First Advisor

Robert M. Strongin

Term of Graduation

Fall 2021

Date of Publication


Document Type


Degree Name

Doctor of Philosophy (Ph.D.) in Chemistry






Fluorescent probes, Thioredoxin, Selenium compounds



Physical Description

1 online resource (xiii, 136 pages)


The identification and quantification of proteins and enzymatic activity in living tissues frequently requires the use of invasive procedures such as biopsy. These techniques can inhibit measurements of protein levels in real-time, and the disruption of tissue can lead to the loss of important information concerning the spatial distribution and differential activities in various cell types. The use of small organic fluorescent probes that rely on activation or accumulation within a tissue embody robust methods of detection that address these issues. This dissertation describes the design, synthesis and evaluation of fluorescent probes that can detect the enzyme thioredoxin reductase (TrxR).

TrxR enzymes in mammals are a group of selenoproteins that incorporate the selenocysteine (Sec) residue on the active site. This gives TrxR greater reactivity and broader substrate specificity compared to other oxidoreductase enzymes. The thioredoxin system is overexpressed in most cancers, where it serves the function of providing reducing equivalents responsible for promoting cell growth and division, whilst inhibiting apoptotic factors.

In this study, turn-on probes were synthesized by conjugating a seminaphthorhodafluor to a quenching moiety that is selective for TrxR. Based on docking simulations, the diselenide quencher had high affinity for the enzyme compared to the disulfide analogues. Probe 1a was selective for TrxR against other biological reductive species. It also had a fast turn-on response and higher fluorescence output in comparison to other published disulde probes and also its disulfide analogue, 1b. 1a also had a low IC50 value, which inspired the design of a new generation of theranostic probes.

A new series of probes (11, 12 and 13) was synthesized by conjugating fluorophores to a selenadiazole ring. The Se-rings were envisioned to covalently bind to the Sec residue of the TrxR active site, thus inhibiting the enzyme activity. Breaking the Se-N bond of the selenadiazole changes the optical properties of the probe and a fluorescence response should be observed. These probes had an absorption in the UV-Vis region in buffer and showed no response with ROS and reductive species. In cells, there is however absorption in the visible region (530 nm, 645 nm emission), which points to a potential reaction of the probes. Low IC50 values were also obtained as predicted; suggesting similar mechanism of action to other selenadiazole drugs that target TrxR.


© 2021 Tendai Joseph Mafireyi

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