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Reports: DNI753287-DNI7: Combinatorial Approaches for the Discovery of Stimulus-Responsive Supramolecular Gels

Jonathan Pokorski, PhD, Case Western Reserve University

This ACS PRF new investigator project aims to use combinatorial chemistry to identify small molecules that will undergo supramolecular polymerization in response to a given stimulus. This proposal was motivated by the many environmental catastrophes that have been at the forefront of the news recently, with the goal of sequestering environmental pollutants from large bodies of water. The main goals of this proposal were to synthesize a split and pool bead-based combinatorial library of small molecules that were sequence defined and had the ability to fibrillize in response to a stimulus, develop a colorimetric assay that allowed for visualization of fibrillization on a solid support, and scale up synthesis of ‘hit’ compounds and test for their ability to provoke the intended gelation response in bulk materials.  

Figure 1: Structure of a peptide backbone versus a peptoid backbone.

In the first goal, we chose to use peptoids as our small molecule platform. Peptoids are similar in nature to the polyamide backbone of natural peptoids, however, the sidechain projects from the nitrogen rather than the a-carbon (Figure 1). This confers proteolytic stability in challenging environments, allows for construction from any primary amine ‘monomer’ and allows for sequence characterization from known peptide sequencing techniques (i.e. Edman degradation). We selected six residues encompassing hydrophobic, hydrophilic, and aromatic residues (Figure 2). Combinatorial synthesis proceeded using a split and pool synthetic procedure that was carried out for 7 cycles. In principle, this should result in ~280,000 individual compounds. The synthesis was performed on TentaGel resin, a resin that is compatible with both organic synthesis and aqueous-based assay development. TentaGel amine resins, however, do not allow for cleavage of the peptoid from the resin. We chose not to include a cleavable spacer that would further complicate synthetic procedures. Instead, we verified synthesis through manual on-bead Edman degradation. Gratifyingly, following Edman degradation LC-MS peaks that corresponded to each of our individual residues were found in all fractions throughout 7 rounds of sequencing.

Figure 2: The six side-chains chosen for incorporation into the split and mix library, encompassing a variety of chemical functionalities.

The next step in the project was to determine ‘hit’ beads, or those that underwent fibrillization under a given stimulus in an aqueous environment. We chose to use dodecyne as our model pollutant, owing to its hydrophobicity, which would mimic an oil spill.  Additionally, the alkyne functionality could be used in the copper-catalyzed azide alkyne cycloaddition reaction should our colorimetric screening method prove difficult. Several individual batches of beads (~10,000/batch) were incubated with varying amounts of dodecyne in a sonicated solution, for dispersion of the organic pollutant. Following incubation, the beads were washed with water and then stained with Congo red. Congo red is an environmentally responsive dye known to stain for fibrillization in protein or peptide fibrils. Beads were screened manually under an optical microscope and beads with the most intense red color were selected as hits and sequestered for sequencing (Figure 3).

Figure 3: Representative optical micrograph of bead-based screen. The darkest red bead in the center of the image was selected as a hit compound and will be sequenced and scaled up.

Fortunately, we were able to identify several beads that appeared to strongly interact with dodecyne to form fibrils on-bead. From here, we are currently synthesizing a peptoid standard which will allow for full sequence characterization of our hit compounds. This purified sequence will be assayed by a commercial vendor that is expert in peptoid sequencing, especially on bead. Once hit peptoid sequences are confirmed the brightest beads will be synthesized in scale to evaluate gelation activity. Briefly, concentration of both components, the peptoid and the pollutant, will be varied to determine gel forming ability under numerous condition, as evaluated through rheology and fluorescent recovery after photobleaching (FRAP) experiments to determine material properties.  

Impact on Students and Career

The PRF has been a very welcome funding source in the early stages of my independent career, allowing our research group to rapidly expand and provide support for a project that was a higher risk project than others funded by start-up funds within my lab. The funding has allowed me to hire a very experienced post-doc, wherein this would not have been possible off of start-up alone. By bringing Richard to the team, he has accelerated the results from my lab but more importantly he is a mentor to younger students. Richard has mentored 2 undergraduates on the PRF project and they have produced astounding results. Stacy Yeh received her B.S. this past year and made outstanding research progress over the course of this project, working closely together with her mentor. She is currently applying to medical school and will surely make for an excellent physician given her critical thinking skills developed within the lab. Once Stacy left, we hired Alex Prossnitz as an undergraduate researcher. In his early stages, he has shown an exceptional command of the research program and is rapidly helping to finish off the project. Alex has plans to go on to graduate school in a chemical science, and thus far it appears that he will be very well suited to be accepted to a top tier institution. As for my career, this has been an invaluable source of funds. By hiring Richard, it gave me a second set of eyes in the lab enabling a very strong start to my independent career. Funding from this grant immediately boosted the experience level of my lab and gave me a senior peer to bounce ideas off of, provided a second source of advice for younger students, and gave a head start on research productivity.