Building addressable libraries: The use of "safety-Catch" linkers on microelectrode arrays

Article abstract of DOI:10.1021/ja109253t

A "safety-catch" linker strategy has been used to site-selectively cleave and characterize molecules from a microelectrode array. The linkers are attached to the array by means of an ester and contain either a protected amine or protected alcohol nucleophile that can be released using acid generated at the microelectrodes.

Full text of DOI:10.1021/ja109253t

Published on Web 11/19/2010
Building Addressable Libraries: The Use of “Safety-Catch” Linkers on
Microelectrode Arrays
Bo Bi,† Karl Maurer,‡ and Kevin D. Moeller*,†
Department of Chemistry, Washington UniVersity in St. Louis, St. Louis, Missouri 63130, United States, and
CustomArray Inc., 6500 Harbor Heights Parkway, Suite 202, Mukilteo, Washington 98275, United States
Received October 14, 2010; E-mail: moeller@wustl.edu
Scheme 1. “Safety-Catch” Linker Strategy
Abstract: A “safety-catch” linker strategy has been used to site-
selectively cleave and characterize molecules from a microelec-
trode array. The linkers are attached to the array by means of an
ester and contain either a protected amine or protected alcohol
nucleophile that can be released using acid generated at the
microelectrodes.
Microelectrode arrays have great potential as a platform for
building “addressable” molecular libraries.1-3 This potential is
derived from the ability to run cyclic voltammetry experiments at
each individual microelectrode in the array. Hence, if molecular
libraries are built or placed on the array such that each individual
member of the library is by a unique, addressable microelectrode,
then the electrodes can be used to monitor the behavior of the
molecules. To this end, site-selective reactions using a variety of
mediators to place or build molecules by individual microelectrodes
have been explored,4-10 a stable porous reaction layer for attaching
molecules to the surface of the arrays has been developed,11 and
proof-of-principle experiments demonstrating the capability of the
arrays for monitoring small-molecule-receptor binding in real time
have been completed.12 However, one of the main barriers to
utilizing the data from any small-molecule library is quality-control.
Is a molecule that gives rise to a signal in a library really the
molecule that one thinks it is? To really understand the data obtained
from a small molecule library, one must have the ability to fully
characterize the molecules in the library. For a small-molecule
library on a microelectrode array, this means characterizing the
molecules that are located next to each of the microelectrodes used
in an analysis. To date, we have shown that the use of a mass-
spectrometry cleavable linker allows TOF-SIMS experiments to be
used for this purpose.13 Yet while this work has been effective, it
is limited in that it “sacrifices” the array and does not provide us
with a handle to examine the chemo-, regio-, or stereoselectivity
of reactions run on the arrays. Since we hope to use the arrays to
probe the three-dimensional binding preferences of biological
receptors, the inability to determine the stereochemistry of molecules
on the arrays is particularly bothersome. Hence, an alternative
approach that both allows us to examine the molecules on the array,
while retaining the array for further biological studies, and allows
us to more fully characterize molecules on the array is of utmost
importance.
can be cleaved using the microelectrode itself. Such a linker would
require two main features: (1) it needs to be stable to the chemistry
used to build molecules libraries but readily cleavable under mild
conditions when needed, and (2) since each microelectrode in the
array has only 20-50 fmole of material on the polymer coating its
surface, it is best if the linker contains a label to aid in detection of
the product by HPLC. The structures of the molecules can be
characterized by independent synthesis. Because reactions run on
microelectrode arrays can be run at larger scales in solution using
identical conditions, characterization can be done for a larger-scale
reaction and then HPLC used to make sure the products on the
array are the same as the products from the solution-phase synthesis.
With these things in mind, it appeared that a “safety-catch” linker
strategy for attaching molecules to the surface of a microelectrode
array might be ideal.14 In this strategy, molecules are attached to
the surface through either an ester or an amide linkage. The linker
contains a protected alcohol or amine that when released can cyclize
onto the ester or amide to form a lactone or lactam, thereby releasing
the molecule from the surface. We report herein, the success of
this strategy for site-selectively removing and characterizing
molecules from the surface of a microelectrode array.
For an initial trial, the linker was attached to the surface of the
array using the ester chemistry employed on numerous occasions
in the past (Scheme 1).5-10,12 The plan is to functionalize this ester
with a substituent (R1) on the R-carbon of the ester that will provide
a handle for attaching fluorescence active groups, small molecules
of interest for binding studies, or both. A protected amine or alcohol
would be located in the γ-position to provide the masked nucleo-
phile for removing the linker and associated molecule from the
array. The use of a protected amine in this position would be
particularly attractive because it would provide a second site (R2)
for locating a biological substrate or fluorescence label.
With a potential strategy in place, we set out to determine if the
cleavage reaction could be accomplished in a site-selective fashion.
Linker 115 (Scheme 2) was selected as an initial substrate. Pyrene
was used as the fluorescent group for monitoring the reactions on
the array, and a t-Boc protected amine was used as the masked
nucleophile.16 For future efforts, such a linker can be converted
into one containing a biological substrate by either using a disub-
stituted pyrene13b or the nitrogen as a site for attaching the ligand
of interest.
One approach to this problem would be to develop linkers that
allow us to site-selectively recover molecules located on the surface
of any microelectrode in an array. To accomplish this requires a
linker between a molecule and the surface of the electrode that
† Washington University in St. Louis.
‡ CustomArray Inc.
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Scheme 2. Initial Trial
Figure 1. HPLC test results. The chromatograms were obtained using a
Discovery HS C18, 5 µm; 25 cm ×4.6 mm column with a flow rate of 1
mL/min; 70% methanol:30% water was used as eluant with a 10.0 µL
injection. A UV detector was used at 254 nm. (a) Independently synthesized
lactam; (b) reaction solution containing 1,2-diphenylhydrazine and tetrabu-
tylammonium hexafluorophosphate prior to electrolysis; (c) reaction solution
following the electrolysis; (d) co-injection of independently synthesized
lactam and the electrolysis product solution.
The experiment was begun by placing the activated ester15
proximal to every microelectrode in an agarose-coated 1k-array
(1024 microelectrodes/cm2). This was accomplished using the
previously developed electrogenerated base procedure. The cleavage
reaction was then performed by site-selectively generating acid on
the array.8,16 To this end, the array was submerged along with a
remote Pt-wire into 1.5 mL of a methanol solution containing both
1,2-diphenylhydrazine and tetrabutylhexafluorophosphate electro-
lyte. Microelectrodes in a checkerboard pattern were then used as
anodes to oxidize the diphenylhydrazine to form diazobenzene and
2 equiv of acid by applying a potential of +3.0 V to the
microelectrode relative to the remote Pt-wire in solution. The
selected microelectrodes were turned on for a period of 0.5 s and
off for a period of 0.1 s for a total of 900 cycles. In this case, the
success of the reaction was determined by a loss of fluorescence
by the selected microelectrodes. As can be seen in the image, a
small amount of fluorescence did remain by the selected electrode,
but most of the substrate on the surface of the electrode was
removed.
Scheme 3. Lactone-Based System
Confinement of the acid to the selected microelectrodes was
accomplished by using excess 1,2-diphenylhydrazine in the reaction.
The excess hydrazine served as a base to neutralize any acid
escaping from the region of the array surrounding the selected
microelectrodes.
To prove that the molecule being released from the array was
the expected lactam derivative, the lactam was independently
synthesized by simply mimicking the chemistry on the array in
solution. This was done by treating the methyl ester derivative of
1 with trifluoroacetic acid to remove the Boc-group and then
triethylamine to trigger the cyclization.15 HPLC analysis was then
used to compare the independently synthesized lactam with the
material being removed from the array (Figure 1). Four HPLC
chromatograms are shown. In Figure 1a, the retention time for the
independently synthesized lactam is shown. Figure 1b shows the
HPLC trace obtained for reaction solution prior to the electrochemi-
cal cleavage reaction. The two peaks observed arise from the
electrolyte used for the reaction (retention time )7.05 min) and
the excess 1,2-diphenylhydrazine (retention time ) 29.5 min) used
as the confining agent for the electrolysis. Figure 1c shows the
HPLC trace obtained for the reaction solution following the
electrolysis reaction. Figure 1d shows the HPLC trace obtained for
coinjection of the solution used for Figure 1c and the independently
synthesized lactam. Clearly, the peak with a retention time of 20.6
min in Figure 1c is the lactam indicating that the electrochemical
cleavage reaction on the array did give rise to the expected lactam.
The peak at 10.7 min in Figure 1c is thought to be a side product
derived from hydrolysis of the agarose surface coating the array.
With the success of the nitrogen-nucleophile based system,
attention was turned toward examining the use of an oxygen
nucleophile based system and establishing the generality of the
method, both in terms of the nucleophile and the type of micro-
electrode array used. In this case, we wanted to put a precursor for
the linker onto the array and then demonstrate that the linker was
compatible with conducting reactions by the electrodes prior to
releasing the molecule from the surface. For this reason, the pyrene
group was added to the linker following its placement on the array.
The plan started by placing 4-tert- butyldimethylsiloxy-2-meth-
ylidene butanoic acid (2) by each of the microelectrodes in an
agarose coated 12k (12,544 microelectrodes/cm2) array (Scheme
3). This was accomplished by again capitalizing on a base-catalyzed
coupling reaction between an activated ester and the agarose surface
coating the array. As in the earlier experiments, the potential was
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held constant relative to the counter electrode. Each enoate on the
array was then used as a substrate for a Heck reaction with
bromopyrene. This placed a fluorescent group by each of the
microelectrodes in the array. The strategy was intriguing because
the same strategy could be used for adding a variety of different
fluorescent groups and/or biological probes to the linker. Using this
chemistry, the nature of the linker on the array can be varied without
building each linker independently and then transferring them one
at a time to the array. Instead, they can be built directly on the
array.
Supporting Information Available: Sample experimental procedure
for the site-selective reactions along with HPLC information and
characterization data for all new compounds 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. ; 1k chips: electrode
diameter ) 92 µm; distance between the Pt-electrodes (rectangular cells)
) 245.3 and 337.3 µm; 12k slide: diameter ) 44 µm; distance between
the Pt-electrodes (square cells) ) 33 µm.
(2) Microelectrode arrays can be purchased from CustomArray, Inc.
(3) 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.;
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(5) For the site-selective use of Pd(0) see: (a) Tian, J.; Maurer, K.; Tesfu, E.;
Moeller, K. D. J. Am. Chem. Soc. 2005, 127, 1392. (b) Hu, L.; Maurer,
K.; Moeller, K. D. Org. Lett. 2009, 11, 1273.
Cleavage of the linker from the array was accomplished using
reaction conditions that were identical to those employed in the
lactam case. In this case, an L-pattern (L for linker) of electrodes
was used to cleave the linker from the surface. The success of the
reaction was monitored using a fluorescence microscope and can
clearly be seen in the image provided.
As in the lactam case, the reaction was checked to make sure it
led to the desired product by independently synthesizing the lactone
and then using the lactone to identify the product in the crude
reaction mixture by HPLC.15
(6) For Pd(II) reactions: (a) Tesfu, E.; Roth, K.; Maurer, K.; Moeller, K. D.
Org. Lett. 2006, 8, 709. (b) Tesfu, E.; Maurer, K.; Ragsdale, S. R.; Moeller,
K. D. J. Am. Chem. Soc. 2004, 126, 6212. (c) Tesfu, E.; Maurer, K.;
McShae, A.; Moeller, K. D. J. Am. Chem. Soc. 2006, 128, 70.
(7) For examples of the site-selective generation of base see reference 4b.
Maurer, K.; McShea, A.; Strathmann, M.; Dill, K. J. Combi. Chem. 2005,
7, 637.
In conclusion, a “safety-catch” linker strategy has been used to
site-selectively cleave molecules from preselected, individual
microelectrodes in a microelectrode array. Both amine and alcohol
nucleophile strategies work well. The chemistry is compatible with
arrays having either 1024 or 12,544 microelectrodes/cm2. The use
of the “safety-catch” linkers will allow for characterization of the
molecules built by the electrodes in the array, and in this way opens
door for doing quality-control analysis of the molecules in addres-
sable molecular libraries.
(8) For the site-selective generation of acid: Kesselring, D.; Maurer, K.; Moeller,
K. D. Org. Lett. 2008, 10, 2501.
(9) For the use of CAN on an array see: Kesselring, D.; Maurer, K.; Moeller,
K. D. J. Am. Chem. Soc. 2008, 130, 11290.
(10) For the site-selective use of Sc(III) see: Bi, B.; Maurer, K.; Moeller, K. D.
Angew. Chem., Int. Ed. 2009, 48, 5872.
(11) Hu, L.; Bartels, J. L.; Bartels, J. W.; Maurer, K.; Moeller, K. D. J. Am.
Chem. Soc. 2009, 131, 16638.
(12) (a) Tesfu, E.; Roth, K.; Maurer, K.; Moeller, K. D. Org. Lett. 2006, 8,
709. (b) Stuart, M.; Maurer, K.; Moeller, K. D. Bioconjugate Chem. 2008,
19, 1514.
(13) (a) Chen, C.; Nagy, G.; Walker, A. V.; Maurer, K.; McShae, A.; Moeller,
K. D. J. Am. Chem. Soc. 2006, 128, 16020. (b) Chen, C.; Lu, P.; Walker,
A. V.; Maurer, K.; Moeller, K. D. Electrochem. Commun. 2008, 10, 973.
(c) Bartles, J. L.; Lu, P.; Walker, A. V.; Maurer, K.; Moeller, K. D. Chem.
Commun. 2009, 5573.
(14) Kenner, G. W.; McDermott, J. R.; Sheppard, R. C. J. Chem. Soc., Chem.
Commun. 1971, 636.
(15) For experimental details please see the Supporting Information.
(16) For the removal of t-Boc groups on microelectrode arrays see: Maurer,
K.; McShea, A.; Strathmann, M.; Dill, K. J. Combi. Chem. 2005, 7, 637.
Acknowledgment. We thank the National Science Foundation
(CHE-9023698) for their generous support of our work. 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.
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