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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 [email protected]
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 transportlimited 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.
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.
Hydrogen bonding then drives
precise assembly in the plane of
the interface.
Air
Water
R1 corresponds to hydrophobic moeity. (Valine)
R2 corrseponds to hydrophilic moeity. (Histidine)
Method
Fig. 1. Schematic view
of amphipathic
polymerization lined
up at the interface
+HOBt
Base
+HBTU
Histidine-Valine Dimer
95% TFA
+
-
Fig.2. Proposed reaction mechanism for the synthesis of
the histidine-valine dimer.
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 HisVal dimer.
Results
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. 3. Mass Spectroscopy Data of solution prior to MTBE
extraction. Peaks around 136 are likely TFA salts.
Fig. 4. Mass Spectroscopy Data of precipitate after MTBE
extraction, show relative high purity of a single species.
The attempts to synthesize and purify the histidinevaline 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.
Conclusions/Future Work
Acknowledgements
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.
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 NSFSTEP program for their support in my research
endeavor.
•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.