Synthesis of a Novel Mn(III) SOD Mimic with a Quinoline- Containing Ligand William A. Gallopp and Steven T. Frey; Department of Chemistry, Skidmore College, Saratoga Springs, NY 12866

IntroductionSuperoxide (O2

• -), a highly reactive oxygen species, is a byproduct of naturally occurring respiratory processes. However, in high concentrations superoxide has been associated with, diabetes, neurodegenerative diseases, tumor formation, and genetic mutations. Fortunately, we have evolved an effective defense system against the toxicity of O2

• -, which includes the superoxide dismutase (SOD) and catalase enzymes. Generally there are three groups of SOD; each having a different metal in the active site. These SODs are known as Cu-ZnSOD, MnSOD and FeSOD.1-3 

In the active site of the MnSOD enzyme a Mn ion is coordinated by three histidine and an aspartate (Figure 1). A fifth coordination site is occupied by water. The mechanism for the dismutation of O2

•- involves the reduction of Mn(III) to Mn(II) by O2

- which is in turn oxidized to O2 (Equation 1). The Mn(II) ion is then oxidized back to Mn(III) by reaction with another equivalent of O2

-, which undergoes reduction to form H2O2 (Equation 2).3  

Mn(III) + O2•- Mn(II) + O2 (1)

 Mn(II) + O2

•- Mn(III) + H2O2 (2)

A number of Mn(III) chelate complexes have been synthesized as SOD mimics. Mn(III) SOD mimic are most suitable since the Mn (III) ion has low toxicity relative to other metal ions and it is less likely than other metal ions to react H2O2 to form OH•- , another harmful ion. 3

The goal of our project is to synthesize a manganese(III) SOD mimic with a tetradentate ligand containing quinoline moieties. We have achieved the synthesis of the tetradentate ligand and its Mn(III) SOD mimic.

Methods

Acknowledgement. Thank you to Skidmore College for the resources and funding for this project. A special thanks to Frey research group for their guidance, support and contributions.

References1. Stroz, P: Reactive Oxygen Species in Tumor Progression. Frontiers in Bioscience. 10, 1881-

1896 (2005)

2. Miriyala, S; Spasojevic, I; Tovmasyan, A; Salvemini, D; Vujaskovic, Z; St. Clair, D; Batinic-Haberle, I: Manganese superoxide dismutase, MnSOD and its mimics. Biochimica et Biophysica Acta. 1822, 794-814 (2012)

3. Iranzo, O: Manganese complexes displaying superoxide dismutase activity: A balance between different factors. Bioorganic Chemistry. 39, 73-87 (2011)

Scheme 1. Synthesis of DQEA ligand

Synthesis of Di-quinoline ethanolamine ligand (DQEA)The synthesis of DQEA was achieved in a one step reaction (Scheme 1). To synthesize the ligand, 5.00g (23.35mmol) 2-(chloromethyl)quinolineHCl were dissolved in 10 mL DI H2O, followed by the drop-wise addition of 1.887g (46.71mmol) NaOH to neutralize any generated HCl. The addition was done in an ice bath to maintain the temperature at 0 °C. The addition of NaOH produced a white/pink solid suspension. Next, 0.714g (11.68mmol) ethanolamine dissolved in 20 mL methylene chloride was added to the suspension. This addition initiated a separation into a clear red-brown organic layer and a clear colorless aqueous layer. The mixture was then refluxed for 1 week, which resulted in a deeper red organic layer. The reaction process was monitored by TLC. The organic layer was then isolated using a separation funnel. The solution was rotary-evaporated to about 10 mL and refrigerated. This lead to the formation of a white precipitate. The crude product was collected by vacuum filtration and washed with cold methylene chloride. The final product was a white crystalline powder weighing 1.305 (Yield: 32.12%).

Synthesis of Mn(III) DQEA SOD Mimic The synthesis of the Mn(III) DQEA SOD mimic was achieved in a one step reaction (Scheme 2). First 0.200 g (0.582mmol) DQEA was dissolved in 20 mL ethanol producing a clear green-yellow solution. Then, 0.156g (0.582mmol) manganese(III) acetate dissolved in 5 mL ethanol was added drop-wise, producing a brown-red solution. The solution was allowed to stir for 1 hr, followed by the removal of ethanol by rotary-evaporation leaving a black solid. Recrystallization was used to purify the solid. The solid was dissolved in a minimal amount of acetonitrile followed by the drop-wise addition of ethyl ether, which acts as a knockout solvent. The mixture was refrigerated for several days and then rotary evaporated to remove the solvents. The final product was a brown-red crystalline weighing 0.109 g (Yield: 32.55%)

Figure 2. NMR spectrum of DQEA ligand

ConclusionIn summary, we believe we have successfully synthesized the DQEA ligand by characterization through H1 NMR and with it a novel Mn(III) complex which exhibits SOD activity as demonstrated by the Fridovich assay.

Results and DiscussionDi-quinoline ethanolamine ligand NMR Spectroscopy

-CH2 Quinoline

-CH2

Ethanolamine

-ArH CH2Cl2

H2O

Scheme 2. Synthesis of Mn(III) DQEA SOD Mimic

Fridovich AssayIn this assay the Mn(III) DQEA SOD mimic will compete with cytochrome c for superoxide. Upon successful interaction with the superoxide, the amount of cytochrome c reduced will decrease, effectively decreasing the change in absorbance over time. The assay revealed that the Mn(III) DQEA SOD mimic exhibited SOD activity as the change in absorbance decreased as the concentration of the SOD mimic increased (Figure 4).

Fridivoich Assay In a cuvette, a 2mL solution was prepared containing phosphate buffer, xanthine, cytochrome c, and catalase. Xanthine oxidase was then added to generate to superoxide. The newly formed superoxide reduces cytochrome c initiating a color change, which can be monitored by UV-Vis spectroscopy. The absorbance was then measured over 2 minutes and the change in absorbance was determined. The assay was repeated with varying concentrations of the Mn(III) DQEA SOD mimic.

Figure 4. Fridovich Assay of Mn(III) DQEA SOD mimicFigure 1. Active site of Mn-SOD Enzyme

Figure 3. IR Characterization of DQEA and Mn(III) DQEA SOD Mimic. This demonstrates incorporation of the DQEA ligand into the Mn(III) SOD mimic.

− Mn (III) SOD Mimic− DQEA ligand