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Enantioselective Enzymatic Reactions in Miniemulsions as Efficient УNanoreactorsФ.

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Angewandte
Chemie
Miniemulsions
DOI: 10.1002/anie.200502854
Enantioselective Enzymatic Reactions in
Miniemulsions as Efficient “Nanoreactors”**
Harald Grger,* Oliver May, Hendrik Hsken,
Sandrine Georgeon, Karlheinz Drauz, and
Katharina Landfester*
The application of enzyme-catalyzed syntheses is of high
industrial interest for the manufacture of enantiomerically
pure compounds.[1] The well-known high selectivity, in
particular enantioselectivity, of enzymes represents a particular advantage; however, conducting enzymatic reactions at
[*] Dr. H. Grger, Dr. O. May, Dipl.-Ing. (FH) H. H"sken,
Dipl.-Ing. (FH) S. Georgeon
Degussa AG
Service Center Biocatalysis
P.O. Box 1345, 63403 Hanau (Germany)
Fax: (+ 49) 6181-592961
E-mail: harald.groeger@degussa.com
Prof. Dr. K. Landfester
University of Ulm
Organic Chemistry III/Macromolecular Chemistry
Albert-Einstein-Allee 11, 89081 Ulm (Germany)
Fax: (+ 49) 731-50-22883
E-mail: katharina.landfester@chemie.uni-ulm.de
Prof. Dr. K. Drauz
Degussa AG
Corporate Center Innovation Management
P. O. Box 1345, 63403 Hanau (Germany)
[**] We thank Dr. F.-R. Kunz and Dr. M. Janik and their teams (Degussa,
AQura GmbH) for carrying out the chiral HPLC analyses and the
NMR-spectroscopic measurements.
Angew. Chem. Int. Ed. 2006, 45, 1645 –1648
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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high substrate concentrations—a key criterion for technical
applications—often proves difficult.[2] A proven methodology
for the accomplishment of (enzymatic) reactions at high
substrate concentrations significantly exceeding the solubility
limit and preferably in the range of > 100 g L 1 is the use of
enzyme-compatible two-phase solvent systems, consisting of
an aqueous phase and an organic solvent. This concept has
been applied successfully for many enzymatic reactions.[3, 4]
Besides high substrate concentrations and engineering advantages, in addition an increase of activity has been often
observed when interface-active enzymes are used.
Nevertheless, alternative concepts to the “classic” twophase solvent system are desirable that would result in
increased homogeneity of the reaction mixture, increased
overall interface area, and improvement of stirrability. These
issues may be addressed by stable miniemulsions, homogenous mixtures in which the organic phase is dispersed in the
form of very small “nanodroplets” with diameters in the range
of 50–500 nm.[5a] The suitability of these liquid “nanoreactors”,[5c] the stable droplets in miniemulsions, for enzymatic
transformations in general has been demonstrated recently by
Landfester and co-workers for the polymerization of lactones.[6] Here, we report the first enantioselective enzymatic
reactions in minemulsions and the suitability of this concept
for the preparation of optically active a- and b-amino acids at
very high substrate concentrations of 500 to > 800 g L 1.
As a benchmark for comparison of the reaction characteristics of miniemulsions with those of the “classic” two-phase
system, we chose the industrially established lipase-catalyzed
hydrolysis of racemic b-amino acid n-propyl esters in methyl
tert-butyl ether (MTBE)/water for the preparation of enantiomerically pure b-amino acids.[7, 8] When racemic b-phenylalanine n-propyl ester, rac-1, was used as a substrate under
standard conditions in the two-phase solvent system, a
conversion of 50 % was obtained within 15 h at a high
substrate concentration of 242 g L 1 [Scheme 1, Eq. (1)].[7]
The reaction proceeds enantioselectively with an E value of
> 100, and the resulting optically active b-amino acid (S)-b-
Scheme 1. Reaction in a two-phase solvent system [Eq. (1)] and in a
miniemulsion [Eq. (2)], both at a substrate concentration of 242 g L 1.
Reaction conditions: a) lipase from Pseudomonas cepacia (Lipase PS
“Amano”, 9 g L 1), H2O/MTBE 1:1; 15 h, pH 8.2, 20 8C; b) lipase from
Pseudomonas cepacia (Lipase PS “Amano”, 9 g L 1), H2O, surfactant
(1 %), hexadecane (1 %), ultrasound; 6 h, pH 8.2, 20 8C.
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phenylalanine, (S)-2, was obtained with an enantiomeric
excess of > 99.4 % ee.[9]
The analogous enantioselective enzymatic reaction in a
miniemulsion as a nanoreactor required a stable miniemulsion of the racemic b-amino acid n-propyl ester, rac-1, in
aqueous media (Scheme 2). The miniemulsion was prepared
by initial addition of a surfactant (Lutensol AT 50, 1 %) and a
Scheme 2. Concept for the formation of miniemulsions for enzymatic
reactions for the preparation of the (S)-b-amino acid (S)-2.
hydrophobic compound (hexadecane, 1 %) to avoid dissolution of the nanodroplets under formation of a “normal” twophase system as a result of aggregation and Ostwald ripening.
Subsequent treatment of this mixture with ultrasound gave a
stable miniemulsion, which contained nanodroplets of a
defined size[6] (typical diameter of 100–150 nm). This stable
miniemulsion was then used as a reaction mixture for the
desired enzymatic reactions.
When the miniemulsion was used as reaction media, the
enantioselective lipase-catalyzed hydrolysis was significantly
faster with a conversion of 49 % after a reaction time of only
6 h. After (nonoptimized[10]) workup the desired product (S)2 was obtained in 38 % yield and with an enantiomeric excess
of > 99.4 % ee [Scheme 1, Eq. (2)]. This accelerated reaction
course also implies that the reaction can be conducted with
smaller amounts of enzymes. When the reaction time was
prolonged to 17 h, the same conversion as that of the
benchmark process was obtained with only 50 % of the
original amount of enzyme. In general, the lipase was added
after treatment of the reaction mixture with ultrasound.
Surprisingly, the reaction also proceeded when the enzyme
was added prior to the ultrasound treatment, although the
conversion was somewhat lower (42 % versus 47 % after a
reaction time of 17 h).
The reaction mixture is significantly more homogeneous
than the two-phase system. The high homogeneity of the
miniemulsion is underlined by a visual comparison (Figure 1).
In comparison with the reaction mixture prior to the
formation of the miniemulsion (left photo), consisting of the
two clearly separated phases water and substrate, just one
“visible” phase with a high degree of homogeneity remains
after formation of the miniemulsion (right photo). In this
connection, the stability of the miniemulsion, in spite of the
precipitation of the desired product (S)-2 during the reaction
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2006, 45, 1645 –1648
Angewandte
Chemie
Figure 1. Homogeneity of the stable miniemulsion (right) in comparison to the reaction mixture prior to formation of the miniemulsion
(left) from the separated phases water and substrate.
(which is subsequently isolated by filtration), is a surprising
effect. Thus, the presence of a solid product that precipitates
during the reaction does not have a negative impact on the
reaction course in the miniemulsion.
The high degree of homogeneity and excellent stirring
properties of the reaction solution prompted us to further
increase the substrate concentration. We were pleased to find
that the enzymatic synthesis of (S)-b-phenylalanine (S)-2
proceeds with high efficiency even at a substrate concentration
of 484 g L 1 ( 2.3 m) with a conversion of 45 % after 17 h. In
addition, a (nonoptimized[10]) yield of 37 % was obtained, and
the enantiomeric excess of the desired (S)-b-phenylalanine (S)-2
was also > 99.4 % ee (Scheme 3). To our knowledge, this is one
of the highest substrate concentrations reported for enantioselective enzymatic reactions so far.[11] A further increase of the
substrate concentration up to 605 g L 1, however, resulted in
somewhat lower conversion (42 % after 24 h).
Scheme 3. Enzymatic synthesis of the (S)-b-amino acid (S)-2 in a
miniemulsion at a substrate concentration of 484 g L 1. Reaction
conditions: a) lipase from Pseudomonas cepacia (Lipase PS “Amano”,
9 g L 1), H2O, surfactant (1 %), hexadecane (1 %), ultrasound; 17 h,
pH 8.2, 20 8C.
Subsequently, we extended this approach to the synthesis
of analogous a-amino acids at high substrate concentrations.
We chose as a model reaction the enzymatic hydrolysis of
racemic phenylalanine esters, which was carried out previously at a substrate concentration of 20 g L 1 in the presence
of a lipase from porcine pancreas (PPL).[12] In our experiments we used the corresponding n-propyl ester rac-3.[13]
Initial experiments with and without miniemulsions at a
substrate concentration of 2.0 m (414 g L 1) were studied with
respect to their reaction rates (pH 7, room temperature, 1 g of
a commercial PPL enzyme preparation per L). Once again a
higher reaction rate was found for the reaction carried out in a
Angew. Chem. Int. Ed. 2006, 45, 1645 –1648
miniemulsion. The reaction rate was 2.4 times higher than
that in the corresponding experiment without the miniemulsion. After subsequent process development, this PPLcatalyzed hydrolysis of rac-phenylalanine n-propyl ester in a
minemulsion proceeded at a substrate concentration of
414 g L 1 with a conversion of ca. 50 % after a reaction time
of 22 h. After workup, the desired a-amino acid l-phenylalanine, (S)-4, was obtained with an enantiomeric excess of
94 % ee and in a yield of 36 % [Scheme 4, Eq. (1)]. Thus,
enantioselective enzymatic reactions in miniemulsions at high
substrate concentrations of > 400 g L 1 can be carried out not
only for the synthesis of optically active b-amino acids, but
also for the synthesis of the analogous a-amino acids. In a
further step the substrate concentration was increased up to
4.0 m rac-3 (827 g per L of aqueous solvent). The reaction also
proceeded efficiently and gave the product l-phenylalanine
(S)-4 in 40 % yield and with an enantiomeric excess of 94 % ee
[Scheme 4, Eq. (2)].
Scheme 4. Enzymatic syntheses of l-phenylalanine (S)-4 in a miniemulsion. Reaction conditions: a) lipase from porcine pancreas
(Lipase PPL, Sigma, 1 g L 1), H2O, surfactant (1 %), hexadecane (1 %),
ultrasound; 22 h, pH 7.0, RT; b) lipase from porcine pancreas (Lipase PPL, Sigma, 5.6 g L 1), H2O, surfactant (1 %), hexadecane (1 %),
ultrasound; 21 h, pH 7.0, RT.
In summary, we have carried out the first enantioselective
enzymatic reactions in miniemulsions as efficient nanoreactors. The methodology proved to be suitable for the preparation of a-amino acids as well as b-amino acids with high
enantiomeric excesses of up to > 99 % ee. A specific key
feature of these reactions, which proceed with high conversions, are the high substrate concentrations of 500 to
> 800 g L 1, which are among the highest substrate concentrations reported so far for enantioselective enzymatic
reactions. We are currently extending this technique to the
reactions of other substrates and addressing its technical-scale
application. Since the re-use of the biocatalyst is also
conceivable in principle, the evaluation of such a biocatalyst
recycling will also be a research topic in the future.
Experimental Section
Lipase-catalyzed enantioselective hydrolysis in miniemulsions (exemplified for the hydrolysis of rac-1 at a substrate concentration of
484 g L 1): A mixture of 1.21 g of the lipase PS “Amano” (lipase from
Pseudomas cepacia; purchased from Amano Enzymes, Inc.) in 40 mL
of a surfactant solution was filtered in order to separate the enzyme
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
1647
Communications
solution from the nonsoluble solid, thus resulting in the enzyme
concentrate. In parallel, a miniemulsion consisting of rac-1 (65.2 g),
hexadecane (1.37 g), and 96 mL of a 1 % surfactant solution (Lutensol
AT 50, BASF AG) was prepared by stirring these components with an
ultrasound tip (4 min at 200 W). Subsequently, the enzyme concentrate and the miniemulsion were mixed. In addition, the pH was
adjusted to pH 8.2 and maintained at this value by automated
titration with a 1m aqueous solution of sodium hydroxide. The
reaction temperature was 20 8C, and the reaction time was 17 h.
During the reaction, a white precipitate consisting of the desired
product (S)-2 forms. After the reaction, 160 mL of acetone were
added in order to complete the precipitation, and the resulting
mixture was stirred for a further 45 min. The solid material was
filtered and washed with a small amount of acetone and 100 mL of
MTBE. At a reaction time of 17 h, a conversion of ca. 45 % was
achieved. The product (S)-2 was isolated after work-up in a yield of
37 % and with an enantiomeric excess of > 99.4 % ee.
[10]
[11]
[12]
[13]
respectively, see e.g.: K. Faber, Biotransformations in Organic
Chemistry, 4th ed., Springer, Berlin, 2000, chap. 2.1.1, pp. 28 – 52.
The workup was analogous to that described for the reaction in
the two-phase solvent system, see Ref. [7]. The development of a
workup protocol specifically for reactions in miniemulsions is
the subject of future work.
Further examples for biocatalytic reactions at high substrate
concentrations are described, e.g., in: a) A. Liese, K. Seelbach,
C. Wandrey, Industrial Biotransformations, Wiley-VCH, Weinheim 2000; b) D. R. Yazbeck, C. A. Martinez, S. Hu, J. Tao,
Tetrahedron: Asymmetry 2004, 15, 2757 – 2763; c) M. Kataoka,
K. Kita, M. Wada, Y. Yasohara, J. Hasegawa, S. Shimizu, Appl.
Microbiol. Biotechnol. 2003, 62, 437 – 445; d) M. Schmidt, H.
Griengl, Top. Curr. Chem. 1999, 200, 193 – 226.
J.-Y. Houng, M.-L. Wu, S.-T. Chen, Chirality 1996, 8, 418 – 422.
The n-propyl ester of rac-phenylalanine, rac-3, is considerably
more stable than the corresponding methyl ester, which undergoes a nonenzymatic hydrolysis to a significant extent.
Received: August 11, 2005
Published online: February 7, 2006
.
Keywords: biphasic catalysis · emulsions · enantioselectivity ·
enzyme catalysis · hydrolysis
[1] Enzyme Catalysis in Organic Synthesis, Vol. 1–3, 2nd ed. (Eds.:
K. Drauz, H. Waldmann), Wiley-VCH, Weinheim 2002.
[2] For a review, see: P. S. J. Cheetham, J. Biotechnol. 1998, 66, 3 –
10.
[3] For selected examples of commercial biotransformations in twophase solvent systems, see: a) M. Kataoka, K. Kita, M. Wada, Y.
Yasohara, J. Hasegawa, S. Shimizu, Appl. Microbiol. Biotechnol.
2003, 62, 437 – 445; b) N. M. Shaw, K. T. Robins, A. Kiener in
Asymmetric Catalysis on Industrial Scale (Eds.: H. U. Blaser, E.
Schmidt), Wiley-VCH, Weinheim, 2004, pp. 105 – 115; c) P.
Poechlauer, W. Skranc, M. Wubbolts in Asymmetric Catalysis
on Industrial Scale (Eds.: H. U. Blaser, E. Schmidt), Wiley-VCH,
Weinheim, 2004, pp. 151 – 164; d) Ref. [7].
[4] A further interesting concept is the use of water-in-oil microemulsions; for selected contributions, see: a) B. Orlich, H.
Berger, M. Lade, R. SchomKcker, Biotechnol. Bioeng. 2000, 70,
638 – 646; b) H. Stamatis, A. Xenakis, F. N. Kolisis, Biotechnol.
Adv. 1999, 17, 293 – 318; c) K. Holmberg, Adv. Colloid Interface
Sci. 1994, 51, 137 – 174.
[5] a) K. Landfester, M. Antonietti in Colloids and Colloid Assemblies (Ed.: F. Caruso), Wiley-VCH, Weinheim, 2004, chap. 6,
pp. 175 – 215; b) The expression “nanoreactors” describes stable
droplets on the nanometer scale in miniemulsions, in which or at
which the desired reaction occurs; for this concept of “nanoreactors” also see: K. Landfester, Abstracts of Papers, 224th ACS
National Meetings, Boston, MA, USA, August 18 – 22, 2002 and
Ref. [5a].
[6] A. Taden, M. Antonietti, K. Landfester, Macromol. Rapid
Commun. 2003, 24, 512 – 516.
[7] H. GrMger, H. Werner (Degussa AG), US Patent 6869781, 2005.
[8] For previous, selected contributions to the hydrolase-catalyzed
resolution of rac-b-amino acid esters and corresponding Nacylated derivatives, see: a) S. G. Cohen, S. Y. Weinstein, J. Am.
Chem. Soc. 1964, 86, 725 – 728; b) M. Prashad, D. Har, O. Repic,
T. J. Blacklock, P. Giannousis, Tetrahedron: Asymmetry 1998, 9,
2133 – 2136; c) S. Katayama, N. Ae, R. Nagata, Tetrahedron:
Asymmetry 1998, 9, 4295 – 4299; d) S. J. Faulconbridge, K. E.
Holt, L. G. Sevillano, C. J. Lock, P. D. Tiffin, N. Tremayne, S.
Winter, Tetrahedron Lett. 2000, 41, 2679 – 2681.
[9] For the definition of the terms “E value” and “ee value ” for the
quantification of the enantioselectivity and enantiomeric excess,
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2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2006, 45, 1645 –1648
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