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40982-GB4
The Use of Crown Ethers as Molecular Shields to Prevent the Formation of Secondary Structures in Solid Phase Peptide Synthesis
This grant contains five major objectives:
i) To provide evidence for the compatibility of crown ethers in the automated synthesis of peptides.
ii) To confirm the formation of supramolecular structures using nuclear magnetic resonance and fast atom bombardment mass spectrometry, both of which have been used in the past to identify these types of structures.
iii) To design derivatized crown ethers that would hydrogen bond with the polyamide backbone and bind non-covalently to target amino acid side chains.
iv) To synthesize and incorporate these derivatized crown ethers into automated peptide synthesis.
v) To provide the opportunity to enhance the laboratory skills of students as a means to expand their scientific education.
The following narrative report will address the five major objectives. Although it was thought that objective number one would be straightforward and easy to accomplish, most of the commercially available crown ethers were to small in size to “thread” the growing peptide chain. It therefore became necessary to synthesize the crown ethers with high purity before any attempt was made at incorporating them into the automated synthesis. Once the material was synthesized attempts to incorporated into the peptide synthesize were thwarted by solubility issues of these crown ethers. It became necessary to heat the reaction vessels to avoid precipitation. Heating the reaction vessel is problematic on several accounts, it decreases the strength of the intermolecular interactions required for the crown “threading”, it also increases the likelihood of adverse side reactions and ultimately changes the parameters of the experiment such that a direct comparison is no longer possible. None the less results by weight indicated incorporation of the crown ethers onto the growing peptide chains after five consecutive amino acid incorporations. Strong cleavage conditions necessary to remove the peptide from the polystyrene bead most often “dethreaded” the peptide chain.
Objective number two did show the presence of crown ethers with the peptide chain but was unable to help elucidate the location of the crown ether. Two possibilities exist for the association of the crown ethers with the peptide chain, a peripheral association with the peptide or a threaded association with the peptide. NMR NOE studies could confirm the crown ether association but these have as of yet, been unsuccessful. The derivatization of the crown ethers has not been attempted to date, which renders objective three and four unattainable.
A total of eight different undergraduates have worked on this project. All of which have presented posters or talks at our in house undergraduate research day. One student presented this work at the Alabama Academy of Science and won third place in his category.
In all, the grant has made research at the undergraduate level not only a possibility but also commonplace at Spring Hill College. All chemistry majors are now using instrumentation, mass spectrometry, nuclear magnetic resonance, and peptide synthesizers that would otherwise be inaccessible. In addition, there is continued hope that once solubility issues are resolved the project will lead to not only improved peptide synthesis but to a better understanding of protein folding and protein dynamics.
