1. 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
Introduction
Superoxide (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.
References
1. 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)quinolineHCl 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:
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
Conclusion
In 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 Discussion
Di-quinoline ethanolamine ligand NMR Spectroscopy
-CH2
Quinoline
-CH2
Ethanolamine
-ArH CH2Cl2
H2O
Scheme 2. Synthesis of Mn(III) DQEA SOD Mimic
Fridovich Assay
In 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 mimic
Figure 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