Building addressable libraries: The use of a mass spectrometry cleavable linker for monitoring reactions on a microelectrode array

Article abstract of DOI:10.1021/ja067194o

Time-of-flight secondary ion mass spectrometry (TOF SIMS) has been used in conjunction with a mass spectrometry cleavable linker to determine the percent conversion of reactions that were conducted site-selectively on an addressable microelectrode array.

Full text of DOI:10.1021/ja067194o

Published on Web 11/24/2006
Building Addressable Libraries: The Use of a Mass Spectrometry Cleavable
Linker for Monitoring Reactions on a Microelectrode Array
Ceng Chen,† Gabriella Nagy,† Amy V. Walker,*,† Karl Maurer,‡ Andy McShea,‡ and
Kevin D. Moeller*,†
Department of Chemistry, Washington UniVersity, St. Louis, Missouri 63130, and CombiMatrix Corporation,
6500 Harbour Heights Parkway, Suite 301, Mukilteo, Washington 98275
Received October 7, 2006; E-mail: moeller@wuchem.wustl.edu
Chip-based molecular arrays are important tools for probing
interactions between libraries of potential ligands and biological
receptors.1,2 Arrays where the members of the library can be
individually addressed are particularly intriguing. For example,
building a molecular library on an addressable array of microelec-
trodes (Figure 1a)3,4 would allow for the “real-time” monitoring of
members in the library using the electrodes.5a But how does one
build a molecular library so that its individual members all reside
proximal to a unique electrode? In order to answer this question,
we have been developing the synthetic methodology needed for
site-selectively synthesizing molecules proximal to individual
microelectrodes in an addressable array.5,6 Recently, we reported
the utility of Pd(II)-initiated reactions for these transformations.
As in all microelectrode array reactions performed to date, the array
chip was first coated with a polyhydroxylated polymer and then
an initial substrate bound to the polymer next to each of the
microelectrodes in the array. For the Pd(II) reactions, selected
electrodes were then used as anodes to convert a Pd(0) substrate
into the desired Pd(II) reagent. Ethyl vinyl ether was added to the
solution above the array as a confining agent in order to consume
any Pd(II) reagent that diffused away from the selected electrodes.
Wacker oxidations (Figure 1b),5b alcohol oxidations,5c and Pd(II)-
catalyzed coumarin syntheses5a were all accomplished in this
manner. In each case, the success of the confinement strategy was
determined using fluorescence techniques.
Yet while the fluorescence techniques used were outstanding for
determining that no reaction occurred at unused electrodes, they pro-
vided little information as to the extent of reaction that occurred
above electrodes that were selected. With this in mind, we turned
our attention toward the use of time-of-flight secondary ion mass
spectrometry (TOF SIMS) techniques for analyzing the microelec-
trode array-reactions.7 In our experiments, a Bi+ beam is used to
ionize molecules on the surface. The ions are then collected and
analyzed by mass spectrometry to gather information as to the nature
of the molecules on the surface. The lateral resolution of
TOF SIMS is approximately 200 nm. Since each microelectrode
in the array illustrated in Figure 1 has a diameter of 95 micrometers,
TOF SIMS can in principle can determine the structure of the
molecules above an individual microelectrode in the array. We
report here that with an appropriate linker, reactions conducted using
the microelectrodes of an addressable array can be directly
monitored with a TOF SIMS experiment.
Figure 1. (a) A “1K-chip” having 1024 addressable electrodes in a 1 cm2
area; (b) a chip utilizing the initially developed Wacker oxidation conditions
at every other electrode. The ketone product was imaged by conversion
into a 2,4-DNP derivative followed by incubation with a fluorescently
labeled anti-2,4-DNP antibody.5b
Scheme 1
cleaving the molecules from the solid support using a preliminary
chemical reaction.7b-d While this approach worked well, it was comp-
licated by the need to keep the molecules cleaved from the surface
of the array close to their initial location. Migration of the molecules
meant a loss in the spatial resolution of the array. An alternative
strategy would be to use a linker for attaching molecules to the
polymer coating the array that would fragment faster than the
polymer under the TOF SIMS reaction conditions. Our initial plan
called for building a linker that would rapidly undergo a McLafferty
fragmentation during the TOF SIMS experiment (Scheme 1).8
To this end, 4-(2′-hydroxyethyl)benzoic acid was converted into
its N-hydroxysuccinimide ester, and then the alcohol coupled to both
the olefin substrate (1) and the ketone product (2) of a Wacker oxida-
tion. The N-hydroxysuccinimide ester was then used to place the
linker and substrate on the chip using the strategy employed in the
earlier studies.3,4 The use of the aromatic linker immediately proved
effective. For an array functionalized with substrate 1, the TOF
SIMS experiment showed a distinct parent peak for the olefin at
m/z ) 183. In addition, the presence of the phenyl ring caused the
entire linker + substrate molecule to be lost from the agarose, giving
rise to a parent peak at m/z ) 331. This second peak proved
particularly useful since the 183 peak fell in a spot consistent with
one of the fragments from agarose. In a similar fashion, the TOF
SIMS experiment arising from the placement of 2 on the array gave
rise to peaks at m/z ) 199 and m/z ) 347. Once again, the m/z )
347 peak proved most useful because of its location relative to
agarose fragmentation peaks.
Initial attempts to use TOF SIMS to directly observe molecules
on an array were not successful. The polyhydroxylated polymer used
for the experiments was agarose, and all that could be consistently
observed in the mass spectrum was fragmentation products from
the agarose. Previous application of the TOF SIMS technique to
the analysis of molecular arrays solved problems of this nature by
At this point, known ratios of the substrate and product were
placed onto the polymer coating the microelectrode array and TOF
SIMS experiments conducted in order to generate a calibration curve
† Washington University.
‡ CombiMatrix Corporation.
9
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J. AM. CHEM. SOC. 2006, 128, 16020-16021
10.1021/ja067194o CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
Fortunately, the development of a mass spectrometry cleavable
linker and the use of the TOF SIMS approach allows for a change
in this strategy. The reactions can be optimized by making changes
in the amount of palladium used in the reactions, the amount and
reactivity of the confining agent employed, the length of time the
positive potential is applied in each cycle, and the number of cycles
used. For example, an increase in the amount of Pd(OAc)2 to 1 mg
and a decrease in the ethyl vinyl ether to 0.5 µL led to an increase
in the percent conversion for the microarray reaction to ap-
proximately 50% (Figure 3c). Increasing the amount of Pd(OAc)2
even further to 3.2 mg led to complete conversion of the olefin
(loss of m/z 331) to the ketone product (m/z 347) (Figure 3d). While
product formation was complete using these conditions, confinement
of the reaction across the chip was not maintained and further
optimization is needed.
Figure 2. Calibration curve for the Wacker oxidation (Ik ) intensity of
ketone, Io ) intensity of olefin, Mk ) molar concentration of ketone, Mo
) molar concentration of olefin).
At this point, it is clear that a combination of fluorescence and
TOF SIMS techniques is needed to truly optimize reactions that
take place on a microelectrode array. In this way, both the
confinement of reagents to selected electrodes on the addressable
array as well as the percent conversion of the reaction that takes
place on the electrodes selected can be monitored. Key to this
approach is the use of a mass spectrometry cleavable linker for
attaching the reaction substrate to the polymer coating the surface
of the microelectrode array. This strategy should be amenable to
the use of a variety of cleavable linkers and the analysis of
molecules on a variety of different surfaces. Work to explore the
scope of mass spectrometry cleavable linkers that can be used on
a microelectrode array is underway.
Figure 3. High-resolution negative ion TOF SIMS mass spectra of Wacker
oxidation with (a) 0.32 mg Pd(OAc)2 and no confining agent;8 (b) 0.32 mg
Pd(OAc)2 and 50 µL confining agent; (c) 1 mg Pd(OAc)2 and 0.5 µL
confining agent; (d) 3.2 mg Pd(OAc)2 and 0.5 µL confining agent.
Acknowledgment. We thank the National Science Foundation
(Grants CHE-0314057 and CHE-0518063) for their generous
support of this work. We also gratefully acknowledge the Wash-
ington University High-Resolution NMR facility, partially supported
by NIH Grants RR02004, RR05018, and RR07155, and the
Washington University Mass Spectrometry Resource Center,
partially supported by NIH Grant RR00954, for their assistance.
for determining the product/starting material ratio over an electrode
(Figure 2). The data obtained showed that TOF SIMS experiments
could be used to provide a good, qualitative assessment of the
percent conversion for a reaction that takes place over a micro-
electrode in an addressable array.
With a calibration curve in place, we were ready to begin explor-
ing the quality of a microelectrode array-based Wacker oxidation. The
reaction was run in a fashion identical to that used in the previously
described Pd(II) mediated reactions.5b For the initial experiment,
0.32 mg of Pd(OAc)2 (in 1.5 mL of a 0.5 M tetraethylammonium
p-toluenesulfonate in 7:1 acetonitrile to water electrolyte solution)8
was used along with no confining agent. Following the reaction, the
microelectrode array was washed, dried, and then a TOF SIMS
experiment was used to show that the starting material (m/z 331)
had been totally consumed and the expected product (m/z 347)
formed (Figure 3a, the peak at m/z ) 339 is from the agarose
polymer).
A second experiment (Figure 3b) was conducted in the same man-
ner5b except in this case 50 µL of the ethyl vinyl ether confining agent
was added. The TOF SIMS experiment showed no signal (m/z 347)
for product formation. This was very surprising since the conditions
used were identical to those employed for the earlier Wacker oxida-
tions. Apparently, the previous reactions only proceeded to a slight
extent. The fluorescence images taken showed a positive reaction on
the electrodes used for Pd(II) generation owing to the very high sensi-
tivity of fluorescent imaging for the product generated. The extremely
high level of confinement observed3b was a result of the confining
agent not only interfering with migration of the Pd(II) reagent to
neighboring electrodes, but also with the reaction on the selected elec-
trodes. Optimization of the process led to the most ideal fluorescent
image possible, but not the most efficient reaction possible!
Supporting Information Available: The procedure for synthesizing
the linker, procedures for conducting the microelectrode array reactions,
and spectral data for all new compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
References
(1) For reviews see: (a) Lipshutz, R. J.; Fodor, S. P. A.; Gingeras, T. R.;
Lockhart, D. J. Nat. Genet. 1999, 21, 20. (b) Pirrung, M. C. Chem. ReV.
1997, 97, 473.
(2) For more recent examples see: (a) Webb, S. M.; Miller, A. L.; Johnson,
B. H.; Fofanov, Y.; Li, T.; Wood, T. G.; Thompson, E. B. J. Steroid
Biochem. and Mol. Biol. 2003, 85, 183. (b) Shih, S.-R.; Wang, Y.-W.;
Chen, G.-W.; Chang, L.-Y.; Lin, T.-Y.; Tseng, M.-C.; Chiang, C.;
Tsao, K.-C.; Huang, C. G.; Shio, M.-R.; Tai, J.-H.; Wang, S.-H.; Kuo,
T.-L.; Liu, W.-T. J. Virol. Methods 2003, 111, 55.
(3) For a description of the chips used here see: Dill, K.; Montgomery, D.
D.; Wang, W.; Tsai, J. C. Anal. Chim. Acta 2001, 444, 69.
(4) For alternative approaches see: (a) Sullivan, M. G.; Utomo, H.; Fagan,
P. J.; Ward, M. D. Anal. Chem. 1999, 71, 4369. (b) Zhang, S.; Zhao, H.;
John, R. Anal. Chim. Acta 2000, 421, 175. (c) Hintsche, R.; Albers, J.;
Bernt, H.; Eder, A. Electroanalysis 2000, 12, 660.
(5) For Pd(II) reactions see: (a) Tesfu, E.; Roth, K.; Maurer, K.; Moeller, K.
D. Org. Lett. 2006, 8 709-712. (b) Tesfu, E.; Maurer, K.; Ragsdale, S.
R.; Moeller, K. D. J. Am. Chem. Soc. 2004, 126, 6212-6213. (c) Tesfu,
E.; Maurer, K.; McShae, A.; Moeller, K. D. J. Am. Chem. Soc. 2006,
128, 70-71.
(6) For a Pd(0) reaction see: Tian, J.; Maurer, K.; Tesfu, E.; Moeller, K. D.
J. Am. Chem. Soc. 2005, 127, 1392-1393.
(7) (a) Pacholski, M. L.; Winograd, N. Chem. ReV. 1999, 99, 2977. (b) Xu,
J.; Szakal, C. W.; Martin, S. E.; Peterson, B. R.; Wucher, A.; Winograd,
N. J. Am. Chem. Soc. 2004, 126, 3902. (c) Xu, J. Y.; Braun, R. M.;
Winograd, N. Anal. Chem. 2003, 75, 6155. (d) Xu, J. Y.; Braun, R. M.;
Winograd, N. Appl. Surf. Sci. 2003, 203-204, 201.
(8) For a synthesis of the linker and experimental details for the electrolyses
reactions please see the Supporting Information.
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