We are interested in the design and synthesis of organic compounds that are able to mediate gated transmembrane ion transport, shown schematically here.

These ion channels will reside in a membrane, but only allow ions to pass following activation by a well-defined signal. Our initial efforts are focused on the energetically favorable transport of ions down their concentration gradient. Later, we will use the knowledge gained to further develop active transport processes, or pumping of ions against their concentration gradient.

So what are these channels actually made of? They are basically large, amphiphilic organic compounds. The components that we are using are shown here. A number of macrocycles have been used in non-gated synthetic channels, such as cyclodextrins, cyclic peptides, calixarenes, or crown ethers. These serve to preorganize the membrane-spanning chains into a channel architecture. The chains that we append to the macrocycle need to serve two purposes: they must favor insertion into a phospholipid membrane, and they must promote ion transport. To favor membrane insertion we are using lipophilic hydrocarbon chains, and the more polar oligoether chains will stabilize ions passing through the channel.

For the actual gating of the channel, we are working on the development of a number of different functionalities that are activated by a variety of signals. For example, we can form a covalent disulfide bond that spans the channel pore, rendering in blocked. The signal in this case is electrolytic reduction to the bis-thiolate resulting in opening of the channel. Alternatively, we may use light to activate the channel. In this example the opening is irreversible, as light results in the cleavage of the benzyl ethers, and the pore-spanning portion is extruded. Lastly, we may use chemical activation as the signal for channel opening. Here we have a nucleophilic phenol hydrogen-bonded to an imidazole ring. It’s difficult to envision in this diagram, but this orientation of the phenol and imidazole should form a hydrophobic plane that serves as the requisite barrier to ion transport. Following the addition of a phosphorylating agent such as ATP, we expect the phenol will be phosphorylated, leading to the formation of this charged ion pair which is significantly more polar than the un-ionized starting structure.

This should provide you with an idea of our project design. Next I’d like to briefly describe how we test for transmembrane ion transport.

Shown here is a schematic representation of the assay for ion transport. We can assemble phospholipid vesicles with our ion channels embedded in the membrane, and a pH sensitive fluorescent dye entrapped in the interior. Initially the salt concentrations inside and outside the vesicle will be identical, and the dye will be protonated and thus non-fluorescent. We then place the suspension of vesicles in a cuvette placed in a fluorimeter, and add a concentrated salt solution. This generates a concentration gradient; however, with our gate intact, this gradient should be maintained. Upon applying the signal for channel opening, if metal cations are able to flow across the membrane, protons will move out of the vesicle to maintain charge balance. This results in an increase in the pH, deprotonation of the dye, and thus fluorescence.

This is a standard technique for testing synthetic ion channels, and a number of other indicators can be utilized. In place of the pH-sensitive dye one can use fluorescent indicators specific for a given electrolyte ion, or you can use a sodium shift reagent. The shift reagent allows the differentiation of sodium ions inside the vesicle from sodium ions outside the vesicle using 23Na NMR.

What I have described so far is independent research that is ongoing in my labs. What is so exciting about participating in this water purification consortium is the opportunity to tailor these materials so that they can actually be incorporated into functional, macroscopic materials. By interacting with experts in the field of water purification as well as various materials gurus we can identify and target ions for transport. Our channels can be modified in many ways to make them both selective for various ions, as well as suitable for incorporation into materials that are compatible with water purification devices. For example, we can vary the size and composition of the macrocyclic pore template to select for different ions. We can alter the chains either in terms of their length or their functionality, perhaps incorporating handles for covalent attachment to a support membrane. Furthermore, the channel gate can be chosen such that the signal employed matches the requirements of a given system. This sort of collaboration is crucial to move from the exploratory stage into real-world applications.