Building addressable libraries: Amino acid derived fluorescent linkers

Article abstract of DOI:10.1021/la2047257

A new amino acid derived fluorescent linker for attaching molecules to the surface of a microelectrode array has been developed. Molecules to be monitored on an array are attached to the C-terminus of the linker, the N-terminus is then used to attach the linker to the array, and the side chain is used to synthesize a fluorescent tag. The fluorescent group is made with a one-step oxidative cycloaddition reaction starting from a hydroxyindole group. The linker is compatible with site-selective Cu(I)-chemistry on the array, it allows for quality control assessment of the array itself, and it is compatible with the electrochemical impedance experiments used to monitor binding events on the surface of the array.

Full text of DOI:10.1021/la2047257

Letter
pubs.acs.org/Langmuir
Building Addressable Libraries: Amino Acid Derived Fluorescent
Linkers
Takamasa Tanabe,† Bo Bi,† Libo Hu,† Karl Maurer,‡ and Kevin D. Moeller*,†
†Department of Chemistry, Washington University in St. Louis, St. Louis, Missouri 63130, United States
‡CustomArray, 6500 Harbor Heights Parkway, Suite 301, Mukilteo, Washington 98275, United States
S
* Supporting Information
ABSTRACT: A new amino acid derived fluorescent linker for
attaching molecules to the surface of a microelectrode array
has been developed. Molecules to be monitored on an array
are attached to the C-terminus of the linker, the N-terminus is
then used to attach the linker to the array, and the side chain is
used to synthesize a fluorescent tag. The fluorescent group is
made with a one-step oxidative cycloaddition reaction starting
from a hydroxyindole group. The linker is compatible with
site-selective Cu(I)-chemistry on the array, it allows for quality
control assessment of the array itself, and it is compatible with
the electrochemical impedance experiments used to monitor
binding events on the surface of the array.
Scheme 1
INTRODUCTION
■
Microarray-based molecular libraries can be powerful tools for
probing the binding preferences of biological targets.1,2
However, current efforts do have several limitations. Efforts
to identify the molecules in the library that bind a receptor
typically involve multiple washing steps, and it is difficult to
fully characterize the molecules in the library. This leads to a
loss of information about weak-binding interactions between
library members and the receptor, and a lack of quality control
with respect to the library itself. One method for potentially
avoiding these difficulties would be to probe the interactions
with the use of a microelectrode array.3−5 In such efforts, each
unique member of the library is placed or synthesized next to a
unique, addressable electrode (or set of electrodes) in the
array.6−9 The electrodes can then be used to both conduct
impedance experiments that detect binding-events between the
molecules in the library and the receptor in “real-time”10 and
reclaim molecules from the library so that they can be
characterized.11
The synthetic strategy used to place or synthesize molecules
on the surface of the microelectrode array is illustrated in
Scheme 1.
Central to this effort is the linker used to connect the
molecules to the polymer coating on the array. The linker
needs to be readily accessible, compatible with both the
synthetic and analytical electrochemistry used to build and
monitor molecules on the arrays, and fluorescent. To date, no
linker has been able to satisfy all three requirements, a fact that
In this example, a Cu(I)-catalyzed coupling reaction is used
to add a molecule with a heteroatomic nucleophile to an
arylbromide12 on the surface of the array.9b The reaction places
the molecule next to selected electrodes in the array by using
the electrodes to generate the Cu(I)-catalyst. Oxygen is then
used to reoxidize the Cu(I)-catalyst in the solution above the
array before the catalyst can migrate to electrodes not selected
for the reaction. For each new molecule to be added to the
array, a new set of electrodes is used to generate the catalyst.
Received: November 30, 2011
Revised: January 6, 2012
Published: January 9, 2012
© 2012 American Chemical Society
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Letter
has been limiting development of the arrays. We report herein a
solution to this problem that capitalizes on amino acid derived
linkers.
synthesizing fluorescent groups from hydroxylated indole
rings.15 The synthesis started with 5-hydroxytryptophan
(Scheme 2).
It is important for the linkers to be fluorescent. Micro-
electrode arrays typically fail when a short-circuit in their wiring
permanently turns on all of the electrodes in a region of the
array. When this occurs, a reaction on the array cannot be
confined. It occurs at both the electrodes selected for
generating the reagent of choice and every electrode accidently
activated by the short-circuit. Since the software used to assess
whether an array is working properly simply checks to see if all
of the electrodes are active, it does not detect this problem. In
addition, all of the electrodes being active does not mean that
they are all working equally well. Does using two different
electrodes to run a reaction on the array really place roughly
equal amounts of material by both of the electrodes? The use of
a fluorescent linker allows one to answer these questions and
assess the quality of the array before investing the time, money,
and effort needed to build and analyze a molecular library.
Our first attempt at building a fluorescent linker took
advantage of a disubstituted pyrene 1 (Figure 1).13,14 The effort
a
Scheme 2.
a
Reagents and Conditions: (a) SOCl2, MeOH, RT. (b) Boc2O,
Na2CO3, Dioxane/H2O, RT. (c) MnO2, Benzyl amine, dry THF, 0
°C−RT, 31% in three steps. (d) LiOH, THF/MeOH/H2O, RT, 92%.
(e) LiAlH4, THF, 45 °C, 84%. (f) DCC, DMAP, DMF, RT, 63%.
Figure 1. Past (1) and newly designed (2) fluorescent linkers.
was partially successful in that it allowed us to assess the quality
of the array. However, the linker was very difficult to synthesize.
Construction of the disubsituted pyrene core of the linker
required the bromination of a pyrenyl butanoic acid derivative,
a reaction that led to a complex mixture of products, generated
only low yields of those products, and could not be scaled up.
Because of these synthetic problems, the compatibility of the
linker with electrochemical signaling experiments on the arrays
could not be determined. A better approach was needed, and it
appeared that an amino acid derived linker like 2 might provide
this opportunity. The plan called for using the C-terminus of
the amino acid to attach molecules for study on the arrays, the
N-terminus of the amino acid to connect the linker to the array,
and the side chain functionality of the amino acid to build the
fluorescent tag. The approach was intriguing because it would
allow for variations in the fluorescent group to be made without
requiring a change in the overall strategy. It would also
capitalize on the very gentle Cu(I)-chemistry outlined in
Scheme 1 to attach the molecules to the array.
Following protection of the C-terminus and N-terminus of
the amino acid, the fluorescent label was constructed from the
hydroxyindol with the use of an oxidative cycloaddition. While
the yield of the cycloaddition was low, it provided a readily
scalable, one-step synthesis of the fluorescent group that could
be varied by simply changing the arylamine used. Next, biotin
was attached to the C-terminus of the amino acid derivative so
that biotin/streptavidin interactions could be used to determine
the compatibility of the linker with electrochemical impedance
experiments on the arrays. This was accomplished by
saponification of 4 followed by esterification of the resulting
acid with alcohol 7.16
Compound 8 was then placed onto an array having 12,544
microelectrodes·cm−1 (Scheme 3). The array was coated with a
diblock copolymer containing a cinnamoyl functionalized
methacrylate block and a poly(p-bromostyrene) block.12 The
cinammoyl groups were photochemically cross-linked to
provide the necessary stability for the surface. At this point,
the t-Boc-protected amine in compound 8 was directly coupled
to the poly(p-bromostyrene) block of the polymer proximal to
selected electrodes by using the chemistry highlighted in
Scheme 1.12,17 To this end, the array was treated with a
CH3CN/DMF/H2O solution containing the substrate, copper
sulfate, triphenylphosphine, and tetrabutylammonium bromide.
The electrodes selected for the reaction were set at a potential
of −1.7 V vs the Pt-counter electrode for 180 s in order to
reduce the copper sulfate to Cu(I) where the catalyst was
needed. Oxygen was used as a solution-phase oxidant to
prevent migration of the Cu(I)-catalyst to remote sites on the
array. Initially, the electrolysis reaction was conducted at 10
RESULTS AND DISCUSSION
■
Two questions about the plan immediately arose. First, what
should the fluorescent group be, and second, would the amino
acid linker and the fluorescent group selected be compatible
with the electrochemical impedance experiments employed for
monitoring binding events on the arrays?
The choice of a fluorescent tag was based on the availability
of the amino acid starting material and the simplicity of the
synthetic route needed for its construction. With this in mind,
we elected to take advantage of an oxidative strategy for
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solution, a cyclic voltammogram was recorded for the iron in
solution at each of the nine selected blocks of electrodes by
sweeping the potential at the electrodes from −700 to 700 mV
relative to the counter electrode.18 A sweep rate of 400 mV/s
was used. Following this experiment, the array was washed
three times with a 10−19 M streptavidin solution and inserted
into the next solution having 10−19 M streptavidin. This was
Scheme 3
done to ensure a consistent concentration of 10−19
M
streptavidin across the surface of the array. The cyclic
voltammetry experiment was then repeated at all nine blocks
of electrodes. The array was then washed three times with a
10−18 M streptavidin solution and inserted into the solution
having 10−18 M streptavidin. The cyclic voltammetry experi-
ment was again repeated at all nine blocks of electrodes. This
process was continued until a cyclic voltammogram for the iron
in solution had been recorded at all nine blocks of electrodes
for every concentration of streptavidin. At this point, the data
recorded at one of the blocks of electrodes having biotin on the
surface (Figure 2) was examined for the potential (25 mV) at
separate sites on the array each containing 12 microelectrodes.
When the reaction was complete, the array was washed with
water and ethanol. The reaction was then repeated, but in this
second case, compound 4 was used in place of 8 as the
substrate and a new set of electrodes was employed. Again, the
reaction was run at 10 separate sites on the array each
containing 12 microelectrodes. The result was an array that
contained both blocks of electrodes that were functionalized
with the linker and biotin (compound 8) and blocks of
electrodes that were functionalized with only the linker
(compound 4). When the reactions were complete, the array
was washed with water and ethanol and then imaged with a
fluorescence microscope. The picture provided in Scheme 3
shows two of the electrode blocks functionalized with the linker
and biotin. The image clearly shows that the array worked
perfectly. The reaction was nicely confined to the selected
electrodes, and roughly equal amounts of material were placed
at each of the electrodes.
Interestingly, solution-phase experiments using these same
reaction conditions showed that Cu(I) did not catalyze the
addition of either a t-Boc protected nitrogen or an indole ring
to the arylbromide. Instead, the reaction conditions led to
removal of the t-Boc group and then direct coupling of the
resulting amine to the arylbromide. The deprotection is not
unexpected, since the reaction on the array is an undivided cell
electrolysis that generates acid at the anode.
Figure 2. Sample voltammograms for one block of electrodes.
which the greatest drop in current had occurred with increasing
streptavidin concentration. The presence of streptavidin causes
a drop in current because it binds the biotin on the surface of
the array and blocks iron from reaching the electrode below.
The current measured at this potential was then used to
compare all of the cyclic voltammograms measured at each
concentration of streptavidin used. This data is summarized in
Figure 3. Each data point in the figure is the average of the
current measured for the three blocks of electrodes used at 25
mV. For example, the data point on the red line at a
concentration of 10−14 M streptavidin represents the average
current measured at 25 mV for this concentration of receptor at
the three blocks of electrodes with linker plus biotin on the
surface. The point on the green line at this concentration
represents the average current measured at 25 mV for the three
blocks of electrodes with only the linker on the surface. The
data has been normalized and the largest change in current
(greatest impedance) observed scaled from zero to one. The
largest current measured for each set of data was given a value
of zero. In this way, the data directly reflects the binding of
streptavidin to the surface of the array. The larger number on
the vertical axis, the greater the binding of the receptor to the
surface.
With the functionalized array in hand, signaling experiments
were conducted by randomly selecting three of the ten
electrode blocks functionalized with the linker plus biotin,
three of the ten electrode blocks functionalized with only the
linker, and three unfunctionalized blocks of 12 electrodes each
from the remainder of the array. The array was then submerged
in a series of solutions containing streptavidin and 8 mM
K4[Fe(CN)4]2/K3[Fe(CN)4]3 in 1× phosphate buffer saline
(PBS). The solutions ranged in concentration of streptavidin
from 10−6 to 10−20 M. The first solution used was the 10−20
streptavidin solution. After submerging the array in this
M
The black line in Figure 3 summarizes the data for the three
blocks of unfunctionalized electrodes taken at random places
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increasing streptavidin concentration becomes almost equal
(parallel slopes in Figure 3).
Since there is only about 20−50 fmol of material by each
electrode in the array, at high concentrations of streptavidin the
biotin becomes completely bound. Hence, any further binding
of streptavidin at these sites results from nonspecific binding of
the streptavidin to the surface. The fact that at these
concentrations the rate of change in the impedance at sites
with biotin is the same as the rate of change for sites without
the biotin indicates that the nonspecific binding of streptavidin
to the surface is the same in both places. Hence, the difference
in impedance between electrodes functionalized with biotin and
electrodes functionalized with only the linker at low
concentrations shows the binding of streptavidin to the biotin.
Clearly, both the polymer surface coating the electrodes and
the fluorescent linker are compatible with the electrochemical
impedance experiments needed to monitor binding events on
the arrays.
In conclusion, the use of an amino acid derived fluorescent
linker provides a convenient handle for site-selectively placing
molecules on the surface of a microelectrode array. The linker
is easy to synthesize and can be rapidly functionalized with
different biological probes to be studied or substrates to be
development on the array’s surface. The linker enables quality
control assessment of the array itself, is compatible with the
analytical experiments used to probe binding events on the
surface of the array, and is stable enough to allow for the
multiple analytical experiments needed to evaluate binding
events in a quantitative manner. The linker design reported
here will be incorporated into all future efforts to capitalize on
the arrays.
Figure 3. Summary of the CV data for all nine blocks of electrodes.
Normalized curves are shown for the impedance measured electrodes
functionalized with the fluorescent linker and biotin (red), electrodes
functionalized with the fluorescent linker and no biotin (green), and
unfunctionalized electrodes (black).
on the array. This data shows that the streptavidin shows no
binding to the unfunctionalized polymer coating the array. The
green line in the figure shows the data for the three blocks of
electrodes functionalized with the linker (4) and no biotin. It
indicates that the streptavidin does bind the surface once it has
been functionalized. The red line shows the binding of
streptavidin to the surface of the electrodes functionalized
with the linker and biotin. The difference between the green
and red lines shows that the presence of the biotin causes a
significant increase in the binding of the streptavidin to the
surface of the electrodes.19 The nature of this difference can be
seen clearly by taking the difference between the lines (Figure
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental details for the synthesis of the fluorescent linker,
for coating the arrays with the diblock copolymer, for site-
selectively functionalizing the polymer, and for conducting the
signaling studies are provided along with the complete ref 15.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: moeller@wustl.edu.
ACKNOWLEDGMENTS
■
We thank the National Science Foundation (CHE-0909723)
for their generous support of this program.
REFERENCES
■
Figure 4. Difference data for the current drops associated with the
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3) and the electrodes functionalized with only the linker (green line in
Figure 3).
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