1. Synthesis of a Novel Mn(III) SOD Mimic
with a Qunioline Containing Ligand
William A. Gallopp* and Steven T. Frey*
*Department of Chemistry, Skidmore College, Saratoga Springs, NY 12866
Abstract
Superoxide ion (O2
-
) is a highly reactive oxygen species that is produced during
metabolism. Superoxide dismutase enzymes (SODs) catalyze the dismutation of
superoxide into hydrogen peroxide and molecular oxygen, thereby protecting cells from
the damaging effects of this radical anion. However, an excess of superoxide ion is
produced by the immune system in response to certain inflammatory diseases or strokes
that can overwhelm the locally available SOD. Therefore, low molecular weight
compounds that mimic SOD activity are of interest as potential pharmaceuticals. The
goal of this study was to synthesize a Mn(III) complex with characteristics of the active
site of Mn-SOD, and to test this complex for its ability to dismutate superoxide ion. With
that in mind, we synthesized a tetradentate ligand based on ethanolamine with two
methylquinoline arms bound to the amine nitrogen. This ligand was characterized by 1
H
NMR and IR. The ligand was then reacted with Mn(III) to produce a complex that we
characterized by IR and UV-vis. We have confirmed the ability of this Mn(III) complex
to mimic SOD activity using the Fridovich assay.
Introduction
Superoxide (O2
• -
), a highly reactive oxygen species (ROS), is a byproduct of naturally
occurring respiratory processes.1
This radical ion has both reducing and oxidizing
properties, which allows superoxide to react with metal ions forming more toxic species,
such as, peroxynitrite which is a strong oxidizing agent.2,3
In high enough concentrations
superoxide has been associated with, diabetes, neurodegenerative diseases, tumor
formation, and genetic mutations.1,4,5
Fortunately, we have evolved an effective defense
system against the toxicity of O2
• -
, which includes the superoxide dismutase (SOD) and
catalase enzymes, which aid in the conversion of superoxide to oxygen (O2).1
As a result
SODs play a crucial role in protecting biological systems for damage associated with
superoxide. Generally there are three groups of SOD; each having a different metal in the
active site. These SODs are known as Copper-Zinc SOD (CuZn-SOD), Manganese SOD
(Mn-SOD), Iron SOD (Fe-SOD) and Nickel SOD (Ni-SOD).1-3
Mammals possess two different classes of SODs to regulate superoxide level to keep
them under control: the CuZn-SOD and the Mn-SOD1
. The CuZn SOD is present in the
nuclear compartments, cytoplasm, and inner membrane of the mitochondria6,7
; the Mn-
SOD is located in the mitochondrial matrix.8
However, there are times were the
production of superoxide is excessive and the SOD enzymes cannot eliminate superoxide
leading to the various diseases states. This imbalance between the generation and

2. elimination of superoxide by the SOD enzyme is known as oxidative stress, and has been
associated with diabetes, neurodegenerative diseases, tumor formation, and genetic
mutations.
Fridovich and his co-workers first discovered the Mn-SOD in 1970.9
In the active site of
the MnSOD enzyme a Mn-ion is coordinated by three histidine and an aspartate. A fifth
coordination site is occupied by water (Figure 1). It is believed that Gln 143 and Tyr 34
play a major role in facilitating proton transfer upon the reduction of superoxide to H2O2.
Gln 134 is also key in controlling the redox potential of the Mn center. The active site is
located in a hydrophobic environment at the end of a tunnel where positively charged
amino acids guide the superoxide to the active site.1
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).1
Mn(III) + O2
•-
à Mn(II) + O2 (1)
Mn(II) + O2
•-
à Mn(III) + H2O2 (2)
A number of Mn(III) chelate complexes have been synthesized as SOD mimics,
including those with macrocyclic10
, salen11
prophrin12
, acylic13
, and peptide based14
ligands. Recent studies have shown that Mn-prophyrin SOD (MnP) complexes can
prevent tumor angiogenesis. MnPs have also been shown to suppress oxidative stress
levels, as well as, decrease incidences and multiplicity of papillomas from skin
carcinogenesis.5
They have also been shown to protect Polϒ against reactive oxygen
species. Mn(III) SOD mimic are the 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.5
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.
Figure 1. Active site of Mn-SOD Enzyme1

3. Methods
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 Ÿ 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. Thin layer chromatography was used
to monitor the reaction process. The organic layer was then isolated using a separation
funnel. The solution was rotary-evaporated to about 10 mL and refrigerated, leading 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%. Selected IR frequencies (cm-1
): 3204
(m, νOH), 3053 (w, νCH), 2897 (m, νCH), 2827 (m, νCH), 1600 (m, νC=C), 1590 (m, νC=C),
1504 (m, νC=C), 1427 (m, νC=C), 826 (s, νC=C),764 (s, νC=C). 1
H NMR (CDCl3 300 MHz):
2.95 (t, 2H), 3.75 (t, 2H), 7.4 -8.1 (m, 12H).
Scheme 2. Synthesis of DQEA ligand
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%. Selected IR frequencies (cm-1
):
3327 (m, νOH), 3065 (w, νCH), 3011 (w, νCH). 2938 (m, νCH), 1562 (s, νC=C),
1411 (s, νC=C), 783 (s, νC=C), 752 (s, νC=C).
+
H2N
OH
N
N
N
N
OH
Cl
2
Ethanolamine 2-(Chloromethyl)qunioline hydrochloride DQEA
4 NaOH
CH2Cl2

4. Scheme 2. Synthesis of Mn(III) DQEA SOD Mimic
Fridovich Assay
In a cuvette, 2 ml solution was prepared in phosphate buffer containing 50 µM
cytochrome c, 50 µM xanthine, and 30 µg/mL catalase. Xanthine oxidase ([Xanthine
Oxidase] = 0.0741 µM) 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 at 550 nm and
the change in absorbance was determined. The assay was repeated with varying
concentrations of the Mn(III) DQEA SOD mimic ([Mn(III) DQEA SOD] = 0.1µm – 1.0
µm).
Results and Discussion
Characterization of the DQEA Ligand
In the Mn-SOD active site the Mn ion is coordinated by three histidines and one
aspartate, which stabilize the Mn ion, and positions the ion, while leaving vacant or
solvent coordinated sites available for reactions with the superoxide ion. Previously
synthesized Mn(III)-SOD mimics utilize ligands containing both nitrogen and oxygen to
best mimic the active site and obtain SOD activity.3
Our first goal was to develop a
tetradentate ligand containing quinoline moieties. This ligand would contain 3 nitrogens
and one oxygen group that will be able to coordinate the Mn(III) ion. DQEA was
synthesized using the procedure outlined in this report. A reaction involving
ethanolamine and 2-chloromethylquinoline-hydrochloride salt with methylene chloride
and under reflux conditions generated DQEA. The final product was isolated as a fine
white crystalline powder.
The H1
-NMR spectrum of DQEA exhibits two triplets, at 2.95 ppm and 3.75 ppm (Figure
2), corresponding to the 4 protons a part of the ethanolamine segment of the ligand. The
triplet at 2.95 ppm corresponds to the protons closest to the OH-, as it is being shielded
and expected to be further down field. The singlet at 4.3 ppm corresponds to the methyl
groups of each quinoline. Finally the multiplets ranging from 7.4 -8.1 ppm correspond to
the aromatic protons associated with the quinoline qroups. These could be due to solvent
impurities from the purification process. The integration of the peaks also provided
evidence of the correctly synthesized product. Looking at the structure of DQEA, the
integration ratio of singlet peaks to triplet peaks should be 2:1, which is seen in the NMR
2 Ac
2
2 H2O+ Mn(Ac)3
Manganese(III) acetate dihydrate
N
N
N
Mn
HO
Ac
N
N
N
OH
DQEA
Mn(III)-DQEA SOD Mimic
EtOH

5. spectrum. It is also known that the multiplet to triplet ratio is 4:1, which is seen in the
spectrum. Although there were peaks corresponding to each of the protons in the
expected compound, impurity peaks were also present and are believed to be water (1.6
ppm) and methylene chloride (0.95 ppm).
Figure 2. 1
H NMR (300 MHz) spectrum of DQEA ligand in CDCl3 (0 – 10.0 ppm range).
The IR spectra f DQEA exhibited a medium peak at 3204 cm-1
corresponding to the –OH
bond stretching do to the ethanolamine (Figure 3). Two medium found at 2897 and 2827
cm-1
are a result of the –CH stretching found in the ethanolamine characteristics of the
ligand. Both the –OH and –CH stretching from the ethanolamine are consistent with
known IR spectra of ethanolamine.15
A weak peak at 3053 cm-1
is from the aromatic –CH
stretching associated with the quinoline groups of the ligand. Two medium peaks were
found at 1600 and 1427 cm-1
suggesting C=C stretching. Two strong sharp peaks were
found at 826 and 764 cm-1
suggesting aromatic –CH bending. Both the 1
H NMR and IR
spectra confirm the successful synthesis of the DQEA ligand.

6. 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.
IR Characterization of the Mn(III) DQEA SOD Mimic
After the successful syntheses of the DQEA ligand the next step was to the synthesize
Mn(III) DQEA SOD mimic. The Mn(III) DQEA SOD mimic was synthesized by
reacting Mn(III) acetate dehydrate and DQEA with ethanol and stirring. The isolated
product was a brown red solid.
The IR spectra f DQEA exhibited a medium peak at 3327 cm-1
corresponding to the –OH
bond stretching do to the ethanolamine (Figure 3). One medium peak found at 2938 cm-1
is a result of the –CH stretching found in the ethanolamine characteristics of the ligand.
There were two very weak peaks at 3065 and 3011 cm-1
from the aromatic –CH stretching
associated with the quinoline groups of the ligand. There were also two very strong sharp
peaks were found at 1562 and 1411 cm-1
suggesting C=C stretching from the quinoline
groups. Two strong sharp peaks were also found at 783 and 752 cm-1
suggesting aromatic
–CH bending and ring torsion.
Upon viewing Figure 3 it can be seen that the spectra of the DEQA ligand and Mn(III)
DQEA SOD mimic have sufficient overlap and similarity, suggesting the successful
synthesis of the Mn(III) DQEA SOD mimic. Although some peaks from the SOD mimic
have been shifted. These shifts can be attributed to Mn(III) which is a Lewis acid and
coordination to the Mn(III) is expected to alter the IR spectrum of the ligand.
Determining SOD Activity Using the Fridovich Assay
A Fridovich assay was utilized to test the SOD activity of the Mn(III) DQEA SOD mimic
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

7. reduced will decrease, effectively decreasing the change in absorbance over time. Several
different concentrations of the Mn (III) DQEA SOD mimic were used in the assay
ranging from 0 µM to 1.0 µM. The change in absorbance was highest at a concentration
of 0 µM (Figure 4). At a concentration of 1.0 µM the change in absorbance was zero. The
sudden decrease to zero when using 1.0 µM of Mn(III) DQEA leads us to believe that the
the IC50 is somewhere between 0.7 µM and 1.0 µM, although further testing must be done
to determine the exact IC50 value The assay revealed that the Mn(III) DQEA SOD mimic
exhibited SOD activity as a result of the decrease in change in absorbance as the
concentration of the SOD mimic increased.
Since the discovery of the SOD enzyme, several groups have synthesized Mn(III) SOD
mimic compounds varying in chemical properties and structure. After discovering the
enzyme Batinic´-Haberle, Fridovich and co-workers began synthesizing various MnP
SOD with great success, having IC50 values ranging from 0.0065 – 0.025 µM.15
Failli and
co-workers synthesized a macrocyclic Mn(III) SOD mimic having a IC50 value of 1.93
µM.11
Several acylic Mn(III) SOD mimics were developed by Durot and co-workers with
IC50 values ranging from 0.87 – 3.7 µM.13
Mn(III) DQEA seems to be more successful
then several acyclic and macrocyclic Mn(III) SOD mimic, however, the MnP are
considerably more efficient.
Figure 3. Fridovich Assay of Mn(III) DQEA SOD mimic
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. Future work includes further

8. characterization of the SOD mimic and optimization of both the DQEA ligand and SOD
mimic.
Acknowledgements
Thank you to Skidmore College for the resources and funding for this project. A special
thanks to the Frey research group for their guidance, support and contributions.
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