Stereo-complementary two-step cascades using a two-enzyme system leading to enantiopure epoxides

Article abstract of DOI:10.1002/adsc.200700027

A novel one-pot, two-step, two-enzyme cascade is described. Pro-chiral α-chloro ketones are stereoselectively reduced to the corresponding halohydrins as an intermediate by a biocatalytic hydrogen transfer process. The intermediate is transformed to the corresponding epoxide by a non-enantioselective halohydrin dehalogenase. Thus, by combining a Prelog- or anti-Prelog alcohol dehydrogenase with a non-selective halohydrin dehalogenase, enantiopure (R)- as well as (S)-epoxides were obtained.

Full text of DOI:10.1002/adsc.200700027

FULL PAPERS
DOI: 10.1002/adsc.200700027
Stereo-Complementary Two-Step Cascades Using a Two-Enzyme
System Leading to Enantiopure Epoxides
Birgit Seisser,a Ivµn Lavandera,a Kurt Faber,b Jeffrey H. Lutje Spelberg,c
and Wolfgang Kroutilb,*
a
Research Centre Applied Biocatalysis, Petersgasse 14, 8010 Graz, Austria, c/o Department of Chemistry, Organic and
Bioorganic Chemistry, University of Graz, Heinrichstrasse 28, A-8010 Graz, Austria
Department of Chemistry, Organic and Bioorganic Chemistry, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria
b
Phone: (+43)-(0)316-380-5350; fax: (+43)-(0)316-380-9840; e-mail: wolfgang.kroutil@uni-graz.at
Julich Chiral Solutions GmbH, Prof.-Rehmstrasse 1, 52428 Jülich, Germany
c
Received: January 16, 2007
Supporting information for this article is available on the WWW under http://asc.wiley-vch.de/home/.
Abstract: A novel one-pot, two-step, two-enzyme Prelog- or anti-Prelog alcohol dehydrogenase with a
cascade is described. Pro-chiral a-chloro ketones are non-selective halohydrin dehalogenase, enantiopure
stereoselectively reduced to the corresponding halo- (R)- as well as (S)-epoxides were obtained.
hydrins as an intermediate by a biocatalytic hydro-
gen transfer process. The intermediate is transformed
to the corresponding epoxide by a non-enantioselec- Keywords: asymmetric catalysis; cascade reactions;
tive halohydrin dehalogenase. Thus, by combining a enzyme catalysis; epoxides; halohydrins; reduction
Introduction
been employed, for example, mono-oxygenation of al-
kenes[11,12] or resolution of epoxides and precursors
Cascade or domino reactions[1,2] involve two or more thereof.[12,13] Among them, transformations employing
sequential transformations without isolation of inter- halohydrin dehalogenases are very promising.[12–15]
mediates using one or more catalysts. Due to their el- These enzymes catalyse the opening of epoxides with
egance, but also due to the more complex system, cas- various nucleophiles[16] such as azide,[14] cyanide[17] and
cade reactions recently gained significant attention.[1–5] nitrite[18] as well as the reversible ring-closure via de-
We have recently described a cascade where in a first halogenation of vicinal halohydrins to yield the corre-
step a-chloro ketones were reduced to the corre- sponding epoxides.[19–21] These transformations usually
sponding halohydrins employing wild-type cells of go in hand with a kinetic resolution, which is limited
Rhodococcus ruber. The halohydrins were trans- to 50% yield for each enantiomer. To overcome this
formed further in a second step to the epoxides under drawback, it seemed reasonable to employ them in a
very basic conditions (pH 12).[6] Unfortunately, these one-pot cascade process starting from the correspond-
reaction conditions applied with the wild-type cells of ing prochiral a-chloro ketones employing an alcohol
Rhodococcus ruber are not suitable either for the dehydrogenase combined with a halohydrin dehaloge-
pure enzyme ADH-ꢀAꢁ or for E. coli cells containing nase.
overexpressed ADH-ꢀAꢁ. Therefore an alternative
was required for an up-scaling of the cascade employ-
ing overexpressed/pure enzyme leading to enantio- Results and Discussion
pure epoxides. The latter are important building
blocks for the synthesis of biologically active com- To access both enantiomers of the epoxide, we plan-
pounds and pharmaceuticals[7] since they can easily ned to use either an (R)- or (S)-selective alcohol de-
react with several types of nucleophiles, affording b- hydrogenase (ADH) for the stereoselective reduction
substituted alcohols. Several chemical approaches of the ketone moiety to the corresponding chiral alco-
have been designed in order to obtain enantiopure hol. The latter represents an intermediate in the cas-
epoxides,[8] such as the Sharpless[9] or Jacobsen[10] ep- cade, which is transformed by ring closure to the cor-
oxidation. Furthermore, biocatalytic methods have responding epoxide catalysed by a halohydrin dehalo-
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Birgit Seisser et al.
FULL PAPERS
Table 1. Enantioselectivities of the HheB-catalysed epoxide
formation from halohydrins 2a–d.
Substrate
Halohydrin
Epoxide
E
[%][a]
ee [%]
[%][a]
ee [%]
2a
2b
2c
2d
91
61
65
87
16 (R)[b]
2 (S)[c]
2 (S)[c]
8 (R)[b]
9
34 (S)[b]
5 (R)[c]
6 (S)[c,d]
7 (S)[b]
2.3
1.1
1.1
1.2
39
35
13
Scheme 1. Cascade reaction to synthesise enantiopure epox-
ides.
[a]
GC yield after 5 h.
[b]
[c]
[d]
Determined by enantioselective GC.
Determined by enantioselective HPLC.
Switch in CIP sequence priority.
genase (Scheme 1). In order to facilitate the
NAD(P)H recycling required for the reduction step,
we used ADHs which are able to catalyse the ketone
reduction and the cofactor recycling at the same
time.[22] In this ꢀcoupled substrateꢁ approach, 2-propa-
nol was employed as a reducing agent via hydrogen
transfer.
Lyophilised cells of E. coli containing the overex-
pressed ADH-ꢀAꢁ from Rhodococcus ruber DSM
44541 (E. coli/ADH-ꢀAꢁ)[23,24] were chosen as a suita-
ble catalyst to perform the first part of the cascade se-
quence since ADH-ꢀAꢁ has shown impressive opera-
tional stability in the presence of high concentrations
of 2-propanol.[25] Excess of 2-propanol is not only
used as hydride donor for cofactor recycling but also
to solubilise the substrate in the aqueous phase.[22]
Among several types of functionalised ketones,
Scheme 2. Cascade process using one enantioselective and
one non-selective enzyme to obtain both product enantio-
mers.
However, it was not known whether HheB would
ADH-ꢀAꢁ reduces a-chloro ketones with excellent ste- tolerate the presence of 2-propanol and the co-prod-
reoselectivity affording the Prelog alcohols.[6] On the uct acetone from the first step. Fortunately, a test ex-
other hand, Lactobacillus brevis alcohol dehydrogen- periment showed that HheB lost only 25% of activity
ase (LBADH) was chosen to access the stereo-com- in up to 10% (v vÀ1) of 2-propanol using (R)-2-
plementary products.[26]
chloro-1-phenylethanol (R)-2a as substrate (Figure 1).
For the second step of the cascade reaction, a halo-
A similar picture was observed for the activity of
hydrin dehalogenase which possesses the matching HheB in the presence of acetone. Subsequently, 5%
enantioselectivity for the halohydrin produced in the (v vÀ1) 2-propanol was the concentration of choice for
first step was required. In a first approach, the halo- the cascade reaction.
hydrin dehalogenase from Mycobacterium sp. GP1
In order to tune the system for the cascade process,
further parameters like enzyme or substrate concen-
(HheB)[27,28] was investigated.
For determination of the enantioselectivity E of the
halohydrin dehalogenase, racemic halohydrins 2a–d
were subjected to the biocatalytic ring closure. E
values were determined from the ee of the halohydrin
and the epoxide (Table 1).
The results showed that the halohydrin dehaloge-
nase HheB converted both enantiomers at almost the
same rate as indicated by the low E value (Table 1).
Although this enzyme is obviously not suitable for a
kinetic resolution of these substrates, HheB is an ex-
cellent candidate for cascade reactions in which the
stereochemistry is created in the first step by an (R)-
or (S)-selective biocatalyst (A or B, Scheme 2) be-
cause no enantiodiscrimination is needed in the
second step. Actually, low or no enantiospecificity is
an advantage, since a single dehalogenase (C) is suffi-
cient and can be combined with ADH-ꢀAꢁ as well as
LBADH leading to either the (R)- or (S)-epoxide.
Figure 1. Conversion of (R)-2a to (R)-3a employing HheB
(t=3 h) at varied concentrations of 2-propanol.
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Stereo-Complementary Two-Step Cascades
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tration, temperature and buffer were optimised for
the formation of (R)-3a from halohydrin (R)-2a using
HheB. Since biotransformations catalysed by halohy-
drin dehalogenases are reversible, the concentration
of the halide ion should be kept low to minimise the
back reaction. For instance, employing Tris-HCl
buffer (50 mM) conversions lower than 10% were ob-
tained (Figure 2). With a non-nucleophilic anion like
sulphate as a counter ion for the buffer (Tris-SO4),
higher concentrations of buffer salt led to enhanced
conversions. For the combined process, 150 mM Tris-
SO4 buffer (pH 7.5) seemed to be appropriate. In an-
other approach, Ag2SO4 was added in the reaction
mixture to bind the chloride and shift the equilibrium,
however in this case no reaction was observed, proba-
bly due to inactivation of the enzyme.
Changes in the temperature or in the amount of
enzyme did not lead to a significant increase of con-
version within three hours (entries 1–4, 8, 9, Table 2).
Lower substrate concentrations led to better apparent
conversions (entries 5–8, Table 2). As a compromise,
a substrate concentration of 45 mM, a temperature of
308C as well as 150 mM Tris-SO4 buffer (pH 7.5)
were chosen for the combined set-up.
For epoxides 3b–3d no spontaneous hydrolysis to
the corresponding diols was found under the reaction
conditions investigated. However, spontaneous chemi-
cal hydrolysis of styrene oxide 3a occurred to a cer-
tain extent (33.5% after 24 h), but at reduced incuba-
tion time (6 h) hydrolysis was negligible.
Finally, the enzymatic reduction via hydrogen trans-
fer was combined with the biocatalytic ring-closure in
a single process. As shown in Table 3, enantiopure ep-
oxides were successfully obtained from prochiral a-
chloro ketones in a one-pot two-enzyme cascade. De-
pending on the ADH employed, the (S)- as well as
the (R)-epoxide were obtained with ee>99%.
Figure 2. Effect of counter ion and buffer concentration on
the conversion of the enzymatic ring-closure of (R)-2a cata-
lysed by HheB at pH 7.5.
Table 2. Optimisation of reaction conditions for HheB cata-
lysed ring-closure of (R)-2a (conversions after 3 h).
Entry Enzyme conc.
[mgmLÀ1]
T (R)-2a conc.
[8C] [mM]
Conv.[a]
[%]
1
2
3
4
5
6
7
8
9
0.04
0.08
30
30
30
r.t.
30
30
30
30
40
75
75
75
75
60
45
30
15
15
11
15
13[c]
8
14
16
29
33
34
0.04+0.04[b]
0.04
0.04
0.04
0.04
0.04
0.04
[a]
[b]
[c]
Determined by GC.
ꢀFreshꢁ enzyme added after 3 h.
Conversion at 6 h.
Table 3. Results of cascade reactions using substrates 1a–d.
Substrate
ADH
Ketone 1
Halohydrin 2
Epoxide 3
[%][a]
Yield [%][a]
ee [%][b]
Yield [%][a]
ee [%]
1a[c]
1b
1c
1d
1b
1c
ADH-ꢀAꢁ
ADH-ꢀAꢁ
ADH-ꢀAꢁ
ADH-ꢀAꢁ
LBADH
LBADH
LBADH
0
1
0
0
86
20
32
90
66
55
56
3
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (S)
>99 (S)
>99 (S)
7
>99 (R)[b]
>99 (R)[d]
>99 (S)[d,e]
>99 (R)[d]
>99 (S)[d]
>99 (R)[d,e]
>99 (S)[d]
33
45
44
11
57
34
26
34
1d
[a]
GC yield after 5 h.
Determined by enantioselective GC or enantioselective HPLC.
3% of diol formed.
[b]
[c]
[d]
In a separate experiment it was proven that ring closure of an enantiopure halohydrin always leads to an enantiopure ep-
oxide. The known mechanism of a halohydrin dehalogenase cannot lead to a decrease of the optical purity.[29]
Switch in CIP priority.
[e]
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Birgit Seisser et al.
FULL PAPERS
Conclusions
Epoxide Formation using HheB at Different Acetone
and 2-Propanol Concentrations
A novel biocatalytic cascade reaction with two en-
zymes was reported starting from prochiral a-halo ke-
tones, which were asymmetrically reduced to the cor-
responding halohydrins. The latter were transformed
further into the corresponding epoxides by a non-se-
lective halohydrin dehalogenase. Although this
enzyme is not suitable for a kinetic resolution, it is a
perfect candidate for a follow-up step in a cascade
process, where the stereoselectivity is fixed in a previ-
ous step. In the first step the biocatalytic asymmetric
reduction via hydrogen transfer by stereoselective
ADHs furnished either the (R)- or the (S)-halohy-
drins, giving the possibility to access both enantiomer-
ic branches. The stereochemical outcome of the pro-
HheB (20 mL, 1 mgmLÀ1) and (R)-2-chloro-1-phenylethanol
(3 mL, 22.5 mmol, 45 mM) were added to a Tris-SO4 buffer
(477 mL, 50 mM, pH 7.5). The appropriate amounts of ace-
tone and 2-propanol were added (5–26 mL acetone or 2-
propanol). The samples were incubated for 3 h at 308C and
120 rpm, extracted twice with ethyl acetate (2”350 mL), and
dried (Na2SO4). The conversion was determined by GC.
Optimisation of Reaction Conditions for HheB
To elucidate the optimal conditions for reactions with HheB
(20 mL, 1 mgmLÀ1) several set-ups were investigated: Varia-
tion of substrate concentration [(R)-2-chloro-1-phenyletha-
nol, 15 mM–75 mM], variation of HheB concentration and
variation of reaction temperature. One sample (Table 2,
cess was controlled by the choice of the appropriate entry 3) was incubated for 3 h at 308C and 130 rpm, then
again 20 mL of HheB (1 mgmLÀ1) were added and the
alcohol dehydrogenase in the first step, while a single
sample was shaken for further 3 h. For all set-ups Tris-SO4
non-selective halohydrin dehalogenase was sufficient
buffer (50 mM, pH 7.5) was used. Samples were taken after
to complete the cascade.
3 h, extracted twice with ethyl acetate (350 mL) and dried
with Na2SO4. The conversion was determined using GC
analysis.
Experimental Section
Enzymatic Ring Closure of (R)-2-Chloro-1-
phenylethanol: Optimisation of the Buffer
HheB (20 mL, 1 mgmLÀ1) was added to Tris-HCl buffer
(475 mL, 50 mM, pH 7.5). The reaction was started by
General Remarks
NMR spectra were recorded in CDCl3 using a Bruker AMX
360 at 360 (1H) and 90 (13C) MHz or a Bruker DMX
Avance 500 at 500 (1H) and 125 (13C) MHz, respectively.
adding (R)-2-chloro-1-phenylethanol (3 mL, 22.5 mmol,
Chemical shifts are reported relative to TMS (d=0.00) and
45 mM) and incubated at 308C and 120 rpm. Samples were
coupling constants (J) are given in Hz. TLC plates were run
taken after 20 min, 40 min, 1 h, 2 h, 3.5 h, 4 h, 6 h and 24 h,
on silica gel Merck 60 F254 and compounds were visualised
extracted twice with ethyl acetate (2”350 mL) and dried
(Na2SO4). The conversion was determined using GC analy-
sis.
The experiment was repeated using (R)-2-chloro-1-phe-
nylethanol (3 mL, 22.5 mmol, 45 mM) and Tris-SO4 buffer
(pH 7.5) at various concentrations (50 mM, 100 mM,
150 mM, 200 mM) and samples were taken hourly during a
period of seven hours. The samples were extracted twice
with ethyl acetate (350 mL) and dried (Na2SO4). The conver-
sion was determined using GC analysis.
either by spraying with Mo reagent [(NH4)6Mo7O24·4H2O
ACHTREUNG
(100 gLÀ1), Ce(SO4)2·4H2O (4 gLÀ1) in H2SO4 (10%)] or by
UV. Optical rotation values ([a]D) were measured on a
Perkin–Elmer polarimeter 341 at 589 nm (Na line) in a one-
dm cuvette.
For anhydrous reactions, flasks were dried and flushed
with dry argon just before use. Standard syringe techniques
were applied to transfer dry solvents and reagents in an
inert atmosphere of dry argon. Anhydrous THF was dis-
tilled from potassium. Petroleum ether (bp 60–908C) and
EtOAc used for chromatography were distilled prior to use.
a-Chloroacetophenone (1a), rac-(2,3-epoxypropyl)-ben-
zene (3b), rac-(2,3-epoxypropyl) phenyl ether (3c), rac-1,2-
epoxyoctane (3d), (S)-1,2-epoxyoctane (S)-3d, and 1-phenyl-
1,2-ethanediol are commercially available.
Enzymatic Ring-Closure of (R)-2-Chloro-1-
Phenylethanol in the Presence of Silver Sulphate
HheB (20 mL, 1 mgmLÀ1) and Ag2SO4 (7.8 mg, 50 mm) were
added to Tris-SO4 buffer (477 mL, 150 mM, pH 7.5) in Ep-
pendorf vials, which were wrapped with aluminium foil. The
reaction was started by adding (R)-2-chloro-1-phenylethanol
(3 mL, 22.5 mmol, 45 mM) and incubated at 308C and
130 rpm. Samples were taken after 2 h, 4 h, 6 h and 24 h, ex-
tracted twice with ethyl acetate (2”350 mL) and dried
(Na2SO4). The conversion was determined using GC analy-
sis.
Synthesis of Substrates and Reference Compounds
1-Chloro-3-phenyl-2-propanone (1b),[6] 3-chloro-1-phenoxy-
2-propanone (1c,[6] 1-chloro-2-octanone (1d),[6] rac-2-chloro-
1-phenylethanol (2a),[30] rac-1-chloro-3-phenyl-2-propanol
(2b),[6] rac-3-chloro-1-phenoxy-2-propanol (2c)[31] as well as
reference compounds (R)-2a–2d and (R)-3a–3c were syn-
thesised as previously described.[6]
Test for Spontaneous Hydrolysis of Styrene Oxide
Styrene oxide (5 mL, 37.5 mmol, 75 mM) and HheB (20 mL,
1 mgmLÀ1) were added to Tris-HCl buffer (500 mL, 50 mM,
pH 7.5) and incubated at 308C and 120 rpm. Samples were
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Adv. Synth. Catal. 2007, 349, 1399 – 1404

Stereo-Complementary Two-Step Cascades
FULL PAPERS
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(Na2SO4). The conversion was determined using GC analy-
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vial (1.5 mL) for 1 h at 308C and 120 rpm. Substrates 1a–d
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A stock solution of LB-ADH (11.1 U mgÀ1, 8 mgmLÀ1) in
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(26 mL, 5% v vÀ1) LB-ADH stock solution (50 mL, 4.44 U)
and HheB (20 mL, 1 mgmLÀ1) were added to Tris-SO4 buffer
(435 mL, 150 mM, pH 7.5, 1 mM NADPH). The sample was
incubated at 308C and 120 rpm. Samples were taken every
hour, extracted twice with ethyl acetate (2”350 mL) and
dried (Na2SO4). The conversion was determined using GC
analysis.
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Acknowledgements
I. L. is funded by the Research Centre Applied Biocatalysis.
Financial support by FFG and the Province ofStyria is grate-
fully acknowledged.
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