In the Laboratory
Synthesis of Superoxide Dismutase (SOD) Enzyme Mimetics A Bioinorganic Laboratory Experiment Graziella Vecchio* and Valeria Lanza Dipartimento di Scienze Chimiche, Università di Catania, viale A. Doria 8 95125, Catania, Italy; *
[email protected] The study of mimetics of metalloenzymes is a fascinating field. Recently, the interest in small inorganic complexes as mimetics of the superoxide dismutase (SOD) enzymes has been growing (1, 2). Superoxide dismutases (SODs) are a family of enzymes that catalyze the dismutation of the superoxide radical anion to hydrogen peroxide and molecular oxygen and thus protect living cells from toxic oxygen metabolites (3). An excess of O2− ∙, which overwhelms the amount of SOD locally available (4), is produced by the immune system in diseases such as inflammation or strokes. This excess damages the surrounding body tissues. Free radical damage has been associated with a growing number of diseases, such as rheumatoid arthritis, cancer, neurodegenerative disorders, diabetic complications, strokes, inflammation, and reperfusion injury (5, 6). The synthesis of a low molecular mass system that mimics SOD has potential for pharmaceutical use (7). Various metal complexes of copper(II) (8), iron(II) (9), manganese(II) (10, 11), or manganese(III) (12) have been characterized as SOD mimetics. Most of the SOD mimetics being developed as drugs are based on manganese. Manganese is favored because decomposition of the catalyst could produce the free metal ion in vivo and the manganese ion is the least toxic to mammalian systems (1). Among the systems reported in the literature, some of them can be easily synthesized and their biological activity investigated. We selected the manganese(III) complexes of salen-type ligands [H2salen; N,N´-bis(salicylidene)ethane-1,2-diamine] (13). These complexes are also described as catalase mimetics for their ability to catalyze the dismutation of hydrogen peroxide. The mechanism for the dismutation of O2− ∙ is the same as that of the natural enzyme and involves the reduction of Mn(III) to Mn(II) by O2−∙, which is oxidized to O2. The Mn(II) is oxidized back to Mn(III) by another molecule of O2−∙, yielding H2O2:
MnIII + O2− •
MnII + O2
MnII + 2H+ + O2 − •
MnIII + H2O2
(1) (2)
The mechanism by which Mn–salens acts as a catalase mimetic involves the oxidation of Mn(III) to oxomanganese by H2O2, releasing water. The oxomanganese is then reduced to Mn(III) by another molecule of H2O2 to form water and oxygen:
MnIII + H2O2 MnVO + H2O2
MnVO + H2O
(3)
MnIII + H2O + O2
(4)
This experiment was introduced into a bioinorganic chemistry lab course and is suitable for the last year of a bachelor’s
degree in chemistry or a more advanced course in a master’s degree program. The synthesis of a ligand, its complexing ability, and its characterization leads to a compound that is a functional mimetic of SOD. The manganese(III) complexes of salen-type ligand are the prototype of a family of mimetics that have been investigated in the literature with various applications and have been tested in vivo on humans. The students learn about some current themes of research such as the role of the reactive oxygen species (ROS) in certain diseases (14), coordination chemistry, synthesis of synthetic enzymes (called “synzyme” in the literature), use of indirect assays, and analysis of results. This experiment consists of three steps: (i) synthesis of salen (15), (ii) synthesis of manganese(III) complex (16), and (iii) characterization of its SOD-like activity (17). A complete description of the experiment is reported in the online material. Experiment Overview This experiment is simple to set up and can be carried out in three, four-hour lab periods, preceded by a two-hour introductory lesson in the classroom. The determination of SOD activity requires logistical organization depending on the number of UV–vis spectrophotometers available during the lab period. Students were organized in groups of two–three people. The first step of this experiment involves the synthesis of the Schiff ’s base of ethylendiamine and salicylaldehyde or other selected aldehydes. Using salicylaldehyde allows the students to compare their results with the data in the literature (7, 18). One or more ligands can be synthesized and isolated. 1H NMR can be used to confirm the identity of the ligand. Electrospray ionization mass spectrometry (ESI-MS) spectra could be also used. The second step involves the synthesis of the metal complex of manganese(III) and could include the acquisition of the UV–vis and ESI-MS spectra to confirm the identity of the compound. This characterization may require an additional lab period. Finally, the SOD-like activity can be determined by a simple indirect assay, the Fridovich assay (19). The superoxide anion is generated at physiological pH using xanthine oxidase in the presence of xanthine (Scheme I). Cytochrome c, which is reduced by the anion O2− ∙, was choosen as the target. The reduction of cytochrome c can be determined by UV–vis spectroscopy. The difference in the molar extinction coefficients at 550 nm between the reduced and oxidized cytochrome c is ~21,000 L mol‒1 cm‒1.
cyt c(FeIII ) + O2− •
cyt c(FeII) + O2
(5)
In the presence of a molecule able to react with superoxide ion, the cytochrome c is protected from the reduction and the ability of the SOD mimetic to react with superoxide can be measured. The assay is carried out in the presence of catalase to eliminate
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In the Laboratory
the H2O2, which is formed as a by-product in the dismutation reaction of the superoxide radical. The H2O2 reacts with the metal complex and can interfere with the assay according to eq 3. The data are collected as variation of absorbance at 550 nm per minute (Δ A/Δ t) in the presence of SOD mimetic (Vc) and they are compared with the variation of absorbance per minute in the absence of the SOD mimetic (V0). A plot of (V0/Vc) − 1 versus the concentration of the manganese complex should yield a straight line. Experimental Procedure Synthesis of H2salen Salicylaldehyde (1.75 mL, 16.4 mmol) was added to ethylendiamine (en, 0.55 mL, 8.22 mmol) in absolute ethanol (20 mL). The reaction was refluxed with stirring. A yellow solid was formed during the reaction. After 3 h, the solid was filtered and washed with ethanol. The yield was ~88%. The structure of the ligand was confirmed by 1H NMR. The UV–vis spectrum of the ligand in ethanol can also be obtained.
H N
O
xanthine oxidase
∙ N H
N
O
H N O N H
O2∙ •
∙
N H
Scheme I. The superoxide anion is generated at physiological pH using the xanthine oxidase in the presence of xanthine.
2.0 1.8
(V0 / Vc) ∙ 1
1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
Concentration [Mn(salen)Ac] / (μmol/L) Figure 1. The SOD-like activity of [Mn(salen)Ac] in the Fridovich assay.
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Superoxide Dismutase Assay The reaction mixture was composed of cytochrome c (50 μM), xanthine (50 μM), and catalase (30 μg/mL) in a phosphate buffer (1.0 × 10‒2 M, pH 7.4, 10% MeOH). An appropriate amount of xanthine oxidase was added to 2 mL reaction mixture to produce a Δ A550nm /Δ t of about 0.025 min‒1. This corresponded to a O2− ∙ production rate of about 1.1 μmol L‒1 min‒1. The cytochrome reduction rate was measured in the presence and in the absence of the investigated complex for 600 s. The I50 value at pH 7.4 was determined. Hazards
Results and Discussion
O HN
A stoichiometric amount of manganese(III) acetate was added to a suspension of the ligand in ethanol and the solution was refluxed. After 3 h the solvent was evaporated and the solid was washed with acetone. The yield was ~75%. The UV–vis and ESI-MS spectra could also be used to characterize the product.
Standard procedures for safe handling of chemicals should be followed. Students should wear lab coat, goggles, and gloves at all the times. All syntheses must be carried out in a fume hood. Ethanol, methanol, salicylaldehyde, ethylendiamine, manganese(III) acetate, and xanthine are irritants and harmful if swallowed, inhaled, or absorbed through the skin. Salicylaldehyde may cause allergic reactions. Methanol and ethanol are flammable.
O HN
Synthesis of [Mn(salen)Ac]
The concentration of the SOD mimetic able to inhibit the reduction of the cytochrome c by 50% is usually determined (I50). The I50 value is the complex concentration for which V0 = 2Vc [(V0/Vc) − 1 = 1]. The data for [Mn(salen)Ac] are shown in Figure 1. The I50 value determined was 2.54 × 10‒6 (±2.2 × 10‒7) M. This experiment can also be carried out in the presence of EDTA or albumin. The albumin is an example of a biological ligand competitor of salen for manganese and EDTA is a chemical ligand competitor. In the case of the [Mn(salen)Ac], the activity is not reduced in the presence of albumin, while it was reduced in the presence of EDTA, because EDTA is able to coordinate the manganese(II) ion (see eq 1) forming [Mn(EDTA)]2−, which does not show SOD activity. The results from this experiment are reproducible and the students were interested in performing this experiment. The students were also interested in the theory of the SOD enzyme mimetics and were energized to discover more about medical inorganic chemistry. In our course, ESI-MS spectra of the ligand and its metal complex were also recorded as an example of application of this technique to characterize organic and inorganic molecules. This was interesting for the students who had studied the technique in other courses. ESI-MS spectra were carried out in our research laboratories. A challenging aspect for the students was the manipulation of proteins in the enzymatic assay and the recording of the UV spectra of native cytochrome c and its reduced form. In one academic year the syntheses of two different complexes, manganese(III) complexes of Schiff ’s bases with ethylendiamine and with propylendiamine, were proposed.
Journal of Chemical Education • Vol. 86 No. 12 December 2009 • www.JCE.DivCHED.org • © Division of Chemical Education
In the Laboratory
The students chose the complex they preferred to examine and at the end of the experiment the data for the different complexes were compared. The students appreciated this flexibility but this kind of organization can be time consuming. If the I50 value is obtained in the presence of albumin or EDTA, students could investigate the role of competitor ligands also in a biological environment. Conclusion The experiment has been developed in a laboratory course of bioinorganic chemistry, but it would be suitable in inorganic course or in integrated laboratory. In addition to the compound described, the students can synthesize different salen-type ligands and compare their results on the SOD activity. The students learn about some current themes of research such as the role of the ROS species in certain diseases (14, 20), coordination chemistry, synthetic enzymes, and use of indirect assay. The experiment could be simplified by purchasing H2salen (Fluka). It is also suitable to extend this experiment to include the determination of the catalase activity of manganese–salen complexes, measuring the oxygen as in ref 21 or with other methods such as the Clark-type electrode (22). Acknowledgments We thank the undergraduate students who tested this experiment during their lab courses. We also thank the Department of Chemical Sciences for funding. Literature Cited 1. Riley, P. D. Chem. Rev. 1999, 99, 2573–2588. 2. Munroe, W.; Kingsley, C.; Durazo, A.; Gralla, E. B.; Imlay, J. A.; Srinivasan, C.; Valentine, J. S. J. Inorg. Biochem. 2007, 101, 1875–1882. 3. McCord, J. M.; Fridovich, I. Free Radical Biol. Med. 1988, 5, 363–369. 4. Moncada, S.; Palmer, R. M. J. L.; Higgs, E. A.; Pharmacol. Rev. 1991, 43, 109–142. 5. McCord, J. M.; Edeas, M. A. Biomed. Pharmacother. 2005, 59, 139–142.
6. Pong, K. Exp. Opin. Biol. Th. 2003, 3, 127–139. 7. Doggrell, S. A. Drugs of the Future 2002, 27, 385–390. 8. Fernandes, A. S.; Gaspar, J.; Cabral, M. F.; Caneiras, C.; Guedes, R.; Rueff, J.; Castro, M.; Costa, J.; Oliveira, N. G. J������������� . Inorg. Bio� chem. 2007, 101, 849–858. 9. Liu, G. F.; Filipovic, M.; Heinemann, F. W.; Ivanovic-Burmazovic, I. Inorg. Chem. 2007, 46, 8825–8835. 10. Masini, E.; Bani, D.; Vannacci, A.; Pierpaoli, S.; Mannaioni, P.; Comhair, S. A. A.; Xu, W.; Muscoli, C������������������������� .; Erzurum, S. C.; Salvemini, D. Free Radical Biology Medicine 2005, 39, 520–531. 11. D’Agata, R.; Grasso, G.; Iacono, G.; Spoto, G.; Vecchio G. Org. Biomol. Chem. 2006, 4, 610–612. 12. Doctrow, S.; Huffman, K.; Marcus, C. B.; Musleh, W.; Bruce, A.; Baudry, M.; Malfroy, B. Adv. Pharmacol. 1997, 38, 247–269. 13. Doctrow, S.; Huffman, K.; Marcus, C. B.; Tocco, G.; Malfroy, E.; Adinolfi, C. A.; Kruk, H.; Baker, K.; Lazarowych, N.; Mascarenhas, J.; Malfroy, B. J. Med. Chem, 2002, 45, 4549–4558. 14. Forman, H. J.; Torres, M. Am. J. Respir. Crit. Care Med. 2002, 166, 4S–8S. 1����������������������������������������������������������������� 5. Haikarainen, A.; Sibila, J.; Pietikainen, P.; Pajunen, A.; Mutikainen, I. J. Chem. Soc., Dalton Trans. 2001, 5, 991–993. 16. Boucher, I. J. J. Inorg. Nucl. Chem. 1974, 36, 531–536. 1���������������������������������������������������������������������� 7. Baudry, M.; Etienne, S.; Bruce, A.; Palucki, M.; Jacobsen, E.; Malfroy B. Biochem. Biophys. Res. Commun. 1993, 192, 964–968. 18. Puglisi, A.; Tabbì, G.; Vecchio, G. J. Inorg. Biochem. 2004, 98, 969–976. 19. Beauchamp, C.; Fridovich, I. Anal. Biochem. 1971, 44, 276–287. 20. Allen, R. G.; Tresini, M. Free Rad. Biol. Med. 2000, 28, 463– 499. 21. Vetter, T. A.; Colombo, D. P. J. Chem. Educ. 2003, 80, 788– 789. 22. Lanza, V.; Vecchio, G. J. Inorg. Biochem. 2009, 103, 381–388.
Supporting JCE Online Material
http://www.jce.divched.org/Journal/Issues/2009/Dec/abs1419.html Abstract and keywords Full text (PDF) with link to cited JCE article Supplement
Student handouts
Instructor note, including spectra
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