Electrochemically Generated Organometallic Reagents and Site

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Electrochemically Generated Organometallic Reagents and SiteSelective Synthesis on a Microelectrode Array Kevin D. Moeller* Department of Chemistry, Washington University, St. Louis, Missouri 63130, United States ABSTRACT: Organometallic reagents and catalysts are powerful tools for site-selectively modifying the surface of a microelectrode array. In this context, the reagents or catalysts are generated at the electrodes in the array and then destroyed again in the solution above the array. In this way, they can be used to conduct reactions that are confined to the regions of the array immediately surrounding the electrodes used. In the review presented, this general strategy is applied to a series of organometallic reactions that serve to illustrate the scope of synthetic chemistry possible.

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INTRODUCTION Microelectrode arrays1−3 contain collections of individually addressable electrodes that can be used to monitor binding events between molecules on the surface of the array and biological receptors in “real time”.4−7 To accomplish this, the molecules on the surface of the array must be built or placed on the array proximal to the electrodes used to monitor their behavior. This challenge was first met in connection with building DNA oligomers on the arrays by scientists at CombiMatrix Corp.8 The chemistry employed in this effort took advantage of the common dimethoxytrityl-protecting group strategy for solid-phase DNA synthesis. After a protected DNA monomer was placed by each of the electrodes in the array, acid was generated by using selected electrodes in the array for an oxidation reaction. The acid generated in this manner cleaved the DMT protecting group from the DNA monomer located by the electrode. The array was submerged in base so that the acid generated by the electrode would be destroyed before it could migrate to sites on the array not selected for the deprotection.9 The strategy worked well, and the deprotected DNA building blocks could be used to selectively build DNA dimers at the sites on the array used for acid generation. Repetition of the strategy allowed for construction of DNA oligomers at the selected electrodes. A nearly identical approach was employed for the synthesis of peptide oligomers.9a This strategy of making a chemical reagent at selected electrodes in an array and then confining the reagent to those electrodes by destroying it in the solution above the array can in principle be applied to a wide variety of chemical reactions. For example, organometallic reagents would appear ideally suited for the method. Organometallic compounds have multiple oxidation states that can be electrochemically manipulated.10,11 Of these oxidation states, one is typically responsible for an observed reaction. A Suzuki or Heck reaction is triggered by Pd(0) but not Pd(II). A Wacker oxidation is triggered by Pd(II) but not Pd(0). Therefore, manipulation of © 2014 American Chemical Society

the oxidation state of an organometallic reagent can turn it on and off with respect to its ability to do a chemical reaction. The manipulation of organometallic reagents on an array in this manner is very intriguing. Organometallic reagents are powerful tools for synthetic chemistry that enable a vast array of transformations. Their availability for use on an array would greatly expand the variety of molecules that could be synthesized on the array, a scenario that would in turn greatly expand the use of microelectrode arrays as analytical tools.

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INITIAL EFFORTS AND THE USE OF PALLADIUM In order to determine the feasibility of using organometallic reagents site-selectively on an array, the use of palladium was selected for study.12,13 As indicated in the preceding paragraph, both Pd(0) and Pd(II) reagents have proven to be effective tools for triggering synthetic reactions. Much is known about the stable oxidation states of palladium and about methods for interconverting them both by electrochemical means and with chemical reagents.10,14,15 This was important, because a “siteselective” reaction on an array must take advantage of both elements: an electrochemical method for generating a specific oxidation state of the metal and a solution-phase method for reversing the electrochemical reaction. This is best understood with an example. Illustrated in Scheme 1 is a site-selective Suzuki reaction that was run on an array having 1028 microelectrodes/cm2.12c,d For this reaction, the surface of the array was coated with a porous agarose coating. The agarose was used to attach substrates to the surface of the electrodes in the array while still allowing chemical reagents to diffuse to the electrodes below. A basecatalyzed esterification reaction between the alcohols in agarose and an activated ester was used to attach an aryl iodide Special Issue: Organometallic Electrochemistry Received: March 5, 2014 Published: June 24, 2014 4607

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Organometallics Scheme 1.

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Scheme 2.

a,1

a

Conditions for an array-based Suzuki reaction with oxygen as the confining agent: (a) − 2.4 V, 0.5 s on and 0.1 s off, 100 cycles; (b) − 1.7 V, 0.5 s on and 0.1 s off, 100 cycles; (c) − 1.4 V, 0.5 s on and 0.1 s off, 100 cycles.

solution in place of the allyl acetate. For all three reactions, every electrode in the array was fuctionalized with the aryl iodide, but only one was used for the Suzuki reaction. All three reactions were run for 100 cycles. The difference between the three reactions shown was the potential drop applied to the electrode selected for the reaction. The array reactions are all “constant current” reactions. This means that during the course of the reaction the current passed through the reaction remains the same. The level of the current passed is directly proportional to the voltage applied (V = iR). Hence, for the three images shown in Scheme 2 the experiment on the left with a potential of −2.4 V relative to the counter electrode passed current through the cell faster than the experiment with a −1.7 V potential drop, which in turn passed current through the cell faster than the experiment with a −1.4 V potential drop. This is important because the current passed through the cell controls the rate of Pd(0) generation at the electrode used. For the experiment using a potential drop of −2.4 V, the rate of Pd(0) generation at the electrode overwhelmed the oxygen confining agent. The bright spot shows destruction of the agarose in the neighborhood of the electrode. More importantly, fluorescence can be seen at every electrode in the array. Remember, each electrode in the array was functionalized with the aryl iodide. Therefore in this case, confinement of the Pd(0) reagent was lost and the Suzuki reaction occurred at each electrode in spite of only one electrode being used for the reduction. As the current being passed through the cell (and hence the rate of Pd(0) generation) was reduced, the rate of oxygen consumption of Pd(0) in the solution above the array was able to keep up with the rate of catalyst production at the electrode and confinement of the Suzuki reaction was established. In the experiment using a potential drop of −1.4 V, the Suzuki reaction occurred at only the single electrode used for the generation of Pd(0). Interestingly, the reverse reaction can be confined using the same strategy. In this case, the arylboronic acid was placed on the array and pyrenyl bromide was used as the solution-phase reagent (Scheme 3). Allyl acetate was used as the confining agent. The reaction was then run by setting every other electrode in the array at a potential of −2.4 V relative to the Pt counter electrode for a period of 0.5 s and then off again for 0.1 s. The experiment was conducted for 600 such cycles. Once again, the reaction was perfectly confined to the microelectrodes used for the reduction of Pd(II). This observation indicated that the Suzuki reaction was fast enough to consume the aryl palladium species generated by oxidative addition to the pyrenyl bromide before it migrated away from the surface of the electrode. This was true even with the faster generation of Pd(0).

substrate for the Suzuki reaction to the surface of every electrode in the array.16 For this reaction, the base was generated at the electrodes by the reduction of vitamin B12. The Suzuki reaction was then run using a checkerboard pattern of electrodes in the array at cathodes. This was accomplished by first treating the array with a DMF/MeCN/water solution containing palladium acetate, pyreneboronic acid, tetra-nbutylammonium bromide (electrolyte), and excess allyl acetate (confining agent). The use of Pd(II) as the source of the metal was chosen because Pd(II) cannot initiate the Suzuki reaction. The Suzuki reaction was then performed at every other electrode in the array by setting those electrodes to a potential of −1.7 V relative to a remote Pt anode for a period of 0.5 s and then off again for 0.1 s. The electrode was cycled 300 times in this fashion. The Pd(0) generated at the electrodes triggered the Suzuki reaction at those sites. The active Pd(0) catalyst was confined to the region of the array surrounding the electrodes selected by the allyl acetate that was added to the solution. Pd(0) rapidly undergoes oxidative addition to allyl acetate in order to form a π-allyl Pd(II) complex that is inactive as a catalyst for the Suzuki reaction. In this way, the allyl acetate reoxidized the Pd(0) generated at the electrodes. The electrodes were cycled as outlined above in order to slow formation of the Pd(0) catalyst at the electrodes so that the confining agent (allyl acetate) in solution could keep up. The success of the reaction was monitored by taking advantage of the pyrene group placed on the array by the Suzuki reaction. In the image provided, it can be clearly seen that the reaction occurred only at electrodes used for the reaction. No fluorescence appeared at electrodes that were not selected for the reaction. The nature of the competition on the array between making the reagent at the electrodes and destroying it again in solution can be seen nicely in Scheme 2. Three images are shown, one each for three separate array-based Suzuki reactions. The reactions were run in a fashion very similar to the reaction shown in Scheme 1, with the major change being the use of oxygen as the confining agent (an oxidant for Pd(0)) in 4608

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vinyl ether. This solution-phase reaction converted all of the Pd(II) in solution to Pd(0). The transformation ensured that no Wacker oxidation would occur on the array without the use of an electrode to regenerate the Pd(II) oxidant needed. After the incubation period, a checkerboard pattern of electrons was set to a potential of +2.4 V relative to a remote Pt electrode for a period of 0.5 s. The electrode was then turned off for 0.5 s. The cycling of electrodes was done for 600 cycles. As with the reduction reaction, the electrodes were cycled in order to slow down formation of the reactive reagent at the electrodes and in so doing optimize confinement of the reagent to the sites of its generation. In this experiment, an indirect oxidation of the Pd(0) was utilized. The positively charged electrodes first oxidized the triarylamine to a radical cation that in turn oxidized the Pd(0). The Pd(II) generated by this process initiated a Wacker oxidation of the olefin substrate attached to the surface of the activated electrodes. Any Pd(II) oxidant that was not consumed by the Wacker oxidation was scavenged by the excess ethylvinyl acetate in solution. The use of triarylamine as a mediator for the Pd oxidation was consistent with the conditions used for the preparative-scale electrochemical Wacker oxidation. In those reactions, use of the triarylamine is thought to prevent palladium deposition on the electrode. The success of the confinement strategy used on the array was determined by converting the ketone product into a 2,4dinitrophenylhydrazine (2,4-DNP) and then treating the array with an anti-2,4-DNP antibody that was labeled with a fluorescent tag. The array was then imaged with a fluorescence microscope. The result of the experiment is shown in Scheme 4. The electrodes selected for the reaction showed the clear fluorescent signature associated with the presence of the ketone product and subsequent hydrazine formation. Electrodes not selected for the oxidation showed no reaction. The confinement strategy worked perfectly. Subsequent studies demonstrated that the reaction did not require the use of a very reactive olefin as the confining agent. In fact, the same substrate placed on the surface of the array served nicely as the solutionphase confining agent.

Scheme 3

An equal but opposite strategy can be used to conduct Pd(II) reactions on the arrays.13 The Torii and Wayner groups have both nicely demonstrated that preparative Wacker oxidations can be run in a catalytic fashion by recycling Pd(II) at an electrode.10b,17 Since electrochemical reactions are readily scalable, it seemed that the same reaction conditions would be applicable to use of the reactions on an array.13b The only additional requirement would be the identification of a confining strategy for the array reaction. This case involved identifying a reducing agent from Pd(II) that would consume in solution any of the reagent generated at the electrodes in the array. Since the Wacker oxidation involves the conversion of an electron-rich olefin into a carbonyl with the use of stoichiometric palladium and water, it seemed that a solutionphase Wacker oxidation might provide a nice way to confine Pd(II) to the sites on an array where it was generated. To this end, ethyl vinyl ether was selected. Ethyl vinyl ether rapidly undergoes the Wacker oxidation to form ethyl acetate, which was easily removed following the reaction. The array reaction was conducted by first placing an olefin substrate by each electrode in a microelectrode array (Scheme 4). As noted earlier, this was accomplished by coupling an activated ester to an agarose polymer, coating the array with the use of base catalysis.16 The array was then treated with an electrolyte solution that contained a catalytic amount of Pd(OAc)2, tris(2-bromophenyl)amine, water, and excess ethyl vinyl ether. Incubation of this solution prior to activating the electrodes on the array led to Wacker oxidation of the ethyl Scheme 4.

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EXPANDING THE SCOPE OF CATHODIC REACTIONS With a strategy in place for conducting both reduction and oxidation chemistry on the arrays in place, attention was turned toward expanding the scope of reactions. This work began by examining the applicability of the strategy for generating and confining Pd(0) on the array to the Heck reaction.12d These efforts were initially puzzling. The first Heck reactions attempted on the arrays ran nicely, but reactions with more complex substrates appeared to be difficult to confine. Consider the experiment illustrated in Scheme 5. In this experiment, a site-selective Heck reaction was attempted as a method for placing a peptide substrate onto a microelectrode array that contained 12544 electrodes/cm2. The array was first coated with agarose, and then the agarose coating was functionalized with an acrylate substrate by two separate sets of electrodes: one being a checkerboard in a box pattern and the second being a series of parallel lines in a box pattern. The Heck reaction was conducted by setting the electrodes in the checkerboard in a box pattern at a potential of −1.7 V relative to the Pt counter electrode. The electrodes were cycled on and off as described previously. The electrodes in the lines in a box pattern were not used for the reaction. A fluorescent label on the peptide

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Scheme 5

Scheme 6.

substrate was then used to track confinement of the reaction (Figure 1). Surprisingly, the Heck reaction appeared to occur

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oxidation reaction at the counter electrode led to the undesired addition of nucleophiles to the agarose coating on the array. The unwanted reactions could be slowed or stopped with the use of a buffer solution that neutralized the acid generated at the anode, but it became soon became clear that a more stable surface for the array was a better alternative. In order to provide a more stable surface for attaching molecules to the electrodes in an array, the diblock copolymer (PBrSt-b-CEMA) shown in Figure 2 was prepared.18 One block

Figure 1.

beautifully at both the electrodes selected for the generation of Pd(0) and the electrodes not selected for the reduction. However, this conclusion was inaccurate, and the example highlighted in Scheme 5 and Figure 1 has been included in this review as a “cautionary tale”. The “loss of confinement” in this case was a result of an unstable agarose surface. When a checkerboard in a box pattern of electrodes was used to place an independently synthesized fluorescent Heck product directly on the array and then a second lines in a box pattern of electrodes used to add a simple acrylate group to the array, treatment of the array with the Heck reaction conditions in the absence of any additional peptide substrate led to migration of the fluorescence from the original pattern to the lines in a box pattern. The reaction equilibrated the esters located at the two sites. The problem with the functionalized agarose surface proved to be the Pd(II) precursor used for generation of the Pd(0) catalyst on the array. Pd(II) can serve as a Lewis acid that catalyzes the generation of oxonium ions from and the addition of nucleophiles to the functionalized agarose polymer. Evidence for this conclusion was obtained by first adding an ester to every electrode in an agarose-coated array. The functionalized array was treated with Pd(II) that was generated and confined to selected electrodes in the array using the conditions developed for the Wacker oxidation. When this was done in the presence of a fluorescently labeled amine, the amine was added to the surface of the array proximal to each electrode used for the generation of Pd(II) (Scheme 6). The problem was observed not only for the use of Pd(II) on the arrays but also for reduction reactions conducted with array formats that have the counter electrode (an anode) too close to the surface of the array. In such cases, the acid generated by the

Figure 2.

of this polymer was used to add cinnamate groups to the polymer. The cinnamate groups were photochemically crosslinked after the polymer was spin-coated onto the array in order to add stability to the surface. The other block of the diblock copolymer was made from a polystyrene with a 4-bromo substituent. This group was used to add molecules to the surface of the electrodes in the array. With the use of the more stable surface, the strategy for generating and confining Pd(0) on the array was general and worked the same way for each reaction attempted. On this surface, the Heck reaction outlined in Scheme 5 was confined perfectly to the selected electrodes. No difference was observed for the confinement of the Heck and Suzuki reactions.12d While the Heck and Suzuki reactions could be run using the same confinement strategy, other Pd(0) reactions did require an individualized approach. For example, the site-selective, Pd(0)-catalyzed allylation reaction illustrated in Scheme 7 cannot be confined with the use of allyl acetate as the solutionphase oxidant.12b The use of allyl acetate in solution would simply lead to a solution-phase allylation reaction with the nucleophilic substrate. This side reaction would both ruin confinement of the catalyst by regenerating Pd(0) in the solution above the array and consume the nucleophile needed for the surface reaction (the confining agent is used in excess). 4610

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Organometallics Scheme 7.

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ion mass spectrometry (TOF-SIMS) experiment was then used to characterize the product formed.20 The TOF-SIMS experiment took advantage of the triazole ring in the linker between the product and the array. Triazoles serve as mass spectrometry cleavable linkers that allow for observation of the product over background decomposition of the agarose coating on the electrode.19a The use of Cu(I) catalysts on the arrays also enabled the addition of heteroatomic nucleophiles to aryl bromides on the surface of the array (Scheme 9).19b,21 Typically, the aryl Scheme 9.

Fortunately, the allylation was nicely confined with the use of quinone as the solution-phase oxidant. Finding an alternative confining agent for the reaction was not hard. Any solutionphase oxidant for Pd(0) has the potential to serve as a confining agent on the array. As in the earlier Suzuki reaction, oxygen did serve as a confining agent for the allylation. However, the rate of Pd(0) oxidation with oxygen is relatively slow (Scheme 2), and it proved easier to optimize the allylation reaction with a faster oxidant. Quinone also worked well as the confining agent for the Suzuki reaction. However, it did not work as a confining agent for the Heck reaction, since it is itself a substrate for the Heck reaction. The lesson was clear. Any solution -phase oxidant for Pd(0) can potentially serve as a confining agent for an array reaction, as it is not itself a substrate for the reaction. The lessons learned by the examination of Pd(0) reactions on the arrays directly translate to other metals. To this end, the use of Cu(I)-catalyzed reactions has been of particular interest.19 The example highlighted in Scheme 8 shows a Scheme 8.

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bromides located proximal to the electrodes in an array are part of the diblock copolymer coating for the array shown in Figure 2. The image shown in Scheme 9 was taken following the addition of an alcohol nucleophile to an array having a density of 12544 microelectrodes/cm2. In this case, a 4-electrode checkerboard pattern of electrodes was employed for the reduction of Cu(II) to Cu(I) by setting the electrodes to a potential of −1.7 V relative to the Pt counter electrode. The reaction was run for 90 s and then turned off. This was repeated four times. As with the earlier Cu(I) reactions on the array, air was used as the solution-phase oxidant for confinement of the Cu(I) generated. While the reaction worked beautifully for the addition of alcohol and amine nucleophiles to the surface of the array, it did not work well in the case of thiol nucleophiles. When a thiol nucleophile was employed using the conditions for the reaction shown in Scheme 9, the reaction proceeded very slowly. Little fluorescence was observed by any electrode in the array. Fortunately, the success of the reaction could be altered greatly by the choice of the ligand used for the Cu(I) catalyst. When the ligand for the array reaction was changed from triphenylphosphine to a 1,3-dicarbonyl (Scheme 10),19b the coupling reaction between a thiol nucleophile and an aryl bromide on the surface of the array proceeded nicely. This

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Cu(I)-catalyzed version of a “Heck reaction” that was performed site selectively on an array.19a In this case, the reaction was conducted on an agarose-coated array that was functionalized at every electrode with the substrate for the reaction. The array was treated with a copper sulfate solution that contained triphenylphosphine as a ligand for the copper and a vinyl iodide. The Cu(II) precursor was then reduced at every other electrode in the array in order to form the Cu(I) catalyst needed for the coupling reaction. The Cu(I) catalyst was confined to the electrodes selected with the use of oxygen (air) as a solution-phase oxidant. The vinyl iodide substrate for the reaction was labeled with pyrene so that the success of the confining strategy could be monitored. As can be seen in the image provided, it worked perfectly. A time of flight, secondary

Scheme 10.

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the oxidation is the agarose coating on the surface of the array. Hence, it is present not only over the electrodes in the array but also between the electrodes. Nevertheless, the only fluorescence seen on the array following the reductive amination is located over the electrodes. No fluorescence is observed between the electrodes, an observation that illustrates just how well the oxidant was confined to the electrodes used for the experiment. The overall strategy for confinement of an oxidant is not restricted to the use of Pd(II). Cerium(IV) is a very versatile oxidant for organic synthesis.22 It can be used for reactions ranging from the generation of N-acyliminium ions from functionalized peptides23 to oxidative condensation reactions that generate bicyclic heterocycles.24 The chemistry highlighted in Scheme 12 shows how ceric ammonium nitrate (CAN) can be used to make reactive N-acyliminium ion intermediates at selected electrodes in an array.25

observation is consistent with what is known about the analogous solution-phase Cu(I)-catalyzed coupling reactions.21b With the use of the 1,3-dicarbonyl ligand, reactions employing alcohol and amine nucleophiles did not proceed as well as they had previously with the use of the triphenylphosphine ligand. However, when these reactions were run with the 1,3-dicarbonyl ligand, the reaction with the amine nucleophile did proceed better than the reaction with the alcohol nucleophile. This observation was again consistent with the rate differences reported for similar solution-phase reactions.21b What was clear from all of these efforts was that general strategies could be developed for array-based reactions that used the microelectrodes in the array as cathodes.

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EXPANDING THE SCOPE OF ANODIC REACTIONS Anodic reactions run on the arrays display a similar generality. For example, the concept of an anodic reaction on the array was introduced above with the Wacker oxidation. In this reaction, the reactive Pd(II) oxidant needed was generated by the oxidation of Pd(0) on the array and then confined to the selected electrodes by placing ethyl vinyl ether and water in the solution above the array. The ethyl vinyl ether served as a solution-phase Wacker substrate that reduced Pd(II) to Pd(0) and hence reversed the reaction conducted at the electrodes in the array. These conditions for confining the Pd(II) oxidant to selected sites on the array also worked beautifully for the oxidation of alcohols to carbonyls on an array. In Scheme 11, the Pd(II) Scheme 11.

Scheme 12.

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In this reaction, the microelectrode array was coated with an agarose polymer and then functionalized by each electrode with a amide substrate using the activated ester−electrochemically generated base chemistry described above. The amide contained a silyl group that can be used to activate the amide for oxidation and N-acyliminium ion formation.23 The array was then treated with solution containing a catalytic amount of ceric ammonium nitrate, electrolyte, 1-pyrenebutanol for trapping any N-acyliminium ion formed, and an excess of a silylated amide. The silylated amide was added to the solution to serve as the confining agent. The reagent chosen for this purpose was the same activated ester substrate used to place the surface-bound silylated amide acid onto the array. The solution was allowed to stand prior to the electrolysis reaction in order to make sure that any Ce(IV) in solution was completely consumed. A checkerboard in a box pattern of electrodes was then used on the array as anodes in order to regenerate Ce(IV) and in so doing trigger the oxidation reaction leading to Nacyliminium ion formation. The fluorescently labeled alcohol in solution then trapped the N-acyliminium ion, a reaction that allowed for the use of fluorescence microscopy to determine the effectiveness of the confinement strategy. The reaction worked nicely and suggested a very easy method for designing new site-selective oxidations. After all, the confining agent needed to isolate the reactions to given sites on the array was just the substrate used on the surface of the array. While this approach works frequently, a different confinement strategy is needed for an oxidation reaction that is part of a multistep sequence. For example, the reaction illustrated in Scheme 13 involves an oxidative condensation reaction.26 The diamine placed on the surface of the array first undergoes

oxidation of the agarose coating on an array is shown.13c The presence of a carbonyl product from each of two reactions on the array was shown by using the carbonyl as a substrate for a subsequent reductive amination reaction. The reductive amination was used to place a fluorescent amine onto the array. Both green and red fluorescent amines were used. The success of the confinement strategy for the Pd(II) oxidant is especially evident in this experiment because the substrate for 4612

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Organometallics Scheme 13.

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acid. Fluorescence microscopy was used to determine the success of the reaction. The image is shown in Scheme 14. Of note, a cleavable linker strategy was used to characterize the Diels−Alder product and show that the reaction ran exactly as intended.28 The overall strategy can be employed to run a variety of similar reactions. We have conducted Pd(II)-catalyzed cycloaddition reactions to form coumarin molecules on an array,13a as well as taken advantage of Os(VIII), TEMPO, and DDQ oxidants in a site-selective fashion.26

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ADVANTAGES TO THE USE OF TRANSITION-METAL REAGENTS ON AN ARRAY While a more complete summary of the types of organometallic reactions that can be done on the array could be given at this point, it is perhaps more instructive to provide an illustration of why the availability of such reactions is important. Consider the challenge of using a microelectrode array to monitor the binding of a peptide-based ligand or family of ligands to a targeted receptor. In such an analysis, the peptide ligands must be placed on the array next to the microelectrodes that will be used to monitor their behavior. One of the most popular methods to add peptide ligands to a variety of solid supports involves the use of a thiol-base Michael type addition. To this end, a cysteine residue is added to either the N or C terminus of the peptide, and then the thiol of the cysteine is used as a nucleophile that adds to an electron-poor olefin on the support. This approach can be used on an array. The addition of a cysteine-labeled peptide containing an RGD sequence to an acrylate moiety on a microelectrode array is illustrated in Scheme 15.29 Since thiol-Michael reactions are base-initiated

condensation with an aldehyde in solution. The resulting azaacetal is then oxidized to an imidazole ring with the use of ceric ammonium nitrate. In this case, the use of excess diamine in solution as the confining agent would lead to formation of the aza-acetal in solution. No aldehyde would remain for the surface reaction. Hence, a confining agent that was inert with respect to the aldehyde was needed. Such a confining agent was not hard to identify. In principle, a molecule that serves as a substrate for ceric ammonium nitrate oxidation can work as a confining agent. As illustrated in Scheme 13, the use of a pmethoxyarylamide worked well to confine CAN to the selected electrodes. A “heart pattern” of electrodes was used on the array for the oxidation. The use of the electrodes in an array as anodes can also trigger catalytic reactions. As in the cathode reactions, the confining agent in these cases serves to destroy the catalytic reaction in solution so that the catalyst can be confined to regions of the array where it is desired. The use of Pd(II) as a site-selective Lewis acid catalyst for the formation of oxonium ions on any array provided a nice example of this strategy (Scheme 6). This strategy can be generally applied. As an example, consider the Lewis acid catalyzed Diels−Alder reaction illustrated in Scheme 14.27 For this reaction, a dienophile was Scheme 14.

Scheme 15.

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placed by every electrode in an agarose-coated array. The array was then treated with a solution that contained a fluorescently labeled diene, a catalytic amount of ScIIIOTf3, and an excess of a thioamine acetal derivative that served as a reducing agent for Sc(III). The reduction of Sc(III) to Sc(I) in the solution above the array prevented catalysis of the Diels−Alder reaction at sites not selected for the reaction. The Diels−Alder reaction was then conducted by using a checkerboard in a box pattern of electrodes to oxidize Sc(I) and regenerate the Sc(III) Lewis

chain reactions, there is no way to confine the Michael reaction to selected microelectrodes in the array. However, placement of the electron-poor olefin onto the array with the use of a basecatalyzed esterification can be confined. In the experiment shown, a base-catalyzed addition of the activated ester to the surface of the array was conducted in the presence of excess 4613

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activated ester. The base catalyst for the reaction was generated by the reduction of vitamin B12 to form a radical anion that then deprotonated alcohols on the surface of the array. The resulting alkoxide reacted with the activated ester. A base can potentially migrate away from the selected electrodes if either the radical anion or an alkoxide on the agarose deprotonates the methanol solvent. However, the methoxide generated from such a reaction would react rapidly with the excess activated ester in solution, preventing it from reaching a neighboring electrode. The success of the approach can be seen in Scheme 15. Initially, an acrylate group was placed on the array in a checkerboard pattern. The array was then incubated with a fluorescently labeled RGD peptide that contained a cysteine group on the C terminus. This led to addition of the thiol nucleophile to the acrylate on the array. The two-step procedure was then repeated using a “dot in a box” pattern of electrodes. The image provided shows how well the confinement strategy worked. The fluorescence associated with the peptide only appears proximal to the electrodes selected for the initial esterification reaction. While the experiment in Scheme 15 demonstrated that the initial esterification reaction was confined to the selected electrodes, the image does not provide information about the stability of the molecules on the surface of the array. Thiolbased Michael reactions are reversible. Was the fluorescence observed for the dot in a box pattern due completely to the second peptide placement reaction, or did some of the fluorescence in the dot in a box pattern come from the original pattern? This is an important question because, if the surface is not stable, then the molecules by any electrode in the array would represent a mixture of compounds. To test this idea, a series of experiments were conducted. In each experiment, an electron-poor olefin was placed on the array in a checkerboard pattern and then the peptide with the RGD sequence added to that site as illustrated for the first reaction in Scheme 15. A second experiment was then conducted to place the Michael acceptor at an alternate site on the array in a dot in a box pattern. No additional peptide was added to the array at this point. Instead, the array was incubated at either pH 7 or pH 4 in order to see if any of the fluorescence from the original checkerboard site moved to the new dot in a box site. The result of one such study is shown in Figure 3. In this experiment, acrylate was used as the electron-poor olefin on the surface of the array, and the second incubation step was performed at a pH of 7.

Scheme 16.

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addition chemistry. This was attractive because it meant that the array strategy could be changed without having to resynthesize the peptide. The conditions described in connection with Scheme 9 for the site-selective addition of a thiol nucleophile to an aryl bromide on an array worked perfectly. The peptide with the RGD sequence was placed by 10 blocks of 12 electrodes. One of the blocks of electrodes is shown in the image provided. Confinement of the reaction with oxygen was perfect. A second peptide without the RGD sequence was placed on the array at alternate sites, and the array was then used to monitor binding events between the peptides and an integrin receptor.29 In a similar fashion, the Cu(II)-catalyzed “Chan−Lam” addition30 of a thiol to a borate ester functionalized surface (PCEMA-b-pBSt) can be used to place molecules siteselectively on an array (Scheme 17).18b In this case, the Scheme 17.

1

Figure 3.

Clearly, at this pH the peptide originally placed in the checkerboard pattern migrated to the dot in a box pattern. The functionalized array was not stable at biologically relevant pHs. What was needed was a covalent, nonreversible method for placing the peptide on the array. To this end, the use of a Cu(I)-catalyzed addition of a thiol to an aryl bromide on the surface of the array proved ideal (Scheme 16).19b,29 The reaction allowed for the use of the same type of cysteinefunctionalized peptide nucleophile as used for the Michael type

confining agent for the reaction is the excess thiol nucleophile in solution. The thiol gets oxidized to a disulfide with Cu(II), a transformation that leads to the formation of Cu(I). The Cu(I) is then oxidized at selected electrodes in the array to regenerate the Cu(II) catalyst needed and trigger the coupling reaction. In the example shown, a fluorescently labeled cysteine was was placed on the array using a “C pattern” of microelectrodes. The success of the confinement strategy for Cu(II) can be seen in the image provided. The reaction enables the irreversible 4614

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NMR facility, partially supported by NIH grants RR02004, RR05018, and RR07155.

placement of biological ligands onto an array with a tunable surface.18b Once a confinement strategy for a metal on an array has been developed, it can typically applied to any reaction using the metal. Hence, the success of the Chan−Lam reaction on the array suggests that any number of Cu(II)-mediated reactions can be conducted on the arrays.

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(1) For a description of the chips used in our laboratories 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 and the power supply for addressing them can be purchased from CustomArray, Inc., 18916 North Creek Parkway, Suite 115, Bothell, WA 98011 (www.CustomArrayInc.com). For a detailed discussion of how the array reactions are run see the Supporting Information in: Bartels, J.; Lu, P.; Maurer, K.; Walker, A. V.; Moeller, K. D. Langmuir 2011, 27, 11199. (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. (d) Gardner, R. D.; Zhou, A.; Zufelt, N. A. Sens. Actuators, B 2009, 136, 177. (e) Beyer, M.; Nesterov, A.; Block, I.; Kö nig, K.; Felgenhauer, T.; Fernandez, S.; Leibe, K.; Torralba, G.; Hausmann, M.; Trunk, U.; Lindenstruth, V.; Bischoff, F. R.; Stadler, V.; Breitling, F. Science 2007, 318, 1888. (f) Devaraj, N. K.; Dinolfo, P. H.; Chidsey, C. E. D.; Collman, J. P. J. Am. Chem. Soc. 2006, 128, 1794. (g) Wassum, K. M.; Tolosa, V. M.; Wang, J.; Walker, E.; Monbouquette, H. G.; Maidment, N. T. Sensors 2008, 8, 5023. (i) Zhang, Y.; Wang, H.; Nie, J.; Zhang, Y.; Shen, G.; Yu, R. Biosens. Bioelectron. 2009, 25, 34. (j) Li, X.; Tian, Y.; Xia, P.; Luo, Y.; Rui, Q. Anal. Chem. 2009, 81, 8249. (k) Chan, E. W. L.; Yousaf, M. N. ChemPhysChem 2007, 8, 1469. (h) Kerkoff, H. G.; Zhang, X.; Mailly, F.; Nouet, P.; Liu, H.; Richardson, A. VLSI Design 2008, DOI: 10.1155/2008/437879. (4) For reviews see: (a) Gyurcsányi, R. E.; Jágerszki, G.; Kiss, G.; Tóth, K. Bioelectrochemistry 2004, 63, 207. (b) Roberts, W. S.; Lonsdale, D. J.; Griffiths, J.; Higson, S. P. J. Biosens. Bioelectron. 2007, 23, 301. (c) Scanning Electrochemical Microscopy, 2nd ed.; Bard, A. J., Mirkin, M. V., Eds.; Marcel Dekker: New York, 2001. (5) For examples using macroscopic electrodes see: (a) Dykstra, P. H.; Roy, V.; Byrd, C.; Bentley, W. E.; Ghodssi, R. Anal. Chem. 2011, 83, 5920. (b) Murata, M.; Gonda, H.; Yano, K.; Kuroki, S.; Suzutani, T.; Katayama, Y. Bioorg. Med. Chem. Lett. 2004, 14, 137. (6) For related references with ion-channel mimic sensors see: (a) Endo, A.; Hyashita, T. J. Ion Exch. 2008, 19, 110. (b) Wirtz, M.; Martin, C. R. Sens. Update 2002, 11, 35. (c) Odashima, K.; Sugawara, M.; Umezawa, Y. ACS Symp. Ser. 1994, 561, 123. (d) Yue, M.; Zhu, X.; Zheng, Y.; Hu, T.; Yang, L.; Wu, X. Electrochim. Acta 2012, 73, 78. (e) Malecka, K.; Grabowska, I.; Radecki, J.; Stachyra, A.; GóraSochacka, A.; Sirko, A.; Radecka, H. Electroanalysis 2012, 24, 439. (f) Zhy, J.; Qin, Y.; Zhang, Y. Anal. Chem. 2010, 82, 436. (g) Xu, Y.; Bakker, E. Langmuir 2009, 25, 568. (h) Kurzatkowska, K.; Dolusic, E.; Dehaen, W.; Sieroń-Stoltny, K.; Sieroń, A.; Radecka, H. Anal. Chem. 2009, 81, 7397. (i) Komura, T.; Yamaguchi, T.; Kura, K.; Tanabe, J. J. Electroanal. Chem. 2002, 523, 126. (j) Odashima, K.; Kotato, M.; Sugawara, M.; Umezawa, Y. Anal. Chem. 1993, 65, 927. (7) For recent examples using the arrays described here, see: (a) Stuart-Fellet, M.; Bartels, J. L.; Bi, B.; Moeller, K. D. J. Am. Chem. Soc. 2012, 134, 16981. (b) Tanabe, T.; Bi, B.; Hu, L.; Maurer, K.; Moeller, K. D. Langmuir 2012, 28, 1689. (c) Bi, B.; Huang, R. Y. C.; Maurer, K.; Chen, C.; Moeller, K. D. J. Org. Chem. 2011, 76, 9053. (8) For examples see: (a) Maurer, K.; Yazvenko, N.; Wilmoth, J.; Cooper, J.; Lyon, W.; Danley, D. Sensors 2010, 10, 7371. (b) Roth, K. M.; Peyvan, K.; Schwarzkopf, D. R.; Ghindilis, A. Electroanalysis 2006, 18, 1982 and references therein. For the lead patent on this work see: Montgomery, D. D. PCT Int. Appl. WO 9801221 A1 19980115, 1998; 91 pp, CODEN PIXXD2. (9) For the use of site-selective acid generation on an array see ref 8 as well as: (a) Maurer, K.; McShea, A.; Strathmann, M.; Dill, K. J.

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CONCLUSIONS Most organometallic catalysts and reagents can be synthesized at an electrode surface. This situation provides an opportunity to use some of the most powerful synthetic tools in modern chemistry to selectively control the surface of a microelectrode array. That development dramatically expands the use of microelectrode arrays as analytical tools for probing biological interactions. After all, it is the nature of the molecules that can be built or placed on a microelectrode array that defines the receptors and therefore the biological problems that can be studied with the arrays. In the work described above, we demonstrate that the electrochemical juggling of transitionmetal oxidation states allows for reactions to be run at selected electrodes in an array. Both oxidative and reductive processes can be confined in this manner. The result is an entirely new method for constructing addressable molecular libraries.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail for K.D.M.: [email protected]. Notes

The authors declare no competing financial interest. Biography

Kevin D. Moeller was born in Scranton, Pennsylvania, in 1958. He received his B.A. degree in Chemistry from the University of CaliforniaSanta Barbara in 1980 and then his Ph.D. in Organic Chemistry from the same institution in 1985 under the direction of Professor R. Daniel Little. After postdoctoral studies as an NIH Fellow with Professor Barry M. Trost at the University of Wisconsin Madison, he joined the faculty at Washington University in St. Louis in 1987, where he is now Professor of Chemistry. His independent research focuses on the use of electrochemistry as a tool for solving synthetic challenges that arise on the chemistry−biology interface.

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ACKNOWLEDGMENTS We thank the National Science Foundation (CBET 1262176) for their generous support of our work. We also gratefully acknowledge the Washington University High Resolution 4615

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Comb. Chem. 2005, 7, 637. (b) Kesselring, D.; Maurer, K.; Moeller, K. D. Org. Lett. 2008, 10, 2501. (10) For pioneering work in the area of organometallic-mediated electrochemical reactions, see: (a) Tsuji, J.; Minato, M. Tetrahedron Lett. 1987, 28, 3683. (b) Inokuchi, T.; Ping, L.; Hamaue, F.; Izawa, M.; Torii, S. Chem. Lett. 1994, 121. (11) For reviews highlighting the utility of organometallic electrochemistry, see: (a) Geiger, W. Organometallics 2007, 26, 5378. (b) Geiger, W. Organometallics 2011, 30, 28. (12) For Pd(0) reactions, see: (a) Tian, J.; Maurer, K.; Tesfu, E.; Moeller, K. D. J. Am. Chem. Soc. 2005, 127, 1392. (b) Tian, J.; Maurer, K.; Moeller, K. D. Tetrahedron Lett. 2008, 49, 5664. (c) Hu, L.; Maurer, K.; Moeller, K. S. Org. Lett. 2009, 11, 1273. (d) Hu, L.; Stuart, M.; Tian, J.; Maurer, K.; Moeller, K. D. J. Am. Chem. Soc. 2010, 132, 16610. (13) For Pd(II) reactions, see: (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. (14) For a beautiful example of electrochemistry as a tool for studying Pd-catalyzed reactions, see: Amatore, C.; Le Duc, G.; Jutand, A. Chem. Eur. J. 2013, 19, 10082. (15) For leading references concerning the chemical recycling of Pd (and Cu) reagents, see: Campbell, A. N.; Stahl, S. S. Acc. Chem. Res. 2012, 45, 851. (16) For an example of the site-selective use of base on an array, see: Stuart, M.; Maurer, K.; Moeller, K. D. Bioconjugate Chem. 2008, 19, 1514. (17) Miller, D. G.; Wayner, D. D. M. Can. J. Chem. 1992, 70, 2485. (18) (a) Hu, L.; Bartels, J. L.; Bartels, J. W.; Maurer, K.; Moeller, K. D. J. Am. Chem. Soc. 2009, 131, 16638. (b) Hu, L.; Graaf, M. D.; Moeller, K. D. J. Electrochem. Soc. 2013, 160, G3020. (19) (a) Bartels, J. L.; Lu, P.; Walker, A.; Maurer, K.; Moeller, K. D. Chem. Commun. 2009, 5573. (b) Bartels, J. L.; Lu, P.; Maurer, K.; Walker, A. V.; Moeller, K. D. Langmuir 2011, 27, 11199. (20) Chen, C.; Walker, A.; Maurer, K.; Moeller, K. D. Electrochem. Commun. 2008, 10, 973. (21) For solution-phase examples of Cu(I)-catalyzed couplings of heteroatom nucleophiles to aryl bromides see: (a) Lu, X.; Bao, W. J. Org. Chem. 2007, 72, 3863. (b) Shafir, A.; Lichtor, P. A.; Buchwald, S. L. J. Am. Chem. Soc. 2007, 129, 3490. (22) Hwu, J. R.; King, K.-Y. Curr. Sci. 2001, 81, 1043. (23) Sun, H.; Martin, C.; Kesselring, D.; Keller, R.; Moeller, K. D. J. Am. Chem. Soc. 2006, 128, 13761. (24) Bahrami, K.; Khodaei, M. M.; Naali, F. J. Org. Chem. 2008, 73, 6835−6837. (25) Kesselring, D.; Maurer, K.; Moeller, K. D. J. Am. Chem. Soc. 2008, 130, 11290. (26) Nguyen, B. H.; Kesserling, D.; Tesfu, E.; Moeller, K. D. Langmuir 2014, 30, 2280. (27) Bi, B.; Maurer, K.; Moeller, K. D. Angew. Chem., Int. Ed. 2009, 48, 5872. (28) Bi, B.; Huang, R. Y.-C.; Maurer, K.; Chen, C.; Moeller, K. D. J. Org. Chem. 2011, 76, 9053. (29) Fellet, M. S.; Bartels, J. L.; Bi, B.; Moeller, K. D. J. Am. Chem. Soc. 2012, 134, 16891. (30) (a) Xu, H.-J.; Zhao, Y.-Q.; Feng, T.; Feng, Y.-S. J. Org. Chem. 2012, 77, 2878. (b) Herradura, P. S.; Pendola, K. A.; Guy, R. K. Org. Lett. 2000, 2, 2019.

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