Date of Award
Doctor of Philosophy (PhD)
Chemical & Biochemical Engineering
Christopher N. Bowman
Kristi S. Anseth
Peptides present an enormous potential for therapeutic and material science applications. However, in their native format, due to a limited number of chemical handles useful for structural manipulation to address stability issues and facilitate inclusion into other materials, the ability to completely access and implement the potential wealth peptides have to offer is restricted. The overall objective of this thesis endeavored to expand the utility of peptides through the development of synthetic amino acids compatible with click chemistry. Specifically, this work investigated the development of maleimide and furan derivatized amino acids for implementation in the Michael addition and Diels-Alder click reactions to manipulate peptide structure, properties and function. Click chemistry handles are typically added or incorporated into a peptide sequence post-synthetically or through the use of side chain protecting groups. By developing amino acids expressing click moieties, the furan and maleimide functionalities could be directly incorporated into a peptide chain during solid phase synthesis reducing both time and expense. The synthetic amino acids were created using amino ethyl glycine as a template to attach furan and maleimide functional groups. Through the addition of orthogonal protecting groups to amino ethyl glycine, it was possible to attach furan and maleimide carboxylic acids to an amine on the backbone molecule by means of well developed coupling chemistry. Additionally, selection of the proper orthogonal protecting groups for the amino ethyl glycine molecule simultaneously made the synthetic maleimide and furan amino acids compatible with standard Fmoc mediated solid phase peptide synthesis. Once developed, the ability to make use of the clickable amino acids to exercise control over peptide structure, properties and function was assessed. To this end, the ability to use the maleimide amino acid as a handle to conduct Michael-type addition reactions for the modification of peptides was first considered. Through the use of a Diels-Alder/retro-Diels-Alder protecting scheme, the possibility to directly incorporate the maleimide amino acid into a growing peptide sequence while maintaining maleimide activity was demonstrated. Due to synthetic difficulties, maleimide functionalities have traditionally been restricted to post-synthetic incorporation into a peptide sequence to afford a reactive moiety. With an active maleimide group present on the peptide, the potential for conformation manipulation was shown by inciting an intramolecular Michael addition between the thiol of a cysteine residue and maleimide to form cyclic structure from a linear sequence. In addition, the maleimide amino acid handle was also exploited to covalently attach peptides to surfaces and into other materials. Finally, the bioactivity of a peptide that had made use of the maleimide functionality as a handle to incorporate the sequence into another material was assessed. The ability to implement the furan amino acid as a handle to influence peptide function was also investigated. The introduction of a furan functionality to a peptide granted the ability of the given sequence to participate in the Diels-Alder reaction. This feature was harnessed to controllable release peptidyl materials from a poly(ethylene glycol) (PEG) hydrogel platform by means of Diels-Alder/retro-Diels-Alder mediated reaction diffusion process. A polymer constructed by off stoichiometrically reacting a tetrafunctional maleimide PEG macromer with various multifunctional thiol crosslinkers such that the thiol species was the limiting reagent allowed for the creation of Diels-Alder tethering sites within the network. The validity of a Diels-Alder release mechanism as well as the ability to predict the release were verified through the use of a reaction-diffusion mathematical model. Furthermore, as the impact of unreacted maleimide and furan species on cell viability is largely unknown, the bioactivity of both the hydrogel release platform containing excess maleimide groups and furan functionalized peptide sequences were evaluated.
Koehler, Kenneth Christopher, "Development and Implementation of Clickable Amino Acids" (2012). Chemical & Biological Engineering Graduate Theses & Dissertations. 31.