New Reductive Mediators for Expanding Array-Based Parallel Synthesis
Authors: Krueger Ruby L , Moeller Kevin D 
Published: July 2021

Microelectrode arrays are powerful analytical tools for monitoring interactions between biological molecules in real-time. By placing members of a molecular library at defined electrodes, one can easily screen interactions with biological targets in the supernatant through simple electrochemical impedance experiments. However, one of the limitations to using arrays with very high electrode densities is the requirement of functionalizing each individual electrode (commonly over 12500/cm2) with a member of the molecular library to be tested. The Moeller Lab seeks to bypass this road block by developing synthetic tools that would allow for synthesis of the combinatorial library directly on the array surface through parallel synthesis techniques.
One of the key features of any parallel synthesis strategy is protecting group orthogonality. The ability to selectively cleave one protecting group in the presence of various other protecting group moieties allows for diversification of a core scaffold in a site-specific manner. To this end, the Moeller Lab has utilized electrochemical methods to site-selectively generate acids, bases, Ce(IV), Ru(VII), Os(VIII), Cr(VI), Pd(0), Pd(II), Cu(I), Cu(II), quinones, H2, and Sc(III) at defined electrodes on a microelectrode array. In order to expand this toolset further, we are investigating protecting groups that are reductively cleavable, such as p-nitrobenzyl carbonates and carbamates. The reduction potential of these groups is readily tunable by modifying the substitution on the aromatic ring, giving rise to a small library of cleavable groups with different reduction potentials. One of the challenges towards using these groups on the arrays, however, is selection of an appropriate reductive mediator to shuttle electrons from the electrode at the array surface to the substrates localized on the outside of a polymer coating over the array. While oxidative mediators are relatively well-characterized and easy to come by, reductive mediators often require multistep syntheses to obtain and are less known than their oxidative counterparts.
We hypothesize that these reductively cleavable protecting groups can be mediated by the aryl ring moiety present in the protecting group (e.g. p-nitrobenzene for a p-nitrobenzyl carbamate-protected amine). Addition of an alkyl substituent (such as the methylene in the carbamate) to an aryl ring makes the reduction potential of the aryl ring more negative by ~200 mV. Thus the free aryl ring should be able to serve as the mediator for reductive cleavage of the protecting group. In support of this hypothesis, we have shown that we can deprotect a p-nitrobenzyl carbamate-protected amine site-selectively on a microelectrode array using p-nitrobenzene as the mediator. In addition, CV studies of a p-nitrobenzyl-protected amine substrate show that p-nitrobenzene is able to transfer electrons to the substrate -- the reduction wave of the mediator disappears when substrate is added. Thus by optimizing these mediated reactions on the microelectrode arrays and in solution, we hope to introduce a new family of reductive mediators and protecting groups to the synthetic community.

Figure 1