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Synthesis of kainic acid derivatives and their application as kainate receptors probes

University of British Columbia

Kainate receptors play important roles in the central nervous system. However, investigating their role in physiology is hampered by the current lack of tools to study them in dynamic cellular contexts. This thesis describes the synthesis of C4-kainoids and their derivatives, which are designed to serve as chemical probes to study kainate receptors in neurobiology. Chapter 1 provides a brief introduction of kainate receptors’ roles in central nervous systems and an overview of existing probes for glutamate receptors. Chapter 2 presented a comprehensive structure/activity analysis for efficient KARs ligands. This chapter reviews all reported kainic acid analogs that have been biologically tested and identifies systematic trends in biological activity of these analogs. These trends combined with protein crystallographic studies led us to identify structural guidelines that must be respected to design the next generation of efficient ligands for kainate receptors. These guidelines informed our synthetic design of the kainoid chemical probes presented in subsequent chapters. Chapter 3 focuses on the development of a synthetic strategy of kainic acid analogs that: (1) is short, (2) is high-yielding and scalable, and (3) allows easy variation of substituents at the C4 position of kainic acid. It resulted in a general stereoselective synthesis of 4-substituted kainoids: kainic acid and its natural isomers were synthesized in 8 to 11 steps from the commercially available 4hydroxyproline. The sequence also enables a late-stage modification of C4 substituents with sp 2 nucleophiles. Stereoselective steps include a cerium-promoted nucleophilic addition and a palladium-catalyzed reduction. A 10-step route to a carboxylic acid derivative was also established to enable ready functionalization of the C4 position. This work represents the first unified synthetic route to C4 derivatives of kainic acid. Chapter 4 describes the synthesis of kainoid derivatives that bear a fluorescent moiety that is used to visualize kainate receptors in living cells. A key intermediate from our general synthesis was exploited to create a novel alkynyl kainoid at the C4 position. This alkynyl kainoid was then coupled with a sulfo-Cy5-azide linker chain using a copper-catalyzed reaction to construct a fluorescent probe. In vitro fluorescence imaging performed by confocal microscopy with this far-red fluorescent probe confirmed the capability of labeling kainate receptors. This work reports the synthesis and use of the first fluorescent probe for kainate receptors. Chapter 5 describes the synthesis of two photo-controllable agonists for kainate receptors: a photocaged and a photoswitchable kainoids. First, our synthesis of one of the most potent unnatural kainoids (phenylkainic acid) was modified to install a photo-cleavable group on the nitrogen atom. The coumarin-type photo-cleavable group can be removed rapidly upon blue light irradiation, which results in the irreversible release of the agonist to trigger kainate receptor activity. Second, a kainoid derivative was synthesized where the C4 side chain integrates an azobenzene that can be isomerized between its cis or trans configuration using light. From the structural analyses presented in Chapter 2, only the trans-azobenzene form should be able to bind to kainate receptors. Since this photoisomerization is reversible using visible light (i.e., photoswitch), this kainoid probe can serve as an on/off switch to control the ion conductance of kainate receptors in cells. Kinetic studies by UV-Vis and 1 H NMR spectroscopy confirmed that the photoswitch does isomerize upon irradiation. Overall, a set of new chemical tools for kainate receptors is reported that expands the range of means to perform neurobiological experiments. Along the way, a practical synthetic route to new analogs of kainic acid was established. The compounds presented in this thesis are now being used by colleagues and collaborators to answer questions about the role of kainate receptors in neurons and glial cells that cannot be addressed using current neurobiology methods.

Text

2019-07-04

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/70906

Chemistry

University of British Columbia

UBCO

http://creativecommons.org/licenses/by-nc-nd/4.0/