Investigation of the coordination capabilities of epinephrine towards Fe2+ and Fe3+ cations and their redox activity

[J. Korać Jačić]

IX, 83, V str.

Hemija - Neorganska hemija / Chemistry - Inorganic chemistry

Epinefrin je kateholamin sa značajnom fiziološkom ulogom. Konformacija ovog molekula utiče na njegovu interakciju sa drugim molekulima i na njegove biološke efekte. Na fiziološkim pH vrednostima, koordinativne sposobnosti epinefrina prema gvožđu i redoks interakcije epinefrina sa gvožđem su od suštinske važnosti za razumevanje dve veoma različite pojave. Prva pojava je štetno dejstvo koje hronični psihološki/sredinski stres izaziva na nivou kardiovaskularnog sistema. Druga pojava je umrežavanje kateholaminima bogatih biopolimera i struktura. Kako bi se rasvetlile uloge rastvarača i vodoničnih veza u interakcijama epinefrina sa gvožđem, proučavana je konformacija epinefrina u vodi i polarnom rastvaraču dimetil sulfoksidu (DMSO). U ovoj disertaciji su predstavljeni rezultati proučavanja mehanizama interakcije epinefrina sa Fe2+ i Fe3+ jonima pri različitim koncentracionim odnosima na pH 7,4, odnosno na pH vrednosti koja odgovara fiziološkim uslovima. U svrhu istraživanja bioloških efekata epinefrina, u ovoj disertaciji su predstavljena ispitivanja efekta Epi-Fe3+ kompleksa na ćelije koje konstitutivno eksprimiraju adrenergičke receptore. Konformacije epinefrina u polarnim rastvaračima, vodi i DMSO, su proučavane metodama H nuklearne magnetne rezonance (1H NMR) kao i dvodimenzionalnim metodama nuklearne magnetne rezonance, i to: 1H - 1H COSY, 1H - 15N HSQC i NOESY. Na NH2 i CH2 grupama epinefrina su uočeni hemijski neekvivalentni protoni prilikom korišćenja DMSO kao rastvarača. Ove pojave nisu uočene kada je rastvarač bila voda. Analizom uticaja korišćenog rastvarača na NMR spektar, i analizom uticaja povećanja temperature uzorka na NMR spektar, dolazi se do zaključka da jedan od protona amino grupe formira jaku intramolekulsku vezu sa alifatičnom hidroksilnom grupom, koja je pak H donor drugoj intermolekularnoj vezi sa DMSO. Pomoću NOESY metode su prikupljeni podaci o prostornoj poziciji protona u bočnom lancu. Na taj način je formiran 3D model konformacije epinefrina u DMSO. Ukratko, epinefrin formira dodatni petočlani prsten koji sadrži bifurkovane intramolekulske/intermolekulske vodonične veze i zauzima strukturu oblika škorpiona, gde kateholni prsten predstavlja telo škorpiona, a bočni lanac predstavlja rep koji je povijen u smeru glave škorpiona. U vodi, kao rastvaraču, konformacija epinefrina ne poseduje intramolekulske vodonične veze pa je tada struktura ovog molekula najverovatnije definisana vodoničnim vezama sa molekulima vode. U okviru ove disertacije ispitivanjima je ustanovljeno da epinefrin sa Fe3+ jonima gradi stabilne visokospinske komplekse čija je stehiometrija 1:1 ili 3:1. Stehiometrija ovog kompleksa zavisi od odnosa koncentracija epinefrina i Fe3+ jona. Na kateholnom prstenu epinefrina atomi kiseonika predstavljaju mesto za formiranje koordinacione veze unutar fiziološki relevantnog bidentatnog 1:1 kompleksa. Fe3+ katjon ima slab uticaj na redoks osobine epinefrina. Međutim, epinefrin i Fe2+ joni grade kompleks koji je jak redukcioni agens. Posledica je redukcija O2, proizvodnja vodonik peroksida i formiranje Epi-Fe3+ kompleksa. U ovom procesu epinefrin se ne oksiduje, odnosno Fe2+ jon nije prenosilac, već donor elektrona. Oksidacija Fe2+ jona koja je katalizovana epinefrinom predstavlja moguće hemijsko objašnjenje za stresom izazvana oštećenja ćelija srca. Takođe, rezultati ovih ispitivanja su u skladu sa prethodnim istraživanjima kateholamina u polimerima i njihovih interakcija sa gvožđem, i upućuju na nove strategije za poboljšavanje efikasnosti umrežavanja kateholaminima bogatih biopolimera i struktura. U stresnim situacijama epinefrin se luči i može interagovati sa labilnim gvožđem koje se nalazi u ljudskoj plazmi. Te interakcije mogu prouzrokovati značajne patofiziološke posledice. Uiv ovoj disertaciji su prikazani rezultati istraživanja prema kojima epinefrin i Fe3+ joni na fiziološkom pH grade stabilni 1:1 bidentatni kompleks. Takođe je pokazano da na fiziološkom pH epinefrin ne degradira u prisustvu gvožđa. Utvrđeno je i da epinefrin i Fe2+ joni grade bezbojni kompleks i da je taj kompleks stabilan pri anaerobnim uslovima. Uočeno je i da epinefrin u prisustvu O2 značajno promoviše oksidaciju Fe2+ jona i formiranje Epi-Fe3+ kompleksa. Pri eksperimentima rađenim metodom ciklične voltametrije, Epi-Fe2+ kompleks je pokazao potencijal E1/2 = -582 mV (u odnosu na standardnu vodoničnu elektrodu). Ovakva vrednost E1/2 potencijala objašnjava katalizovanu oksidaciju. Interakcije sa gvožđem mogu uticati na biološke efekte/efikasnost epinefrina. Uticaj vezivanja gvožđa na biološko ponašanje epinefrina je ispitivan metodom nametnute voltaže na deliću membrane u konfiguraciji cela ćelija, u kulturi ćelija koje konstitutivno eksprimiraju adrenergičke receptore. Epinefrin je samostalno, bez značajnog prisustva gvožđa, uzrokovao povećanje amplitude struja usmerenih ka spoljašnosti ćelija, tj. povećanje izlaznih struja. Kompleks epinefrina sa Fe3+ nije izazivao slične posledice. Ovim se nameće zaključak da formiranje kompleksa sa gvožđem sprečava vezivanje epinefrina za adrenergičke receptore i njihovu posledičnu aktivaciju. Prooksidativna aktivnost Epi-Fe2+ kompleksa možda predstavlja vezu između hroničnog stresa i kardiovaskularnih problema, a labilno gvožđe u plazmi je potencijalni modulator bioloških aktivnosti liganda.

Epinephrine (Epi) is a catecholamine with important physiological roles. Interactions with other molecules and associated biological effects of Epi are controlled by its molecular conformation. Coordinate interactions of epinephrine with iron at physiological pH and their redox activity are crucial for understanding two distinct phenomena. First, the adverse effects that chronic stress causes to cardiovascular system. Second, the cross-linking of biopolymers and frameworks which are rich in catecholamines. Conformation of epinephrine in polar solvents, namely in dimethyl sulfoxide (DMSO) and water, was investigated in order to shed light on effects solvents and hydrogen bonds exert on interactions of epinephrine with iron. Mechanism of epinephrine interactions with Fe2+ and Fe3+ ions was studied at different concentration ratios, at physiological pH (pH 7.4), and the results are presented in this dissertation. For the sake of exploration of biological effects of epinephrine, this dissertation also contains the results of examination of effects Epi-Fe3+ complex has on cell culture with constitutive expression of adrenergic receptors. Conformation of epinephrine in polar solvents, namely in dimethyl sulfoxide (DMSO) and water, was investigated using 1H NMR, 1H - 1H COSY, NOESY and 1H - 15N HSQC methods. When DMSO was used as a solvent, chemical and magnetic nonequivalence of protons was spotted at NH2 and CH2 groups on epinephrine. Characteristics of hydrogen bonds in DMSO were determined by studying effect which temperature rise has on NMR spectra and also analyzing influences of solvent substitution on NMR spectra. Results have shown that epinephrine induces strong intramolecular bond between one of the protons of NH2 group and the OH group on the side chain. On the other hand, the OH group on the side chain, i.e. the aliphatic OH group, presents a proton donor for intermolecular bond between epinephrine and DMSO. This phenomenon was not noticed when water was used as a solvent. 3D modelling of epinephrine molecule structure was based on information about spatial arrangement of protons, which in turn was obtained using NOESY method. Obtained 3D model shows that epinephrine in DMSO has a rigid structure that resembles the shape of a scorpion, in which the catechol ring presents the body of the scorpion and the side chain presents the tail of the scorpion. This structure is a consequence of formation of an additional five–membered ring limited by inter/intra–molecular bonds. If water is used as a solvent (instead of DMSO), epinephrine takes different and non-rigid conformation which does not possess the aforementioned intramolecular hydrogen bond. In this case, conformation of epinephrine is determined by hydrogen bonds with solvent molecules. Examinations conducted in the scope of this dissertation showed that epinephrine and Fe3+ form stable high-spin complexes in 1:1 and 3:1 stoichiometry. Stoichiometry of these depends on concentration ratio of epinephrine and Fe3+. Results acquired using Raman spectroscopy have shown that 1:1 bidentate Epi–Fe3+ complex is formed by coordinative bonding of Fe3+ ions to epinephrine molecule through O atoms on the catechol ring. Effect of Fe3+ and Fe2+ ions on redox properties of epinephrine was studied using method of cyclic voltammetry. It was observed that Fe3+ ions do not significantly affect redox properties of epinephrine, but epinephrine with Fe2+ ions forms strong reducing agent. Fe2+ ion presents electron donor that in the presence of epinephrine reduces O2 and causes production of H2O2. Specific hemism of epinephrine, which includes oxidation of Fe2+ ions, may present a mechanism that explains stress-induced cardiotoxicity and heart diseases. Also, these results can be used for improvement of synthesis and development of biopolymers.vi In stressful situations epinephrine is released and it may interact with labile iron in human blood plasma. These interactions can have potentially important (patho)physiological effects. In this dissertation, it is shown that at physiological pH epinephrine and Fe3+ build stable 1:1 high-spin bidentate complex. It is also shown that in presence of iron, at physiological pH, epinephrine does not degrade. It was observed that epinephrine and Fe2+ build colorless complex, which was stable under anaerobic conditions. In presence of O2, epinephrine significantly catalyzed oxidation of Fe2+ ions and formation of Epi-Fe3+ complex. Cyclic voltammetry results showed that the mid-point potential of Epi-Fe2+ complex equals -582 mV (vs. standard hydrogen electrode). This value of mid-point potential explains the oxidation promotion. Biological effects/efficiency of epinephrine are influenced by its interaction with iron. Iron binding effects on biological performance of epinephrine were examined using patch clamping in cell culture with constitutive expression of adrenergic receptors. Epinephrine, on its own, induced an increase of outward currents, whereas Epi-Fe3+ complex did not evoke similar phenomenon. These imply that the binding of epinephrine to adrenergic receptors and their activation is inhibited by the formation of the complex of Epi with iron. Oxidative promoting activity of Fe2+ in the presence epinephrine may represent a basis for cardiovascular problems caused by chronic stress. The results obtained in this dissertation indicate that the labile iron pool may have a new function that represents a modulation of the activity of biologically significant ligands/molecules.Epinephrine (Epi) is a catecholamine with important physiological roles. Interactions with other molecules and associated biological effects of Epi are controlled by its molecular conformation. Coordinate interactions of epinephrine with iron at physiological pH and their redox activity are crucial for understanding two distinct phenomena. First, the adverse effects that chronic stress causes to cardiovascular system. Second, the cross-linking of biopolymers and frameworks which are rich in catecholamines. Conformation of epinephrine in polar solvents, namely in dimethyl sulfoxide (DMSO) and water, was investigated in order to shed light on effects solvents and hydrogen bonds exert on interactions of epinephrine with iron. Mechanism of epinephrine interactions with Fe2+ and Fe3+ ions was studied at different concentration ratios, at physiological pH (pH 7.4), and the results are presented in this dissertation. For the sake of exploration of biological effects of epinephrine, this dissertation also contains the results of examination of effects Epi-Fe3+ complex has on cell culture with constitutive expression of adrenergic receptors. Conformation of epinephrine in polar solvents, namely in dimethyl sulfoxide (DMSO) and water, was investigated using 1H NMR, 1H - 1H COSY, NOESY and 1H - 15N HSQC methods. When DMSO was used as a solvent, chemical and magnetic nonequivalence of protons was spotted at NH2 and CH2 groups on epinephrine. Characteristics of hydrogen bonds in DMSO were determined by studying effect which temperature rise has on NMR spectra and also analyzing influences of solvent substitution on NMR spectra. Results have shown that epinephrine induces strong intramolecular bond between one of the protons of NH2 group and the OH group on the side chain. On the other hand, the OH group on the side chain, i.e. the aliphatic OH group, presents a proton donor for intermolecular bond between epinephrine and DMSO. This phenomenon was not noticed when water was used as a solvent. 3D modelling of epinephrine molecule structure was based on information about spatial arrangement of protons, which in turn was obtained using NOESY method. Obtained 3D model shows that epinephrine in DMSO has a rigid structure that resembles the shape of a scorpion, in which the catechol ring presents the body of the scorpion and the side chain presents the tail of the scorpion. This structure is a consequence of formation of an additional five–membered ring limited by inter/intra–molecular bonds. If water is used as a solvent (instead of DMSO), epinephrine takes different and non-rigid conformation which does not possess the aforementioned intramolecular hydrogen bond. In this case, conformation of epinephrine is determined by hydrogen bonds with solvent molecules. Examinations conducted in the scope of this dissertation showed that epinephrine and Fe3+ form stable high-spin complexes in 1:1 and 3:1 stoichiometry. Stoichiometry of these depends on concentration ratio of epinephrine and Fe3+. Results acquired using Raman spectroscopy have shown that 1:1 bidentate Epi–Fe3+ complex is formed by coordinative bonding of Fe3+ ions to epinephrine molecule through O atoms on the catechol ring. Effect of Fe3+ and Fe2+ ions on redox properties of epinephrine was studied using method of cyclic voltammetry. It was observed that Fe3+ ions do not significantly affect redox properties of epinephrine, but epinephrine with Fe2+ ions forms strong reducing agent. Fe2+ ion presents electron donor that in the presence of epinephrine reduces O2 and causes production of H2O2. Specific hemism of epinephrine, which includes oxidation of Fe2+ ions, may present a mechanism that explains stress-induced cardiotoxicity and heart diseases. Also, these results can be used for improvement of synthesis and development of biopolymers.vi In stressful situations epinephrine is released and it may interact with labile iron in human blood plasma. These interactions can have potentially important (patho)physiological effects. In this dissertation, it is shown that at physiological pH epinephrine and Fe3+ build stable 1:1 high-spin bidentate complex. It is also shown that in presence of iron, at physiological pH, epinephrine does not degrade. It was observed that epinephrine and Fe2+ build colorless complex, which was stable under anaerobic conditions. In presence of O2, epinephrine significantly catalyzed oxidation of Fe2+ ions and formation of Epi-Fe3+ complex. Cyclic voltammetry results showed that the mid-point potential of Epi-Fe2+ complex equals -582 mV (vs. standard hydrogen electrode). This value of mid-point potential explains the oxidation promotion. Biological effects/efficiency of epinephrine are influenced by its interaction with iron. Iron binding effects on biological performance of epinephrine were examined using patch clamping in cell culture with constitutive expression of adrenergic receptors. Epinephrine, on its own, induced an increase of outward currents, whereas Epi-Fe3+ complex did not evoke similar phenomenon. These imply that the binding of epinephrine to adrenergic receptors and their activation is inhibited by the formation of the complex of Epi with iron. Oxidative promoting activity of Fe2+ in the presence epinephrine may represent a basis for cardiovascular problems caused by chronic stress. The results obtained in this dissertation indicate that the labile iron pool may have a new function that represents a modulation of the activity of biologically significant ligands/molecules.

epinefrin, Fe2+ i Fe3+ joni, struktura, kompleks, koordinacione veze, redoks potencijal, biološka aktivnost

epinephrine, Fe2+ and Fe3+ ions, conformation, complex, coordinate bond, redox potential, biological activity

546.722/.723-386:577.175.5:543.242(043.3)

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