Building addressable libraries: Site selective coumarin synthesis and the "real-time" signaling of antibody-coumarin binding

Article abstract of DOI:10.1021/ol052891r

The feasibility of using active semiconductor chips containing addressable arrays of microelectrodes for the "real-time" monitoring of biologically relevant binding events has been demonstrated by detecting the binding of a coumarin substrate by an antico

Full text of DOI:10.1021/ol052891r

ORGANIC
LETTERS
2006
Vol. 8, No. 4
709-712
Building Addressable Libraries: Site
Selective Coumarin Synthesis and the
“Real-Time” Signaling of
Antibody−Coumarin Binding
Eden Tesfu,† Kris Roth,‡ Karl Maurer,‡ and Kevin D. Moeller*,†
Department of Chemistry, Washington UniVersity, St. Louis, Missouri 63130,
CombiMatrix Corporation, 6500 Harbor Heights Pkwy, Suite 301,
Mukilteo, Washington 98275
moeller@wuchem.wustl.edu
Received November 29, 2005
ABSTRACT
The feasibility of using active semiconductor chips containing addressable arrays of microelectrodes for the “real-time” monitoring of biologically
relevant binding events has been demonstrated by detecting the binding of a coumarin substrate by an anticoumarin antibody. The coumarin
substrate was synthesized proximal to predetermined electrodes on the chip with the use of a Pd(II) reagent that was itself generated by using
the selected electrodes. Once the coumarin was synthesized, its binding to the anticoumarin antibody was detected by monitoring the current
associated with a ferrocene
electrode.
−ferrocinium ion redox cycle that was established between the electrodes on the chip and a remote auxiliary
Chip-based molecular libraries can provide a valuable tool
for screening molecular interactions involving a variety of
biomolecules.1 Of particular interest are libraries having the
molecules located proximal to individually addressable
electrodes on an active-semiconductor microarray.2,3 In
principle, the molecules in such a library can be monitored
by using the electrodes thereby providing a “real-time”
measure of how the molecules interact with biological
receptors. With this in mind, we have been working to
develop the synthetic methodology needed for synthesizing
organic molecules at preselected sites on electrochemically
addressable microarrays.4,5 Taking our lead from initial
efforts to use electrochemically generated acid and base on
† Washington University.
‡ CombiMatrix Corporation.
(1) For reviews see: (a) Lipshutz, R. J.; Fodor, S. P. A.; Gingeras, T.
R.; Lockhart, D. J. Nature Genetics 1999, 21, 20. (b) Pirrung, M. C. Chem.
ReV. 1997, 97, 473. For more recent examples see: (c) Webb, S. M.; Miller,
A. L.; Johnson, B. H.; Fofanov, Y.; Li, T.; Wood, T. G.; Thompson, E. B.
J. Steroid Biochem. Mol. Biol. 2003, 85, 183. (d) 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.
(2) 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.
(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.;
Bernt, H.; Eder, A. Electroanalysis 2000, 12, 660.
(4) For the use of Pd(0) catalysts see: Tian, J.; Maurer, K.; Tesfu, E.;
Moeller, K. D. J. Am. Chem. Soc. 2005, 127, 1392.
10.1021/ol052891r CCC: $33.50
© 2006 American Chemical Society
Published on Web 01/28/2006

the arrays to build DNA and peptide libraries,6,7 this work
has utilized selected electrodes in the arrays to synthesize
both Pd(0)4 and Pd(II)5 reagents for effecting chemical
transformations on molecules tied to a polymer coating above
the electrodes. By utilizing a chemical reagent in the solution
above the array that destroys the reagent generated, the
desired reactions can be confined to the region of the array
immediately surrounding a selected electrode.
interactions with the receptor, the microelectrode array
experiment seeks to accelerate the rate at which ligands can
be screened by placing the ligands proximal to the electrodes
and then probing their interactions with a solution phase
biomolecule in a parallel fashion. Second and more worri-
some, the current microarray based chemistry does not attach
the molecules to the electrode surface, but rather binds the
molecule to a porous polymer covering the entire surface of
the chip. Will extra distance between the electrode and
ligand/receptor interaction result in a situation where the
binding event no longer interferes with the current associated
with a secondary redox couple? If the answer is yes, then
how do we change the overall approach so that the molecules
can be directly bound to the electrodes? If the answer is no,
then what distance between the electrode and the molecule
being monitored can be tolerated?
To begin addressing these questions and demonstrate the
feasibility of the approach, we decided to locate coumarins
proximal to the electrodes of an addressable microarray. The
ability of the array to signal a binding event would then be
determined by treating the array with commericially avail-
able, coumarin specific antibodies. With this in mind, our
attention turned toward a site-selective synthesis of coumarins
on the microarrays. To this end, the synthetic protocol
outlined in Scheme 1 was followed.
With a strategy for exploring new reactions in place and
a number of new microarray-based synthetic methods in
development, attention has turned toward determning how
the microelectrodes in the array could be used to monitor
the “real-time” binding of small molecules attached to the
array’s surface. To date, most efforts to monitor the behavior
of molecules on electrode microarrays have focused on either
fluorescence based approaches8 or electrochemical amplifica-
tion techniques.9 Both techniques require washing the array
following incubation of the library with a desired biological
receptor and then capitalizing on an appropriate antibody
bioconjugate to effect signaling. While these approaches can
be very effective, the washing step is potentially problematic.
For example, if one is using a library containing conforma-
tionally constrained ligands to probe the three-dimensional
requirements of a receptor, then weak binding interactions
can provide important information suggesting that a particular
conformation is close to being correct. A separate washing
step could “wash away” this information thereby changing
a positive binding event into a false negative and affording
inaccurate information.
Scheme 1
For this reason, we sought a method that would allow the
electrodes to directly signal a binding event on the array’s
surface. The overall strategy taken for the development of
electrochemical sensors appeared ideal.10 In this approach,
a biological receptor is placed on the surface of a gold
electrode. A redox couple is then cycled between the
electrode and a remote auxiliary electrode creating a current
that can be monitored. The heterogeneous electron-transfer
rate is a function of the surface coverage of electroinactive
species11 and therefore as molecules bind to surface bound
receptors, the observed current decreases. Several aspects
of this approach were of concern with respect to its
application to chip-based microarrays. First, rather than attach
a large biological receptor to the electrode and then screen
a series of solution phase ligands one at a time for their
(5) For the use of Pd(II) see: (a) Tesfu, E.; Maurer, K.; Ragsdale, S. R.;
Moeller, K. D. J. Am. Chem. Soc. 2004, 126, 6212. (b) Tesfu, E.; Maurer,
K.; Moeller, K. D. J. Am. Chem. Soc. 2006, 128, 70.
(6) For the lead patent on DNA related work see: Montgomery, D. D.
PCT Int. Appl. 1998, 91 pp, CODEN: PIXXD2 WO 9801221 A1 19980115.
(7) For peptide based libraries see: Rossi, F. M.; Montgomery, D. D.
PCT Int. Appl. 2000, 52 pp, CODEN: PIXXD2 WO 0053625 A2 20000914.
For recent work see: Oleinikov, A. V.; Gray, M. D.; Zhao, J.; Montgomery,
D. D.; Ghindilis, A. L.; Dill, K. J. Proteome Res. 2003, 2, 313 as well as
ref 2 above.
The synthesis began by coupling a phenol substrate to the
surface of the array with use of an amine based porous
polymer.12 The phenol was placed on the entire surface of
(8) For examples, see refs 2 and 4-7 above.
(9) (a) Dill, K.; Montgomery, D. D.; Ghindilis, A. L.; Schwarzkopf, K.
R.; Ragsdale, S. R.; Oleinikov, A. V. Biosens. Bioelectron. 2004, 20, 736.
(b) Dill, K.; Montgomery, D. D.; Ghindilis, A. L.; Schwarzkopf, K. R. J.
Biochem. Biophys. Methods 2004, 59, 181.
(12) (a) For a previous use of this polymer see: Maurer, K.; McShea,
A.; Strathmann, M.; Dill, K. J. Comb. Chem. 2005, 7, 637. (b) Chips coated
with this polymer either as a “blank chip” or a synthesized DNA array are
commercially available and can be purchased from CombiMatrix Corpora-
tion, 6500 Harbour Heights Pkwy., Suite 310, Muilteo, WA 98275; http://
www.combimatrix.com.
(10) Murata, M.; Gonda, H.; Yano, K.; Kuroki, S.; Suzutani, T.;
Katayama, Y. Bioorg. Med. Chem. Lett. 2004, 14, 137.
(11) Bard, A. J.; Faulkner, L. R. Electrochemical Methods, Fundamentals,
and Applications, 2nd ed.; John Wiley and Sons: New York, 2001.
710
Org. Lett., Vol. 8, No. 4, 2006

the chip supporting the array. The coumarin synthesis then
capitalized on a Pd(II)-catalyzed cycloaddition between the
phenol substrate and an acetylene in the solution above the
array.13 This chemistry used the methodology developed
previously for doing site-selective Pd(II)-mediated oxidations
on the arrays.5 In this way, Pd(0) was generated in the
solution above the array by a Wacker oxidation involving
Pd(OTf)2 and ethyl vinyl ether. The Pd(0) was then converted
to the desired Pd(II) reagent at selected electrodes by using
them as anodes to oxidize an amine and generate a radical
cation, which in turn oxidized the Pd(0). The Pd(II) catalyst
generated in this fashion was confined to the region of the
chip surrounding the electrode with the use of excess ethyl
vinyl ether. The success of this strategy was analyzed by
imaging the array with the use of a biotinylated anticoumarin
antibody14 followed by incubation with a Texas red conju-
gated steptoavidin.15 Figure 1 shows a fluorescent microscope
Scheme 2
(n ) 4) having an aminoethoxyethyl terminating group. On
the third block, the coumarin was linked to the polymer by
using 15 thymidine units (n ) 14) having an aminoethoxy-
ethyl terminating group. The entire electrode array schemati-
cally illustrated in Scheme 2 was then submerged in a
solution of an iron redox couple (ferrocene acetic acid/
ferrocinium acetic acid) and electrolyte. A platinum counter
electrode having an area of 0.75 cm2 was then placed over
the chip. The distance between the chip and the counter
electrode was kept at approximately 650 to 800 µm with
the use of an O-ring.17 Cyclic voltammetery was performed
from -0.4 to 1.2 V (this potential represents the cell potential
and is reported as the potential of the working electrode
relative to the Pt counter electrode) at a sweep rate of 100
mV s-1 by using the electrodes on the chip as anodes and
the remote auxiliary electrode as the cathode. The addition
of an anticoumarin antibody was expected to interfere with
Fc+ (ferrocinium acetic acid) transport to the anode on the
chip reducing the total current flow.
Figure 1. “Zigzag” pattern of electrodes used for site-selective
coumarin synthesis.
From the start, only a small signal could be observed for
the blocks of electrodes having the coumarins linked to the
surface with 5 thymidine units while no signal could be
observed for the blocks of electrodes having 15 thymidine
units. However, a clear signal could be observed for the block
of electrodes having the coumarin linked to the surface with
a single thymidine. The results of this experiment are
illustrated in Figure 2. The initial current measurement was
made in the absence of any antibody and is represented by
the black curve. The array was then treated with an anti-
2,4-DNP antibody and the current measured giving rise to
the red curve illustrated in Figure 2. No significant drop in
current was measured indicating that there was minimal
nonspecific interaction between the antibody and the array’s
surface. Following this control experiment, the chip was
treated with the anticoumarin antibody used in the imaging
experiment illustrated in Figure 1. The current was measured
and gave rise to the green curve illustrated in Figure 2.
Clearly, the overall strategy employed is capable of providing
a “real time” signal in response to a small molecule-antibody
binding event. Even with the current level of sensitivity, the
use of only 121 electrodes on a 12K chip suggests that
libraries of approximately 100 molecules can be rapidly
screened.
image of an array where a “zigzag” pattern of electrodes
(blocks of 32) were used as anodes. The effectiveness of
the confinement strategy is readily apparent. In direct analogy
to the use of site-selective catalytic Pd(0) reactions on the
arrays,3 it was clear that the strategy for effecting site-
selective Pd(II) reactions was not just effective for oxidation
reactions requiring a stoichiometric Pd(II) reagent, but also
for transformations utilizing catalytic Pd(II).
Having effectively located coumarins proximal to elec-
trodes on the surface of a chip, attention was turned to the
signaling experiments. For this work, an electrode microarray
having ca. 12 000 electrodes/cm2 was utilized.16 Of the ca.
12 000 electrodes, three blocks of 121 electrodes were
employed as anodes for the coumarin synthesis. On the first
block, the coumarin was linked to the polymer through a
single thymidine unit having an aminoethoxyethyl terminat-
ing group (Scheme 2, n ) 0). On the second block, the
coumarin was linked to the polymer by using 5 thymidines
(13) Oyamada, J.; Jia, C.; Fujiwara, Y.; Kitamura, T. Chem. Lett. 2002,
380.
(14) Available from Vector Laboratories, catalogue no. BA-0606.
(15) Available from Molecular Probes, catalogue no. S872.
(16) For these experiments, a CombiMatrix 12K CustomArray DNA chip
with a variable length polythymidine strand of DNA terminated with a 5′-
aminoethoxyethyl modifier (Glen Research) was used. For ordering
information see ref 11.
(17) The distance between the electrodes was dependent upon the nature
of the O-ring material and the pressure used to hold the plates together.
Org. Lett., Vol. 8, No. 4, 2006
711

electrochemical sensors. However, in the case of the array
the coumarins were attached to a polymer covering the
surface of the chip and not directly attached to the electrodes
themselves. The presence of this polymer did not prevent
detection of the binding event; however, the stregnth of the
signal is dependent on the distance between the coumarin
and the electrode used for monitoring its behavior. At the
present time, the level of sensititivity obtained would allow
for the rapid monitoring of small libraries of molecules.
Current work is focusing on optimizing the sensitivity of
the system, probing the size of the library that can be
monitored, and examining the use of the overall strategy for
differentiating between the binding of an antibody in solution
with different substrates on the surface of the chip.
Acknowledgment. We thank the National Science Foun-
dation (CHE-0314057) and CombiMatrix for their generous
support of this 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.
Figure 2. Cyclic voltammetry of ferrocene acetic acid on Pt
electrodes with coumarin linked to a polymer through a single
thymidine having an amino ethoxy ethyl terminating group. All
measurements were performed in PBS buffer and the concentration
of the ferrocene acetic acid was 8 mM.
Note Added after ASAP Publication. There were errors
in the Abstract graphic and Scheme 2 in the versions
published ASAP January 28 and 31, 2006; the corrected
version was published ASAP February 1, 2006.
In conclusion, we have demonstrated that the same
confinement strategy for isolating Pd(II)-mediated oxidations
to site-selective locations on a chip can also be employed
for conducting site-selective cycloaddition reactions that
utilize a Pd(II) catalyst. This reaction scheme was utilized
to synthesize coumarins on the surface of an addressable
electrode array. The coumarins were used to probe an
electrochemical method for monitoring ligand/receptor bind-
ing events on the arrays. The method for detecting binding
on the array surface was designed in analogy to existing
Supporting Information Available: Experimental details
for the synthesis of 1, sample procedures for the chip-based
experiments, and CV data with 5 and 15 thymidine linkers
on the polymer. This material is available free of charge via
the Internet at http://pubs.acs.org.
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Org. Lett., Vol. 8, No. 4, 2006