Enantioselective Michael addition of water

Article abstract of DOI:10.1002/chem.201405579

The enantioselective Michael addition using water as both nucleophile and solvent has to date proved beyond the ability of synthetic chemists. Herein, the direct, enantioselective Michael addition of water in water to prepare important β-hydroxy carbonyl compounds using whole cells of Rhodococcus strains is described. Good yields and excellent enantioselectivities were achieved with this method. Deuterium labeling studies demonstrate that a Michael hydratase catalyzes the water addition exclusively with anti-stereochemistry.

Full text of DOI:10.1002/chem.201405579

DOI: 10.1002/chem.201405579
Full Paper
&
Biocatalysis
Enantioselective Michael Addition of Water
Bi-Shuang Chen,[a] Verena Resch,[a, b] Linda G. Otten,[a] and Ulf Hanefeld*[a]
Abstract: The enantioselective Michael addition using water
as both nucleophile and solvent has to date proved beyond
the ability of synthetic chemists. Herein, the direct, enantio-
selective Michael addition of water in water to prepare im-
portant b-hydroxy carbonyl compounds using whole cells of
Rhodococcus strains is described. Good yields and excellent
enantioselectivities were achieved with this method. Deuteri-
um labeling studies demonstrate that a Michael hydratase
catalyzes the water addition exclusively with anti-stereo-
chemistry.
Introduction
part of the primary metabolism where perfect substrate specif-
icity is required. This very high substrate selectivity severely
limits their practical applicability in organic synthesis.[2a]
A
The direct addition of water to C=C bonds is a highly attractive
transformation, yielding (chiral) alcohols.[1] However, the enan-
tioselective addition of water to a,b-unsaturated carbonyl (Mi-
chael) acceptors still represents a chemically very challenging
reaction,[2] due to the poor nucleophilicity of water and its
small size, which make regio- and stereoinduction difficult.
Equally, the often unfavorable equilibrium of water-addition re-
actions remains to be solved. Although this reaction benefits
from its simplicity and excellent atom economy, no protocol
with broad applicability has to date been developed. Indirect
methods[3] using complex catalysts[4] or strong alternative nu-
cleophiles[5] have been employed. Some of the described
methods require either cumbersome catalyst preparation or re-
ductive/oxidative follow-up chemistry. Selective direct methods
have been reported by Roelfes and co-workers, applying DNA-
based CuII catalysts[6] or the use of a protein as chiral ligand.[7]
However, they are limited to a,b-unsaturated 2-acyl imidazoles
as substrates and yield the corresponding alcohols in moder-
ate enantiomeric purities. The only chemocatalytic process run
on industrial scale was the addition of water to acrolein.[1d]
Nevertheless, due to its poor selectivity and productivity, even
this seemingly straightforward reaction has been replaced by
a fermentative process.[1d,8]
recent report on an asymmetric hydration of hydroxystyrene-
type substrates catalyzed by phenolic acid decarboxylases
showed that a broader flexibility in the substrate spectrum for
hydratases is possible.[10] In order to broaden the biocatalytic
toolbox of hydratases, the work represented herein is dedicat-
ed to the search for a Michael hydratase with a more relaxed
substrate specificity.
In our search for a straightforward approach for the prepara-
tion of b-hydroxy carbonyl compounds via the direct Michael
addition of water, it was noted that whole cells of Rhodococcus
rhodochrous ATCC 17895 convert 3-methylfuran-2(5H)-one 1a
into (S)-3-hydroxy-3-methylfuranone 2a; as briefly described in
1998.[11] Neither substrate 1a nor product 2a are part of the
primary metabolism indicating the involvement of a putative
Michael hydratase with possibly a broader substrate scope.
Since whole cells were used in this transformation, the hydra-
tase activity needed to be critically evaluated.[11–14] Instead of
a direct addition of water, the conversion of 1a to 2a could
also occur via a two-step approach (Scheme 1). Indeed, the
enantioselective hydroxylation of a range of THF and THP de-
rivatives was reported for R. rhodochrous strains.[15] Therefore, it
is of high interest to probe whether the conversion of 1a to
In contrast, enzymes such as fumarase, malease, citraconase,
aconitase, and enoyl-CoA hydratase have been successfully
used on industrial scale, and their excellent (enantio-) selectivi-
ties are highly valued.[1d,9] Unfortunately, most hydratases are
[a] B.-S. Chen, Dr. V. Resch, Dr. L. G. Otten, Prof. Dr. U. Hanefeld
Technische Universiteit Delft
Gebouw voor Scheikunde, Afdeling Biotechnologie
Julianalaan 136, 2628 BL Delft (Netherlands)
E-mail: u.hanefeld@tudelft.nl
[b] Dr. V. Resch
University of Graz, Organic and Bioorganic Chemistry
Institute of Chemistry, Heinrichstrasse 28, 8010 Graz (Austria)
Scheme 1. Biotransformation of 1a to 2a by R. rhodochrous by Michael addi-
tion of water or alternatively by a reduction–oxidation stepwise ap-
proach.[11,15]
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201405579.
Chem. Eur. J. 2014, 20, 1 – 12
1
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
&
These are not the final page numbers! ÞÞ

Full Paper
2a is actually a Michael addition of water and how broadly it
is applicable.
occurs. In previous studies[2b,14] we were able to show that
a chemically catalyzed addition reaction occurs when 2-cyclo-
hexenone (1h) is used as a substrate. Therefore, any undesired
background reaction needed to be ruled out. Heat-denatured
cell preparations in control experiments (Table 1, control 2)
clearly showed that there is no chemically catalyzed reaction
taking place; thus the reaction is effected by the active
enzyme.
Herein we report the results of screening several Rhodococ-
cus strains as promising biocatalysts for the enantioselective
Michael addition of water to a variety of a,b-unsaturated car-
bonyl compounds.
Encouraged by the complete conversion after 17 h, we eval-
uated the rate of the reaction with 330 mgmLꢀ1 of wet cells.
This revealed an almost linear increase in product formation
during the first 6 h of the reaction and 2a was formed in 75%
yield (Figure 1A). Complete conversion based on the consump-
Results and Discussion
Optimization
To fully assess the potential of the putative Michael addition of
water, the previously reported conversion of 3-methylfuran-
2(5H)-one (1a)[11] was repeated and optimized. 1a was synthe-
sized using a modified literature procedure (see the Support-
ing Information, S3).[16] Whole cells of R. rhodochrous ATCC
17895 were used in two different concentrations (100 mgmLꢀ1
and 330 mgmLꢀ1 of wet cells; Table 1). The reaction with
Table 1. Influence of the catalyst concentration on the conversion.
Catalyst
Catalyst
conc. (wet
cells)
Substrate Conversion[a] Yield[a] ee[a]
of 1a [%]
of
of
2a[b]
[%]
2a
[%]
this
study
resting
cells
resting
cells
100 mgmLꢀ1
330 mgmLꢀ1
100 mgmLꢀ1
330 mgmLꢀ1
1a
1a
1a
3a
1a
69
99
55
–
57
87
91
Figure 1. Time course (A), temperature profile at reaction time 6 h (B), pH
profile at reaction time 6 h (C) and Michaelis–Menten kinetics (D, based on
the yield of 2a) of the putative Michael addition catalyzed using whole cells
of R. rhodochrous ATCC 17895. For reaction conditions, see the Experimental
Section. Conversion, yield, and ee values were determined by GC. Filled cir-
cles represent ee of 2a. Filled triangles represent consumption of 1a. Filled
squares represent yield of 2a. Empty triangles represent consumption of 1a
in blank reactions. Empty squares represent yield of 2a in blank reactions (in
A and D, blank reaction was carried out with heat-denatured cells; in C,
blank reaction was carried out without the addition of cells).
90
ref. [11] resting
cells
control 1 resting
cells[c]
control 2 denatured 330 mgmLꢀ1
cells[d]
55
95
<3
<3
n.d.[f]
n.d.[f]
12[e]
[a] Conversion, yield, and ee values were determined by GC; [b] absolute
configuration of 2a has been established by converting 2a into the corre-
sponding methyl ester [methyl S-(ꢀ)-3,4-dihydroxy-3-methylbuta-
noate];[12,13] [c] reaction with 3a was carried out to rule out possible oxida-
tion; [d] reaction with heat-denatured cells was carried out to ensure no
background reaction is taking place; [e] conversion is caused by the ring
opening of lactone 1a, no water addition product (2a) was detected;
[f] n.d.=not determined.
tion of 1a was obtained after 9 h. No significant changes of
the product ee within the first 9 h were observed (from 99%
to 95%; see the Supporting Information, S11 for GC chromato-
graphs). It should be noted that, since the desired Michael ad-
dition products (2a) are highly soluble in water, the choice of
the organic solvent for extraction is crucial. For example, using
ethyl acetate or dichloromethane as the extraction solvent
only allowed recovery of 30% of the product (data not
shown). In extraction studies, isoamyl alcohol gave the best
result for extraction of (S)-3-hydroxy-3-methylfuranone (2a).
However, due to the similar polarity of isoamyl alcohol and
water-addition product 2a, severe problems, such as separa-
tion issues during purification, arose. Therefore, for prepara-
tive-scale experiments, reaction mixtures were always continu-
ously extracted overnight using a liquid–liquid extractor and
ethyl acetate as the organic solvent (see the Supporting Infor-
100 mgmLꢀ1 cells gave a maximum conversion of 69% after
17 h and, even after a prolonged reaction time (4 days), no fur-
ther increase in conversion was observed. Furthermore, an ee
of 91% was determined, which is in agreement with the previ-
ously reported study.[11] An increase of the cell concentration
to 330 mgmLꢀ1 of wet cells resulted in full conversion after
17 h, while ee values remained unchanged (90%). When using
3a as substrate under aerobic conditions (Table 1, control 1),
no conversion to 2a was detected, indicating that no oxidation
&
&
Chem. Eur. J. 2014, 20, 1 – 12
www.chemeurj.org
2
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Paper
mation, S9). This procedure had no influence on the ee values
of the product (data not shown).
R. rhodochrous ATCC 17895 was shown to be much more
closely related to R. erythropolis than to R. rhodochrous.[17] For
this reason, strains R. erythropolis DSM 43296, R. erythropolis
CCM 2595, R. erythropolis NBRC 100887, and R. erythropolis
DSM 43066 were evaluated (Table 2). Experiments for compar-
ing the different organisms were carried out under conditions
optimized for R. rhodochrous ATCC 17895. Gratifyingly, in each
case, 3-methylfuran-2(5H)-one (1a) was converted into (S)-3-
hydroxy-3-methylfuranone (2a) with good yields and excellent
enantioselectivities (see the Supporting Information, S12 for
GC chromatographs). Encouraged by these results, the less
closely related strain R. rhodochrous DSM 43241 was also
tested for water addition activity. Interestingly, the enantiomer-
ically enriched water-addition product (S)-3-hydroxy-3-methyl-
furanone 2a was also obtained in 75% yield and with an
86% ee, which was slightly lower than that with R. erythropolis
strains. All the results suggest that this promising hydratase ac-
tivity is not limited to R. rhodochrous ATCC 17895 but may be
a general feature in several Rhodococcus strains. Taking the
conversion, enantioselectivity, and available genome sequence
into account, we decided to continue to use strain R. rhodo-
chrous ATCC 17895 for all further studies.
The temperature profile of the reaction was evaluated as
well. Temperatures ranging from 188C to 488C were tested.
Conversions and ee values at different temperatures are sum-
marized in Figure 1B. When increasing the temperature above
288C a decrease in enzyme activity was observed. At 488C,
a yield of only 5% was detected (an additional 12% was
brought about by ring opening of lactone 1a). Due to the low
amount of product at 488C, no reliable ee determination was
possible. Taking both the conversion and enantioselectivity
into account, the best results were achieved at 288C. These re-
sults are in agreement with the reported optimal growth tem-
perature of 268C for R. rhodochrous.[17]
Since water serves not only as the reaction medium but also
as a substrate, the pH needs to be considered as a very impor-
tant parameter. To quantify this effect, the reaction system was
tested at different pH values using potassium phosphate
buffer (pH 5.2–8.2) and citrate/phosphate buffer (pH 4.2) to
control the pH of the reaction medium. The results from this
study clearly show the dependence on pH. The conversion in-
creased with increasing pH (Figure 1C, filled triangles), as ex-
pected from our previous study,[2b] demonstrating that the hy-
dration reaction is generally base-catalyzed. However, at neu-
tral and slightly basic conditions (pH 7.2 and pH 8.2), signifi-
cant ring opening of lactone 1a took place (Figure 1C, empty
triangles), which explains the rather poor product yield (Fig-
ure 1C, filled squares). This effect can be explained by the
spontaneous hydrolysis of the lactone in basic aqueous
medium, which is an often observed phenomenon.[18] To con-
firm this, the blank reaction mixtures at pH 7.2 and 8.2 were
acidified with conc. HCl to pH 1. This leads to complete recov-
ery of the substrate 1a, validating our hypothesis. No desired
enantioselective Michael addition product 2a was detected in
the blank reactions (Figure 1C, empty squares) indicating that
chemical/base-catalyzed Michael addition does not occur
within the measured pH ranges. Therefore, the conversion in
the blank reactions (which is based on the decrease in amount
of substrate) is caused by the hydrolysis of 1a. Moreover, prod-
uct 2a showed good stability under strongly acidic conditions
and only 10% yield was lost overnight. Comparing the mass
balance and reaction rate, slightly acidic conditions (pH 6.2)
represented the optimal pH for this substrate.
Substrate scope and limitations
Since the very limited substrate scope of the known hydratases
is one of the challenging factors for their broad application,
we were interested in the scope of the Michael hydratase from
R. rhodochrous ATCC 17895. Neither substrate 1a nor product
2a are known to be part of primary metabolic pathways,
therefore the substrate scope of the hydratase from R. rho-
dochrous ATCC 17895 might be more relaxed than that for
other known hydratases. Hence we tested a set of different
substrates to evaluate the limitations of the enzyme (Table 3).
For a,b-unsaturated lactones (X=O; Table 3, entries 1–3) with
substituents in the b-positions, the reaction proceeded
smoothly in all cases to yield the corresponding hydration
products, whereas for R1 =H (X=O; Table 3, entries 4 and 5),
no water addition product was obtained. This result is surpris-
ing, as the tertiary alcohols obtained are sterically much more
demanding than the secondary alcohols, and suggests that
substituents in the b-position might play an important role for
proper orientation of the lactones in the enzyme’s active site.
However, the enzyme did not accept substrates with substitu-
ents in both b- and g-positions, such as 1 f (Table 3, entry 6),
which is probably due to its more bulky structure. Products 2a
and 2b are tertiary alcohols, representing a class of com-
pounds that are difficult to prepare by chemical methods, to
date only accessible via this route.[11] The enantioselectivity
was measured using a chiral Ivadex 7/PS086 GC column and,
Control experiments (Table 1 and Figure 1A–C) confirmed
that the formation of 2a is based on an enzymatic reaction
with high enantioselectivity and that no chemical background
reaction occurred. Therefore, the kinetic parameters Km, Vmax
,
and Vmax/Km were determined with the optimized conditions.
The Michaelis–Menten Plot (Figure 1D) allowed calculation of
the affinity constant Km as 1.7ꢁ10ꢀ2
m
and Vmax as
6.9 nmolsꢀ1 gꢀ1 (wet cells), providing further support for one
enzymatic reaction, rather than a sequence of reactions
(Scheme 1).
in parallel, the ee was confirmed by analysis of H and 19F NMR
1
spectra of their corresponding Mosher esters (see the Support-
ing Information, S4, S5, and S27–S29). In both cases, results
1
To establish the distribution of the enzymatic activity over
different organisms, we proceeded with testing different close-
ly related Rhodococcus strains. The selection was based on
phylogenetic analysis (Table 2). The previously reported strain
from H and 19F NMR spectra of the Mosher esters and chiral
GC analysis of the alcohols were comparable, showing excel-
lent enantioselectivities. The absolute configuration of the
product was established by converting 2a into the corre-
Chem. Eur. J. 2014, 20, 1 – 12
www.chemeurj.org
3
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
&
These are not the final page numbers! ÞÞ

Full Paper
Table 2. Comparison of closely related Rhodococcus strains. Phylogenetic tree based on 16 rRNA
Entry[a]
Catalysts
Conversion[b] of 1a [%]
Yield[b] of 2a [%]
ee[b] of 2a [%]
Genome sequence
1
2
3
4
5
6
7
R. rhodochrous ATCC 17895
R. erythropolis DSM 43296
R. erythropolis CCM 2595
R. erythropolis NBRC100887
R. erythropolis DSM 43066
R. rhodochrous DSM 43241
908C heat-denatured cells of R. rhodochrous ATCC 17895
87
82
88
80
90
87
12
75
70
76
68
78
95
93
95
93
95
+
ꢀ
+
+
ꢀ
ꢀ
75
<3
86
n.d.
[a] List of entries comparing activities using different organisms. [b] Conversion, yield, and ee values were determined by GC.
sponding methyl ester [methyl S-(ꢀ)-3,4-dihydroxy-3-methyl-
butanoate].[12,13]
For a,b-unsaturated ketones (X=C), substrates without sub-
stituent in the b-position were surprisingly accepted by the
putative Michael hydratase (Table 3, entries 7–9) but no activity
towards the b-substituted 3-methylcyclohex-2-enone and
3-methylcyclopent-2-enone was found (Table 3, entries 10 and
11). This might lead to the conclusion that b-substituted
a,b-unsaturated ketones may be challenging for Michael addi-
tion of water using R. rhodochrous, although the opposite is
true for a,b-unsaturated lactones. The a,b-unsaturated ketones
1g–i, were mostly reduced into ketones 3g–i (75%, 76%, and
80% yields, respectively), which explains the rather poor yield
of the water-addition reaction (Table 3, entries 7–9). Experiments
to rule out the reduction–oxidation as a possible reaction path-
way were performed for these cyclic ketones (Scheme 2). Reac-
tion using 1h as substrate was performed under a nitrogen at-
mosphere to exclude air as a potential oxygen source. Even so,
22% yield of 2h was obtained with 65% ee, ruling out the in-
volvement of O2 as an active species in the reaction. Further-
more, when 3h was used as a substrate directly under aerobic
conditions, no product 2h was detected. These two control ex-
periments demonstrate that the alcohol 2h was the result of
the enantioselective Michael addition of water to 1h. The com-
peting reduction reaction to 3g, 3h, and 3i is most likely due
to an ene reductase also present in the Rhodococcus cells.
Interestingly, the hydration of substrate 1c (Table 3, entry 3)
gave access to the natural product mevalonolactone 2c, the
salt form of which represents an intermediate in the pathway
leading to terpenoids.[19] Absolute configuration of (R)-2c was
determined by comparison with previously reported optical ro-
tation data.[20] Mevalonate is a product of acetate metabolism
and thus a key building block in secondary metabolism.[21] To
identify whether the putative Michael hydratase is a promiscu-
ous enzyme of the mevalonate pathway, bioinformatics studies
were performed. We have sequenced and annotated the
genome of R. rhodochrous ATCC 17895 in a previous study.[17]
Looking for annotated hydratases in this genome only showed
known hydratases with their narrow substrate specificity, em-
phasizing that the hydratase of this study has not been de-
scribed before. This enzyme could therefore be one of the
many unknown gene functions in the genome, or a promiscu-
ous activity of a known enzyme. Screening through all three
sequenced Rhodococci genomes (Table 2, entry 1, 3 and 4), we
unexpectedly found that most of the typical enzymes from the
mevalonate pathway were missing. Instead the full deoxyxylu-
lose phosphate (mevalonate-independent) pathway for terpe-
noid biosynthesis was present.
&
&
Chem. Eur. J. 2014, 20, 1 – 12
www.chemeurj.org
4
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Paper
To further probe whether the putative Michael hydra-
tase also accepts acyclic a,b-unsaturated carbonyl com-
pounds, methyl crotonate (1l), crotonic acid (1m),
(Z)-ethyl-4-hydroxy-3-methylbut-2-enoate (1n), and benzy-
lideneacetone (1o) were subjected to the resting cell sus-
pensions (Table 3, entries 12–15). Gratifyingly, the enzyme
readily accepted acyclic a,b-unsaturated ester (1l), al-
though no activity was observed for acyclic a,b-unsaturat-
ed carboxylic acid 1m, ester 1n, or ketone 1o. Notably, in
many water-addition reactions to carbon–carbon double
bonds, the equilibrium can be on the side of the starting
material although the reaction is performed in wa-
ter.[1d,6c,22] The unfavorable equilibrium might impede the
Michael addition of water; for example, the equilibrium
yield of 3-hydroxycyclohexanone (2h) was determined to
be 25% (Table 3, entry 8),[2b,23] corresponding with the
measured yield of 22%.
Table 3. Substrate scope for the enantioselective Michael addition of water.
Finally we tested the scalability of the developed reac-
tion system. Therefore the reaction was scaled to gram
scale using 1a (2 g, 20 mmol, 200 g of wet cells) to give
2a in 69% isolated yield after column chromatography
and an ee of 90% was determined.
Entry Substrate Product Conversion[a] Yield[a] ee of Enantio-
Equilibrium
of 1 [%]
of 2
2
preference yield of 2
[%]
[%]
[%]
1
2
3[b]
4
5
6
7
8
1a
1b
1c
1d
1e
rac-1 f
1g
1h
1i
1j
1k
1l
1m
1n
1o
2a
2b
2c
2d
2e
2 f
2g
2h
2i
2j
2k
2l
2m
2n
2o
87
80
75
12
32
12
93
98
95
<3
<3
42
<3
<3
<3
75
68
62
<3
<3
<3
18
95[c,d] S[e]
94[d]
>97[i]
[j]
S
–
73[c] R[f]
n.d. n.d.
n.d. n.d.
n.d. n.d.
22[c] R[g]
65[c] R[g]
20[c] R[h]
n.d. n.d.
n.d. n.d.
48[c] R[f]
n.d. n.d.
n.d. n.d.
n.d. n.d.
–
–
–
–
–
[j]
[j]
[j]
[j]
[j]
Recyclability and enzyme investigation
One of the most important characteristics of a catalyst is
its operational stability and reusability over an extended
period of time, to ensure a practical application.[26] From
the viewpoint of process economics, the higher the
number of cycles that an enzyme remains stable, the
more efficiently a process can be run. Experiments were
performed to examine this recyclability of the whole cells
of strain R. rhodochrous ATCC 17895 for the Michael addi-
tion of water to 3-methylfuran-2(5H)-one (1a). Based on
the results summarized in Table 1, every reaction was car-
ried out in a 50 mL Erlenmeyer flask at 288C with 180 rpm
for 23 h. At the end of the reaction, the cells were centri-
fuged, washed twice with potassium phosphate buffer
(100 mm, pH 6.2), and then reused for the next cycle.
Whole cells showed high activity and complete conversion
for 4 cycles (Figure 2). Only a slight decrease was observed
in cycle 5, whereas 27% lower conversion was detected in
the cycle 6. However, even after 9 consecutive cycles, the
whole cells retained 20% of the initial activity. Notably, no
significant changes in enantioselectivities of the water-ad-
dition reactions were detected during the 9 cycles (see the
Supporting Information, S12 for GC chromatography).
22
15
25[k]
[j]
9
–
–
–
–
–
–
–
[j]
10
11
12
13
14
15
<3
<3
40
<3
<3
<3
[j]
[j]
[j]
[j]
[j]
[a] Conversion and yield were determined by GC; [b] reaction was performed at
pH 5.2 to suppress ring open of lactone 1c at pH 6.2; [c] ee was determined by
GC; [d] ee was determined by 1H and 19F NMR of the corresponding Mosher
ester; [e] changing CIP priorities; [f] (R)-enantiomers commercially available;
[g] absolute stereochemistry was determined by converting them into litera-
ture-known derivatives, following a procedure established earlier in our labora-
tory;[24] [h] absolute configuration was determined by comparison of the reten-
tion times using the same GC column with a reported method;[25] [i] reverse re-
action with 2a as substrate was performed, analysis of this sample showed no
dehydration and no decrease of the ee; [j] no literature values available; [k] see
references [2b,23]
.
To isolate the putative Michael hydratase for further investi-
gation, we first broke the whole cells of R. rhodochrous ATCC
17895. The desired hydratase activity (yielding 2a) was only
found in the cell pellets, rather than in the cell-free extract,
when 1a was used as a substrate (Figure 3). Furthermore, no
significant difference was found between the initial rate of
whole cells and pelleted cell debris (Figure 3). Additionally,
with 1h as a substrate, most of the ene reductase activity
(Table 3, entries 7–9) was retained in the cell-free extract,
whereas only minor activity was still detectable in the cell pel-
Scheme 2. Control experiments to confirm that 2h was formed by enzy-
matic water addition, rather than a reduction–oxidation sequence.
Chem. Eur. J. 2014, 20, 1 – 12
www.chemeurj.org
5
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
&
These are not the final page numbers! ÞÞ

Full Paper
formed by reduction of 1a. Therefore 3a was synthesized (see
the Supporting Information, S5) according to a standard proce-
dure.[27a] When using 3a with a resting cell suspension, no cor-
responding oxidation product was detected under aerobic
conditions (Scheme 3). These two control experiments demon-
strated that the enantiomerically enriched tertiary alcohol 2a
was the result of the direct enantioselective Michael addition
of water to 1a.
Figure 2. Repeated water-addition reactions catalyzed by whole cells of
R. rhodochrous ATCC 17895. Conversion, yield, and ee values were deter-
mined by GC.
Scheme 3. Control experiments to confirm that 2a was formed by enzymat-
ic water addition, rather than a reduction–oxidation sequence.
The stereochemical course of this water-addition reaction
was further evaluated by carrying out the biotransformation in
D2O using lyophilized cells as catalyst. The reaction in D2O was
found to be slower than that in H2O, which might be due to
activity loss caused by the lyophilization or an isotope effect.
However, upon elongation of the reaction time to 24 h, deute-
rium oxide-addition product 4a was found at a conversion of
90%. After extraction with ethyl acetate and column purifica-
tion, compound 4a, containing the optically active OD group,
was exchanged back into an OH group, which is an often-ob-
served phenomenon.[28] NMR and GC-MS measurements
showed that the obtained compound (4b) contained one deu-
Figure 3. Michael hydratase activities in different biocatalyst preparations
(whole cells, pelleted cell debris, and cell-free extract). Conversion was deter-
mined by GC.
1
terium at the a carbon (Figure 4A). In the H NMR spectrum of
lets. These results indicate that the putative Michael hydratase
is not a soluble protein but bound to either the membrane or
cell wall, whereas the reductase activity apparently resides
within another enzyme that is soluble. This natural immobiliza-
tion of the putative Michael hydratase explains the high reusa-
bility of the whole cells (Figure 2).
2a, the geminal coupling constant between the two a protons
is 17.6 Hz, whereas the H NMR spectrum of 4b showed only
1
one a proton, which indicates one deuterium at the a carbon.
Comparing with the singlet signal of 2a in the 13C NMR spec-
trum, the triplet signal (coupling constant of 19.75 Hz) of 4b
again indicates one deuterium at the a carbon. GC-MS spectra
also show 4b to be one unit heavier than 2a (for full spectra,
see the Supporting Information, S21–S23). A control experi-
ment was performed by shaking pure 3-hydroxy-3-methylfura-
none (2a) in D2O. Analysis of this sample showed that no deu-
terium was incorporated at the a-position; hence, the 2H-la-
beled product 4b must have resulted from enzymatic water
addition. This further supports a one-step hydration mecha-
nism, via a Michael addition reaction.
Mechanistic studies
As mentioned above, Rhodococci have been shown to mediate
the hydroxylation of unactivated CꢀH bonds on selected THF
and THP derivatives.[15] To clearly rule out the possibility of de-
tecting a hydroxylation reaction instead of a hydration, we
turned our attention to the mechanism, including the stereo-
chemical course of the reaction. The reaction was performed
under a nitrogen atmosphere to exclude air as a potential
oxygen source. Under these conditions, 87% conversion with
95% enantioselectivity was still obtained, ruling out that O2 is
involved as an active species in the reaction. The second ap-
proach included the use of substrate 3a, which might be
According to the NMR measurements, the reaction of sub-
strate 1a and lyophilized R. rhodochrous ATCC 17895 cells in
D2O yielded monodeuterated 4b as a sole diastereoisomer
(Figure 4A; for full spectra see the Supporting Information, S21
and S22). The observation brought us to investigate the dia-
stereospecificity of the Michael addition of water, which until
&
&
Chem. Eur. J. 2014, 20, 1 – 12
www.chemeurj.org
6
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Paper
Figure 4. Diastereoselective Michael addition of water catalyzed by lyophilized cells of R. rhodochrous ATCC 17895.
Chem. Eur. J. 2014, 20, 1 – 12
www.chemeurj.org
7
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
&
These are not the final page numbers! ÞÞ

Full Paper
now had been reported to show, depending on the enzyme,
either syn or anti preference.[29a] For example, in the case of
enoyl-CoA hydratase, selectivity towards syn-addition was ob-
served,[29b] whereas enzymes belonging to the aspartase/fu-
marase superfamily, such as fumarase, aconitase, or enolase,
showed anti preference.[29c]
product formed. The biocatalytic reaction system was carefully
optimized for gram-scale synthesis, resulting in good conver-
sions and excellent enantioselectivities. Under the optimized
conditions, whole cells could be reused for 4 cycles without
significant loss of activity while maintaining up to 90% ee. Our
study suggests that this promising Michael hydratase is not
soluble but membrane-bound or cell wall-associated. In sum-
mary, whole cells from Rhodococcus strains are able to catalyze
the enantioselective Michael addition of water to several differ-
ent substrates using water as both solvent and substrate
under mild conditions. This opens up an entirely new
approach to the synthesis of chiral b-hydroxy carbonyl
compounds.
Nuclear Overhauser effect (NOE) experiments unfortunately
did not give conclusive results on the stereochemical course of
the water addition (see the Supporting Information, S32 and
S33). To further probe the stereoselectivity of the addition of
water, an anti E2 elimination of the deuterium oxide addition
product 4b was performed.[30] The reaction was accomplished
with acetic anhydride/triethylamine in the presence of a catalyt-
ic amount of 4-dimethylaminopyridine (DMAP) and the corre-
sponding product was measured directly by NMR and GC-MS
without further purification. The results showed 1a as the only
elimination product, HDO being expelled during the elimina-
Experimental Section
Material and Methods
1
tion process (Figure 4B). H NMR spectroscopy showed the ap-
All chemicals were purchased from Sigma–Aldrich (Schnelldorf,
Germany) and were used without further purification unless other-
wise specified. The culture media components were obtained from
BD (Becton, Dickinson and Company, Germany).
pearance of signal for a proton at 5.91 ppm, indicating that
HDO was eliminated via an anti E2 elimination. The appear-
ance of a singlet (which was visible as a triplet at 43.6 ppm
previously in 4b) at 116.2 ppm for the a carbon in the 13C NMR
spectrum also proved the loss of molecular HDO. The D-NMR
spectrum shows only one peak at 2.60 ppm, which belongs to
the unconverted 4b, but no peak at 5.91 ppm, again proving
that HDO was eliminated. This elimination was further con-
firmed by GC-MS analysis (for full spectra, see the Supporting
Information, S24 and S25). Since the E2 elimination always
occurs exclusively in anti fashion, and removed a-D and b-OH
groups, the enzymatic D2O addition must have proceeded
with exclusive anti stereochemistry. Our results are supported
by the findings of Mohrig et al.,[29a,d] who described that the
stereopreference of water addition depends on the position of
the abstracted proton: If the proton is in the a-position to the
carboxylate group, as in the case of our studies, anti-selectivity
is observed; abstracted protons that are in the a-position to
the carbonyl group of the thioester lead to syn-selectivity.[29a,b]
1H, 2H, 13C, and 19F NMR spectra were recorded with Bruker Ad-
vance 400 or Varian 300 (400 MHz, 61.4 MHz, 100 MHz and
376.33 MHz, respectively) instrument and were internally refer-
1
enced to residual solvent signals. Data for H NMR are reported as
follows: chemical shift (d ppm), multiplicity (s=singlet, d=doublet,
t=triplet, q=quartet, m=multiplet), integration, coupling con-
stant (Hz) and assignment. Data for 13C NMR and 19F NMR are re-
ported in terms of chemical shift. Optical rotations were obtained
at 208C with a PerkinElmer 241 polarimeter (sodium D line).
Column chromatography was carried out with silica gel (0.060–
0.200 mm, pore diameter ca. 6 nm) and with mixtures of petroleum
ether (PE) and ethyl acetate (EtOAc) as solvents. Thin-layer chroma-
tography (TLC) was performed on 0.20 mm silica gel 60-F plates.
Organic solutions were concentrated under reduced pressure with
a rotary evaporator.
Conversion of substrates and yield of products were quantified by
GC using calibration lines with dodecane as an internal standard
(specifications and temperature programs given in the Supporting
Information, S2) and the optical purity of the products [excepted
for 2b] were determined using chiral GC (specifications and tem-
perature programs given in the Supporting Information, S2). The
Conclusion
1
enantiomeric excess (ee) of 2b was determined by H and 19F NMR
b-Hydroxy carbonyl compounds represent an important class
of compounds that is often found as a structural motif in natu-
ral products. Although the molecules themselves look rather
simple, their synthesis can be challenging. A straightforward
route for the preparation of chiral b-hydroxy carbonyl com-
pounds was established, employing whole cells from several
Rhodococcus strains harboring a Michael hydratase. They cata-
lyzed the enantioselective Michael addition of water in water
with good yields and excellent enantioselectivities. Compared
to the very narrow substrate scope of known hydratases, the
particularly intriguing feature and advantage of this new hy-
dratase is its broad substrate range; a,b-unsaturated lactones
with substituents in b-position (1a, 1b and 1c), a,b-unsaturat-
ed cyclic ketones with no substituent in b-position (1g, 1h
and 1i), and an a,b-unsaturated ester (1l). A series of control
experiments and deuterium labeling studies demonstrate that
the reaction is diasterospecific, with only the anti hydration
spectroscopy of the corresponding Mosher ester (see the Support-
ing Information, S4, S5, and S27–S29).
Microorganisms and culture conditions
Rhodococcus rhodochrous ATCC 17895 was purchased from ATCC
(American Type Culture Collection, Manassas, USA). Rhodococcus
erythropolis DSM 43296, Rhodococcus erythropolis DSM 43060, Rho-
dococcus erythropolis DSM 43066, and Rhodococcus rhodochrous
DSM 43241 were purchased from DSMZ (Germany). Rhodococcus
erythropolis PR4 NBRC 100887 was purchased from NBRC (Biologi-
cal Resource Centre, Chiba, Japan). The organisms were maintained
on agar plates at 48C and these were subcultured at regular inter-
vals. The medium used for cultivation[11] contained Solution A
(980 mL) with potassium dihydrogen phosphate (0.4 g), dipotassi-
um hydrogen phosphate (1.2 g), peptone (5 g), yeast extract (1 g),
glucose (15 g), final pH 7.2, sterilized at 1108C in an autoclave; Sol-
ution B (10 mL) with magnesium sulphate (0.5 g), filter-sterilized;
&
&
Chem. Eur. J. 2014, 20, 1 – 12
www.chemeurj.org
8
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Paper
Solution C (10 mL) with iron(II) sulphate (0.3 g), filter sterilized. Sol-
utions were mixed before inoculation to make 1 L medium with
a buffer concentration of 3 mm. A loop of bacteria was used to in-
oculate 1 L medium in a 2 L Erlenmeyer flask. This culture was
shaken reciprocally at 288C for about 72 h to an optical density
(OD600) of around 6.3. The cells of Rhodococcus strains were har-
vested by centrifugation at 10000 rpm and at 48C for 20 min. The
supernatant was removed and the cells were rinsed with potassi-
um phosphate buffer (100 mm, pH 6.2) and centrifuged again. The
supernatant was discarded and the pellets were stored at ꢀ208C.
When needed, the wet pellets were freeze dried overnight and col-
lected as lyophilized cells.
reactions the setup was the same but without the addition of
whole cells. For substrate recovery studies, experiments were per-
formed by dissolving 3-methylfuran-2(5H)-one (1a; 6.5 mg,
0.07 mmol) in buffer (pH 7.2 or pH 8.2, 2 mL, 100 mm) and shaken
at 288C for 6 h (the same conditions as for the reaction), then
1 mL of the mixture was extracted directly while the remaining
1 mL was acidified with HCl to pH 1.0 before extraction. Workup
and analysis were as described in General biotransformation pro-
cedure for rate measurement (Figure 1C).
Enzyme kinetic study
The reaction setup for the enzyme kinetic study was the same as
for the rate determination. Reactions were performed in potassium
phosphate buffer (100 mm, pH 6.2) at 288C for 2 h with various
substrate concentrations (1, 2, 4, 5, 8, 10, 15, 20, 25, 30, 35, 40, 50,
60, 70, 80, 90, 100 mm) of 1a and with of wet cells (330 mgmLꢀ1).
For the blank reaction the setup was the same but heat-denatured
cells (908C, 30 min) were used. Workup and analysis were as de-
scribed in General biotransformation procedure for rate mea-
surement (Figure 1D).
General biotransformation procedure for catalyst concentra-
tion study
Cells in the culture age of OD600 =6.3 were harvested by centrifu-
gation, washed twice with 100 mm potassium phosphate buffer
(pH 6.2). Around 100 mgmLꢀ1 or 330 mgmLꢀ1 of the cells were re-
suspended in the same buffer (15 mL) containing 33 mm 3-methyl-
furan-2(5H)-one (1a; 50 mg, 0.51 mmol). The resting cell reactions
were carried out in screw-capped Erlenmeyer flasks. Reactions
were shaken at 288C overnight (17 h). For the blank reaction the
setup was the same but heat-denatured cells (908C, 30 min) were
used. For workup, the cells were removed by centrifugation and
1 mL of the supernatant was saturated with NaCl followed by ex-
traction with 2ꢁ0.5 mL of isoamyl alcohol (containing internal
standard) by shaking for 5 min. The combined organic layer was
dried over Na2SO4 and measured by GC for conversion, yield, and
ee (Table 1).
General procedure for organisms activity screening
The reaction setup for organisms activity screening was the same
as for the rate determination. Reactions were performed in 30 mL
of potassium phosphate buffer (100 mm, pH 6.2) containing 1a
(100 mg, 1.02 mmol) with whole cells of given organisms at 288C
for 6 h. For the blank reaction the setup was the same but heat-de-
natured cells (908C, 30 min) were used. Workup and analysis were
as described in General biotransformation procedure for rate
measurement (Table 2).
General biotransformation procedure for rate measurement
The reaction setup for rate determination was the same as for the
catalyst concentration study. Duplicate experiments were per-
formed respectively in potassium phosphate buffer (100 mm,
90 mL, pH 6.2) containing 3-methylfuran-2(5H)-one (1a; 33 mm,
300 mg, 3.06 mmol) and resting cells (330 mgmLꢀ1). For the blank
reaction, the setup was the same but heat-denatured cells (908C,
30 min) were used. Reactions were allowed to proceed at 288C.
Every 1 hour, a 1.5 mL sample was taken from the reaction mixture.
Cells were removed by centrifugation and then 1 mL of the super-
natant was saturated with NaCl followed by extraction with 2ꢁ
0.5 mL of isoamyl alcohol (containing internal standard) by shaking
for 5 min. The combined organic layer was dried over Na2SO4 and
measured with GC for conversion, yield, and ee (Figure 1A).
General procedure for substrate screening
Reactions were carried out as described in the General bio-transfor-
mation procedure for rate measurement using the same concen-
tration for each substrate. After extraction with isoamyl alcohol
(2ꢁ0.5 mL) samples were dried over Na2SO4 and crude samples
were analyzed by GC when product reference material was avail-
able or GC-MS (Varian FactorFour VF-1 ms column [25 mꢁ
0.25 mmꢁ0.4 mm] and He as carrier gas) when product reference
material was not commercially available (Table 3).
General procedure for recyclability
Reactions were carried out with substrate 1a (50 mg, 0.51 mmol)
in 15 mL of potassium phosphate buffer (100 mm, pH 6.2) and
330 mgmLꢀ1 of wet cells, shaken at 288C for 23 h. At the end of
the reaction, cells were centrifuged at 4000 rpm for 20 min to be
separated from the reaction mixture, then washed by potassium
phosphate buffer (100 mm, pH 6.2), and resuspended in 15 mL of
the same buffer containing the same substrates. The reaction mix-
ture (1 mL of supernatant separated from cells) was saturated with
NaCl and then extracted with 2ꢁ0.5 mL of isoamyl alcohol (con-
taining internal standard) by shaking for 5 min. The combined or-
ganic phase were dried over Na2SO4 and crude samples were ana-
lyzed by GC (Figure 2).
Reaction temperature study
The reaction setup for the temperature study was the same as for
the rate determination. Reactions were performed in potassium
phosphate buffer (100 mm, 15 mL, pH 6.2) containing 1a (50 mg,
0.51 mmol) and wet cells (330 mgmLꢀ1) at given temperatures for
6 h. Workup and analysis were as described above in General bio-
transformation procedure for rate measurement (Figure 1B).
pH study
The reaction setup for the pH profile was the same as for the rate
determination. Reactions were performed in buffer (15 mL) con-
taining 1a (50 mg, 0.51 mmol) and wet cells (330 mgmLꢀ1) at
given pH values (pH 5.2–8.2 were prepared as potassium phos-
phate buffers and pH 4.2 was prepared as citrate/phosphate buffer,
all at a buffer strength of 100 mm) at 288C for 6 h. For the blank
Activities comparison using pelleted cell debris and cell free
extract
15 g of cells in the culture age of OD600 =6.3 were harvested by
centrifugation, washed twice with 100 mm of potassium phosphate
Chem. Eur. J. 2014, 20, 1 – 12
www.chemeurj.org
9
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
&
These are not the final page numbers! ÞÞ

Full Paper
1
buffer (pH 6.2) and resuspended in the same buffer (45 mL). The
cells were incubated first with lysozyme (1 mgmLꢀ1, 48C, 1 h) and
subsequently disrupted using a French press (2.05 kBar, 2 shots).
Cell-free extract and cell debris were separated by centrifugation
for 40 min at 10000 rpm at 48C. Substrate 1a (150 mg, 1.53 mmol)
was added to the supernatant (cell-free extract) and shaken at
288C (reaction A). Cell debris was resuspended in potassium phos-
phate buffer (45 mL, 100 mm, pH 6.2) containing the same concen-
tration of substrates (reaction B). Every 1 hour, a 1.5 mL sample
was taken from both reaction A and B. For workup, the cells were
removed by centrifugation and 1 mL of the supernatant was satu-
rated with NaCl followed by extraction with 2ꢁ0.5 mL of isoamyl
alcohol (containing internal standard) by shaking for 5 min. The
combined organic layer was dried over Na2SO4 and measured with
GC for conversion, yield and ee (Figure 3).
24.96, 43.29 (t, JC,D =19.8 Hz, CD), 74.61, 79.80, 176.49 ppm; m/z:
117 (M +, 2), 89 (5), 86 (4), 74 (3), 60 (9), 59 (100), 58 (20), 57 (4), 44
(36), 43 (87), 42 (10), 41 (4), 40 (7).
Dehydration of deuterium oxide addition product (4b)
To a solution of alcohol 4b (30 mg, 0.26 mmol) in EtOAc (2 mL)
was slowly added acetic anhydride (35 mL) and triethylamine
(60 mL), followed by 4-dimethylaminopydridine (50 mL of 3 mgmLꢀ1
solution in EtOAc). The reaction was allowed to proceed for 30 min
at room temperature and was stopped by the addition of 0.5 mL
of water. The phases were separated and the organic layer was
dried over Na2SO4 and evaporated. The crude product was mea-
sured by NMR and GC-MS which showed the elimination product
was 3-methylfuran-2(5H)-one (1a). 1H NMR (400 MHz, CDCl3) d=
1.96 (s, 3H), 4.00 (s, 2H), 5.94 ppm (s, 1H); 13C NMR (100 MHz,
CDCl3) d=14.02, 73.86, 116.23, 166.32, 174.1 ppm (in accordance
with literature[27]); m/z: 98 [M+] (26), 71 (16), 70 (62), 69 (100), 68
(13), 67 (3), 55 (3), 54 (3), 53 (6), 52 (3), 51 (3), 50 (6), 45(10), 44 (5),
43 (19), 42 (56), 41 (98), 40 (71).
(S)-3-hydroxy-3-methylfuranone (2a); preparative scale)
For isolation and characterization of the Michael addition product,
the reaction was carried out on preparative scale. Pelleted cells
from 20 L medium were resuspended in potassium phosphate
buffer (600 mL, 100 mm, pH 6.2), and substrate 3-methylfuran-
2(5H)-one (1a; 2 g, 20.38 mmol) was added. Reaction was incubat-
ed at 288C and shaken at 180 rpm for 24 h. Then the cells were re-
moved by centrifugation and the supernatant was saturated with
NaCl. Due to the high solubility of the resulting alcohols in water,
continuous extraction with ethyl acetate was performed overnight.
The extract was then concentrated under reduced pressure and
purified by flash column chromatography on silica gel (eluent: PE/
EtOAc 1:1) to yield 2a (1.63 g, 14.06 mmol, 69%) as a colorless oil;
½aꢁ2D0 = +46.6 (c 0.96 in CHCl3)[11] [a]D +53.92 (c 0.96 in CHCl3)];
1H NMR (400 MHz, CDCl3): d=1.48 (s, 3H), 2.57 (d, J=17.6 Hz, 1H),
2.63 (d, J=17.6 Hz, 1H), 3.71 (s, 1H), 4.14 (d, J=9.6 Hz, 1H),
4.27 ppm (d, J=9.6 Hz, 1H); 13C NMR (100 MHz, CDCl3): d=24.94,
43.06, 74.70, 79.82, 176.27 ppm (in accordance with literature[11]).
Acknowledgements
The authors thank Dr. K. Djanashvili and Dr. J. Martinelli for
help with NMR measurements and analysis. We also thank M.
Gorseling and R. van Oosten for technical assistance and Prof.
S. de Vries for helpful discussions. A senior research fellowship
of China Scholarship Council–Delft University of Technology
Joint Program to B.S.C. is gratefully acknowledged. V.R. thanks
the Austrian Science Fund (FWF) for an “Erwin-Schroedinger”
Fellowship (J3292).
Keywords: biocatalysis
Michael addition · water
· enantioselectivity · hydratases ·
(S)-3-hydroxy-3-ethylfuranone (2b; preparative scale)
For isolation and characterization of the Michael addition product,
the reaction was carried out on preparative scale. Using the bio-
transformation procedure described for 2a, reaction of 3-ethylfur-
an-2(5H)-one (1b; 1 g, 8.92 mmol) gave 2b (0.75 g, 5.80 mmol,
[1] a) J. E. McMurry, Organic Chemistry, 8th ed., Cengage Learning, Mel-
bourne, Australia, 2012; b) K. Schwetlick, Organikum, 23rd ed., Wiley-
VCH, Weinheim, 2009; c) J. Clayden, N. Greeves, S. Warren, Organic
Chemistry, 2nd ed., Oxford University Press, Oxford, 2012; d) V. Resch, U.
Hanefeld, Catal. Sci. Technol. 2014, DOI: 10.1039/C4CY00692E.
[2] a) J. Jin, U. Hanefeld, Chem. Commun. 2011, 47, 2502–2510; b) V. Resch,
C. Seidler, B.-S. Chen, I. Degeling, U. Hanefeld, Eur. J. Org. Chem. 2013,
7697–7704.
65%) as
a
colorless oil; ½aꢁ2D0 =+49.6 (c 0.75 in CHCl3),[11]
1
[a]D =+48.9 (c 0.72 in CHCl3)]; H NMR (400 MHz, CDCl3) d=0.98 (t,
J=7.6 Hz, 3H), 1.72 (q, J=7.4 Hz, 2H), 2.49 (s, 1H), 2.53 (s, 2H),
4.13 (d, J=9.6 Hz, 1H), 4.21 (d, J=9.6 Hz, 1H); 13C NMR (100 MHz,
CDCl3) d=8.70, 31.69, 42.36, 78.05, 79.26, 176.82 ppm (in accord-
ance with literature[11]).
[3] a) C. F. Nising, S. Brꢂse, Chem. Soc. Rev. 2012, 41, 988–999; b) C. F.
Nising, S. Brꢂse, Chem. Soc. Rev. 2008, 37, 1218–1228.
[4] a) X. Feng, J. Yun, Chem. Commun. 2009, 6577–6579; b) L. Xue, B. Jia, L.
Tang, X. F. Ji, M. Y. Huang, Y. Y. Jiang, Polym. Adv. Technol. 2004, 15, 346–
349; c) S. Wang, Z. Zhang, C. Chi, G. Wu, J. Ren, Z. Wang, M. Huang, Y.
Jiang, React. Funct. Polym. 2008, 68, 424–430.
[5] a) C. D. Vanderwal, E. N. Jacobsen, J. Am. Chem. Soc. 2004, 126, 14724–
14725; b) E. Hartmann, D. J. Vyas, M. Oestreich, Chem. Commun. 2011,
47, 7917–7932.
[6] a) A. J. Boersma, R. P. Megens, B. L. Feringa, G. Roelfes, Chem. Soc. Rev.
2010, 39, 2083–2092; b) R. P. Megens, G. Roelfes, Chem. Commun.
2012, 48, 6366–6368; c) A. J. Boersma, D. Coquiere, D. Geerdink, F.
Rosati, B. L. Feringa, G. Roelfes, Nat. Chem. 2010, 2, 991–995.
[7] J. Bos, A. Garcꢃa-Herraiz, G. Roelfes, Chem. Sci. 2013, 4, 3578–3582.
[8] E. Celinska, Biotechnol. Adv. 2010, 28, 519–530.
[9] G. Agnihotri, H. W. Liu, Bioorg. Med. Chem. 2003, 11, 9–20.
[10] a) C. Wuensch, J. Gross, G. Steinkellner, K. Gruber, S. M. Glueck, K. Faber,
Angew. Chem. Int. Ed. 2013, 52, 2293–2297; Angew. Chem. 2013, 125,
2349–2353; b) C. Wuensch, J. Gross, S. M. Glueck, K. Faber (Acib Gmbh,
Karl-Franzens Universitꢂt Graz), WO186358, 2013.
[2-D]-3-hydroxy-3-methylfuranone (4b)
Lyophilized cells (3 g) were resuspended in D2O (100 mL) contain-
ing 4 drops of potassium hydroxide solution (100 mm; final pD 6.5,
corresponds to pH 6.1). 1a (330 mg, 3.40 mmol) was added. The
reaction mixture was shaken at 180 rpm, 288C for 24 h, then centri-
fuged and the supernatant was saturated with NaCl and continu-
ously extracted with ethyl acetate (200 mL) overnight. The extract
was dried over Na2SO4 and evaporated under reduced pressure.
The crude product mixture was purified using flash chromatogra-
phy on silica gel (eluent: PE/EtOAc 1:1) yielding deuterium oxide-
addition
product
(S)-[2-D]-3-hydroxy-3-methylfuranone
4b
(265 mg, 2.28 mmol, 67%) as a colorless oil. 1H NMR (400 MHz,
CDCl3): d=1.51 (s, 3H), 2.64 (s, 1H), 2.96 (s, 1H), 4.15 (d, J=9.6 Hz,
1H), 4.28 ppm (d, J=9.6 Hz, 1H); 13C NMR (100 MHz, CDCl3): d=
&
&
Chem. Eur. J. 2014, 20, 1 – 12
www.chemeurj.org
10
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Paper
[11] H. L. Holland, J.-X. Gu, Biotechnol. Lett. 1998, 20, 1125–1126. In this
study, the absolute stereochemistry was assigned as R, based on the
conversion of 2a into the corresponding triol. However, the optical ro-
[23] M. Stiles, A. Longroy, Tetrahedron Lett. 1961, 2, 337–340.
[24] a) S. K. Karmee, R. van Oosten, U. Hanefeld, Tetrahedron: Asymmetry
2011, 22, 1736–1739; b) B.-S. Chen, U. Hanefeld, J. Mol. Catal. B. Enzym.
2013, 85-86, 239–242.
20
tation {[a]D +0.96 (c 5 in EtOH)} of this triol is very small and, there-
fore, impurities can easily lead to errors. Moreover, no experimental de-
tails and reaction conditions were given. Since our experiments with all
the other substrates gave the opposite orientation of the hydroxy
[25] O. Lifchits, M. Mahlau, C. M. Reisinger, A. Lee, C. Fares, I. Polyak, G. Go-
pakumar, W. Thiel, B. List, J. Am. Chem. Soc. 2013, 135, 6677–6693.
[26] a) N. N. Rao, S. Lꢄtz, K. Seelbach, A. Liese in Industrial Biotransformations,
2nd ed. (Eds.: A. Liese, K. Seelbach, C. Wandrey), Wiley-VCH, Weinheim,
2006, pp. 135–138; b) M. Schrewe, M. K. Julsing, B. Bꢄhler, A. Schmid,
Chem. Soc. Rev. 2013, 42, 6346–6377.
[27] a) V. Sharma, G. T. Kelly, C. M. H. Watanabe, Org. Lett. 2008, 10, 4815–
4818; b) M. Egi, Y. Ota, Y. Nishimura, K. Shimizu, K. Azechi, S. Akai, Org.
Lett. 2013, 15, 4150–4153.
[28] a) N. J. Patel, G. Britton, T. W. Goodwin, Biochim. Biophys. Acta. 1983,
760, 92–99; b) W. G. Niehaus, A. Kisic, A. Torkelson, D. J. Bednarczyk,
G. J. Schroepfer, J. Biol. Chem. 1970, 245, 3790–3797; c) D. H. Flint, Arch.
Biochem. Biophys. 1994, 311, 509–516; d) O. Gawron, T. P. Fondy, J. Am.
Chem. Soc. 1959, 81, 6333–6334.
group, we converted 2a into its literature-known methyl ester,[12] which
20
gave an optical rotation of ½aꢁ2D0 =ꢀ9.3 (c 2.15 in CHCl3; Ref. [13]: ½aꢁD
=
+10.26 for the R-enantiomer). Therefore we reassigned the absolute
stereochemistry of 2a to be S (for further details see the Supporting In-
formation, S6 and S31).
[12] See the Supporting Information for experimental details.
[13] A. Kutner, M. Chodynski, S. J. Halkes, Synth. Commun. 1996, 26, 1175–
1182.
[14] V. Resch, J. Jin, B.-S. Chen, U. Hanefeld, AMB Express 2014, 4, 30–37.
[15] E. O’Reilly, S. J. Aitken, G. Grogan, P. P. Kelly, N. J. Turner, S. L. Flitsch, Beil-
stein J. Org. Chem. 2012, 8, 496–500.
[16] A. Eisenfꢄhr, P. S. Arora, G. Sengle, L. R. Takaoka, J. S. Nowick, M. Famu-
lok, Bioorg. Med. Chem. 2003, 11, 235–249.
[17] B.-S. Chen, L. G. Otten, V. Resch, G. Muyzer, U. Hanefeld, Stand. Genomic.
Sci. 2013, 9, 175–184.
[18] S. Kara, D. Spickermann, J. H. Schrittwieser, A. Weckbecker, C. Leggewie,
I. W. C. E. Arends, F. Hollmann, ACS Catal. 2013, 3, 2436–2439.
[19] K. M. Byrne, S. K. Smith, J. G. Ondeyda, J. Am. Chem. Soc. 2002, 124,
7055–7060.
[20] T. Sugai, H. Kakeya, H. Ohta, Tetrahedron 1990, 46, 3463–3468.
[21] a) D. E. Wolf, C. H. Hoffman, P. E. Aldrich, H. R. Skeggs, L. D. Wright, K.
Folkers, J. Am. Chem. Soc. 1956, 78, 4499–4499; b) P. M. Dewick, Medici-
nal Natural Products: A Biosynthetic Approach, Wiley, Hoboken, 2nd ed.,
2001, pp. 167–175.
[29] a) J. R. Mohrig, Acc. Chem. Res. 2013, 46, 1407–1416; b) B. J. Bahnson,
V. E. Anderson, G. A. Petsko, Biochemistry 2002, 41, 2621–2629; c) H. M.
Holden, M. M. Benning, T. Haller, J. A. Gerlt, Acc. Chem. Res. 2001, 34,
145–157; d) J. R. Mohrig, K. A. Moerke, D. L. Cloutier, B. D. Lane, E. C.
Person, T. B. Onasch, Science 1995, 269, 527–529.
[30] a) J.-M. Adam, J. Foricher, S. Hanlon, B. Lohri, G. Moine, R. Schmid, H.
Stahr, M. Weber, B. Wirz, U. Zutter, Org. Process Res. Dev. 2011, 15, 515–
526; b) J. S. Dickschat, C. A. Citron, N. L. Brock, R. Riclea, H. Kuhz, Eur. J.
Org. Chem. 2011, 3339–3346.
Received: October 9, 2014
[22] R. M. Bock, R. A. Alberty, J. Am. Chem. Soc. 1953, 75, 1921–1925.
Published online on && &&, 0000
Chem. Eur. J. 2014, 20, 1 – 12
www.chemeurj.org
11
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
&
These are not the final page numbers! ÞÞ

Full Paper
FULL PAPER
Water as substrate and solvent: The
Michael hydratase from Rhodococcus
rhodochrous ATCC 17895 enables the
direct enantioselective synthesis of sec-
ondary and tertiary alcohols in good
yields and excellent enantioselectivities
by a direct Michael addition of water in
water. Deuterium labeling studies dem-
onstrate that the water addition pro-
ceeds exclusively with anti-stereochem-
istry.
&
Biocatalysis
B.-S. Chen, V. Resch, L. G. Otten,
U. Hanefeld*
&& – &&
Enantioselective Michael Addition of
Water
&
&
Chem. Eur. J. 2014, 20, 1 – 12
www.chemeurj.org
12
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!