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Development of Dynamic, Bioorthogonal Peptide Cross-Linking Methods

Haney, Conor (2015) Development of Dynamic, Bioorthogonal Peptide Cross-Linking Methods. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Short 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.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Haney, Conorcmh88@pitt.eduCMH88
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee MemberDeiters, Alexanderdeiters@pitt.eduDEITERS
Committee MemberLiu, Xinyuxinyuliu@pitt.eduXINYULIU
Committee MemberWetzel, Ronaldrwetzel@pitt.eduRWETZEL
Committee ChairHorne, W. Sethhorne@pitt.eduHORNE
Date: 13 January 2015
Date Type: Publication
Defense Date: 14 August 2014
Approval Date: 13 January 2015
Submission Date: 1 August 2014
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Number of Pages: 231
Institution: University of Pittsburgh
Schools and Programs: Dietrich School of Arts and Sciences > Chemistry
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: peptides; protein folding; protein–protein interactions; schiff bases; side-chain staples; cross-linking
Date Deposited: 13 Jan 2015 18:43
Last Modified: 15 Nov 2016 14:22


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