Date of Award

5-2019

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical and Chemical Engineering and Sciences

First Advisor

Nasri Nesnas

Second Advisor

Joel Olson

Third Advisor

Yi Liao

Fourth Advisor

Eric Guisbert

Abstract

The spatiotemporal regulation of biomolecules in their physiological environment is an essential aspect of biological research. In neuroscience, the activity of neurotransmitters can be regulated by protecting the molecules with photoresponsive protecting groups (PPGs), also known as cages. Diverse set of PPGs have been developed for caging of biomolecules. PPGs can be categorized based on their absorbing wavelengths (ranging from UV to near-IR) and photochemical properties (quantum yield, photo-cross section, solubility). PPGs absorb light and undergo intramolecular electron transitions and bond rearrangements that result in the release of the caged substrates. Hence, the caging of a specific substrate is unique and comes with its own set of challenges. Glutamate is an important neurotransmitter that has been used as a substrate for caging. One of the most efficient caged Glu in use is the CDNI-Glu (4- carboxymethoxy-5,7-dinitroindolinyl glutamate). Despite its superior photophysical qualities, the ability to use CDNI-Glu in neuroscience experiments is limited by the challenges of its intricate synthetic process. We devised an improved synthetic procedure to access CDNI-Glu that reduced the total number of steps (from 10 to 4 steps) and the process time from 200 h to 40 h. This also resulted in an increased overall yield (from 10 to 20%). The photochemical properties of the resulting CDNI-Glu were experimentally determined via 1H NMR and UV/Vis spectroscopy. The CDNI-Glu was uncaged in an in vitro mouse hippocampal brain slice with two-photon radiation to evaluate its performance in physiological conditions. CDNI-Glu generates off-target effects with GABAA receptors in the synapse (GABAA inhibition), limiting the concentrations at which this molecule is useful. It has been shown that the structure of CDNI-Glu molecule is partly responsible for the GABA inhibition effects. Therefore, we designed variations of CDNI-Glu by which the PPG was attached to the other two functional groups. We named these novel structures of caged Glu α-CDNI-Glu and the N-CDNI-Glu, referring to the position of protection. We compared their photochemical properties to those of the conventional CDNI-Glu. Furthermore, a completely different form of CDNI-Glu was designed by tethering a modified β-cyclodextrin via click chemistry (CDNIGlu-β-CD). The bulkiness of β-cyclodextrin was expected to enhance the solubility of the CDNI-Glu further while also preventing GABAA inhibition. The technology of activating genetically modified neuronal receptors with designer drugs is known as chemogenetics/pharmacogenetics. There are two main classes of designer drugs based on the type of receptor they target. The GPCR (Gprotein coupled receptor) targeting drug DREADD 21 and LGIC (ligand-gated ion channel) targeting drug PSEM 89 were caged with NVOC-Cl (4,5-Dimethoxy-2- nitrobenzyl chloroformate). Calcium ions (Ca2+) perform key roles in biological systems such as intracellular signaling pathways. The BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'- tetraacetic acid) based exogenous molecules are using in chelating Ca2+ ions. A visible light (470-490 nm) sensitive thiol coumarin derivative was employed in caging the BAPTA. One of the exposed carboxylic groups in the BAPTA was photo-protected to eliminate the Ca2+ chelating ability. The caged BAPTA will only chelate Ca2+ions, upon irradiation with blue light. The organocatalyst proline was caged with CDNI to form a pro-catalyst CDNIPro. The inactive catalyst was activated via UV light (350 nm) to catalyze aldol and Mannich type reactions. The CDNI-pro was applied in a biological assay to generate a biocide from self-aldol condensation of butyraldehyde.

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