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 1H

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

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

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 � 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). 1H 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

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

NN

NMn

HO

Ac

N

N

N

OH

DQEA Mn(III)-DQEA SOD Mimic

EtOH

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. 1H 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 1H NMR and IR spectra confirm the successful synthesis of the DQEA ligand.

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

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

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. Literature Cited

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