Synthesis of a PbTx-2 photoaffinity and fluorescent probe and an alternative synthetic route to photoaffinity probes
Doctor of Philosophy (PhD)
First Advisor's Name
Dr. Kathleen Rein
First Advisor's Committee Title
Second Advisor's Name
Dr. Watson Lees
Second Advisor's Committee Title
Third Advisor's Name
Dr. Kevin O'Shea
Third Advisor's Committee Title
Fourth Advisor's Name
Dr. John Makemson
Fourth Advisor's Committee Title
Fifth Advisor's Name
Dr. Xiaotang Wang
Fifth Advisor's Committee Title
brevetoxin, photoaffinity probe, red tide, NSP, labeling, alexa fluor, polyether ladder toxins, thiol-michael addition, diazirines, biotin, click chemistry, voltage-gated sodium channels
Date of Defense
A natural phenomenon characterized by dense aggregations of unicellular photosynthetic marine organisms has been termed colloquially as red tides because of the vivid discoloration of the water. The dinoflagellate Karenia brevis is the cause of the Florida red tide bloom.
K. brevis produces the brevetoxins, a potent suite of neurotoxins responsible for substantial amounts of marine mammal and fish mortalities. When consumed by humans, the toxin causes Neurotoxic Shellfish Poisoning (NSP). The native function of brevetoxin within the organism has remained mysterious since its discovery. There is a need to identify factors which contribute to and regulate toxin production within K. brevis. These toxins are produced and retained within the cell implicating a significant cellular role for their presence.
Localization of brevetoxin and identification of a native receptor may provide insight into its native role as well as other polyether ladder type toxins such as the ciguatoxins, maitotoxins, and yessotoxins. In higher organisms these polyether ladder molecules bind to transmembrane proteins with high affinity. We anticipated the native brevetoxin receptor would also be a transmembrane protein.
Photoaffinity labeling has become increasingly popular for identifying ligand receptors. By attaching ligands to these photophors, one is able to activate the molecule after the ligand binds to its receptor to obtain a permanent linkage between the two. Subsequent purification provides the protein with the ligand directly attached.
A molecule that is capable of fluorescence is a fluorophore, which upon excitation is capable of re-emitting light. Fluorescent labeling uses fluorophores by attaching them covalently to biologically active compounds.
The synthesis of a brevetoxin photoaffinity probe and its application in identifying a native brevetoxin receptor will be described. The preparation of a fluorescent derivative of brevetoxin will be described and its use in localizing the toxin to an organelle within K. brevis. In addition, the general utility of a synthesized photoaffinity label with other toxins having similar functionality will be described.
An alternative synthetic approach to a general photoaffinity label will also be discussed whose goal was to accelerate the preparation and improve the overall synthetic yields of a multifunctional label.
Cassell, Ryan T., "Synthesis of a PbTx-2 photoaffinity and fluorescent probe and an alternative synthetic route to photoaffinity probes" (2014). FIU Electronic Theses and Dissertations. 1612.
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