New Synthetic Methododology/Organic Synthesis

Because electrochemistry enables the selective manipulation of molecular oxidation states, the generation of highly reactive intermediates, and the reversal of functional group polarity, it provides an ideal method for discovering and exploring new, synthetically useful reactions. For instance, consider the generalized reaction in Scheme 1. In this transformation, an electron-rich, normally nucleophilic enolate equivalent is oxidized leading to the formation of an electrophilic radical cation. The radical cation traps a second nucleophile leading to the formation of a new bond and ring.

To date, a number of such reactions have been developed as synthetic tools. Several recent examples are illustrated in Scheme 2. The first two reactions illustrated are being employed in the synthesis of arteannuin M, the third in the construction of new quaternary carbons, the fourth in the synthesis of new constrained amino acid derivatives, and the fifth in the construction of glycopeptides. Each reaction reverses the polarity of an electron-rich olefin in order to accomplish a transformation that is otherwise not possible. The electrochemical reactions do not require the use of highly specialized equipment. All of the reactions shown can be performed in a three neck round bottom flask using a 6V-lantern battery as a power supply and a total reaction setup (minus the flask) costing less that $6.

An important aspect of ongoing work in this area is to demonstrate that the new reactions developed can be used as tools for the total synthesis of molecular targets. Key to this effort is developing both an understanding of what controls product formation in the anodic cyclizations and efficient overall strategies for using the reactions in conjunction with more traditional synthetic methods. To date, this work has involved a number of mechanistic studies examining the nature and reactivity of radical cation intermediates as well as the total synthesis of multiple constrained peptidomimetics and natural products.

Current efforts are focused on exploring the generality of Curtin-Hammett control of radical cation reactions, exploring the utility of tandem radical cation derived cyclizations, utilizing competition studies to probe the chemoselectivity of the cyclizations, and probing the chemistry of new radical cation intermediates. Several current target molecules of interest to our group are shown below in Scheme 3.

New Analytical Tools for Probing Molecular Interactions

While the chemistry outlined above has proven very useful for solving a number of structural challenges in synthesis, not all synthetic challenges in organic chemistry are of a “structural-variety”. Instead some are of a logistical nature.

For example, as part of a long standing effort to “map” the preferred three-dimensional binding motifs of biological receptors, we recently became engaged in an effort to develop methods for monitoring ligand – receptor binding events in “real-time”. To accomplish this goal, potential ligands (small molecules, peptidomimetics, glycoproteins, etc.) will be placed or synthesized on microelectrode arrays so that each unique molecular ligand is located proximal to a unique, individually addressable electrode in the array. The electrodes in the array will then be used to monitor binding events between the potential ligands and various biological receptors. The monitoring experiments will be conducted using electrochemical impedance as outlined in Figure 1. In this experiment, the microelectrodes in the array are used as anodes in order to oxidize an iron species (typically ferrocene or ferricyanide) in the solution covering the array. The iron species is re-reduced at a remote Pt-wire cathode setting up a measurable current at each microelectrode. When a receptor (M1R) is added to the solution above the array and binds to ligands on the array (M1, M2, etc.) the iron species is impeded from reaching the neighboring microelectrodes. This causes the current measured at the microelectrode next to the ligand that binds to drop, an event that can be readily detected. In this way, each molecular ligand on the surface of the array can be monitored for its binding to a biological receptor at the same time and as the events occur.

While the plan seems straight forward, it leads to the logistical synthetic challenge mentioned earlier. With the microelectrode arrays used for the signaling experiments having a density of 12,544 electrodes per square centimeter, how does one place or build a molecular library so that each unique member of the library is located next to only one unique electrode? How can synthetic chemistry be accomplished in a site-selective fashion. One answer to these questions is to take advantage of the electrodes themselves to trigger chemical reactions. This can be done by capitalizing on what electrochemical reactions do best. They reverse the reactivity of groups by either adding or subtracting electrons. On an array, this ability can be used to setup a competition between an electrochemical reaction that synthesizes a chemical reagent and a solution phase reaction that destroys the reagent before it can migrate away from the site of its origin. The result is a net reaction that is highly selective for individual electrodes in an array. An example of one such reaction is illustrated in Scheme 4. In this example, a site-selective Diels-Alder reaction is accomplished by using selected electrodes in the array as anodes to generate a Sc(III) Lewis acid from Sc(I) in the solution above the array. The Lewis acid generated in this manner is then promptly destroyed again by the presence of a reducing agent in the solution. By increasing the rate of Sc(III) generation at any given microelectrode in the array, the formation of the Lewis acid can overwhelm the reducing agent in the region surrounding the electrode. The presence of the Lewis acid by the electrode triggers a Diels-Alder cycloaddition at that site. Of course, the concentration of the Lewis acid fall off as the distance from the electrode increases allowing the solution phase reducing agent to destroy the Sc(III) before it migrates to a non-selected electrode. The success of this approach is shown for arrays having both 1028 microelectrodes cm-2 and 12,544 microelectrodes cm-2.

The overall plan for conducting site-selective reactions on the arrays has proven to be general. The strategy has been used to site-selectively run acid and base catalyzed reactions, transition metal reactions using Pd(0), Pd(II), and Cu(I), and oxidation reactions using both Pd(II) and ceric ammonium nitrate. These reactions can be very useful. The use of a base on the arrays has allowed us to place peptide substrates proximal to the microelectrodes using Michael reaction chemistry.

The ability to place peptides on the arrays allows us to explore the electrochemical impedance approach to monitoring binding events as outlined in Figure 1. For example, consider the study highlighted in Figure 2. In this experiment, two RGD peptides were placed onto a microelectrode array having 12,544 microelectrodes cm-2 (a 12K-array). Both peptides were located proximal to a block of ten microelectrodes. The first peptide (sequence = KGGRGDSPC) is known to bind tightly to the integrin receptor αIIbßIII (GPIIb/IIIa). The second peptide (sequence = KGGRADSPC) is known to bind weakly to the integrin receptor. The microelectrode array was then inserted into a solution containing potassium ferricyanide and a Pt-counter electrode. A cyclic voltammogram was run using the microelectrodes located next to both the tight binding peptide (the CV on the left) and the weak binding peptide (the CV on the right) giving rise to the red line in both cases. This line represents the background current associated with ferricyanide in the absence of the receptor. The integrin receptor was then added to the solution and the CV experiments repeated giving rise to the blue line in both cases. Clearly, a difference in binding was observed with the current measured at the microelectrodes proximal to the tight binding peptide dropping off significantly more than the current measured at the microelectrodes proximal to the weak binding peptide.

Work on this project is continuing along several paths. New site-selective synthetic methods are being explored, site-selectively cleavable linkers are being developed for characterizing molecules on the surface of an array, strategies for determining in “real-time” the relative binding of molecular ligands to various biological receptors are being studied, new custom polymers for coating the arrays and controlling the surface used for the subsequent synthetic and analytical experiments are being synthesized, and studies aimed at expanding the utility of the microelectrode arrays as bioanalytical tools are being pursued.