Development of Dynamic, Bioorthogonal Peptide Cross-Linking MethodsHaney, Conor (2015) Development of Dynamic, Bioorthogonal Peptide Cross-Linking Methods. Doctoral Dissertation, University of Pittsburgh. (Unpublished)
AbstractShort peptide sequences typically lack well-defined structure when removed from the context of a larger protein. In order to constrain peptides into well-defined structures, nature often relies on the formation of disulfides between cysteine residues. Chemists have leveraged alternative chemoselective reactions to constrain peptides into defined folded conformations. Chemoselective cyclization strategies typically require extensive synthetic manipulation or the use of conditions which are incompatible with biological systems. This work encompasses the development of alternative peptide cyclization strategies that can be utilized under conditions compatible with biological systems. The strategies we have focused on offer the possibility of implementing cross-linking in a dynamic and reversible manner in aqueous solutions. We have chosen to take advantage of the bioorthogonal and reversible nature of Schiff base formation in the development of bioorthogonal and dynamic side chain cross-linking. We have demonstrated that an oxime formed by a cyclization reaction between aminooxy- and aldehyde-functionalized side chains in peptide sequences proceeds rapidly and in high yield. These cross-links have been shown to affect peptide folding and are capable of dynamic covalent exchange. Oxime cross-links have been shown to stabilize a model protein-protein interaction, with a dependence upon the sequence context and structure of the cross-link formed. Analysis of a high-resolution structure of the cross-linked oligomer demonstrates that the oxime side chain cross-link is well accommodated into an α-helical fold. We have also demonstrated that the product distribution of a cross-linking reaction in which multiple cross-linked regioisomers can be generated is dependent upon the folded state of the peptide at the time of cross-link formation. An alternative strategy which employs the use of an intermolecular cyclization between a bifunctionalized peptide and dialdehyde functionalized small molecules has also been explored. The intermolecular cyclization strategy has been shown to affect peptide folded structure and be capable of dynamic covalent exchange. Intermolecular cyclization offers a generally applicable and convergent manner to synthesize multiple cross-linked species in a combinatorial manner. This strategy has then been employed to template formation of different cross-linked structures based on the presence of an expressed protein receptor. Share
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