Isolation, chemical modification and applications of flax cyclolinopeptides
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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.
DegreeDoctor of Philosophy (Ph.D.)
DepartmentFood and Bioproduct Sciences
SupervisorReaney, Martin J.
CommitteeSammynaiken, Ramaswami; Tanaka, Takuji; Yang, Jian; Tyler, Robert T.
Copyright DateJune 2013
Cyclolinopeptides, Isolation, Characterization, Flax, Chemical modification, Fluorescence, Affinity Chromatography and Antibodies