Document Type

Thesis

Degree Name

Master of Science (MSc)

Department

Chemistry

Faculty/School

Faculty of Science

First Advisor

Masoud Jelokhani-Niaraki

Advisor Role

Primary Supervisor

Second Advisor

Matthew Smith

Advisor Role

Secondary Supervisor

Abstract

Cell-penetrating peptides (CPPs) are a family of peptides that have the ability to penetrate biological membranes. They were discovered in the late 1980s and have been the topic of many studies. Much of the interest in CPPs has been due to their ability to translocate biological membranes, and the possibility that they could offer a novel drug delivery method by conjugation to biologically active molecules. Linear CPPs can be modified to form cyclic structures. This change in structure has been observed to enhance the stability and penetrative ability of the CPPs which have been studied. The current thesis focuses on the biophysical properties of CPPs modified to allow for cyclization. Penetratin (primary sequence: RQIKIWFQNRRMKWKK) and the Tat peptide (primary sequence: YGRKKRRQRRR) are two examples of linear CPPs that interact with and translocate across biological membranes. Fmoc based solid-phase peptide synthesis (SPPS) was used to synthesize cyclizable analogs of these two peptides by adding glycine and cysteine residues to the peptides’ termini. Reversed-phase high performance liquid chromatography (RP-HPLC) was used to purify and analyze the homogeneity of the protected linear analogs of the Tat peptide and penetratin (pTatL and pPenL, respectively). Fluorescence emission spectroscopy and circular dichroism (CD) spectroscopy were used to observe the conformational differences between the cyclizable analogs, and their original peptide constructs in different (aqueous, organic solvent and lipidic) environments. Dithiothreitol was required to measure the fluorescence and CD spectra of the peptides in the presence of lipid vesicles, to prevent the precipitation which might occur in the absence of the reducing agent. The CD and fluorescence spectra of pPenL were similar to the previously reported spectra of the native penetratin peptide; it displayed an unordered conformation in aqueous environments and α-helical conformations in organic solvents hexafluoroisopropanol and lipidic phosphatidylcholine/phosphatidylglycerol and phosphatidylethanolamine/phosphatidylglycerol (PC/PG and PE/PG) environments. The Tat peptide has been reported to maintain an unordered secondary structure in all environments and when conjugated to biologically active molecules. The spectra of pTatL in aqueous buffer agreed with the previous reports, but the spectra in HFIP displayed partial α-helical conformations, likely due to the modifications made to the sequence to enable cyclization.

Convocation Year

2024

Convocation Season

Spring

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