Cyclolinopeptides (CLs) are hydrophobic cyclic peptides found in flaxseed. They show immunosuppressive activity, but the biological function of these compounds is largely unknown. This thesis presents the results of studies that were conducted to determine whether CLs could act as antioxidants. In the first study, flaxseed oil was passed over a silica adsorbent column to remove polar compounds. The polar compounds were then eluted from the silica absorbant using a series of increasingly polar solvents. Individual polar fractions were then added back to the silica-treated flaxseed oil and the oxidative stability index of these samples was determined at 100 °C. A polar fraction containing mainly CLA, β/γ- and δ-tocopherol increased the induction time of silica-treated flaxseed oil from 2.3 ± 0.28 h to 3.2 ± 0.41 h. A positive effect of the polar fraction containing a mixture of CLA and CLD-CLG on the oxidative stability of oil was also observed. The antioxidant mechanism of CLs was investigated in several model systems using electron spin resonance spectroscopy. The concentration of radicals in a DMPO (5,5-dimethyl-1-pyrroline-N-oxide) radical-CLs reaction mixture was monitored. All CLs exhibited dose dependent scavenging activities. CLA–CLC reactions with DMPO-OH at a concentration of 5 mM resulted in a 24–30% decrease in electron paramagnetic resonance (EPR) signal intensity. The reaction of CLs and the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH•) revealed a more complex interaction than simple radical scavenging. Peptides (CLG and CLG") that contained both tryptophan and methionine showed stronger radical scavenging activity than did CLs containing methionine or methionine sulfoxide but not tryptophan (CLB and CLC). Irradiation of the reaction mixture of DPPH• and peptide with UV light also affected the radical scavenging behaviour. Scavenging activities of DPPH• by CLB, CLC and CLA were enhanced by light, whereas scavenging of DPPH• by the tryptophan containing peptides CLG and CLG″ was not affected. High-performance liquid chromatography with mass spectrometry (HPLC-MS) analysis of the reaction mixtures after a radical scavenging reaction was used to determine the impact of radical scavenging on the peptides. These reactions revealed new masses that were identified and characterized. It was established that DPPH• reacted with the methionine of CLB and with tryptophan in CLG and CLG, by formation of a new covalently-bonded species. Covalent linkages between these amino acids (alone or in peptides) and DPPH• have not been reported previously.

Oil from flaxseed (Linum usitatisssimum L.) contains hydrophobic cyclic peptides or cyclolinopeptides (CLs) comprising eight or nine amino acids. These bioactive compounds have potential therapeutic applications and may be used as scaffolds for increased utility. Two steps were undertaken to increase the potential utility of these compounds. Initially multigram quantities of flax CLs were highly enriched from flax oil. Subsequently new synthetic procedures were developed for modification of the CLs through the methionine group (Met). Finally, the utility of the modified CLs was tested in a number of applications. CLs were recovered from a crude oil extract that contain five CLs (CLA, CLC, CLE, CLJ and CLK). Oxidation of this mixture reduced the complexity of the mix to just three CLA, CLJ and CLK. CLJ and CLK were enriched then characterized by NMR and MS-MS methods. CLs containing methionine sulfoxide groups (Mso), CLC and CLE were isolated from crude mixture then selectively reduced to afford Met containing analogs: CLB and CLE'. The Met of modified CLs was used as a point for attachment of tags and couplers for various applications. Cyclic peptide modification through Met groups has not been reported previously. Synthetic methods were devised to introduce activating functional groups such as -CN, -COOH, -OH and -NH2 to the sulfur atom of Met. The modified CL conjugates were characterized using spectrometric techniques including 1D and 2D NMR spectrometry, as well as mass spectrometry. After activation the CLs were covalently linked to molecules or materials of interest including fluorescence tags (coumarin), affinity chromatography media and bovine serum albumin (BSA) for production of polyclonal antibodies. Fluorescence studies were performed in methanol, ethanol, dimethylformamide and acetonitrile to study the solvent effect. CLs attached to solid affinity matrix showed specific binding to apolipoprotein A1 after incubation with chicken serum. These CLs also act as hapten and have been used to couple BSA to produce polyclonal antibodies. Met modification was a satisfactory approach to produce a range of useful peptide products where more conventional methods of molecule attachment are not available.

The thesis consists of two parts targeting the characterization of chromatographic behavior of linear tryptic and cyclic peptides containing oxidized methionine (Met) in reversed-phased chromatography. The retention order of methionine-containing peptide analogues was observed to be the same in both studies: Met oxide < Met dioxide < Met. For linear tryptic peptides, the magnitude of the retention time shift may vary dramatically: from –9 % to +0.36 % acetonitrile. Particularly, large negative retention time shifts are found mostly associated with methionine being in the hydrophobic face of an amphipathic helix. Contrary to previously reported observations, I demonstrate for the first time that methionine oxidation may increase peptide hydrophobicity, this occurs only when methionine is in the N3 position of the N-capping stabilization motif preceding an amphipathic helix. In the second study, the effect of peak splitting was observed for some Met oxide-containing cyclolinopeptides, which most likely appear due to diastereomerization.