Microelectrode arrays and ceric ammonium nitrate: A simple strategy for developing new site-selective synthetic methods

Article abstract of DOI:10.1021/ja804661h

Conditions for a site-selective ceric ammonium nitrate oxidation have been developed. The reactions proceed nicely on both 1K- and 12K-microelectrode arrays. The procedure for developing the reactions was very simple and demonstrated that the same reagents used for a solution-phase reaction can be used for a related site-selective reaction on a microelectrode array. Copyright

Full text of DOI:10.1021/ja804661h

Published on Web 08/05/2008
Microelectrode Arrays and Ceric Ammonium Nitrate: A Simple Strategy for
Developing New Site-Selective Synthetic Methods
David Kesselring,† Karl Maurer,‡ and Kevin D. Moeller*,†
Department of Chemistry, Washington UniVersity, St. Louis, Missouri 63130 and CombiMatrix Corporation,
6500 Harbor Heights Parkway, Suite 301, Mukilteo, Washington 98275
Received June 18, 2008; E-mail: moeller@wustl.edu
The construction of a molecular library on a microelectrode array1,2
Scheme 1
opens up the possibility for monitoring the binding of molecules in
the library with biological receptors in “real time”.3 Accomplishing
this task means that each unique member of the library must be built
next to a unique, individually addressable microelectrode in the array,
even when the array has a density of 1024 or 12 554 microelectrodes
cm-2. For this reason, we have been developing tools for site-
selectively synthesizing4-7 and characterizing8 molecules on micro-
electrode arrays. From a synthetic standpoint, each new site-selective
reaction requires the development of both an electrochemical means
for generating the reagent used to effect the transformation and a
solution phase substrate for destroying the reagent generated before it
migrates away from the electrode used in its synthesis. To date, much
Scheme 2
of this work has focused on the use of palladium reactions in a site-
selective fashion.4,5 This was done because palladium can be easily
cycled between its nucleophilic Pd(0) and electrophilic Pd(II) oxidation
states, a fact that had already led to the development of effective
preparative reactions that recycle palladium at an electrode.9 In addition,
the availability of a variety of Pd(0) and Pd(II) reagents and a broad
knowledge of how to chemically oxidize and reduce them (most
stoichiometric reactions involving Pd either oxidize or reduce the
corresponding reagent) made adapting Pd chemistry to the microelec-
trode arrays straightforward. However, not all synthetic reactions
needed for building molecular libraries have such readily identifiable
reduction-oxidation cycles and available reagents. For example, ceric
ammonium nitrate is a very useful oxidant for a wide range of chemical
reactions.10 We have found that the cleavage of a silylelectroauxiliary
substituted amino acid using ceric ammonium nitrate provides an ideal
method for inserting N-acyliminium ions into peptides (Scheme 1).11
Hence, the use of ceric ammonium nitrate in a site-selective fashion
might provide a pathway for building addressable libraries of func-
tionalized peptidomimetics. But how would one utilize ceric am-
monium nitrate in a site-selective fashion? What is the reduced cerium
reagent that one can oxidize at a microelectrode to site-selectively form
a Ce(IV) oxidant, and what is the chemical reducing agent that can be
used in solution to confine the ceric ammonium nitrate generated to
the region surrounding a preselected microelectrode?12 We report here
a very simple strategy for developing new site-selective reactions and
its application to the site-selective use of Ce(IV).
this in mind, a silylelectroauxiliary-substituted amide was constructed
as a test substrate and then placed by each of the microelectrodes in
a 1 cm2 array having 1024 electrodes (a 1K array, Scheme 2).
Placement of the substrate on the microelectrode array took advantage
of an electrochemically generated base-catalyzed esterification
reaction.3-7 In this reaction, the microelectrodes were used as cathodes
to generate the radical anion from vitamin B12 and in turn catalyze an
esterification reaction between the agarose polymer coating the
microelectrode array and the activated ester in substrate 6. Use of the
electrogenerated base ensured that the substrate was placed proximal
to the electrodes (7).
With 7 in hand, attention was turned to the ceric ammonium nitrate
oxidation (Scheme 3). Our plan for developing a site-selective Ce(IV)
oxidation called for the simplest possible approach. The reduced cerium
reagent needed for the reaction was generated by incubating a catalytic
amount of the commercially available ceric ammonium nitrate with
an excess of a solution phase substrate that was identical to the substrate
placed on the surface of the microelectrode array for the site-selective
reaction. The resulting solution was then placed above the array, and
selected microelectrodes were used as anodes to regenerate a Ce(IV)
oxidant where it was needed. The hope was that the excess solution
The oxidative cleavage of a silylelectroauxiliary substituted amino
acid was an ideal reaction for probing site-selective ceric ammonium
nitrate reactions because a method for determining the site-selective
formation of an N-acyliminium ion on a microelectrode array was
already available.7 In addition, the substrate attached to the agarose
polymer coating the microelectrode array is insulated from the electrode
surface by the polymer. Hence, no background oxidation of the
substrate in the absence of the Ce(IV) mediator is possible.13 With
† Washington University.
‡ CombiMatrix Corporation.
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11290 J. AM. CHEM. SOC. 2008, 130, 11290–11291
10.1021/ja804661h CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
Scheme 3
oxidation on the 1K array was also effective for developing site-
selective Ce(IV) oxidations on the more dense 12K microelectrode
array.
In conclusion, we have found that Ce(IV) oxidations can be
performed in a site-selective fashion on both 1K- and 12K-microelec-
trode arrays. Additionally, it was found that the site-selective reactions
could be performed using the same reagents employed for a solution-
phase reaction. This allowed for a very simple approach to developing
site-selective reactions. To this end, a solution phase oxidation was
modified by removing the stoichiometric amount of ceric ammonium
nitrate normally used and replacing it with a catalytic amount of the
oxidant. The mixture was stirred overnight to allow for complete
consumption of the oxidant. An electrolyte was added followed by
insertion into the solution of a microelectrode array functionalized with
a substrate for the oxidation. Selected microelectrodes in the array were
then turned on as anodes to regenerate the oxidant exclusively at sites
where it was desired. The excess substrate in the reaction served as
the confining agent. Both the use of ceric ammonium nitrate on the
microelectrodes and the overall strategy used in its development should
prove extremely useful for the construction of addressable molecular
libraries in the future. Efforts along these lines are currently underway.
phase substrate would serve as the confining agent in solution needed
for preventing the migration of any Ce(IV) reagent generated to remote
locations on the array. The result would be an approach for developing
a site-selective microelectrode-array oxidation that capitalized on the
same reagents and substrates used for the solution phase reaction. One
would simply take the solution-phase reaction, reduce the amount of
oxidant used to a catalytic amount, add an electrolyte, and then use
the solution on the microelectrode array to effect the site-selective
reaction.
In practice, 1 equiv of ceric ammonium nitrate and 10 equiv of
activated ester 6 were added to dichloromethane and stirred overnight.
To this mixture were added tetrabutylammonium hexafluorophosphate
as an electrolyte for the electrolysis and pyrene butanol in order to
trap any N-acyliminium ion generated. The surface functionalized
microelectrode array 7 was then inserted into the solution and a
checkerboard pattern of microelectrodes used as anodes at a potential
of +2.0 V relative to a remote platinum wire cathode. The reaction
was run for 300 cycles where the selected microelectrodes were turned
on for 0.5 s and off for 0.1 s. It was found that turning the current on
for longer periods of time led to a deterioration in the amount of current
that could be passed. Following the reaction, the array was removed
from the solution, washed to remove any unbound pyrene butanol,
and then imaged using a fluorescence microscope (Figure 1a).
Acknowledgment. This work is generously supported by the
National Science Foundation (CHE-9023698). We also gratefully
acknowledge the Washington 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 NIHRR00954, for their assistance.
Supporting Information Available: Spectral data for all new
compounds are provided along with sample procedures for conducting the
microelectrode array reactions. This material is available free of charge
via the Internet at http://pubs.acs.org.
References
(1) 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. Electrode diameter
) 92 µm: Distance between the Pt electrodes (rectangular cells) ) 245.3
and 337.3 µM.
(2) 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.;
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(4) For Pd(II) reactions, see ref 3a as well as: (a) Tesfu, E.; Maurer, K.;
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A checkerboard pattern of fluorescence on the microelectrode
array was obtained indicating site selective formation of the
N-acyliminium ion intermediate from the silyl-substituted amide.
No loss of confinement was observed. Clearly, the simple strategy
taken for generating a reduced cerium substrate for the electrolysis
and confining Ce(IV) to the selected microelectrodes was very
effective.
The site-selective ceric ammonium nitrate oxidation could also be
accomplished using an array having 12 544 microelectrodes cm-2 (a
12K array). This was important because the 12K arrays are used in
electrochemical signaling experiments.3 Using the same conditions
employed for the 1K array, the image in Figure 1b was generated using
a 12K array by employing a checkerboard pattern of electrodes inside
a box as anodes for the oxidation. Once again, a high level of
confinement of the reaction to the selected electrodes was observed.
The very simple strategy used for developing the site-selective Ce(IV)
(8) Chen, C.; Nagy, G.; Walker, A. V.; Maurer, K.; McShae, A.; Moeller,
K. D. J. Am. Chem. Soc. 2006, 128, 16020. as well as ref 5c.
(9) For example, see: (a) Inokuchi, T.; Ping, L.; Hamaue, F.; Izawa, M.; Torii,
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(10) Hwu, J. R.; King, K.-Y. Curr. Sci. 2001, 81, 1043.
(11) Sun, H.; Martin, C.; Kesselring, D.; Keller, R.; Moeller, K. D. J. Am. Chem.
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(12) For a preparative-scale, CAN-mediated oxidation of aromatic hydrocarbons
in aqueous HNO3/hydrocarbon solvent emulsions containing a phase-transfer
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Electrochim. Acta 1988, 33, 509.
(13) To date, the direct electrolysis of any substrate attached to the agarose
polymer coating the microelectrode array has not been observed.
Figure 1. Site-selective Ce(IV) oxidation reactions. (a) Site-selective pattern
on a 1K microelectrode array. (b) Site-selective pattern on a 12K array.
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