The long-term goal of exploring macrocycles is to be able to produce drugs that can interfere with certain protein-protein interactions within cells. This strategy could have the potential to change the way scientists think about drug design. Aspartic acid is a particularly useful to incorporate because it is one of the top five amino acids that contribute to binding at protein-protein interfaces. The acid sidechain of aspartic acid presents significant challenge because of the potential for side reactions. This research has established that an aspartic acid macrocycle can be synthesized quickly in three steps. The route is remarkably efficient and has the characteristics of those that could be used to make drugs. This poster details the chemical synthesis and characterization of this molecule, discusses potential side reactions, and identifies the next steps in advancing this project.

N-terminal acetylation plays an important role in the stability, activity, and targeting of proteins in eukaryotes. Most proteins expressed in bacteria are not acetylated, although the N-terminal acetylation is critical for the activities of a handful of biologically important proteins. Therefore, it is of practical significance to control N-terminal acetylation of recombinant proteins in bacteria. This study is aimed to alter the substrate specificity of RimJ, a protein N-terminal aminotransferase (NAT) that is known to acetylate a few recombinant proteins including the Z-domain in E. coli. The RimJ-mediated protein acetylation occurs at a higher rate when the substrate’s N-terminal amino acid is small. Because of this narrow substrate specificity, RimJ is not applicable for a broad range of recombinant proteins. Based on the AlphaFold-predicted structure of E. coli RimJ (AF-P0A948_F1), we predicted that five amino acids (Y106, M142, N144, Y170, and L171) may recognize substrate proteins in the active site. We created RimJ variants, in which one or two of these five amino acids are changed to alanine, a small neutral amino acid, so that the active site becomes larger to accommodate substrate proteins containing bigger N-terminal amino acid residues. Then, the substrate specificity of RimJ was investigated by co-expressing two Z-domain variants T2I and S3K, which were not acetylated by the wild-type RimJ. The expressed Z-domain variants were purified by immobilized metal affinity chromatography and subsequently analyzed by mass spectrometry, by which a 42-Da mass increment indicates the presence of an N-terminal acetyl group. The RimJ single mutants such as N144A, M142A, and Y106A showed little acetylation on both T2I and S3K Z-domain variants. In contrast, the RimJ double mutants, Y106A M142A, Y106A N144A, and Y170A L171A showed higher acetylation rates on the Z-domain T2I variants. Little acetylation was observed for the Z-domain S3K variant by any of these double mutants. We also created more RimJ variants in which three different amino acids located on the other side of the active site were changed to alanine. These variants will be used to co-express the Z-domain variants, whose N-terminal acetylation patterns will be analyzed by mass spectrometry.

In the lab, molecules used as drugs are made either in solution (wherein the reactive agents dissolve) or on solid supports referred to as 'beads' (wherein reactive agents are washed over beads and become attached only to be liberated later). The virtue of bead-based synthesis comes with the savings in time and energy normally required to purify the reaction products. That is, solution phase synthesis is work intensive. Here, a route to cyclic molecules synthesized on beads is described. The molecules produced by these bead-based methods have already been prepared in solution for comparison. In addition to evaluating the relative efficiencies of these two routes, the bead-based method can be used to rapidly make 100s-1000s of cyclic molecules. Such numbers are not possible using solution phase methods due to the burdens of purification. The effort relies on tethering an acetal to a reactive bead, followed by a protection and deprotection sequence, the addition of an amino acid using standard peptide coupling strategies and a reaction with a core group that offers the potential for the attachment of 100s-1000s of different groups. Cleavage of this linear molecule from the bead leads to spontaneous cyclization to the desired products. The products will be characterized by NMR spectroscopy and mass spectrometry as well as be assayed for biological activity in a disease model of breast cancer.

Tissue engineering encompasses many important medical applications that pertain to the repair and regeneration of various tissues throughout the human body that have been adversely affected by disease or injury. Through combining the body’s cells with synthetic scaffolds, tissue engineering promotes proliferation of cells at damaged sites. Recent advances have demonstrated that using biocompatible materials such as alginate hydrogels—polymer networks derived from brown algae—are a cheap and environmentally-friendly approach to this. Alginate hydrogels are effective because they mimic the extracellular matrix of tissues, which provides structural support to cells that comprise human tissues.
One necessary modification to these scaffold materials is to load them with drugs that can facilitate healing. More complex designs can ideally deliver more than one therapeutic species simultaneously. In addition to hydrogels, drugs can also be loaded into a material known as porous silicon (pSi). pSi nanoparticles can be physically entrapped inside alginate hydrogels to create a two-system drug delivery mechanism with sustained release. This allows drugs such as growth factors, substances that stimulate cell growth, to be released at different times as the pSi and alginate hydrogel degrade.
This project entails the construction of alginate hydrogels that incorporate model dye-loaded pSi particles. The release of two dye molecules known as curcumin and rhodamine were monitored to assess the efficacy of the two-system drug delivery mechanism. It was first found that curcumin was too hydrophobic of a dye to achieve significant loading in the pSi. Rhodamine was found to be released from the pSi/alginate hydrogel system in a more incremental (sustained) manner over time compared to a relatively large initial ‘burst’ release observed for the release of rhodamine from pSi only. Sustained release in drug delivery is important to ideally reduce the amount of drug necessary and contrasts a burst release where large amounts of the loaded molecules are released prior to achieving a stable release profile. Furthermore, the localization of pSi in the alginate hydrogels was achieved by inserting loaded pSi membranes into pre-gelled alginate hydrogels, which is important to control the spatial delivery of the loaded molecule from pSi. Overall, it is believed that this pSi/alginate hydrogel material can greatly benefit the field of tissue engineering by creating dual delivery platforms with more diverse control over drug release.

It is estimated that 50 million individuals worldwide live with Alzheimer’s disease (AD), a neurodegenerative progressive disorder that, along with other chronic dementias, cost the United States $355 billion in 2021. Previous research links AD with amyloid beta (A𝛽) aggregation in the brain. Possible therapeutic drugs, including antioxidants and metal chelating agents, need efficient delivery systems that can cross the blood-brain barrier and release drugs appropriately. Recent discoveries in nanoscale materials as targeted drug delivery and controlled release agents have shown that such materials can release therapeutic drugs in a slow manner and increase efficacy. Chief among these carriers are porous materials with high surface areas because of their tunable pore structure, surface chemistry and drug loading capacity. This project focuses on using porous silicon derivatives as a carrier because, in addition to the above properties, it is a known biocompatible material.
This research deals with developing efficient protocols for loading mesoporous silica (pSiO2) with selected metal ion binding agents through systematic manipulation of external variables in order to achieve the highest percentage of loading. Once this has been determined, release and complexation studies are conducted. Known spectrophotometric methods are used to monitor diffusion over time and evaluate the profile of the sustained release. Different derivatives of chelating agents are tested and compared to determine the best suited candidates. The macrocyclic molecule Pyclen was the first tested candidate, followed by its dimer form, and finally a halogen substituted derivative. Stoichiometric complexation ratios with copper ions are measured followed by testing their success of inhibiting amyloid beta aggregation. Developing a slow and steady rate at which drugs capable of inhibiting neurotoxic A𝛽 aggregates in the brain can be released should be more effective and lead to more promising solutions for AD.