ORCID Identifier(s)

0000-0002-3851-0810

Graduation Semester and Year

2018

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Chemistry

Department

Chemistry and Biochemistry

First Advisor

Frederick M MacDonnell

Abstract

Herein, we report the ruthenium (II) polypyridyl complexes (RPCs), [(phen)₂Ru(tatpp)] ²⁺ (3²⁺) and [(phen)₂Ru(tatpp)Ru(phen)₂] ⁴⁺ (4⁴⁺) are shown to cleave DNA in cell-free studies in the presence of a mild reducing agent, i.e. glutathione (GSH), in a manner that is enhanced upon lowering the [O₂] . Reactive oxygen species (ROS) are involved in the cleavage process as hydroxy radical scavengers attenuate the cleavage activity. Cleavage experiments in the presence of superoxide dismutase (SOD) and catalase reveal a central role for H₂O₂ as the immediate precursor for hydroxy radicals. A mechanism is proposed which explains the inverse [O₂] dependence and ROS data and involves redox cycling between three DNA-bound redox isomers of 3²⁺ or 4⁴⁺. Cultured non-small cell lung cancer cells (H358) are sensitive to 3²⁺ and 4⁴⁺ with IC₅₀ values of 13 and 15 μM, respectively, and xenograft H358 tumors in nude mice show substantial (~ 80%) regression relative to untreated tumors when the mice are treated with enantiopure versions of 3²⁺ and 4⁴⁺.(Yadav et.al. Mol Cancer Res, 2013, 12, 643) Fluorescent microscopy of H358 cells treated with 15 μM 44+ reveals enhanced intracellular ROS production in as little as 2 h post treatment. Detection of phosphorylated ATM via immunofluorescence within 2 h of treatment with 4⁴⁺ reveals initiation of the DNA damage repair machinery due to the ROS insult and DNA double strand breaks (DSBs) in the nuclei of H358 cells and is confirmed using the γH2AX assay. The cell data for 3²⁺ is less clear but DNA damage occurs. Notably, cells treated with [Ru(diphenylphen)3} 2+ (IC₅₀ 1.7 μM) show no extra ROS production and no DNA damage by either the pATM or γH2AX even after 22 h. The enhanced DNA cleavage under low [O₂] (4 μM) seen in in cell-free cleavage assays of 3²⁺ and 4⁴⁺ is only partially reflected in the cytotoxicity of 3²⁺ and 4⁴⁺ in H358, HCC2998, HOP-62 and Hs766t under hypoxia (1.1 % O₂) relative to normoxia (18% O₂). Cells treated with RPC 3²⁺ show up to a two-fold enhancement in the IC₅₀ under hypoxia whereas cells treated with RPC 4⁴⁺ gave the same IC₅₀ whether under hypoxia or normoxia. We also report herein method development for semi-preparative scale HPLC enantiomeric separation and purification of the ruthenium (II) polypyridyl complex [Ru(phen)₂ phendione} ²⁺.The results of the retention factor vs. methanol/acetonitrile ratio plot indicated different interactions take place at different polar organic solvent compositions. Interestingly, the results of the selectivity, retention factor, and resolution vs. Methanol/ acetonitrile ratio plot showed no significant change in selectivity as the retention factor and resolution increased. This allowed the focus for semi-prep scale method optimization to be on retention time. Finally, we report the method development and optimization of six Ru(II) dyads derived from thiophene (1T) and oligothiophenes (4T) for chiral separation and enantiomeric purification in polar organic mode using HPLC. The results of the retention factor vs. methanol/acetonitrile ratio plot indicated different interactions take place at different polar organic solvent compositions. Although selectivity and resolution remained relatively constant the retention factors increased with increasing amounts of methanol. However, the retention factor increases significantly with increasing retention times, allowing the focus of optimization to be on shortening the retention while maintaining resolution. These results are important for later use in semi-preparative enantiomeric purification by allowing the focus to be on optimal retention time.

Keywords

RPCs, Enantiomeric separation, Cell, ROS, Cell-free studies

Disciplines

Chemistry | Physical Sciences and Mathematics

Comments

Degree granted by The University of Texas at Arlington

Included in

Chemistry Commons

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