1.68 (s, 3H),
1.67-1.58 (m, 2H), 1.30 (s, 3H), 1.25 (s, 3H), 1.21 (t, J =
7.0 Hz, 3H); 13C NMR (126 MHz, CDCl3): δ 139.0, 121.7,
67.2, 65.7, 64.2, 58.5, 36.3, 27.3, 25.0, 18.9, 16.6, 15.4
ppm.
chemoenzymatic access to (2R,5R)-linalool oxide.
Experimental Section
General
Starting materials were purchased from Sigma Aldrich and
were used without further treatment. Unless stated
otherwise, solvents were purchased from VWR Chemicals
or Biosolve and were used as such. Cyclohexane (cHex)
was purified by distillation before use. Celite 512 medium
was purchased from Sigma Aldrich. Column
chromatography was performed on Silica-P Flash Silica
Gel (40-63 μm, pore diameter 60 Å) from Silicycle. TLC
was performed using TLC plates F254 (silica gel 60 on
rac-(E)-3-(5-(Allyloxy)-3-methylpent-3-en-1-yl)-2,2-
dimethyloxirane (rac-3).
Flash column chromatography (c-Hex/EtOAc, 6:1)
provided rac-3 as a colorless liquid (0.67 g, 55%).
7
This article is protected by copyright. All rights reserved.

10.1002/adsc.201801094
Advanced Synthesis & Catalysis
1H NMR (500 MHz, CDCl3): δ 5.92 (ddt, J = 17.2, 10.3,
5.7 Hz, 1H), 5.40 (ddp, J = 6.7, 5.4, 1.4 Hz, 1H), 5.31-5.10
(m, 2H), 4.04-3.89 (m, 4H), 2.70 (t, J = 6.3 Hz, 1H), 2.29-
2.06 (m, 2H), 1.68 (s, 3H), 1.67-1.62 (m, 2H), 1.29 (s, 3H),
1.25 (s, 3H); 13C NMR (126 MHz, CDCl3): δ 139.4, 135.1,
121.5, 117.2, 71.3, 66.6, 64.1, 58.5, 36.3, 27.3, 25.0, 18.9,
16.7 ppm.
7.1, 1.5 Hz, 2H), 1.29 (d, J = 1.7 Hz, 3H), 1.25 (d, J = 1.9
Hz, 3H), 1.18 (d, J = 2.0 Hz, 9H). 13C NMR (126 MHz,
CDCl3) δ 178.69, 140.77, 119.46, 64.02, 61.33, 58.52,
38.85, 36.27, 27.33, 27.21, 24.99, 18.88, 16.61 ppm.
NMR data in accordance with literature.[27]
Reference material synthesis
Standard Procedure for acid catalyzed hydrolysis of
epoxides[7b]
rac-(E)-8-(3,3-Dimethyloxiran-2-yl)-6-methyloct-5-en-2-
one (rac-5a)[25]
Epoxide (1 mmol) was hydrolyzed in a mixture of water (6
mL) and THF (6 mL) under acidic conditions (6 N H2SO4,
5 drops). The solution was extracted twice with EtOAc.
The combined organic layers were dried over Na2SO4 and
concentrated in vacuo. The crude product was purified by
flash column chromatography. Complete conversion was
obtained for substrates 2, 4, 6a and 6b.
Flash column chromatography (c-Hex/EtOAc, 3:1)
provided rac-5a as a colorless liquid (0.86 g, 71%).
1H NMR (300 MHz, CDCl3): δ 5.16 (t, J = 7.2 Hz, 1H),
2.70 (t, J = 6.2 Hz, 1H), 2.48 (t, J = 7.4 Hz, 2H), 2.39-2.22
(m, 2H), 2.15 (s, 3H), 2.21-2.05 (m, 2H), 1.68-1.58 (m,
2H), 1.65 (s, 3H), 1.32 (s, 3H), 1.27 (s, 3H).
NMR data in accordance with literature.[25]
rac-(E)-8-Ethoxy-2,6-dimethyloct-6-ene-2,3-diol (rac-2).
Flash column chromatography (c-Hex/EtOAc, 1:1)
provided rac-2 (173 mg, 0.80 mmol, 80%) as a colorless
viscous oil.
rac-(Z)-8-(3,3-Dimethyloxiran-2-yl)-6-methyloct-5-en-2-
one (rac-5b)[25]
Flash column chromatography (c-Hex/EtOAc, 3:1)
provided rac-5b as a colorless liquid (0.83 g, 68%).
1H NMR (300 MHz, CDCl3) δ 5.08 (t, J = 6.9 Hz, 1H),
2.67 (t, J = 6.3 Hz, 1H), 2.42 (t, J = 7.3 Hz, 2H), 2.23 (q, J
= 7.3 Hz, 2H), 2.17-2.11 (m, 2H), 2.09 (s, 3H), 1.65 (d, J =
1.2 Hz, 3H), 1.62-1.52 (m, 2H), 1.27 (s, 3H), 1.23 (s, 3H).
NMR data in accordance with literature.[25]
1H NMR (500 MHz, CDCl3): δ 5.41 (tq, J = 6.7, 1.3 Hz,
1H), 4.03-3.90 (m, 2H), 3.48 (q, J = 7.0 Hz, 2H), 3.34 (dd,
J = 10.6, 1.9 Hz, 1H), 2.38-2.22 (m, 1H), 2.16-1.99 (m,
3H), 1.68 (s, 3H), 1.61 (dddd, J = 13.7, 9.8, 6.5, 2.0 Hz,
1H), 1.48-1.42 (m, 1H), 1.23-1.19 (m, 6H), 1.15 (s, 3H);
13C NMR (126 MHz, CDCl3): δ 139.8, 121.5, 78.3, 73.2,
67.2, 65.8, 36.8, 29.6, 26.6, 23.3, 16.4, 15.4 ppm.
rac-(E)-5-(3,3-Dimethyloxiran-2-yl)-3-methylpent-2-en-
1-ol (rac-9)
rac-(E)-8-(Allyloxy)-2,6-dimethyloct-6-ene-2,3-diol (rac-
4).
Flash column chromatography (c-Hex/EtOAc, 1:1)
provided rac-4 as a colorless viscous oil.
(E)-5-(3,3-dimethyloxiran-2-yl)-3-methylpent-2-en-1-ol
(20 mmol) was dissolved in CH2Cl2 (100 mL). The
mixture was then cooled to 0 °C and m-CPBA (1.1 eq.)
was added. The mixture was allowed to warm to room
temperature and the reaction was monitored with TLC.
After the reaction was complete Na2SO4 was added to dry
the reaction mixture, followed by addition of Na2CO3.
After filtration the product was purified with flash column
chromatography (c-Hex/EtOAc, 2:1), which provided rac-
9a as a colorless liquid (0.89 g, 5.2 mmol, 28%).
1H NMR (500 MHz, CDCl3): δ 5.96 (ddt, J = 17.3, 10.4,
5.8 Hz, 1H), 5.45 (tq, J = 6.7, 1.3 Hz, 1H), 5.36-5.16 (m,
2H), 4.05-3.98 (m, 4H), 3.39 (dd, J = 10.6, 1.9 Hz, 1H),
2.38-2.30 (m, 1H), 2.14 (ddd, J = 14.8, 9.6, 6.7 Hz, 1H),
1.72 (s, 3H), 1.65 (dddd, J = 13.7, 9.8, 6.5, 2.0 Hz, 1H),
1.48 (dddd, J = 13.7, 10.5, 9.4, 5.2 Hz, 1H), 1.23 (s, 3H),
1.19 (s, 3H); 13C NMR (126 MHz, CDCl3): δ 140.2, 135.0,
121.3, 117.3, 78.3, 73.1, 71.3, 66.6, 36.8, 29.6, 26.6, 23.3,
16.7 ppm.
1H NMR (500 MHz, CDCl3) δ 5.44 (tdt, J = 5.5, 2.7, 1.4
Hz, 1H), 4.15 (d, J = 6.8 Hz, 2H), 2.71 (t, J = 6.2 Hz, 1H),
2.16 (ddt, J = 29.8, 14.5, 7.3 Hz, 2H), 1.69 (s, 3H), 1.65
(ddd, J = 8.7, 4.7, 1.6 Hz, 2H), 1.30 (s, 3H), 1.25 (s, 3H).
13C NMR (126 MHz, CDCl3) δ 138.78, 124.06, 64.17,
59.41, 58.54, 36.37, 27.27, 24.99, 18.88, 16.41 ppm.
NMR data in accordance with literature.[26]
rac-(E)-9,10-Dihydroxy-6,10-dimethylundec-5-en-2-one
(rac-6a)
Flash column chromatography with gradient eluent
(EtOAc (0% → 50%) in c-Hex) provided rac-6a as a
colorless viscous oil.
1H NMR (500 MHz, CDCl3): δ 5.16 (t, J = 6.9 Hz, 1H),
3.33 (dd, J = 10.4, 1.7 Hz, 1H), 2.47 (t, J = 7.1 Hz, 2H),
2.29-2.16 (m, 2H), 2.14 (s, 3H), 2.10-2.03 (m, 2H), 1.63 (s,
3H), 1.61-1.55 (m, 1H), 1.44-1.36 (m, 1H), 1.20 (s, 3H),
1.16 (s, 3H); 13C NMR (90 MHz, CDCl3): 209.0, 136.4,
123.5, 78.3, 73.1, 43.8, 36.9, 30.1, 29.7, 26.6, 23.4, 22.6,
16.1.
rac-(E)-5-(3,3-Dimethyloxiran-2-yl)-3-methylpent-2-en-
1-yl pivalate (rac-13c)
(E)-3,7-dimethylocta-2,6-dien-1-yl pivalate (7.1 mmol)
was dissolved in CH2Cl2 (40 mL). The mixture was then
cooled to 0 °C and m-CPBA (1 eq.) was added. The
mixture was allowed to warm to room temperature and the
reaction was monitored with TLC. After the reaction was
complete Na2SO4 was added to dry the reaction mixture,
followed by addition of NaHCO3. After filtration the
product was purified with flash column chromatography
(c-Hex/EtOAc, 19:1), which provided rac-13c as a
colorless liquid (1.45 g, 5.7 mmol, 80%).
NMR data in accordance with literature.[25]
rac-(Z)-9,10-Dihydroxy-6,10-dimethylundec-5-en-2-one
(rac-6b)
Flash column chromatography with gradient eluent
(EtOAc (0% → 100%) in c-Hex) provided rac-6b as a
colorless viscous oil.
1H NMR (500 MHz, CDCl3) δ 5.37-5.33 (m, 1H), 4.56 (d,
J = 6.8 Hz, 2H), 2.69 (td, J = 6.2, 1.6 Hz, 1H), 2.17 (ddt, J
= 29.9, 14.5, 7.4 Hz, 2H), 1.71 (s, 3H), 1.65 (pd, J = 8.0,
8
This article is protected by copyright. All rights reserved.

10.1002/adsc.201801094
Advanced Synthesis & Catalysis
1H NMR (500 MHz, CDCl3): δ 5.07 (t, , J = 6.9 Hz, 1H),
3.31 (dd, J = 10.8, 2.1 Hz, 1H), 2.53-2.48 (m, 2H), 2.47-
2.34 (m, 1H), 2.33-2.24 (m, 2H), 2.23-2.15 (m, 1H), 2.14
(s, 3H), 1.67 (s, 3H), 1.63-1.56 (m, 1H), 1.42-1.34 (m, 1H),
1.20 (s, 3H), 1.16 (s, 3H); 13C NMR (90 MHz, CDCl3):
209.6, 136.1, 124.5, 77.8, 73.1, 43.8, 30.1, 29.4, 28.6, 26.5,
23.4, 23.2, 22.2.
See rac-2 for NMR data.
(S,E)-8-(Allyloxy)-2,6-dimethyloct-6-ene-2,3-diol (S)-4.
AD-mix-α (1.41 g) was dissolved in t-BuOH/H2O (1:1, 10
mL) at rt and stirred for 1.5 h. The reaction mixture was
cooled to 0 °C and MeSO2NH2 (95 mg, 1 mmol) was
added. After 5 min. (E)-1-allyloxy-3,7-dimethylocta-2,6-
diene was added and stirred for 38 h at 0 °C. The reaction
was quenched with Na2S2O3 (5.0 g) and stirred until the
mixture was colorless. The product was extracted three
times with CH2Cl2 (30 mL) and the combined fractions
were dried with Na2SO4. After filtration the extract was
concentrated in vacuo. Flash column chromatography with
gradient eluent (EtOAc (0% → 50%) in c-Hex) provided
(S)-4 (43 mg, 0.18 mmol, 18%) as a colorless viscous oil.
See rac-4 for NMR data.
NMR data in accordance with literature.[25]
rac-(E)-3,7-Dimethyloct-2-ene-1,6,7-triol (rac-10)
To a solution of epoxide (0.32 mmol) in THF (0.64 mL)
was added KHSO4 (1.0 M, 0.64 mL) at room temperature.
After 2 h the starting material was consumed and the
reaction mixture was applied on a silica gel. The mixture
was purified by flash column chromatography
(cHex/EtOAc, 1:1) to provide rac-10 as a white waxy solid
(42 mg, 0.23 mmol, 70%).
1H NMR (400 MHz, CDCl3) δ 5.47 (dt, 1H), 4.16 (d, J =
6.9 Hz, 2H), 3.35 (dd, J = 10.6, 2.0 Hz, 1H), 2.30 (ddd, J
= 14.5, 9.6, 5.3 Hz, 1H), 2.15-2.06 (m, 1H), 1.69 (s, 3H),
1.61 (tdd, J = 9.2, 6.8, 3.3 Hz, 1H), 1.49-1.39 (m, 1H),
1.21 (s, 3H), 1.17 (s, 3H).
(S,E)-9,10-Dihydroxy-6,10-dimethylundec-5-en-2-one
(S)-6a:
AD-mix-α (0.14 g) was dissolved in t-BuOH/H2O (1:1, 1.0
mL) at rt and stirred for 1.5 h. The reaction mixture was
cooled to 0 °C and MeSO2NH2 (9.5 mg, 0.1 mmol) was
added. After 5 min. (E)-6,10-dimethylundeca-5,9-dien-2-
one (20 mg, 0.10 mmol) was added and stirred for 63 h at
0 °C. The reaction was quenched with Na2S2O3 (sat. aq.,
1.0 mL) and stirred until the mixture was colorless. The
product was extracted three times with CH2Cl2 (5 mL) and
the combined fractions were dried with Na2SO4. After
filtration the extract was concentrated in vacuo. Flash
column chromatography with gradient eluent (EtOAc (0%
→ 50%) in c-Hex) provided (S)-6a (5 mg, 22 µmol, 20%)
as a colorless viscous oil.
NMR data in accordance with literature.[26]
rac-(E)-6,7-Dihydroxy-3,7-dimethyloct-2-en-1-yl
pivalate (rac-14c).
To a solution of rac-13c (1.0 mmol) in THF (2.5 mL) was
added KHSO4 (100 mM, 2.5 mL) at rt. The reaction was
vigorously stirred for 16h. When the conversion was
complete, NaHCO3 (5 mL, sat. aq.) was added and the
mixture was diluted with EtOAc (10 mL). The aqueous
phase was extracted three times with EtOAc (25 mL) and
the combined organic fractions were dried over Na2SO4.
Concentration in vacuo provided the crude product, which
was purified by flash column chromatography
(cHex/EtOAc, 1:1) to provide rac-14c as a white waxy
solid (233 mg, 0.86 mmol, 86%).
See rac-6a for NMR data.
(R,E)-9,10-Dihydroxy-6,10-dimethylundec-5-en-2-one
(R)-6a:
AD-mix-β (0.71 g) was dissolved in t-BuOH/H2O (1:1, 5.0
mL) at rt and stirred for 1.5 h. The reaction mixture was
cooled to 0 °C and MeSO2NH2 (48 mg, 0.5 mmol) was
added. After 5 min. (E)-6,10-dimethylundeca-5,9-dien-2-
one (97 mg, 0.5 mmol) was added and stirred for 8 days at
0 °C. The reaction was quenched with Na2S2O3 (sat. aq., 5
mL) and stirred until the mixture was colorless. The
product was extracted three times with CH2Cl2 (5 mL) and
the combined fractions were dried with Na2SO4. After
filtration the extract was concentrated in vacuo. Flash
column chromatography with gradient eluent (EtOAc (0%
→ 50%) in c-Hex) provided (R)-6a (8 mg, 35 µmol, 7%)
as a colorless viscous oil.
1H NMR (500 MHz, CDCl3) δ 5.37 (ddd, J = 8.1, 5.0, 3.4
Hz, 1H), 4.56 (dd, J = 6.9, 2.9 Hz, 2H), 3.33 (dd, J = 10.5,
2.0 Hz, 1H), 2.32-2.08 (m, 4H), 1.71 (s, 3H), 1.60 (dddd, J
= 13.8, 9.1, 6.8, 2.0 Hz, 1H), 1.47-1.41 (m, 1H), 1.19 (s,
3H), 1.18 (s, 9H), 1.15 (s, 3H); 13C NMR (126 MHz,
CDCl3) δ 178.82, 141.67, 119.37, 78.11, 73.15, 61.40,
38.88, 36.70, 29.59, 27.33, 26.59, 23.41, 16.60. HRMS
(ESI): m/z calcd. for C15H28NaO4 ([M+Na]+) 295.1880,
found 295.1882.
Stereoselective dihydroxylation of alkenes:[1a]
(S,E)-8-Ethoxy-2,6-dimethyloct-6-ene-2,3-diol (S)-2.
AD-mix-α (1.41 g) was dissolved in t-BuOH/H2O (1:1, 10
mL) at rt and stirred for 2 h. The reaction mixture was
cooled to 0 °C and MeSO2NH2 (95 mg, 1 mmol) was
added. After 5 min. (E)-1-ethoxy-3,7-dimethylocta-2,6-
diene was added and stirred for 63 h at 0 °C. The reaction
was quenched with Na2S2O3 (sat. aq., 10 mL) and stirred
until the mixture was colorless. The product was extracted
three times with CH2Cl2 (30 mL) and the combined
fractions were dried with Na2SO4. After filtration the
extract was concentrated in vacuo. Flash column
chromatography with gradient eluent (EtOAc (10% →
50%) in c-Hex) provided (S)-2 (72 mg, 0.32 mmol, 32%)
as a colorless viscous oil.
See rac-6a for NMR-data.
(S,Z)-9,10-Dihydroxy-6,10-dimethylundec-5-en-2-one
(S)-6b:
AD-mix-α (1.41 g) was dissolved in t-BuOH/H2O (1:1, 10
mL) at rt and stirred for 2 h. The reaction mixture was
cooled to 0 °C and MeSO2NH2 (95 mg, 1.0 mmol) was
added. After 5 min. (Z)-6,10-dimethylundeca-5,9-dien-2-
one (389 mg, 2.0 mmol) was added and stirred for 8 days
at 0 °C. The reaction was quenched with Na2S2O3 (sat. aq.,
10 mL) and stirred until the mixture was colorless. The
product was extracted three times with CH2Cl2 (30 mL)
and the combined fractions were dried with Na2SO4. After
9
This article is protected by copyright. All rights reserved.

10.1002/adsc.201801094
Advanced Synthesis & Catalysis
filtration the extract was concentrated in vacuo. Flash
column chromatography with gradient eluent (EtOAc (0%
→ 50%) in c-Hex) provided (S)-6b (128 mg, 0.56 mmol,
56%) as a colorless viscous oil.
product was extracted twice with EtOAc and dried over
Na2SO4. The crude product was purified by flask column
chromatography (EtOAc) to afford rac-8a.
1H NMR (300 MHz, CDCl3): δ 5.24 (t, J = 7.1 Hz, 1H),
3.87-3.77 (m, 1H), 3.36 (dd, J = 10.4, 1.7 Hz, 1H), 2.14-
2.07 (m, 4H), 1.65 (s, 3H), 1.58-1.36 (m, 4H), 1.21 (s, 6H),
1.17 (s, 3H).
See rac-6b for NMR-data.
(S,E)-3,7-Dimethyloct-2-ene-1,6,7-triol (S)-10:
AD-mix-α (1.41 g) was dissolved in t-BuOH/H2O (1:1, 10
mL) at rt and stirred for 3 h. The reaction mixture was
cooled to 0 °C and MeSO2NH2 (95 mg, 1.0 mmol) was
added. After 5 min. (Z)-6,10-dimethylundeca-5,9-dien-2-
one (264 µL, 1.5 mmol) was added and stirred for 7 days at
0 °C. The reaction was quenched with Na2S2O3 (sat. aq.,
10 mL) and stirred until the mixture was colorless. The
product was extracted three times with CH2Cl2 (30 mL)
and the combined fractions were dried with Na2SO4. After
filtration the extract was concentrated in vacuo. Flash
column chromatography with gradient eluent (EtOAc (0%
→ 100%) in c-Hex) provided (S)-10 (174 mg, 0.93 mmol,
93%) as a waxy solid.
NMR data in accordance with literature.[28]
rac-(Z)-8-(3,3-Dimethyloxiran-2-yl)-6-methyloct-5-en-2-
ol rac-8b
8b was obtained similarly to 8a, from acidic hydrolysis of
7b:
1H NMR (300 MHz, CDCl3): δ 5.15 (t, J = 6.9 Hz, 1H),
3.82-3.71 (m, 1H), 3.34-3.18 (m, 4H), 2.46-1.99 (m, 4H),
1.65 (s, 3H), 1.63-1.23 (m, 4H), 1.18 (dd, J = 5.2, 2.6 Hz,
6H), 1.13 (s, 3H).
NMR data in accordance with literature.[28]
Stereoselective synthesis of epoxides:
See rac-10 for NMR data.
(R,Z)-8-(3,3-Dimethyloxiran-2-yl)-6-methyloct-5-en-2-
one (R)-5b (and (R)-5a as contaminant)
To a solution of (S)-6b (23 mg, 0.1 mmol) in CH2Cl2 (1
mL) was added Et3N (56 µL, 0.4 mmol) at rt. The mixture
was cooled to 0 °C and MsCl (15 µL, 0.2 mmol) was
added. After allowing reaching rt and stirring for 8h, the
reaction was complete as judged by TLC. The reaction was
quenched with NH4Cl (sat. aq., 2 mL) and extracted three
times with CH2Cl2 (5 mL). The combined organic fractions
were dried over Na2SO4 and concentrated in vacuo. Flash
column chromatography (cHex/EtOAc, 2:1) afforded the
intermediate mesylate (14 mg, 46 µmol, 46%), which was
converted to (R)-5b in MeOH (0.5 mL) with K2CO3 (20 mg,
140 µmol) in 2 h at rt. The product was extracted three
times with Et2O (5 mL) after neutralizing with NH4Cl (sat.
aq., 2 mL). The combined organic fractions were dried
over Na2SO4 and concentrated in vacuo after filtration.
Flash column chromatography (cHex/EtOAc, 9:1) afforded
(R)-5b (2.0 mg, 9 µmol, 9% over two steps).
Hydride reduction of ketones:
rac-(E)-8-(3,3-Dimethyloxiran-2-yl)-6-methyloct-5-en-2-
ol rac-7a
To a mixture of epoxide rac-5a (105 mg, 0.5 mmol) in
MeOH (1.5 mL) was added NaBH4 (23 mg, 0.6 mmol) in
small portions at 0 °C. When all of NaBH4 was added, the
ice-bath was removed and stirred for 15 min. Then 10%
HCl (2.5 mL) was added and the product was extracted
with Et2O. The organic phase was dried over NaSO4 and
concentrated in vacuo. The crude product was purified by
flash column chromatography (cHex/EtOAc, 3:1) to afford
rac-7a.
1H NMR (300 MHz, CDCl3): δ 5.21 (t, J = 7.1 Hz, 1H),
3.89-3.76 (m, 1H), 2.71 (t, J = 6.2 Hz, 1H), 2.27-2.01 (m,
4H), 1.66 (s, 3H), 1.64-1.42 (m, 4H), 1.32 (s, 3H), 1.28 (s,
3H), 1.21 (d, J = 6.2 Hz, 3H).
NMR data in accordance with literature.[25]
See rac-5b for NMR data.
rac-(Z)-8-(3,3-Dimethyloxiran-2-yl)-6-methyloct-5-en-2-
ol rac-7b
(S,Z)-8-(3,3-Dimethyloxiran-2-yl)-6-methyloct-5-en-2-
one (S)-5b (and (S)-5a as contaminant).
To a mixture of epoxide rac-5b (2.0 mg, 10 µmol) in
MeOH (200 µL) was added a spatula tip NaBH4 at 0 °C.
The ice-bath was removed and stirred for 15 min. Then the
reaction mixture was poured into a mixture of sat. aq.
NH4Cl (5 mL) and CHCl3 (10 mL), from which the
product was extracted with CHCl3 (3 x 20 mL). The
combined organic fractions were dried over NaSO4 and
concentrated in vacuo. The crude product was purified by
flash column chromatography (cHex/EtOAc, 2:1) to afford
rac-7b (1.1 mg, 5 µmol, 50%).
To a solution of (R)-6b (23 mg, 0.1 mmol) in CH2Cl2 (1
mL) was added Et3N (56 µL, 0.4 mmol) at rt. The mixture
was cooled to 0 °C and MsCl (12 µL, 0.15 mmol) was
added. After 1h the mixture was allowed to reach rt and
stirring was continued for 22 h. The reaction was quenched
with NH4Cl (sat. aq., 2 mL) and extracted three times with
CH2Cl2 (5 mL). The combined organic fractions were dried
over Na2SO4 and concentrated in vacuo. Flash column
chromatography (cHex/EtOAc, 2:1) afforded the
intermediate mesylate (3.0 mg, 10 µmol, 10%), which was
converted to (S)-5b in MeOH (250 µL) with K2CO3 (4 mg,
30 µmol) in 2 h at rt. The reaction mixture was directly
applied on silica gel column. Flash column
chromatography (cHex/EtOAc, 4:1) followed by a second
flash column chromatography (cHex/EtOAc, 9:1) afforded
(S)-5b (1.8 mg, 8 µmol, 8% over two steps).
1H NMR (300 MHz, CDCl3): δ 5.20 (t, J = 7.2 Hz, 1H),
3.89-3.74 (m, 1H), 2.74 (dd, J = 10.0, 4.0 Hz, 1H), 2.29-
2.08 (m, 4H), 1.72 (s, 3H), 1.71-1.44 (m, 4H), 1.33 (s, 3H),
1.29 (s, 3H), 1.20 (d, J = 6.2 Hz, 3H).
NMR data in accordance with literature.[25]
rac-(E)-8-(3,3-Dimethyloxiran-2-yl)-6-methyloct-5-en-2-
ol rac-8a
See rac-5b for NMR data.
rac-7a was hydrolyzed in a mixture of THF/water (1:1) (4
mL), which was acidified with H2SO4 (6 N, 2 drops). The
10
This article is protected by copyright. All rights reserved.

10.1002/adsc.201801094
Advanced Synthesis & Catalysis
(R,E)-6,7-Dihydroxy-3,7-dimethyloct-2-en-1-yl pivalate
(R)-14c
2-[(2R,5R)-5-Methyl-5-vinyltetrahydrofuran-2-
yl]propan-2-ol (2R,5R)-11[22]
E-4 (1.5 g, whole cell lyophilisate of DSM 44539) was
rehydrated for 1 h in phosphate buffer (2x 25 mL, 100mM,
pH 8.0) in two plastic Greiner tubes at 30 °C. Subsequently
rac-13c (254 mg, 1 mmol) was added and the conversion
was carefully monitored by GC until (S)-13c was
consumed. After 40 h the products were extracted three
times with EtOAc, dried over Na2SO4 and concentrated
after filtration. The crude product mixture was then
redissolved in THF (5 mL) and treated with KHSO4 (5 mL,
0.1 M). When the starting material was consumed after 1.5
h, NaHCO3 (20 mL, sat.aq.) was added and the product
was extracted three times with EtOAc (25 mL). The
combined organic fractions were dried over Na2SO4,
filtered and concentrated in vacuo. Flash column
chromatography (cHex/EtOAc, 3:1) afforded (R)-14c (143
mg, 0.52 mmol, 52%) in high optical purity (97% ee).
See rac-14c for NMR data.
To a solution of (S)-TunePhos (31 mg, 49 µmol) in
anhydrous THF (2.0 mL) was added (R)-14c (62 mg, 216
µmol). Subsequently Pd2dba3 (12 mg, 13 µmol) was added
followed by Et3N (60 µL, 446 µmol). The reaction mixture
was then heated to 65 °C under N2-atmosphere for 29 days.
Five aliquots (0.2 mL) were taken over the course of the
reaction, which left 50% of the reaction mixture. After
evaporation of the solvent the crude reaction mixture was
applied on a silica gel column and purified by flash column
chromatography, which provided (2R,5R)-11 (13.8 mg, 81
µmol, 75%) as a colorless oil.
1H NMR (500 MHz, CDCl3) δ 5.88 (dd, J = 17.3, 10.7 Hz,
1H), 5.19 (dd, J = 17.3, 1.5 Hz, 1H), 4.99 (dd, J = 10.6,
1.5 Hz, 1H), 3.80 (t, J = 7.2 Hz, 1H), 1.90 (ddd, J = 10.9,
8.3, 6.1 Hz, 1H), 1.83 (q, J = 7.3 Hz, 2H), 1.73 (ddd, J =
10.9, 8.3, 6.1 Hz, 1H), 1.32 (s, 3H), 1.23 (s, 3H), 1.13 (s,
3H). 13C NMR (126 MHz, CDCl3) δ 143.85, 111.47, 85.70,
83.20, 71.27, 37.63, 27.39, 26.97, 26.46, 24.31.
NMR data in accordance with literature.[22]
Determination of absolute configuration of 7b
Enzymatic hydrolysis of rac-5b delivered enantioenriched
(S)-5b using E-12 (Table 4), which was further reduced by
ADH of known selectivity (Prelog or anti-Prelog) to
produce 7b, allowing attribution of absolute configuration
to enantiomers of 7b on chiral GC:
Relevant NMR spectra and GC methods and traces can be
found in the supporting information.
(S,Z)-8-(3,3-Dimethyloxiran-2-yl)-6-methyloct-5-en-2-
Acknowledgements
one
(S)-5b
and
(R,Z)-9,10-dihydroxy-6,10-
The Amsterdam Institute for Molecules Medicines and Systems
(AIMMS) and the University of Graz are gratefully acknowledged
for financial support. We thank Elwin Janssen (VUA) for
technical assistance, Dr. Andreas W. Ehlers (VUA) for NMR
maintenance and Barbara Grischek (UG) for the production of
the enzyme preparations. Dr. Thomas Netscher is thanked for the
kind gift of starting materials. Prof. Michael Arand (University of
Zürich, Switzerland) and Elisabeth Blee (University of
Strasbourg, France) are thanked for the kind gift of epoxide
hydrolase samples.
dimethylundec-5-en-2-one (R)-6b:
To a rehydrated suspension of E-12 (NRRL4671, 300 mg,
whole cell) in Tris-Buffer (50 mM, pH 8.0, 10 mL) was
added rac-5b (14 mg, 67 µmol). After incubation of 27 h
the suspension was extracted two times with EtOAc (10
mL), dried over Na2SO4, filtered and concentrated in vacuo.
Flash column chromatography (cHex/EtOAc, 9:1) afforded
(S)-5b, which was used for further reductive
biotransformation, and (R)-6b.
Biocatalytic reduction of ketones:
References
(S,E)-8-[(S)-3,3-Dimethyloxiran-2-yl]-6-methyloct-5-en-
2-ol (3S,10S)-7b.
[1] K. B. Sharpless, W. Amberg, Y. L. Bennani, G. A.
Crispino, J. Hartung, K. S. Jeong, H. L. Kwong, K.
Morikawa, Z. M. Wang, J. Org. Chem. 1992, 57,
2768-2771; b) H. C. Kolb, M. S. Van Nieuwenhze, K.
B. Chem. Rev. 1994, 94, 2483-2547.
One aliquot of (S)-5b obtained from enzymatic hydrolysis
reaction (see above) was added to a mixture of ADH-A (10
µL) in phosphate Buffer (500 µL, 100 mM, pH 8.0) with
NADH (0.1 mL, 200 mM). After incubation of 24 h, the
product was extracted two times with EtOAc (1 mL), dried
over Na2SO4 and analysed by GC for assignment of
enantiomer peak to (3S,10S)-7b.
[2] E. N. Jacobsen, Acc. Chem. Res., 2000, 33, 421-431.
[3] a) S. Lin, G. P. Horsman, Y. Chen, W. Li, B. Shen, J.
Am. Chem. Soc. 2009, 131, 16410-16417; b) N. S. Kim,
J. R. Choi, J. K. Cha, J. Org. Chem. 1993, 58, 7096-
7099; c) Y. Kobayashi, A. D. William, Y. Tokoro, J.
Org. Chem. 2001, 66, 7903-7906.
Linalool oxide synthesis:
2-[(5S)-5-Methyl-5-vinyltetrahydrofuran-2-yl]propan-
2-ol (11*)
According to literature procedure[18], [{Ir(cod)Cl}2] (3.4
mg, 5 µmol) was dissolved with phosphoramidite (S)-L1
(10.2 mg, 20 µmol) and m-ClC6H4CO2H (16 mg, 100
µmol) in anhydrous ClCH2CH2Cl (0.4 mL) at rt. After
stirring for 1 h, rac-9 was added and the mixture was
heated to 50 °C for 1 day. After sampling of the reaction
mixture, GC analysis showed that the cycloetherification
proceeded with very low selectivity. Crude NMR showed
signals of both trans- and cis-isomers (data not shown)
which correspond to literature data.[22]
[4] a) M. Tokunaga, J. F. Larrow, F. Kakiuchi, E. N.
Jacobsen, Science 1997, 277, 936-938; b) M. R.
Monaco, S. Prévost, B. List, Angew. Chem. Int. Ed.
2014, 53, 8142-8145.
[5] R. Wohlgemuth, in Epoxide Hydrolysis, Biocatalysis in
Organic Synthesis, Science of Synthesis, vol. 2 (Eds.: K.
Faber, W.-D. Fessner, N. J. Turner), Thieme, Stuttgart,
2015, pp. 529-555.
11
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10.1002/adsc.201801094
Advanced Synthesis & Catalysis
[6] R. V. A. Orru, K. Faber, Curr. Opin. Chem. Biol. 1999,
3, 16-21.
[26]A. Masuyama,K. Fukuoka, N. Katsuyama, M. Nojima,
Langmuir, 2004, 20, 82-89.
[7] a) S. Wu, A. Li, Y. S. Chin, Z. Li, ACS Catal. 2013, 3,
752-759; b) K. Edegger, S. F. Mayer, A. Steinreiber, K.
Faber, Tetrahedron 2004, 60, 583-588.
[27] B. M. Smith, E. J. Skellam, S. J. Oxley, A. E. Graham,
Org. Biomol. Chem., 2007, 5, 1979-1982.
[28] M. Miyazawa, H. Nankai, H. Kameoka, J. Agric.
Food Chem., 1996, 44, 1543-1547.
[8] S. F. Mayer, A. Steinreiber, R. V. A. Orru, K. Faber,
Tetrahedron: Asymmetry 2001, 12, 41-43.
[9] M. Fuchs, Y. Simeo, B. T. Ueberbacher, T. Netscher,
K. Faber, Eur. J. Org. Chem. 2009, 833-840.
[10] R. V. A. Orru, B. Groenendaal, J. van Heyst, M.
Hunting, C. Wesseling, R. F. Schmitz, S. F. Mayer, K.
Faber, Pure Appl. Chem. 2003, 75, 259.
[11] O. Lopez, J. G. Fernandez-Bolanos, M. V. Gil, Green
Chem., 2005, 7, 431-442.
[12] a) S. Pedragosa-Moreau, C. Morisseau, J. Zylber, A.
Archelas, J. Baratti, R. Furstoss, J. Org. Chem. 1996,
61, 7402-7407. b) W. Kroutil, M. Mischitz, P.
Plachota, K. Faber, Tetrahedron Lett. 1996, 37, 8379-
8382.
[13] A. J. J. Straathof, J. A. Jongejan, Enzyme Microb.
Technol., 1997, 21, 559-571.
[14] G. Vidari, A. Di Rosa, G. Zanoni, C. Bicchi,
Tetrahedron: Asymmetry 1999, 10, 3547-3557.
[15] J. H. Brewster, J. Am. Chem. Soc. 1956, 78, 4061-
4064.
[16] J. Logan, N. Stanczyk, A. Hassanali, J. Kemei, A.
Santana, K. Ribeiro, J. Pickett, A. J. Mordue, Malaria
J. 2010, 9, 239.
[17] a) W. Stampfer, B. Kosjek, C. Moitzi, W. Kroutil and
K. Faber, Angew. Chem., Int. Ed., 2002, 41, 1014-
1017; b) V. Prelog, Pure Appl. Chem. 1963, 9, 119-
130.
[18] M. Roggen, E. M. Carreira, Angew. Chem. Int. Ed.
2011, 50, 5568-5571.
[19] J. L. L. Rakels, A. J. J. Straathof, J. J. Heijnen,
Enzyme Microb. Technol. 1993, 15, 1051-1056.
[20] F. G. Bordwell, Acc. Chem. Res. 1988, 21, 456-463.
[21] P. Christ, A. G. Lindsay, S. S. Vormittag, J.-M.
Neudörfl, A. Berkessel, A. C. O'Donoghue, Chem.
Eur. J. 2011, 17, 8524-8528.
[22] K. K. Wan, J. P. Litz, D. A. Vosburg, Tetrahedron:
Asymmetry 2010, 21, 2425-2428.
[23] M. Arthuis, R. Beaud, V. Gandon, E. Roulland,
Angew. Chem. Int. Ed. 2012, 51, 10510-10514.
[24] a) T. Ema, S. Maeno, Y. Takaya, T. Sakai, M. Utaka,
J. Org. Chem. 1996, 61, 8610-8616; b) K.
Kaewprapan, J. Wongkongkatep, W. Panbangred, P.
Phinyocheep, E. Marie, A. Durand, P. Inprakhon, J.
Biosci. Bioeng. 2011, 112, 124-129.
[25] A. Gliszczynska, C. Wawrzenczyk, J. Mol. Catal. B.:
Enzym., 2008, 52-53, 40-48.
12
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FULL PAPER
Enantioselective Bio-Hydrolysis of Geranyl-
Derived rac-Epoxides: A Chemoenzymatic Route
to trans-Furanoid Linalool Oxide
Adv. Synth. Catal. Year, Volume, Page – Page
Matthijs J. van Lint, Aysegül Gümüs, Eelco
Ruijter, Kurt Faber, Romano V.A. Orru* and
Mélanie Hall*
13
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