Abstract

1. Synthesis of Amphipathic Amino Acid Dimer for Studying Interfacial Growth of Synthetic Peptide Matthew Harris1,2, Matthew Kubilius1, Dr. Raymond Tu1 1 Department of Chemical Engineering, City College of New York, New York, NY 2 malbert.harris@gmail.com Bio Matthew Harris is a junior in Chemical Engineering at City College. He transferred to CCNY two years ago after studying welding at SUNY Delhi and working in an art foundry. Mr. Harris has a deep interest in understanding the physical and chemical processes that govern nature. He believes these processes along with the principles of engineering play a concrete role in driving all of human interaction and production in our society. This sentiment is what motivated him to pursue a degree in Chemical Engineering at CCNY. After completing his undergraduate studies in chemical engineering Mr. Harris intends to pursue a Ph.D. in Chemical Engineering and Materials Science. His primary interests regarding lines of research concern energy resources and the ecologic/economic issues that surround them. Specifically, he is interested in studying the extraction and separation of rare earth elements and their applications in energy technologies and impacts on the economic and ecologic spheres. Mr. Harris has conducted research in amphipathic peptide design in Professor Tu’s lab under Matthew Kubilius since January 2013. Over the summer, he participated in a CENSES REU program to study heat effects on thin films of topological insulators in the lab of Dr. Lia Krusin. Abstract Synthetic peptides have the potential to generate novel functionalities in biological systems that mimic certain aspects of biological proteins and peptides. Biological peptides are often characterized by certain properties that define their functionality and govern their extreme specificity, such as well-defined molecular weight and secondary structure. Synthetic peptides are often lacking those aspects that incur such specificity to biological molecules. However, amphipathic peptide design and synthesis can minimize these limitations and generate synthetic peptides with lower polydispersity index (PDI) and greater affinity for guided self-assembly at an interface. The goal of amphipathic design is to generate conditions in which a synthetic dimer will undergo a condensation polymerization in solution that becomes increasingly transport-limited as the reaction proceeds. To this end, a two-amino acid dimer in which one residue is hydrophilic and one is hydrophobic must be synthesized and then polymerized. As the polymer chain grows, the molecule will incur increasing amphipathic character. In the presence of an interface, the polymer will preferentially sequester itself to that interface, creating a transport barrier to further polymerization. This type of transport-mediated polymerization will generate a polypeptide that has a narrower molecular weight distribution than expected from a condensation polymerization and a higher propensity for self-assembly, particularly into β-sheets. The present work focused on the synthesis and characterization a histidine-valine dimer with the goal towards studying the behavior of the dimer in a transport-mediated condensation polymerization using multi-angle light scattering to investigate polydispersity and circular dichroism to examine structure. Polymerized and aligned naturally on the interface, hydrophobic groups populate one side of the interface while hydrophilic groups populate the other. Hydrogen bonding then drives precise assembly in the plane of the interface. R1 corresponds to hydrophobic moeity. (Valine) R2corrseponds to hydrophilic moeity. (Histidine) Air Water Method The synthesis of the histidine-valine dimer was performed entirely in solution. The protected amino acids were dissolved in DMF. To this solution the coupling reagents, HOTB and HBTu, were added. When the reaction was completed the solvent was removed and replaced with small quantities of methanol/ethanol and filtered to remove the excess reagent. A 95:5 solution of TFA and water was used to remove the protecting groups from the dimer. The excess acid was removed by evaporation and the resulting residue was dissolved in methanol and filtered the remove solid impurities. The solution was then neutralized using a combination of piperidine and solid NaCO3H to break the dimer-TFA complex. After a series of filtrations the solution was sampled for mass spectrometry to determine the components of the resulting solution. The mass spectrometry data consistently revealed that the TFA salt along with additional ionic impurities remained in solution with the dimer despite numerous purification steps, as in Fig. 3. Ultimately, an attempt was made to precipitate the dimer out of solution using MTBE and centrifugation. This step yielded a solution with relatively high purity in a single component, but the molecular mass did not match the predicted mass of the His-Val dimer. Introduction The goal of this project is to synthesize a pure sample of a histidine-valine dimer in order to investigate its behavior in a condensation polymerization in the presence of an interface. The principle behind the choice of these specific residues follows from the hypothesis that a dimer composed of one hydrophilic residue, histidine, and one hydrophobic, valine, will generate a peptide with increasing amphipathic character when it undergoes polymerization. In the presence of an interface, it is expected that the peptide will reach a limit where it will sequester itself to the interface creating a transport barrier to further reaction. To the end of investigating this molecule under these conditions, a method of synthesis and purification has first to be developed. Fig. 1. Schematic view of amphipathic polymerization lined up at the interface +HOBt +HBTU Base Histidine-Valine Dimer 95% TFA + - - Fig.2. Proposed reaction mechanism for the synthesis of the histidine-valine dimer. Results The attempts to synthesize and purify the histidine-valine dimer did not yield a sample of the predicted species at high enough purity to proceed to polymerization and characterization of the behavior in the presence of an interface. Fig. 3, on the left, shows mass spectroscopy data of the solution prior to the MTBE extraction. The graph reflects low purity of the solution and no clear indication that the target species is present. Fig. 4, on the right, shows the resulting mass spectroscopy data of the species precipitated in the MTBE extraction. The peak at 441 amu indicates a species present in relatively high concentration compared to the residual impurities and background noise. This species, however, does not correspond in molecular weight to the target dimer. Single peak at 441.1 amu corresponds to relatively pure species in solution after MTBE extraction. Species likely involves dimer complexed with ionic species. Peaks around 135 amu likely correspond to large amount of persistent TFA salt impurities Target species should show a well-defined peak at 254 amu. Fig. 4. Mass Spectroscopy Data of precipitate after MTBE extraction, show relative high purity of a single species. Fig. 3. Mass Spectroscopy Data of solution prior to MTBE extraction. Peaks around 136 are likely TFA salts. Acknowledgements I would like to thank Matthew Kubilius and Dr. Raymond Tu for their guidance and input. I would additionally like to thank the GSOE OSRS and the NSF-STEP program for their support in my research endeavor. • Conclusions/Future Work • The methodology employed to synthesize a dimer of histidine and valine proved to be ineffective in creating a high purity sample of the desired species. There is a necessity to reexamine the properties of histidine in solution and redesign the synthesis scheme based on an evaluation of ionic interactions and pH response of the histidine residue. • Synthesis of dimers from additional amino acid pairs will be attempted, specifically Gln-Val and Asn-Val. • Further attempts to synthesize His-Val will be made.