Asymmetric organocatalysis and analysis on a single microfluidic nanospray chip

Article abstract of DOI:10.1002/anie.201102331

Organocatalysis meets chip MS: By integrating asymmetric organocatalysis and analysis on a chip (see picture), optimization of reactions can be achieved with minimal consumption of resources in a short period of time.

Full text of DOI:10.1002/anie.201102331

DOI: 10.1002/anie.201102331
Lab on a Chip
Asymmetric Organocatalysis and Analysis on a Single Microfluidic
Nanospray Chip**
Stefanie Fritzsche, Stefan Ohla, Petra Glaser, David S. Giera, Marcel Sickert,
Christoph Schneider, and Detlev Belder*
The miniaturization of chemical processes onto so-called lab-
on-a-chip devices has gained significant importance in differ-
ent fields of chemistry. Besides the advantages of enhanced
portability, reduced reagent consumption, and improved
safety, a characteristic feature of miniaturized platforms is
the possibility to achieve higher reaction and analysis rates.[1]
From their roots in analytical sciences, microfluidic systems
have received much attention over the past decade. At
present, microfluidics is becoming increasingly popular in
inorganic and organic chemistry, as syntheses are performed
on chip-based microreactors[2] or capillary-based microflow
reactors.[3] While diverse reactions have been performed in
microfluidic chip devices with impressive results,[4] the
analytical characterization is, however, usually carried out
off-chip by conventional macroscopic instruments. However,
this approach does not exploit the promise and the full
potential of chip technology, namely, the integration of
different functionalities such as chemical synthesis and
analysis on one single device. Thus, it is desirable to develop,
in analogy to microelectronics, integrated chemical circuits[5]
as new chemical tools, for example, for catalyst screening[6] or
for online monitoring of biological processes.[7]
In previous work we demonstrated a first approach for
integrating chemical reactions and analysis on a single
microchip for the screening of enantioselective biocatalysts.[8]
Nevertheless, this system was limited to aqueous media and
native fluorescent molecules.[9]
Therefore we intended to develop an advanced chip
system with a wider applicability in synthetic chemistry,
including the utilization of non-aqueous reaction media and a
more general detection system. In this context, the coupling to
mass spectrometry appears to be very attractive,[10] as it
provides additional structural information for substance
identification.
Odedra and Seeberger[12] described a first miniaturized
approach, performing organocatalysis in a microfluidic flow
reactor chip. The reaction products were, however, analyzed
offline by traditional HPLC.
Herein we present the, to our knowledge, first asymmetric
organocatalytic reaction on a single chip with integrated
analysis. As a model system we chose the enantioselective
vinylogous Mannich reaction published in 2008, which is
catalyzed by chiral phosphoric acid.[13] The alcoholic solvent
mixture and the nonfluorescent reaction products in this
synthesis are quite challenging for chip integration. Thus, it
was necessary to adapt the reaction media to the aqueous
separation electrolyte on-chip to enable an undistorted
electrophoretic analysis of the reaction products.
Therefore, we developed a new microfluidic chip design
containing different functionalities, including a reaction
structure for the organic synthesis, a structure for aqueous
dilution of the reaction mixture, a cross section for injection,
and a separation channel for chip electrophoresis. Further-
more, the chip layout includes an integrated nanoelectrospray
emitter with makeup-flow channels for dead-volume free
coupling to mass spectrometry at the end of the separation
channel.[14] A schematic drawing of the chip with 50 mm wide
channels and a photo is shown in Figure 1.
Reaction and analysis processes on the single chip were
carried out as follows: initially, the reaction structure was
filled with the alcoholic solvent mixture for synthesis; then
the remaining structure was replenished with the aqueous
separation electrolyte. Afterwards, each reactant solution
To demonstrate our concept, we focused on organocatal-
ysis, which has been one of the most innovative research fields
in synthetic chemistry over the past years.[11] Recently,
[*] S. Fritzsche, S. Ohla, P. Glaser, Prof. Dr. D. Belder
Institute of Analytical Chemistry, University of Leipzig
Linnꢀstrasse 3, 04103 Leipzig (Germany)
E-mail: belder@uni-leipzig.de
D. S. Giera, Dr. M. Sickert, Prof. Dr. C. Schneider
Institute of Organic Chemistry, University of Leipzig
Johannisallee 29, 04103 Leipzig (Germany)
[**] This work was partly supported by the Deutsche Forschungsge-
meinschaft (SPP 1179 “Organokatalyse,” Schn 441/7-1)
Figure 1. Schematic drawing (top) and photograph (bottom) of the
chip layout with reaction, separation, and mass spectrometric detec-
tion by nanoelectrospray (nanoES) ionization.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201102331.
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Communications
(8 mL) was placed in the respective microvial of the chip, and
The resulting reconstructed electropherogram of an asym-
metric on-chip catalysis with enantiomer separation as well as
a schematic drawing of the second microchip is depicted in
Figure 3.
the reaction in the mixing structure was initialized by applying
pressure and voltages. The organic reaction phase was
subsequently diluted with the separation electrolyte consist-
ing of 0.1 vol% acetic acid and 25% methanol (v/v) in the
second serpentine structure. Thereafter, the aqueous alco-
holic reaction mixture was guided to the separation channel
by applying a voltage-controlled pinched injection.[15] The
separation was carried out by electrophoresis, and at the end
of the separation channel, the analytes were transferred into
the mass spectrometer via the integrated nanospray emitter.
Further information and details of the working principle are
described in the Supporting Information.
The MS electropherogram in Figure 2 exemplarily shows
the result of the integrated process with on-chip catalysis and
analysis. Since the reaction was carried out in 3 min and the
Figure 3. Electropherogram reconstructed from the extracted mass
traces showing the chiral separation of on-chip reaction products.
This setup enabled the rapid determination of the
enantiomeric excess of an asymmetric reaction. For the
example given in Figure 3, we obtained 55% ee. The reaction
yields can be evaluated by adding an internal standard to
ensure exact quantization. To test our system as a screening
tool, we performed the reaction with different binol-based
phosphoric acids and compared the results to traditional
batch experiments with external HPLC analysis (Table 1).
The given ee values for chip experiments are mean values
of triplicate determination with standard deviation of less
than 5%. These data show that the enantioselectivity induced
by the investigated catalysts can be reliably determined. In
comparative batch experiments, it was found that the
enantioselectivities can be considerably increased by reducing
the temperature to À308C (Table 1). Furthermore, ee values
above 90% could be obtained in optimized solvent mixtur-
es.[13e]
In comparison to macroscopic synthesis the microfluidic
experiments were performed at adapted conditions to
increase the robustness of the microfluidic processes. Never-
theless, the data show that rapid and unequivocal identifica-
tion of the best hits is possible. Beside the variation of the
catalyst, we also investigated different imines, which resulted
in good agreement between traditional macroscopic and chip-
based synthesis. As an example, the reaction of (E)-N-(4-
ethylbenzylidene)-4-methoxyaniline under catalysis of 3,3’-
dimesityl-substitued binol-based phosphoric acid is shown in
Scheme 1.
Figure 2. Total ion electropherogram and extracted mass traces with
respective mass spectra of the on-chip organocatalysis. TIC=total ion
current.
separation with MS-detection was performed in 1.5 min, the
total time for synthesis and analysis took less than 5 min. With
the help of the mass spectra, the reaction product 3a as well as
the starting material 1a could be clearly identified, whereby
the imine immediately hydrolyzes in aqueous media and was
detected as the corresponding primary amine.
After successful integration of on-chip catalysis and
analysis, the next challenge was the enantiomeric separation
of the reaction product in order to determine the ee-value of
the synthesis. This can be achieved by chiral modified chip
electrophoresis.[16] In automated capillary electrophoresis
experiments, the chiral selector hydroxylproyl-g-cyclodextrin
was found to be a suited additive for the separation electro-
lyte. However, the transfer to the chip scale was difficult
because of the short separation length of 7.5 cm. Thus, we
developed a second microchip with an elongated separation
length of 23 cm to facilitate the separation of the enantiomers.
To our knowledge, this enantiomer separation is the first to be
performed on a microchip with mass spectrometric detection.
The organocatalytic synthesis was carried out by pipetting
and mixing the reactant solutions directly in one microvial.
The high process speed achievable with our microfluidic
nanospray chips allows us to monitor reactions instantane-
ously by mass spectrometry. As model system we chose the
organocatalytic transformation of 1a from Figure 1. For that
purpose we employed a microchip with several integrated
makeup-flow channels allowing inflows at two positions (2.1
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Table 1: Catalyst screening on a microchip compared to batch experi-
ments with external analysis.[a]
No.
1
R
4
Method
ee [%][b]
2,4,6-(CH3)3C6H2
4a
chip/RT
batch/RT
55Æ3
62
batch/À308C
73
2
3
2,4,6-(iPr)3C6H2
4b
4c
chip/RT
40Æ4
batch/RT
26
Figure 4. Investigation of reaction mechanism by microsynthesis-nano-
electrospray mass spectrometry.
batch/À308C
48
4-tBu-2,6-(CH3)3C6H2
chip/RT
66Æ4
batch/RT
76
batch/À308C
85
should reduce the residence time down to the subsecond
scale, thus enabling the detection and identification of short-
lived species.
[a] Reaction conditions: 1a (1 equiv), 2 (3 equiv), 4 (5 mol%), room
temperature, H2O (1 equiv), chip: 22 mm in iPrOH/tBuOH/2-Me-2-
BuOH (1:1:1), batch: 0.1m in iPrOH/tBuOH/2-Me-2-BuOH (1:1:1).
[b] On-chip with chiral selector, batch results by HPLC on chiral
stationary phases.
We demonstrated the first combination of enantioselec-
tive organocatalysis, enantiomer separation, and mass spec-
trometric detection on a single microchip. This chip system
was successfully applied to screen catalysts in asymmetric
synthesis. The obtained results showed a very good correla-
tion with comparative batch experiments. Additionally, the
first enantiomer separation on a microchip with mass
spectrometric detection could be realized. Furthermore, the
utilized microchip design offered the possibility to study
reactions by mass spectrometry directly after initialization.
This feature enables the detection and identification of
reaction intermediates and thus exhibits great potential for
the investigation of reaction mechanisms.
Received: April 4, 2011
Revised: May 13, 2011
Scheme 1. Asymmetric organocatalytic conversion of (E)-N-(4-ethylben-
zylidene)-4-methoxyaniline with comparison of ee values from chip and
batch experiments.
Published online: August 30, 2011
Keywords: electrophoresis · high-throughput screening ·
.
lab on a chip · mass spectrometry · organocatalysis
and 0.5 cm) ahead of the nanospray needle (Figure 4). As
depicted, the reaction was initialized in the upper inlets and
the reaction mixture was electrokinetically pumped toward
the nanospray emitter, with subsequent dilution at the front
channels to enhance the ionization. The analysis in the mass
spectrometer was achieved within one minute after the
reaction start. We clearly observed the contact ion pairs of
4a with 1a and 3a as well as the protected catalysts TBS-4a
(TBS = tert-butyldimethylsilyl). Although ion suppression is
reduced in comparison to traditional electrospray,[14b] an exact
quantification is, of course, difficult. However, the detected
species were in good agreement with the postulated reaction
mechanism.[13g] Beside the detection of 3a and 1a, the
hydrolysis of 1a to 5 was also monitored. Further optimiza-
tion of the chip layout by shortening channel lengths, pressure
assistance, and the utilization of fast mass spectrometers[17]
[1] a) G. M. Whitesides, Nature 2006, 442, 368 – 373; b) D. Janasek,
J. Franzke, A. Manz, Nature 2006, 442, 374 – 380; c) A. J.
deMello, Nature 2006, 442, 394 – 402; d) M. Grumann, A.
Geipel, L. Riegger, R. Zengerle, J. Ducrꢀe, Lab Chip 2005, 5,
560 – 565; e) F. Schçnfeld, V. Hessel, C. Hofmann, Lab Chip
2004, 4, 65 – 69; f) K. Jꢁhnisch, V. Hessel, H. Lçwe, M. Baerns,
Angew. Chem. 2004, 116, 410 – 451; Angew. Chem. Int. Ed. 2004,
43, 406 – 446; g) M. Brivio, W. Verboom, D. N. Reinhoudt, Lab
Chip 2006, 6, 329 – 344; h) M. Kakuta, F. G. Bessoth, A. Manz,
Chem. Rec. 2001, 1, 395 – 405.
[2] a) A. Arora, G. Simone, G. B. Salieb-Beugelaar, J. T. Kim, A.
Manz, Anal. Chem. 2010, 82, 4830 – 4847; b) A. Abou-Hassan, O.
Sandre, V. Cabuil, Angew. Chem. 2010, 122, 6408 – 6428; Angew.
Chem. Int. Ed. 2010, 49, 6268 – 6286; c) P. Watts, S. J. Haswell,
Chem. Soc. Rev. 2005, 34, 235 – 246; d) A. Popp, J. J. Schneider,
Angew. Chem. 2008, 120, 9092 – 9095; Angew. Chem. Int. Ed.
Angew. Chem. Int. Ed. 2011, 50, 9467 –9470
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org 9469

Communications
2008, 47, 8958 – 8960; e) H.-I. Ryoo, J. S. Lee, C. B. Park, D.-P.
[9] a) P. Schulze, M. Ludwig, F. Kohler, D. Belder, Anal. Chem.
2005, 77, 1325 – 1329; b) W. Hellmich, C. Pelargus, K. Leffhalm,
A. Ros, D. Anselmetti, Electrophoresis 2005, 26, 3689 – 3696;
c) M. Schꢂttpelz, C. Mꢂller, H. Neuweiler, M. Sauer, Anal.
Chem. 2006, 78, 663 – 669; d) W. Hellmich, D. Greif, C. Pelargus,
D. Anselmetti, A. Ros, J. Chromatogr. A 2006, 1130, 195 – 200.
[10] a) S. L. S. Freire, A. R. Wheeler, Lab Chip 2006, 6, 1415 – 1423;
b) L. M. Fidalgo, G. Whyte, B. T. Ruotolo, J. L. P. Benesch, F.
Stengel, C. Abell, C. V. Robinson, W. T. S. Huck, Angew. Chem.
2009, 121, 3719 – 3722; Angew. Chem. Int. Ed. 2009, 48, 3665 –
3668; c) T. Sikanen, S. Franssila, T. J. Kauppila, R. Kostiainen, T.
Kotiaho, R. A. Ketola, Mass Spectrom. Rev. 2010, 29, 351 – 391;
d) J. S. Rossier, N. Youhnovski, N. Lion, E. Damoc, S. Becker, F.
Reymond, H. H. Girault, M. Przybylski, Angew. Chem. 2003,
115, 55 – 60; Angew. Chem. Int. Ed. 2003, 42, 53 – 58.
Kim, Lab Chip 2011, 11, 378 – 380; f) J. E. Kreutz, A. Shukhaev,
W. Du, S. Druskin, O. Daugulis, R. F. Ismagilov, J. Am. Chem.
Soc. 2010, 132, 3128 – 3132; g) F. E. Valera, M. Quaranta, A.
Moran, J. Blacker, A. Armstrong, J. T. Cabral, D. G. Blackmond,
Angew. Chem. 2010, 122, 2530 – 2537; Angew. Chem. Int. Ed.
2010, 49, 2478 – 2485; h) H. R. Sahoo, J. G. Kralj, K. F. Jensen,
Angew. Chem. 2007, 119, 5806 – 5810; Angew. Chem. Int. Ed.
2007, 46, 5704 – 5708.
[3] a) O. Trapp, S. K. Weber, S. Bauch, W. Hofstadt, Angew. Chem.
2007, 119, 7447 – 7451; Angew. Chem. Int. Ed. 2007, 46, 7307 –
7310; b) N. Wang, T. Matsumoto, M. Ueno, H. Miyamura, S.
Kobayashi, Angew. Chem. 2009, 121, 4838 – 4840; Angew. Chem.
Int. Ed. 2009, 48, 4744 – 4746; c) O. Trapp, Electrophoresis 2010,
31, 786 – 813.
[11] Recent reviews: a) D. W. C. MacMillan, Nature 2008, 455, 304 –
308; b) Topics in Curr. Chem. (Ed.: B. List), 291, Springer,
Berlin, 2010; c) M. Terada, Synthesis 2010, 1929 – 1982; d) Chem.
Rev. (Guest Editor: B. List), 2007, 107, Nr. 12 “Special issue on
organocatalysis”; e) P. Melchiorre, M. Marigo, A. Carlone, G.
Bartoli, Angew. Chem. 2008, 120, 6232 – 6265; Angew. Chem. Int.
Ed. 2008, 47, 6138 – 6171; f) C. Grondal, M. Jeanty, D. Enders,
Nat. Chem. 2010, 2, 167 – 178; g) A. Zamfir, S. Schenker, M.
Freund, S. B. Tsogoeva, Org. Biomol. Chem. 2010, 8, 5262 – 5276;
h) S. Bertelsen, K. A. Jorgensen, Chem. Soc. Rev. 2009, 38,
2178 – 2189; i) Z. Zhang, P. R. Schreiner, Chem. Soc. Rev. 2009,
38, 1187 – 1198; j) S. J. Connon, Synlett 2009, 354 – 376; k) D.
Enders, A. A. Narine, J. Org. Chem. 2008, 73, 7857 – 7870.
[12] A. Odedra, P. H. Seeberger, Angew. Chem. 2009, 121, 2737 –
2740; Angew. Chem. Int. Ed. 2009, 48, 2699 – 2702.
[13] a) T. Akiyama, Chem. Rev. 2007, 107, 5744; b) M. Terada, Chem.
Commun. 2008, 4097; c) T. Akiyama, J. Itoh, K. Yokota, K.
Fuchibe, Angew. Chem. 2004, 116, 1592 – 1594; Angew. Chem.
Int. Ed. 2004, 43, 1566 – 1568; d) D. Uraguchi, M. Terada, J. Am.
Chem. Soc. 2004, 126, 5356 – 5357; e) M. Sickert, C. Schneider,
Angew. Chem. 2008, 120, 3687 – 3690; Angew. Chem. Int. Ed.
2008, 47, 3631 – 3634; f) D. S. Giera, M. Sickert, C. Schneider,
Org. Lett. 2008, 10, 4259 – 4262; g) M. Sickert, F. Abels, M. Lang,
J. Sieler, C. Birkemeyer, C. Schneider, Chem. Eur. J. 2010, 16,
2806 – 2818.
[14] a) P. Hoffmann, U. Hꢁusig, P. Schulze, D. Belder, Angew. Chem.
2007, 119, 5000 – 5003; Angew. Chem. Int. Ed. 2007, 46, 4913 –
4916; b) P. Hoffmann, M. Eschner, S. Fritzsche, D. Belder, Anal.
Chem. 2009, 81, 7256 – 7261.
[15] S. C. Jacobsen, R. Hergenrçder, L. B. Koutny, R. J. Warmack,
J. M. Ramsey, Anal. Chem. 1994, 66, 1107 – 1113.
[16] a) S. Nagl, P. Schulze, S. Ohla, R. Beyreiss, L. Gitlin, D. Belder,
Anal. Chem. 2011, 83, 3232 – 3238; b) S. Nagl, P. Schulze, M.
Ludwig, D. Belder, Electrophoresis 2009, 30, 2765 – 2772.
[17] S. Fritzsche, P. Hoffmann, D. Belder, Lab Chip 2010, 10, 1227 –
1230.
[4] a) J. P. McMullen, K. F. Jensen, Annu. Rev. Anal. Chem. 2010, 3,
19 – 42; b) B. P. Mason, K. E. Price, J. L. Steinbacher, A. R.
Bogdan, D. T. McQuade, Chem. Rev. 2007, 107, 2300 – 2318;
c) K. Geyer, J. D. C. Codꢀe, P. H. Seeberger, Chem. Eur. J. 2006,
12, 8434 – 8442; d) P. Kuhn, J. Puigmarti-Luis, I. Imaz, D.
Maspoch, P. S. Dittrich, Lab Chip 2011, 11, 753 – 757; e) K.-I.
Min, T. H. Lee, C. P. Park, Z.-Y. Wu, H. G. Girault, I. Ryu, T.
Fukuyama, Y. Mukai, D.-P. Kim, Angew. Chem. 2010, 122, 7217 –
7221; Angew. Chem. Int. Ed. 2010, 49, 7063 – 7067; f) M. A. M.
Gijs, F. Lacharme, U. Lehmann, Chem. Rev. 2010, 110, 1518 –
1563; g) M. Brivio, R. H. Fokkens, W. Verboom, D. N. Rein-
houdt, N. R. Tas, M. Goedbloed, A. van den Berg, Anal. Chem.
2002, 74, 3972 – 3976.
[5] D. Belder, Angew. Chem. 2009, 121, 3790 – 3791; Angew. Chem.
Int. Ed. 2009, 48, 3736 – 3737.
[6] a) M. Rasheed, T. Wirth, Angew. Chem. 2011, 123, 374 – 376;
Angew. Chem. Int. Ed. 2011, 50, 357 – 358; b) J. P. McMullen,
M. T. Stone, S. L. Buchwald, K. F. Jensen, Angew. Chem. 2010,
122, 7230 – 7234; Angew. Chem. Int. Ed. 2010, 49, 7076 – 7080;
c) E. R. Murphy, J. R. Martinelli, N. Zabarenko, S. L. Buchwald,
K. F. Jensen, Angew. Chem. 2007, 119, 1764 – 1767; Angew.
Chem. Int. Ed. 2007, 46, 1734 – 1737; d) B. Gutmann, J.-P. Roduit,
D. Roberge, C. O. Kappe, Angew. Chem. 2010, 122, 7255 – 7259;
Angew. Chem. Int. Ed. 2010, 49, 7101 – 7105; e) Y. Wang, W.-Y.
Lin, K. Liu, R. J. Lin, M. Selke, H. C. Kolb, N. Zhang, X.-Z.
Zhao, M. E. Phelps, C. K. F. Shen, K. F. Faull, H.-R. Tseng, Lab
Chip 2009, 9, 2281 – 2285; f) C. de Bellefon, N. Pestre, T.
Lamouille, P. Grenouillet, V. Hessel, Adv. Synth. Catal. 2003,
345, 190 – 193.
[7] a) P. Sichler, S. Bꢂttgenbach, L. Baars-Hibbe, C. Schrader, K.-H.
Gericke, Chem. Eng. J. 2004, 101, 465 – 468; b) S. Bꢂttgenbach,
R. Wilke, Anal. Bioanal. Chem. 2005, 383, 733 – 737.
[8] D. Belder, M. Ludwig, L.-W. Wang, M. T. Reetz, Angew. Chem.
2006, 118, 2523 – 2526; Angew. Chem. Int. Ed. 2006, 45, 2463 –
2466.
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