Building addressable libraries: A site-selective click-reaction strategy for rapidly assembling mass spectrometry cleavable linkers

Article abstract of DOI:10.1039/b910577h

A click-reaction was site-selectively carried out on 1000 and 12000 microelectrode arrays and characterized using TOF-SIMS.

Full text of DOI:10.1039/b910577h

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Building addressable libraries: a site-selective click-reaction strategy
for rapidly assembling mass spectrometry cleavable linkersw
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a
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Jennifer L. Bartels, Peng Lu, Amy Walker, Karl Maurer and Kevin D. Moeller*
Received (in Austin, TX, USA) 29th May 2009, Accepted 23rd July 2009
First published as an Advance Article on the web 17th August 2009
DOI: 10.1039/b910577h
A click-reaction was site-selectively carried out on 1000
and 12 000 microelectrode arrays and characterized using
TOF-SIMS.
especially worrisome because both linkers must be assembled
ahead of time, bound to the substrate, and then placed on the
array in a separate step. Furthermore, the placement of both
linker 1 and linker 2 on an array employs synthetic chemistry
that we would like to use in subsequent synthetic steps. Linker
1 is placed on the array using a base-catalyzed addition
to an activated ester, a strategy that rules out the use of
base-cleavable protecting groups in the substrate. Linker 2 is
placed on the array with the use of a Heck reaction, a strategy
that rules out the use of Pd(0)-catalyzed reactions to further
develop a substrate.
Recently, we have been developing the tools necessary for
building molecular libraries on addressable microelectrode
arrays that contain between 1024 (a 1K-array) and 12 544
2
1–7
(
a 12K-array) electrodes per cm .
In this work, the
microelectrodes are used to effect chemical reactions in a
site-selective fashion by generating the reagents or catalysts
needed for the synthetic transformations. The reagents or
catalysts are then destroyed by a confining agent in solution
so that they cannot migrate to non-selected regions of the
arrays. In this way, the same chemistry typically used to build
molecular libraries on solid-phase supports can in principle be
used to assemble them on the arrays. Once the molecules are
synthesized on the arrays, the microelectrodes can be used to
monitor interactions between the molecules in a library and a
The optimal TOF-SIMS cleavable linker would avoid these
issues by using unique chemistry that rapidly assembles the
linker on the array from simple functional groups while
tolerating a wide variety of molecules and functional groups.
1
0
To this end, the use of ‘‘click-chemistry’’ appears ideal.
‘‘Click-reactions’’ are compatible with a wide range of
substrates and have proven very useful for attaching bio-
11
3a,5a
biological receptor in ‘‘real-time’’.
One critical aspect of
molecules to surfaces. Since the chemistry conducted on a
this work is the development of TOF-SIMS cleavable linkers
for attaching molecules to the porous polymer coating the
microelectrode array is essentially the same as normal solution
and solid-phase chemistry (the microelectrodes are used to
generate the same catalysts), the use of a ‘‘click-reaction’’ on a
microelectrode array should also allow for the attachment of a
wide variety of biomolecules to the surface of the array. In
addition, one can anticipate the triazole product fragmenting
nicely in a TOF-SIMS experiment. ‘‘Click-reactions’’ are well
suited for development on a microelectrode array because they
are typically accomplished with the use of a Cu(I)-catalyst that
8
,9
surface of the array. The use of a linker that cleaves under
TOF-SIMS conditions is important for characterizing the
molecules built on the arrays. Without a cleavable linker,
TOF-SIMS experiments on the arrays only show the polymer
8
coating on the array. In other words, the signal to noise of the
side-chain relative to the polymer is too small to observe the
side-chain. With a cleavable linker, this is not a problem. Since
TOF-SIMS experiments can probe surfaces with a resolution
of the order of 50 microns and the microelectrodes on a
1
0
is generated in situ by reducing Cu(II) with sodium ascorbate.
If the sodium ascorbate is removed from the reaction, then the
microelectrodes in the array can in principle be used to reduce
Cu(II) and generate the catalyst selectively at specific locations
on the array. This possibility was first recognized by Chidsey,
Collman, and co-workers who utilized the ‘‘click-reaction’’ to
selectively functionalize a pair of gold interdigitated array
1
K-array are 94 microns and a 12K-array are 45 microns in
diameter, a TOF-SIMS cleavable linker enables characterization
of the molecules associated with any given electrode in the
array. Hence, the use of a cleavable linker provides a method
for conducting quality control analyses of libraries built on the
arrays. To date, the two linkers shown in Fig. 1 have shown
1
2
band electrodes. Selectivity for one electrode over the other
was obtained by using one of the electrodes as a cathode in
order to generate the desired Cu(I) catalyst from a Cu(II)
precursor in the solution above the electrodes and the second
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considerable promise along these lines.
While useful in TOF-SIMS studies, both of these linkers are
problematic. They are tricky to prepare, require multiple-step
syntheses, and are only available in small quantities. This is
a
Department of Chemistry, Washington University in St. Louis,
St. Louis, MO 63130, USA. E-mail: moeller@wustl.edu;
Fax: +1 314-935-4481; Tel: +1 314-935-4270
CombiMatrix Corporation, 6500 Harbor Heights Parkway,
b
Suite 301, Mukilteo, WA 98275, USA
w Electronic supplementary information (ESI) available: Experimental
procedures for microelectrode array reactions, TOF-SIMS and
synthesis and characterization of all compounds used. See DOI:
10.1039/b910577h
Fig. 1 TOF-SIMS fragmentation sites.
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Chem. Commun., 2009, 5573–5575 | 5573

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Fig. 2 (a) A ‘‘click-reaction’’ with every microelectrode turned on, on
a 1K-array. (b) A site-selective ‘‘click-reaction’’ on a 1K-array.
inserted into an identical 7 : 2 : 1 acetonitrile–dimethyl-
formamide–water solution containing pyrene methylazide 5,
copper sulfate, disodium bis(bathophenanthroline) disulfonate
1
3
ligand, and tetrabutylammonium bromide. In this case, the
reaction was not degassed and not protected from the air in
the hope that oxygen would serve as a scavenger for any Cu(I)
generated at selected microelectrodes during the reaction. A
checkerboard pattern of electrodes in the array was then
used to reduce the Cu(II)-species in solution by cycling the
microelectrodes on for 0.5 s at À2.4 V relative to a remote Pt
wire anode and off again for 0.1 s. Four hundred cycles were
completed. Microelectrodes that were not selected for the
experiment were left off for the entire reaction. The micro-
electrode array was then washed with water and 95% ethanol
and examined using a fluorescence microscope to give the
image in Fig. 2b. From this image, it is clear that the reaction
only occurred at microelectrodes that were turned on. No
reaction was observed at microelectrodes not used for the
experiment, microelectrodes that were proximal to acetylene
substrate. Hence, the use of oxygen to confine the reaction was
very successful and no ‘‘bleed-over’’ of Cu(I) from the selected
microelectrodes to neighboring electrodes was found.
Scheme 1
as an anode to destroy any active Cu(I) catalyst above its
surface. But how does this chemistry translate to a microelectrode
2
array having a density of over 12 000 microelectrodes per cm ,
especially when the arrays do not have the capability of
employing some microelectrodes as cathodes and others as
anodes at the same time? Is there a confining agent that can be
added to the solution above an array to stop the migration of
the Cu(I)-catalyst generated at one microelectrode to the
neighboring microelectrodes? If the reactions can be run
site-selectively on a microelectrode array, then will the linkers
cleave cleanly in TOF-SIMS experiments in a manner that
allows for the observation of molecules on the surface of the
array? We report here that ‘‘click-reactions’’ can be run
site-selectively on both 1K- and 12K-microelectrode arrays
with the use of air as a confining agent, and that the triazole
product generated is a useful TOF-SIMS cleavable linker.
The feasibility of the microelectrode array reaction was first
examined using a 1K-array. To this end, the activated ester 3
was placed on the surface of the array proximal to the
electrodes using the previously developed electro-generated
The exact same set of conditions could be utilized to
perform a site-selective ‘‘click-reaction’’ on a 12K-array
(
Fig. 3). In this case, a checkerboard inside of a box pattern
of microelectrodes was used for generating the Cu(I)-catalyst.
Once again, the oxygen present in the solution over the
microelectrode array served as an excellent confining agent
for the reaction. No reaction was observed at microelectrodes
that were not used as cathodes.
With the site-selective ‘‘click-reaction’’ in place, attention
was turned toward examining the utility of the triazole linker
prepared. First, the cleavage properties of the triazole linker
under TOF-SIMS conditions were examined. When the
2
–7
base conditions (Scheme 1).
The base was generated by
using the microelectrodes in the array as cathodes in order to
reduce vitamin B12. This was done using every microelectrode
in the array.
functionalized array 6 was placed in a primary ion beam at
m+
The functionalized array was then inserted into a 7 : 2 : 1
acetonitrile–dimethylformamide–water solution containing
pyrene methylazide 5, copper sulfate, tetrabutylammonium
bromide, and disodium bis(bathophenanthroline) disulfonate
2
5 keV (generated using a Bi
n
(n = 1–7, m = 1, 2) liquid
metal ion gun) and the positive ions generated collected for
1
3
ligand. In order to confirm that the reaction described above
placed the acetylene by each microelectrode in the array,
the ‘‘click-reaction’’ was first performed by cycling every
microelectrode in the array on for 0.5 s at À2.4 V relative to
a remote Pt wire anode and off again for 0.1 s. Four hundred
cycles were completed. As expected, this reaction led to
the generation of Cu(I)-catalyst and hence a successful
‘
‘click-reaction’’ at each microelectrode (Fig. 2a).
With the control experiment in place, a second array
(
functionalized in an identical manner to the first) was then
574 | Chem. Commun., 2009, 5573–5575
Fig. 3 A site-selective reaction on a 12K-array.
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In conclusion, the ‘‘click-reaction’’ provides an ideal
method for attaching molecules to the surface of an addressable
microelectrode array. The reactions can be performed
site-selectively on both 1K- and 12K-arrrays using the same
reagents employed in solution phase reactions and oxygen as a
confining agent. Furthermore, the triazole product formed is
readily cleaved in TOF-SIMS experiments and can therefore
be used to gather information about molecules attached to the
surface of the array. At the present time, it appears clear that
the use of ‘‘click-chemistry’’ derived linkers will play a pivotal
role in the development of future addressable microelectrode
array supported molecular libraries.
Scheme 2
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.
Notes and references
1
For
D. D. Montgomery, W. Wang and J. C. Tsai, Anal. Chim. Acta,
001, 444, 69–78. 1K chips: electrode diameter = 92 mm; distance
between the Pt-electrodes (rectangular cells) = 245.3 mm and
37.3 mm; 12K slide: diameter = 44 mm; distance between the
a description of the chips used here see: K. Dill,
2
3
Pt-electrodes (square cells) = 33 mm.
For alternative approaches see: (a) M. G. Sullivan, H. Utomo,
P. J. Fagan and M. D. Ward, Anal. Chem., 1999, 71, 4369–4375;
2
3
(
1
b) S. Zhang, H. Zhao and R. John, Anal. Chim. Acta, 2000, 421,
75–187; (c) R. Hintsche, J. Albers, H. Bernt and A. Eder,
Electroanalysis (N. Y.), 2000, 12, 660–665.
For Pd(II) reactions: (a) E. Tesfu, K. Roth, K. Maurer and
K. D. Moeller, Org. Lett., 2006, 8, 709–712; (b) E. Tesfu,
K. Maurer, S. R. Ragsdale and K. D. Moeller, J. Am. Chem.
Soc., 2004, 126, 6212–6213; (c) E. Tesfu, K. Maurer, A. McShae
and K. D. Moeller, J. Am. Chem. Soc., 2006, 128, 70–71.
For Pd(0) reactions: (a) J. Tian, K. Maurer, E. Tesfu and
K. D. Moeller, J. Am. Chem. Soc., 2005, 127, 1392–1393;
Fig. 4 (a) TOF-SIMS for agarose. (b) TOF-SIMS for agarose
functionalized with compound 6. For full spectra see the ESIw.
4
5
6
(
3
b) F. Tang, C. Chen and K. D. Moeller, Synthesis, 2007,
411–3420.
analysis, it was found that the triazole linker cleaved along two
major pathways (Scheme 2).
For examples of the site-selective generation of base: (a) M. Stuart,
K. Maurer and K. D. Moeller, Bioconjugate Chem., 2008, 19,
1514–1517; (b) K. Maurer, A. McShea, M. Strathmann and
K. Dill, J. Comb. Chem., 2005, 7, 637–640.
For the site-selective generation of acid: D. Kesselring, K. Maurer
and K. D. Moeller, Org. Lett., 2008, 10, 2501–2504.
7 For the use of CAN in a site-selective fashion: D. Kesselring,
The first observed cleavage pathway (7, m/z = 281)
resulted from a b-elimination of the triazole ring from
the ester attaching the linker to the agarose polymer. This
cleavage may well be a characteristic of the specific substrate
used on the array for the ‘‘click-reaction’’. The second
fragmentation pathway (8, m/z = 242) resulted from a
retro [3+2]-cycloaddition involving the triazole itself. This
fragmentation should be independent of the alkyl substituents
on the triazole and hence represent a general cleavage
mechanism for the linker that can be used to characterize
molecules placed or constructed on the surface of the array.
Cleavage of the linker along both pathways allowed for the
presence of the pyrene group on the surface of the array to be
detected in the presence of the agarose polymer coating the
array (Fig. 4).
K. Maurer and K. D. Moeller, J. Am. Chem. Soc., 2008, 130,
11290–11291.
C. Chen, G. Nagy, A. V. Walker, K. Maurer, A. McShae and
K. D. Moeller, J. Am. Chem. Soc., 2006, 128, 16020–16021.
9 C. Chen, P. Lu, A. Walker, K. Maurer and K. D. Moeller,
Electrochem. Commun., 2008, 10, 973–976.
0 H. C. Kolb, M. G. Finn and K. B. Sharpless, Angew. Chem., Int.
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8
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