Date of Award

2015

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Chemistry

First Advisor

Amos B. Smith

Abstract

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.

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