Stapling and Unstapling Peptides and Proteins With S-Tetrazine

This thesis will focus on the design, synthesis, and validation of synthetic techniques to introduce s-tetrazine into peptides and proteins. The s-tetrazine molecule is effective for restricting peptides/proteins to macrocyclic conformations (i.e. stapling). Importantly, the incorporated s-tetrazine chromophore will undergo photodisassociation upon absorp-tion of a photon permitting the release of the restricted conformations (i.e. unstapling). In chapter one, we aimed to study a fundamental folding process known as the helix-coil transition by phototriggering coupled with transient two-dimensional infrared spec-troscopy (2D IR). The s-tetrazine molecule possesses the photochemical properties of an ideal phototrigger, as such, s-tetrazine was employed towards the development of tech-niques capable of capturing the fastest structural transitions of biomolecules with both high spatial and high temporal resolution. Tripeptide linchpins, containing the s-tetrazine phototrigger, were prepared by solid-phase peptide synthesis. The latter were then em-ployed toward the construction kinked helices near equilibrium via a fragment coupling procedure. The relaxation of the kinked helical structures were observed by pump/probe transient 2D IR spectroscopy. In chapter two, new synthetic protocols have been developed and validated for the in-troduction of s-tetrazine into peptides and proteins to staple and unstaple the confor-mations. Conditions for the introduction of s-tetrazine into cysteine sulfhydryl groups of unprotected peptides conducted with aqueous biphasic conditions, permitting the con-struction of macrocyclic peptides with a wide range of functionally and ring topology, bridging from one to 27 amino acid residues adjoining the cysteines. Importantly, the sta-pled conformations were released photochemically to their thiocyanate counterparts, and in turn the resulting thiocyanates removed to regenerate the native peptide. To the best of our knowledge s-tetrazine comprises the first example of a readily removable peptide sta-ple. Finally, the stapling and unstapling protocol has been extended to include thioredox-in as an example of a protein with an incorporated s-tetrazine construct that can also serve a useful role in conjugation strategies.

Author dc:creator

• Brown, Stephen P

Advisor dc:contributor.advisor

• Amos B. Smith

Statement dc:rights

• Stephen P. Brown

Language dc:language

en

Repository record dc:identifier.uri

https://repository.upenn.edu/handle/20.500.14332/28455

OAI identifier oai:identifier

oai:repository.upenn.edu:20.500.14332/28455