Enantioselective one-pot two-step synthesis of hydrophobic allylic alcohols in aqueous medium through the combination of a Wittig reaction and an enzymatic ketone reduction

Article abstract of DOI:10.1002/ejoc.200700647

A one-pot two-step process for the enantioselective synthesis of hydrophobic allylic alcohols was developed, which comprises ketone formation by the Wittig reaction and their enzymatic in situ biotransformation into the desired target products. By means of this combined Wittig reaction and bioreduction, the allylic alcohols were prepared with conversions of up to 90 %, and with excellent enantioselectivities of >99 % ee. Wiley-VCH Verlag GmbH & Co. KGaA, 2007.

Full text of DOI:10.1002/ejoc.200700647

SHORT COMMUNICATION
DOI: 10.1002/ejoc.200700647
Enantioselective One-Pot Two-Step Synthesis of Hydrophobic Allylic Alcohols
in Aqueous Medium through the Combination of a Wittig Reaction and an
Enzymatic Ketone Reduction
Marina Kraußer,[a] Werner Hummel,[b] and Harald Gröger*[a]
Keywords: Alcohols / Chemoenzymatic synthesis / Enzyme catalysis / Reduction / Wittig reactions
A one-pot two-step process for the enantioselective synthesis
of hydrophobic allylic alcohols was developed, which com-
prises ketone formation by the Wittig reaction and their en-
zymatic in situ biotransformation into the desired target
products. By means of this combined Wittig reaction and bio-
reduction, the allylic alcohols were prepared with conver-
sions of up to 90%, and with excellent enantioselectivities of
Ͼ99% ee.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2007)
organic synthetic processes. Thus, biocatalytic steps are
often treated as “isolated” steps within a multistep organic
Introduction
The biocatalytic reduction of ketones represents a highly chemical synthesis, whereas combination of organic chemi-
efficient method for the asymmetric synthesis of chiral cal reaction steps to multistep one-pot syntheses is widely
alcohols.[1,2] Excellent enantioselectivities in combination known.[4,5] Certainly for a broader application range of bio-
with an easily available (bio)catalyst and excellent produc- catalysis in organic synthesis the development of compati-
tivity data make this technology highly attractive for large- ble chemical and enzymatic reactions is of crucial impor-
scale applications; thus, it is competitive even with the No- tance. Whereas successful examples of chemoenzymatic
bel prize technology of metal-catalyzed asymmetric hydro- one-pot processes have been already reported for the syn-
genation[3] as an industrially well-established, highly ef- thesis of a range of water-soluble organic molecules in aque-
ficient and widely applied reduction route. Surprisingly, ous media[6] and dynamic kinetic resolution of hydrophobic
however, in spite of its high potential and attractiveness for alcohols and amines in organic media using hydrolases,[7,8]
large-scale processes, the current application range of enzy- there is still a lack of chemoenzymatic syntheses of hydro-
matic reductive methods (and in general biocatalytic routes) phobic molecules in aqueous media as the most preferred
in organic chemistry is still restricted. One reason might be media for biotransformations. In the following, we report
their lack of compatibility with reaction media for “typical” this type of reaction. As a representative example for many
Scheme 1.
conceivable reactions, we describe the one-pot synthesis of
[a] Institute of Organic Chemistry, University of Erlangen-Nurem-
hydrophobic, water-immiscible alcohols by the oxidore-
berg,
ductase-catalyzed reduction of ketones prepared in situ by
the Wittig reaction starting from aldehydes and phosphorus
ylides. The concept of such a chemoenzymatic two-step
one-pot synthesis is shown in Scheme 1. To the best of our
Henkestr. 42, 91054 Erlangen, Germany
E-mail: harald.groeger@chemie.uni-erlangen.de
[b] Institute of Molecular Enzyme Technology at the Heinrich-
Heine University of Düsseldorf, Research Centre Jülich,
Stetternicher Forst, 52426 Jülich, Germany
Eur. J. Org. Chem. 2007, 5175–5179
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
5175

M. Kraußer, W. Hummel, H. Gröger
SHORT COMMUNICATION
knowledge this chemoenzymatic reaction also represents
the first example of a one-pot synthesis of chiral alcohols
by the asymmetric alcohol dehydrogenase catalyzed re-
duction of ketones, which were prepared in situ prior to the
enzymatic reduction by means of a non-enzymatic “typical”
organic chemical reaction.
Results and Discussion
Scheme 2.
For the combination of biocatalytic and chemical reac-
tions in one-pot processes, we envisioned the design of
chemical and biocatalytic processes, which are compatible
with each other. Thus, the in situ preparation of ketones 3
was planned to be carried out in reaction media that would
be suitable for subsequent enzymatic reduction. Since we
chose to recycle the cofactor in the enzymatic reduction
process with 2-propanol as a cosubstrate (according to
Scheme 1; for this concept of cofactor regeneration, see
ref.[1b]), the aqueous buffer/2-propanol reaction media also
needed to be suitable for the preparation of ketones 3 by
the Wittig reaction. Recently, Bergdahl and coworkers re-
ported a Wittig reaction in pure water as a solvent for the
synthesis of α,β-unsaturated esters and ketones of type 3
(e.g., 3a,d) to achieve significantly superior results in water
than in organic solvents.[9] In our studies we found that the
Bergdahl method was broadly applicable for the synthesis
of several other ketones of type 3 (e.g., 3b,c) and led to both
high conversion (Ͼ95%) and E/Z ratio (Ն95:5). For some
ketones 3, optimization of the reaction time and tempera-
ture was carried out (data not shown).
We were in particular interested to learn if the Bergdahl
method could also proceed with the same high efficiency
with the use of water/2-propanol and aqueous buffer/2-pro-
panol mixtures as reaction media (Scheme 2). When the
Wittig synthesis of 4-methylbenzylidene acetone (3c) was
carried out in mixtures of water/2-propanol with a 2-propa-
nol content of 25% (v/v) and 50% (v/v), respectively, both
reactions proceeded with a conversion of Ͼ95% and a high
E/Z ratio of Ͼ95:5 after a reaction time of 1 h at room
temperature, followed by 1 h at 50 °C, thus being in the
same range relative to the reaction in pure water (Ͼ95%
conversion, Scheme 2). A further experiment in a phosphate
buffer/2-propanol [25% (v/v)] mixture also gave an excellent
result (Ͼ95% conversion; E/Z, Ͼ95:5), which shows that a
highly efficient Wittig synthesis is also possible in aqueous
buffer/2-propanol mixtures. Thus, a negative impact of the
organic cosolvent was not observed as one might expect
from the literature-known decreased reaction rates when or-
ganic solvents are used as reaction media.[9a]
substrates 3, prepared by the Wittig reaction, were found
(data not shown). Subsequently, the preparative biotrans-
formations were carried out on the basis of a substrate-
coupled cofactor-regeneration method. Therein, the alcohol
dehydrogenases reduce the ketone with formation of the de-
sired alcohol in an enantioselective manner under con-
sumption of the cofactor NAD(P)H, which is used in a low
catalytic amount only (according to the concept shown in
Scheme 1). The in situ recycling of the cofactor by means
of an oxidation of 2-propanol with formation of acetone
is catalyzed by the same alcohol dehydrogenase. Thus, 2-
propanol acts as a cosubstrate for the alcohol dehydroge-
nase, and is oxidized to acetone, which makes the reduction
of corresponding ketone 3 to desired alcohol 4 possible.
When carrying out this biotransformation with the nonsub-
stituted and para-substituted benzylidene acetone sub-
strates 3a,c,d, the reactions proceed under formation of al-
lylic alcohols 4a,c,d, respectively, with conversions in the
range of 44–82% and excellent enantioselectivities of up to
Ͼ99% ee (Scheme 3). Besides its function as a cosubstrate,
a further advantage of 2-propanol as a cosolvent might be
the improved solubility of the hydrophobic ketone in the
reaction mixture.
After having prepared ketones 3 in a reaction medium
that is also suitable for enzymatic reductions, we focused
on the enzymatic reduction of α,β-unsaturated ketones. As
enzymes we used an alcohol dehydrogenase (ADH) from
Lactobacillus kefir[10] as an (R)-enantioselective biocatalyst
and an ADH from Rhodococcus sp.[11] as an (S)-enantiose-
Scheme 3.
With respect to the desired combination of the Wittig
lective biocatalyst.[12] In the preliminary photometer tests, reaction with the biocatalytic reduction, we studied if the
activities of both enzymes for the α,β-unsaturated ketone presence of ylide 2 (used as the substrate in the Wittig reac-
5176
www.eurjoc.org
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2007, 5175–5179

One-Pot Synthesis of Hydrophobic Allylic Alcohols in Aqueous Medium
tion in slight excess) might have a negative impact on the tivity was influenced and gave a somewhat lower activity in
activity of the alcohol dehydrogenase as a result of “cross the presence of ylide 2, we were pleased to find that at an
inhibition”. This study was done by means of a photometer ylide concentration of 0.6 m, which is close to the solubil-
assay (according to the assay conditions described in ref.[13]
)
ity limit, 33% of the enzyme activity remained (relative to
with the use of (S)-ADH from Rhodococcus sp. as an en- the enzyme activity in the absence of ylide 2), and thus it is
zyme and 4-chloroacetophenone as the “standard sub- in a range expected to be sufficient for preparative conver-
strate” for this enzyme (Scheme 4). Albeit the enzyme ac- sions (Scheme 4).
Scheme 4.
Table 1. One-pot two-step synthesis of allylic alcohols.
[a] The synthetic protocol is given in the experimental section. [b] A reduced catalytic amount of 50% was used in this experiment. [c]
The enantiomeric excess was determined by chiral HPLC [Daicel Chiralcel OD column, hexane/2-propanol (95:5) or (99.5:0.5)]. The
absolute configuration of (S)-3a (Entry 1) was determined by comparison of the sign of the optical rotation with the literature data
(ref.[14]), and it was in accordance with the expected absolute configuration known from the usual (S) selectivity of this (S)-ADH. The
absolute configurations of alcohols 3b–d (Entries 2–6) were assigned accordingly.
Eur. J. Org. Chem. 2007, 5175–5179
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5177

M. Kraußer, W. Hummel, H. Gröger
SHORT COMMUNICATION
In the subsequent step, the Wittig reaction and the enzy- which were prepared in situ prior to the enzymatic re-
matic reaction, which both were adapted to a compatible duction by means of a “typical” organic chemical reaction.
reaction media, were combined into a one-pot process. We Further process development is planned including the in
were pleased to find that starting from 4-methylbenzalde- situ removal of acetone to shift the equilibrium to the direc-
hyde (1c) the one-pot two-step synthesis of allylic alcohol tion of the products, which would further improve the con-
4c proceeded successfully under the same reaction condi- versions. In addition, the development of other chemoen-
tions that were applied before for the separated steps (i) zymatic one-pot multistep reactions is currently in progress.
ketone synthesis and (ii) reduction. In this one-pot two-step
synthesis, desired allylic alcohol 4c was also formed with an
Experimental Section
excellent enantioselectivity of Ͼ99% ee (Table 1, Entry 3).
However, the conversion did not exceed 32% after a reac-
tion time of 16 h; thus, the conversion was somewhat lower
relative to the single-step process starting from purified
ketone 3c (60% conversion). This result is in accordance
Protocol for the Spectrophotometer Assay for the Measurement of
the Enzyme Activity in the Presence of Ylide 2: The enzyme activity
(see also Scheme 4) was determined spectrophotometrically meas-
uring the consumption of NADH by oxidation to NAD+ at 340 nm
with the above-mentioned observed inhibition of the en- (ε340 = 6.3 m–1 cm–1) in the presence of 4-chloroacetophenone (5)
as the substrate and ylide 2 as an additive. The concentration of the
ylide was varied between 0 and 0.6 m. For the spectrophotometric
measurement of the enzyme activity, a cuvette (1 mL) was filled
with a buffered solution of 4-chloroacetophenone (960 µL, 5;
10 m; phosphate buffer: pH 7.0, 100 m) containing ylide 2 in
different concentrations, and a buffered solution of NADH (20 µL,
NADH: 12.5 m; phosphate buffer: pH 7.0, 100 m). The reaction
was started by the addition of a 1:100 diluted enzyme solution
(20 µL) of (S)-ADH from Rhodococcus sp. (partially purified;
NADH-dependent; volumetric activity: 488 UmL–1 referring to 4-
zyme in the presence of ylide 2, albeit other effects might
play a role as well. Subsequent process optimization, for
example, of the reaction time and adaptation of the biocata-
lyst amount, led to an improved conversion of 48% after
42 h and an excellent enantioselectivity of Ͼ99% ee when
the (S)-ADH from Rhodococcus sp. was used as the catalyst
with 4-methylbenzaldehyde (1c) as the substrate for this
chemoenzymatic one-pot two-step synthesis (Table 1, En-
try 4).
As a next step, the substrate range of this new one-pot chloroacetophenone as a standard substrate), and subsequently the
consumption of NADH was detected. The relative activities were
determined by comparison of the detected spectrophotometrical
activities in UmL–1 with the one in the experiment without the
addition of ylide 2 (which was regarded as the reference experi-
ment; relative activity: 100%).
two-step synthesis of hydrophobic allylic alcohols was in-
vestigated. A high conversion of 90% was obtained with 4-
nitrobenzaldehyde (1d) and ylide 2 in the presence of the
(S)-ADH from Rhodococcus sp. as the biocatalyst. Subse-
quent downstream processing gave resulting allylic alcohol
(S)-4d in 75% yield and with an enantioselectivity of Ͼ99%
ee (Table 1, Entry 6). Notably, good results were obtained
with other substrates as well (Table 1, Entries 1,2), which
indicates that this chemoenzymatic one-pot two-step syn-
thesis proceeds smoothly with a broad range of substrates.
The desired allylic alcohol products (S)-4a–d were formed
with conversions of 48–90% and with excellent enantio-
selectivities of Ͼ99% ee independent of the substitution
Protocol for the One-Pot Two-Step Synthesis of Allylic Alcohols 4
as Exemplified for the Synthesis of (S)-4d: A 20-mL round-bottom
flask, fitted with a magnetic stir bar, was charged with ylide 2
(0.6 mmol), 4-nitrobenzaldehyde (1d; 0.5 mmol), phosphate buffer
(1.875 mL, 100 m; pH 7.0) and 2-propanol (0.625 mL). The mix-
ture was stirred at room temperature for 2 h. Subsequently, cofac-
tor NADH (0.02 mmol) and an aqueous solution of (S)-ADH from
Rhodococcus sp. (40 µL, partially purified; NADH-dependent; vol-
umetric activity: 488 UmL–1 referring to 4-chloroacetophenone as
pattern. The (R)-enantioselective one-pot synthesis of al- a standard substrate) were added, and the reaction mixture was
stirred for a further 42 h at room temperature. The heterogeneous
mixture was extracted with ethyl acetate, dried with magnesium
sulfate, filtered and concentrated in vacuo. The resulting crude
product, which showed a conversion of 90% for desired product
(S)-4d, was purified by flash chromatography (silica gel 60 Å; ø
1.5 cm; length 22 cm; cyclohexane/ethyl acetate, 4:1) to afford iso-
lated allylic alcohol (S)-4d in 75% yield and with Ͼ99% ee (see
also Table 1, Entry 6).
lylic alcohol (R)-4c was carried out with (R)-ADH from
Lactobacillus kefir as a biocatalyst leading to desired prod-
uct (R)-4c in 31% conversion and with a high enantio-
selectivity of Ͼ99% ee (Table 1, Entry 5).
Conclusions
A one-pot two-step process for the enantioselective syn-
thesis of hydrophobic allylic alcohols was developed, which
comprises ketone formation by the Wittig reaction followed
by their enzymatic in situ biotransformation. By means of
this combined Wittig reaction and bioreduction, the desired
target products were prepared with moderate-to-good con-
versions of 31–90% and with excellent enantioselectivities
of Ͼ99% ee. To the best of our knowledge this chemoen-
zymatic reaction also represents the first example of a one-
pot synthesis of chiral alcohols by an asymmetric alcohol
dehydrogenase catalyzed reduction starting from ketones,
Acknowledgments
The authors would like to thank Mr. Andreas Latza for experimen-
tal assistance, and they are grateful to Dr. Harald Maid, Dr.
Michael Brettreich and Ms. Katja Maurer for their technical sup-
port. The authors also would like to thank OYC International for
donating cofactor samples.
[1] For reviews, see: a) M. Kataoka, M. Kita, M. Wada, Y. Yaso-
hara, J. Hasegawa, S. Shimizu, Appl. Microbiol. Biotechnol.
2003, 62, 437–445; b) S. Buchholz, H. Gröger in Biocatalysis
5178
www.eurjoc.org
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2007, 5175–5179

One-Pot Synthesis of Hydrophobic Allylic Alcohols in Aqueous Medium
[7] For selected examples, see: a) A. L. E. Larsson, B. A. Persson,
in the Pharmaceutical and Biotechnology Industries (Ed.: R. N.
Patel), CRC Press, New York, 2006, ch. 32, pp. 757–790.
[2] For selected recent contributions of efficient biocatalytic
ketone reductions, see: a) M. Wolberg, W. Hummel, C. Wand-
rey, M. Müller, Angew. Chem. 2000, 112, 4476–4478; Angew.
Chem. Int. Ed. 2000, 39, 4306–4308; b) N. Kizaki, Y. Yasohara,
J. Hasegawa, M. Wada, M. Kataoka, S. Shimizu, Appl. Micro-
biol. Biotechnol. 2001, 55, 590–595; c) A. Matsuyama, H. Yam-
amoto, Y. Kobayashi, Org. Process Res. Dev. 2002, 6, 558–561;
d) W. Stampfer, B. Kosjek, C. Moitzi, W. Kroutil, K. Faber,
Angew. Chem. 2002, 114, 1056–1059; Angew. Chem. Int. Ed.
2002, 41, 1014–1017; e) H. Pfründer, M. Amidjojo, U. Kragl,
D. Weuster-Botz, Angew. Chem. 2004, 116, 4629–4631; Angew.
Chem. Int. Ed. 2004, 43, 4529–4531; f) M. Amidjojo, M. Weus-
ter-Botz, Tetrahedron: Asymmetry 2005, 16, 899–901; g) H.
Gröger, F. Chamouleau, N. Orologas, C. Rollmann, K. Drauz,
W. Hummel, A. Weckbecker, O. May, Angew. Chem. 2006, 118,
5806–5809; Angew. Chem. Int. Ed. 2006, 45, 5677–5681; h) H.
Gröger, C. Rollmann, F. Chamouleau, I. Sebastien, O. May, W.
Wienand, K. Drauz, Adv. Synth. Catal. 2007, 349, 709–712; i)
T. Ema, N. Okita, S. Ide, T. Sakai, Org. Biomol. Chem. 2007,
5, 1175–1176; j) G. de Gonzalo, I. Lavandera, K. Faber, W.
Kroutil, Org. Lett. 2007, 9, 2163–2166.
J. E. Bäckvall, Angew. Chem. 1997, 109, 1256–1258; Angew.
Chem. Int. Ed. Engl. 1997, 36, 1211–1212; b) B. Martin-Matute,
M. Edin, K. Bogar, J. E. Bäckvall, Angew. Chem. 2004, 116,
6697–6701; Angew. Chem. Int. Ed. 2004, 43, 6535–6539; c)
B. A. Persson, A. L. E. Larsson, M. Le Ray, J. E. Bäckvall, J.
Am. Chem. Soc. 1999, 121, 1645–1650; d) N. Kim, S. B. Ko,
M. S. Kwon, M. J. Kim, J. Park, Org. Lett. 2005, 7, 4523–4526;
e) S. Wuyts, K. De Temmerman, D. E. De Vos, P. A. Jacobs,
Chem. Eur. J. 2005, 11, 386–397; f) A. Berkessel, M. L. Sebas-
tian-Ibarz, T. N. Müller, Angew. Chem. 2006, 118, 6717–6720;
Angew. Chem. Int. Ed. 2006, 45, 6567–6570.
[8] For a review, see: O. Pamies, J.-E. Bäckvall, Chem. Rev. 2003,
103, 3247–3261.
[9] a) J. Dambacher, W. Zhao, A. El-Batta, R. Anness, C. Jiang,
M. Bergdahl, Tetrahedron Lett. 2005, 46, 4473–4477; b) Very
recently, this Wittig reaction in water was successfully applied
in a chemoenzymatic one-pot reaction for the synthesis of
water-soluble -amino acids by the Rétey group, see ref.[6c]
[10] a) W. Hummel, M.-R. Kula (FZ Jülich GmbH), EP 456107,
1991; b) C. W. Bradshaw, W. Hummel, C.-H. Wong, J. Org.
Chem. 1992, 57, 1532–1536; c) A. Weckbecker, W. Hummel,
Biocatal. Biotransform. 2006, 24, 380–389.
[11] The (S)-alcohol dehydrogenase from Rhodococcus sp., recombi-
nant in E. coli, was developed in the research group of Prof.
Dr. Werner Hummel and is available as a commercial enzyme
formulation from EVOcatal GmbH, Merowinger Platz 1a,
40225 Düsseldorf, Germany; internet: http://www.evocatal.com
[12] For other alcohol dehydrogenases suitable as a catalyst for the
reduction of α,β-unsaturated ketones, see, for example, ref.[1b]
[13] H. Gröger, W. Hummel, C. Rollmann, F. Chamouleau, H.
Hüsken, H. Werner, C. Wunderlich, K. Abokitse, K. Drauz, S.
Buchholz, Tetrahedron 2004, 60, 633–640.
[3] For a review, see: R. Noyori, T. Okhuma, Angew. Chem. 2001,
113, 40–75; Angew. Chem. Int. Ed. 2001, 40, 40–73.
[4] For selected very recent examples underlining the power of
chemocatalytic one-pot syntheses, see: a) D. Enders, H. R. M.
Hüttl, C. Gronau, G. Raabe, Nature 2006, 441, 861–863; b) D.
Enders, M. R. M. Hüttl, J. Runsink, G. Raabe, B. Wendt, An-
gew. Chem. 2007, 119, 471–473; Angew. Chem. Int. Ed. 2007,
46, 467–469; c) A. Carlone, S. Cabrera, M. Marigo, K. A.
Jørgensen, Angew. Chem. 2007, 119, 1119–1122; Angew. Chem.
Int. Ed. 2007, 46, 1101–1104.
[5] For reviews, see: a) L. F. Tietze, G. Brasche, K. M. Gericke,
Domino Reactions in Organic Synthesis, Wiley-VCH,
Weinheim, 2006; b) D. Enders, C. Grondal, M. R. M. Hüttl,
Angew. Chem. 2007, 119, 1590–1601; Angew. Chem. Int. Ed.
2007, 46, 1570–1581.
[6] For selected examples, see: a) H. J. M. Gijsen, C.-H. Wong, Tet-
rahedron Lett. 1995, 36, 7057–7060; b) R. Schoevaart, T. Kie-
boom, Tetrahedron Lett. 2002, 43, 3399–3400; c) C. Paizs, A.
Katona, J. Rétey, Eur. J. Org. Chem. 2006, 1113–1116.
[14] a) H. C. Brown, G. G. Ganesh, J. Org. Chem. 1985, 50, 1384–
1394; b) T. Itoh, Y. Matsushita, Y. Abe, S.-h. Han, S. Wada, S.
Hayae, M. Kawatsura, S. Takai, M. Morimoto, Y. Hirose,
Chem. Eur. J. 2006, 12, 9228–9237.
Received: July 13, 2007
Published Online: September 20, 2007
Eur. J. Org. Chem. 2007, 5175–5179
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5179