Enantioselective catalysis and analysis on a chip

Article abstract of DOI:10.1002/anie.200504205

A chip off the old block: A prototype of a miniaturized catalysis laboratory is successfully applied for screening enantioselective biocatalysts created by directed evolution of enzymes. For this purpose an integrated microfluidic catalysis/analysis chip has been developed (see scheme SO: sample outlet, SI: alternative sample inlet, BI: buffer outlet). (Chemical Equation Presented)

Full text of DOI:10.1002/anie.200504205

Angewandte
Chemie
Chip Laboratories
products was, however, performed offline utilizing traditional
GC/MS.[8]
We have now implemented a novel integrated approach
DOI: 10.1002/anie.200504205
which combines a microfluidic reactor and analysis unit on a
single device. We have utilized this reaction/analysis chip for
testing the enantioselectivity of enzyme mutants created by
directed evolution of the enzymes.[9] As a model system, the
epoxide hydrolase catalyzed hydrolytic kinetic resolution of
glycidyl phenyl ether[10] was integrated with chiral electro-
phoresis on a chip.
Enantioselective Catalysis and Analysis on a
Chip**
Detlev Belder,* Martin Ludwig, Li-Wen Wang, and
Manfred T. Reetz*
The working principle of the chip we developed for this
purpose is shown in Figure 1. Substrate and catalyst are
The application of microsystems technology to chemistry
enables miniaturization of chemical processes on so-called
microfluidic chips, where reactions are performed in micro
channels and cavities rather than in flasks and tubes. The
ambitious aim is to shrink chemical laboratories to so called
lab-on-a-chip systems. Besides the advantages of miniatur-
ization, such as improved portability, safety, and reduced
reagent consumption, one of the most promising features of
integrated palmtop laboratories is the accelerated speed of
reaction and analysis,[1] with corresponding potential for high-
throughput screening. While the introduction of classical
high-throughput screening systems based on robotics and
massive parallelization has revolutionized chemical synthesis,
miniaturization and integration on chip size would constitute
a quantum leap.
Currently, significant progress is being made in micro-
fluidics and its application for down-sizing chemical reac-
tors.[2] One of the most successful applications of microfluidics
in analytical chemistry is microchip electrophoresis (MCE),
which can be regarded as a miniaturized version of classical
capillary electrophoresis. One of the most prominent features
of MCE is the extraordinary high speed of analysis, which
could also be successfully applied to the separation of
enantiomers by us[3] and others.[4] By applying MCE to the
enantioselective separations of amino acids in less than a
second we have already reported the fastest chiral separa-
tion.[5] In previous work we laid the foundation for the general
applicability of chip electrophoresis to chiral high-throughput
screening of enantioselective catalysts.[6]
The ultimate potential of chip technology in chemistry can
only be exploited fully by the integration of chemical
synthesis and analysis. While complex integrated lab-on-a-
chip systems have recently been described for biochemical
applications, such as genetic analysis of the influenza virus,[7]
similar achievements remain to be made in synthetic chemis-
try. Recently, a miniaturized T-reactor was impressively
applied in asymmetric synthesis, chiral analysis of the reaction
Figure 1. Schematic drawing of the working principle of the integrated
catalysis/analysis chip. SO: sample outlet, SI: alternative sample inlet,
BI: buffer outlet.
placed in respective microvials on the chip. Upon applying
pressure or electrical fields, enzyme and substrate solutions
are merged through microfluidic channels, the mixing and
reaction are aided by means of a meandering channel. The
reaction products are then guided to the separation channel
by applying a voltage-controlled pinched injection.[11] The
actual electrophoretic separation of products and educts
occurs in an electrical field in the separation channel.
A photo of such a microfluidic chip made from fused-
silica[12] together with a typical example of a separation of
(+/À) substrate and (+/À) product is shown in Figure 2. This
prototype contains four microvials for dispensing different
substrates or catalysts, enabling, for example, three catalysts
acting on one substrate to be tested in a single set of
experiments.
For separation of the compounds a 50 mmolLÀ1 borate
buffer at pH 8.5 containing 15 mmolLÀ1 heptakis-6-sulfato-b-
cyclodextrin as a chiral selector was utilized. Under these
conditions product and educt can simultaneously be sepa-
rated into the respective enantiomers in less than 90 seconds
(Figure 2). For the detection of the analytes we applied native
fluorescence detection, using a deep-UV laser (Nd:YAG, l =
266 nm); a technique that we only recently introduced in chip
analysis[13] and which requires UV-transparent fused-silica
chips, rather than the commonly applied borofloat glass as
chip material.
[*] Dr. D. Belder, Dr. M. Ludwig, L.-W. Wang, Prof. Dr. M. T. Reetz
Max-Planck-Institut für Kohlenforschung
Kaiser-Wilhelm-Platz 1
45470 Mülheim an der Ruhr (Germany)
Fax: (+49)208-306-2982
E-mail: belder@mpi-muelheim.mpg.de
[**] We acknowledge financial support from the Fonds der Chemischen
Industrie and the DFG (SSP1170) and L.-W. Wang acknowledges
support from the DAAD for a doctoral stipend.
This new system was successful in testing the enantiose-
lectivity of new enzyme mutants created by directed evolu-
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chiral HPLC (Table 1). The agreement is good, which shows
in principle the applicability of our miniaturized integrated
system. The data for the chip experiments in Table 1 are mean
values of triplicate determinations. The relative standard
Table 1: Comparison of experimental results for determination of
conversion, enantiomeric excess (ee), and E value obtained with the
catalysis/analysis chip or with conventional reaction in bench-scale
format and subsequent HPLC-Analysis.[a]
Enzyme
Method
Conv [%]
ee diol [%]
E
wild type
chip
38
18
22
23
43
40
28
37
41
48
49
58
90
82
84
84
93
89
95
95
4
4
bench-scale/HPLC
chip
bench-scale/HPLC
chip
bench-scale/HPLC
chip
bench-scale/HPLC
chip
LW080
LW086
LW144
LW202
23
13
21
20
37
29
101
115
Figure 2. Photo of the microfluidic chip in comparison with a 2-Euro
coin together with the reaction scheme (top) and the electrophoretic
data (bottom) of the separated enantiomers of product and precursor.
tion. The data of on-chip electrophoretic separations after on-
chip reaction of different epoxide hydrolase mutants[10b] from
Aspergillus niger, as well as from the wild type,[10a] are shown
in Figure 3. Reaction on-chip was possible using purified
bench-scale/HPLC
[a] The data are mean values from triplicate experiments.
deviations (RSD) are in a quite acceptable range, especially in
view of the fact that these are data from a prototype. For the
ee value determinations we obtained RSDs of 1–3% and for
the E values RSD of 3–21% were determined. Although the
RSDs for the E values are somewhat high, unequivocal
identification of the best mutants is still possible.
In the experiments described above the mixing process
and reaction of substrates and enzymes occurred in micro-
fluidic channels on a chip. The liquids were forced through the
reaction channel by the application of vacuum; alternatively
this was also carried out by applying an electrical field. Due to
the long channel geometry the voltage driven reaction step
was, however, more time consuming (about 25 min) than the
vacuum transport (10 min). By integration of a more effective
and faster micromixer it can be expected that the reaction and
mixing times can be reduced considerably.[15]
Instead of merging substrate and enzyme in a microfluidic
channel, mixing and reaction can be performed much faster
and straightforward by pipetting the compounds together in a
microvial. The reaction will then start instantly, which
however may be disadvantageous for testing large libraries
of catalysts because different conversions are then to be
expected. On the other hand, this instant-start effect can be
exploited to study the conversion and selectivity in real time
by utilizing the fast and integrated analysis part of the chip.
This approach can be put into practice by pipetting substrate
and enzyme into the microvial labeled BI (Figure 1) and
applying a so-called gated injection[16] by repetitive switching
between defined electrical potentials. This results in sequen-
tial injections of reactants into the separation channel from a
continuous sample flow. As a result stacked electrophero-
grams are obtained which can be used to follow the kinetics of
the reaction. (Figure 4).
Figure 3. Application of the system for testing the wild type as well as
different epoxide hydrolase mutants created by directed evolution.
Electrophoretic separation of product and educt enantiomers after on-
chip catalysis. The E values were calculated from the corresponding
single measurements.
enzymes in the case of the wild type or cell lysates and with
whole cells. No interfering signals for the enzymes or cell
lysates or cells where obtained under the experimental
conditions employed. As reaction medium a 10 millimolar
phosphate buffer at pH 7.2 containing 7.5% (v/v) acetonitrile
was employed. With this approach it is possible to reliably
differentiate the enzymes with respect to their selectivities,
(Figure 3). While a selectivity factor[14] of E = 4 was obtained
for the wild type, an E value of 118 was determined for the
best mutant LW202 in the respective single measurements.
The results obtained with the miniaturized catalysis/
analysis chip were compared with those determined by the
traditional procedure, namely reaction on bench-scale format
and subsequent analysis of the supernatant with conventional
The respective data together with the extracted values of
conversion and E value are displayed in Figure 5. These
experiments were carried out with 10 mL of a 0.02 mgmLÀ1
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Herein we have demonstrated the first example of an
integrated chip-based system applied in synthetic chemistry
and catalysis which integrates a microfluidic reactor with
chemical analysis. In this initial study the device was success-
fully applied for testing enantioselective biocatalysts created
by directed evolution of enzymes; the device also has great
potential for high-throughput screening. Integration of chem-
ical reaction and analysis on a chip results in reduced reagent
consumption and allows the speed of the whole screening
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Received: November 25, 2005
Published online: January 27, 2006
Keywords: directed evolution · electrophoresis · enantiomer
.
separation · high-throughput screening · microreactors
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