Dinitrosyl iron complexes with natural thiol-containing ligands in aqueous solutions: Synthesis and some physico-chemical characteristics (A methodological review).

Two approaches to the synthesis of dinitrosyl iron complexes (DNIC) with glutathione and l-cysteine in aqueous solutions based on the use of gaseous NO and appropriate S-nitrosothiols, viz., S-nitrosoglutathione (GS-NO) or S-nitrosocysteine (Cys-NO), respectively, are considered. A schematic representation of a vacuum unit for generation and accumulation of gaseous NO purified from the NO2 admixture and its application for obtaining aqueous solutions of DNIC in a Thunberg apparatus is given. To achieve this, a solution of bivalent iron in distilled water is loaded into the upper chamber of the Thunberg apparatus, while the thiol solution in an appropriate buffer (рН 7.4) is loaded into its lower chamber. Further steps, which include degassing, addition of gaseous NO, shaking of both solutions and formation of the Fe2+-thiol mixture, culminate in the synthesis of DNIC. The second approach consists in a stepwise addition of Fe2+ salts and nitrite to aqueous solutions of glutathione or cysteine. In the presence of Fe2+ and after the increase in рН to the physiological level, GS-NO or Cys-NO generated at acid media (pH < 4) are converted into DNIC with glutathione or cysteine. Noteworthy, irrespective of the procedure used for their synthesis DNIC with glutathione manifest much higher stability than DNIC with cysteine. The pattern of spin density distribution in iron-dinitrosyl fragments of DNIC characterized by the d7 electronic configuration of the iron atom and described by the formula Fe+(NO+)2 is unique in that it provides a plausible explanation for the ability of DNIC to generate NO and nitrosonium ions (NO+) and the peculiar characteristics of the EPR signal of their mononuclear form (M-DNIC).

The binuclear form of dinitrosyl iron complexes with thiol-containing ligands in animal tissues.

It has been established that treatment of mice with sodium nitrite, S-nitrosoglutathione and the water-soluble nitroglycerine derivative isosorbide dinitrate (ISDN) as NO donors initiates in vivo synthesis of significant amounts of EPR-silent binuclear dinitrosyl iron complexes (B-DNIC) with thiol-containing ligands in the liver and other tissues of experimental mice. This effect is especially apparent if NO donors are administered to mice simultaneously with the Fe2+-citrate complex. Similar results were obtained in experiments on isolated liver and other mouse tissues treated with gaseous NО in vitro and during stimulation of endogenous NO synthesis in the presence of inducible NO synthase. B-DNIC appeared in mouse tissues after in vitro treatment of tissue samples with an aqueous solution of diethyldithiocarbamate (DETC), which resulted in the transfer of iron-mononitrosyl fragments from B-DNIC to the thiocarbonyl group of DETC and the formation of EPR-detectable mononitrosyl iron complexes (MNIC) with DETC. EPR-Active MNIC with N-methyl-d-glucamine dithiocarbamate (MGD) were synthesized in a similar way. MNIC-MGD were also formed in the reaction of water-soluble MGD-Fe2+ complexes with sodium nitrite, S-nitrosoglutathione and ISDN.

Redox activities of mono- and binuclear forms of low-molecular and protein-bound dinitrosyl iron complexes with thiol-containing ligands.

EPR, optical, electrochemical and stopped-flow methods were used to demonstrate that Fe(NO)2 fragments in paramagnetic mononuclear and diamagnetic binuclear forms of dinitrosyl iron complexes with glutathione are reversibly reduced by a two-electron mechanism to be further transformed from the initial state with d(7) configuration into states with the d(8) and d(9) electronic configurations of the iron atom. Under these conditions, both forms of DNIC display identical optical and EPR characteristics in state d(9) suggesting that reduction of the binuclear form of DNIC initiates their reversible decomposition into two mononuclear dinitrosyl iron fragments, one of which is EPR-silent (d(8)) and the other one is EPR-active (d(9)). Both forms of DNIC produce EPR signals with the following values of the g-factor: g⊥=2.01, g||=1.97, gaver.=2.0. M-DNIC with glutathione manifest an ability to pass into state d(9), however, only in solutions with a low content of free glutathione. Similar transitions were established for protein-bound М- and B-DNIC with thiol-containing ligands.

A simple protocol for the synthesis of dinitrosyl iron complexes with glutathione: EPR, optical, chromatographic and biological characterization of reaction products.

The diamagnetic binuclear form of dinitrosyl iron complexes (B-DNIC) with glutathione can be easily synthesized in the air at ambient temperature. The synthetic protocol includes consecutive addition to distilled water of glutathione, which decreases the pH of the test solution to 4.0, a bivalent iron salt (e.g., ferrous sulphate) and sodium nitrite at the molar ratio of 2:1:1, with a subsequent increase in pH to neutral values. Under these conditions, the amount of B-DNIC formed is limited by initial nitrite concentration. In the novel procedure, 20mM glutathione, 10mM ferrous sulfate and 10mM sodium nitrite give 2.5mM B-DNIC with glutathione, while 5mM glutathione remains in the solution. Bivalent iron (5mM) is precipitated in the form of hydroxide complexes, which can be removed from the solution by passage through a paper filter. After the increase in рН to 11 and addition of thiols at concentrations exceeding that of DNIC tenfold, B-DNIC are converted into a mononuclear EPR-active form of DNIC (M-DNIC) with glutathione. B-DNIC preparations synthesized by using new method contain negligible amount of nitrite or S-nitrosoglutathione as a contaminations. All the steps of DNIC synthesis were characterized by using optical, EPR and HPLC methods. A long-lasting hypotensive action of DNIC formed was demonstrated.

Redox conversions of dinitrosyl iron complexes with natural thiol-containing ligands.

Using the electron paramagnetic resonance (EPR) and optical spectrophotometric methods, it has been established that biologically active, water-soluble dinitrosyl iron complexes (DNIC) with glutathione are predominantly represented by the diamagnetic binuclear form (B-DNIC) even in the presence of a 10-fold excess of glutathione non-incorporated into DNIC at neutral pH. With the increase in рН to 10-11, B-DNIC are fully converted into the paramagnetic mononuclear form (М-DNIC) with a characteristic EPR signal at g⊥=2.04, g‖=2.014 and gaver.=2.03. After treatment with a strong reducing agent sodium dithionite, both М- and B-DNIC are converted into the paramagnetic form with a characteristic EPR signal at g⊥=2.01, g‖=1.97 and gaver.=2.0. Both forms display similar absorption spectra with absorption bands at 960 and 640nm and a bend at 450nm. After oxidation by atmospheric oxygen, this situation is reversed, which manifests itself in the disappearance of the EPR signal at gaver.=2.0 and complete regeneration of initial absorption spectra of М- or B-DNIC with characteristic absorption bands at 390 or 360 and 310nm, respectively. Treatment of bovine serum albumin (BSA) solutions with gaseous NO in the presence of Fe(2+) and cysteine yields BSA-bound М-DNIC (М-DNIC-BSA). After treatment with sodium dithionite, the latter undergo transformations similar to those established for low-molecular М-DNIC with glutathione. Based on the complete coincidence of the optical and the EPR characteristics of sodium dithionite-treated М- and B-DNIC and other findings, it is suggested that sodium dithionite-reduced B-DNIC are subject to reversible decomposition into М-DNIC. The reduction and subsequent oxidation of М- and B-DNIC are interpreted in the paradigm of the current concepts of the initial electronic configurations of М- and B-DNIC (d(7) ({Fe(NO)2}(7)) and d(7)-d(7) ({Fe(NO)2}(7)-{Fe(NO)2}(7)), respectively).

Dinitrosyl iron complexes with glutathione as NO and NO⁺ donors.

It has been found that heating of solutions of the binuclear form of dinitrosyl iron complexes (B-DNIC) with glutathione in a degassed Thunberg apparatus (рН 1.0, 70°Ð¡, 6 h) results in their decomposition with a concomitant release of four gaseous NO molecules per one B-DNIC. Further injection of air into the Thunberg apparatus initiates fast oxidation of NO to NO2 and formation of two GS-NO molecules per one B-DNIC. Under similar conditions, the decomposition of B-DNIC solutions in the Thunberg apparatus in the presence of air is complete within 30-40 min and is accompanied by formation of four GS-NO molecules per one B-DNIC. It is suggested that the latter events are determined by oxidation of B-DNIC iron and concominant release of four nitrosonium ions (NO⁺) from each complex. Binding of NO⁺ to thiol groups of glutathione provokes GS-NO synthesis. At neutral рН, decomposition of B-DNIC is initiated by strong iron chelators, viz., о-phenanthroline and N-methyl-d-glucamine dithiocarbamate (MGD). In the former case, the reaction occurs under anaerobic conditions (degassed Thunberg apparatus) and is accompanied by a release of four NO molecules from B-DNIC. Under identical conditions, MGD-induced decomposition of B-DNIC gives two EPR-active mononuclear mononitrosyl iron complexes with MGD (MNIC-MGD) able to incorporate two iron molecules and two NO molecules from each B-DNIC. The other two NO molecules released from B-DNIC (most probably, in the form of nitrosonium ions) bind to thiol groups of MGD to give corresponding S-nitrosothiols. Acidification of test solutions to рН 1.0 initiates hydrolysis of MGD and, as a consequence, decomposition of MNIC-MGD and the S-nitrosated form of MGD; the gaseous phase contains four NO molecules (as calculated per each B-DNIC). The data obtained testify to the ability of B-DNIC with glutathione (and, probably, of B-DNIC with other thiol-containing ligands) to release both NO molecules and nitrosonium ions upon their decomposition. As far as nitrosyl iron complexes with non-thiol-containing ligands predominantly represented by the mononuclear mononitrosyl iron form (MNIC) are concerned, their decomposition yields exclusively NO molecules.

Nitrosonium Cation as a Cytotoxic Component of Dinitrosyl Iron Complexes with Thiol-containing Ligands (based on the Experimental Work on MCF7 Human Breast Cancer Cell Culture).

Here we demonstrate that binuclear dinitrosyl iron complexes with thiol-containing ligands (glutathione and mercaptosuccinate, B-DNIC-GSH and B-DNIC-MS, respectively) exert cytotoxic effects on MCF7 human breast cancer cells. We showed that they are mediated by nitrosonium cations released from these complexes (NO+). This finding is supported by the cytotoxic effect of both B-DNICs on MCF7 cells evidenced to retain or was even promoted in the presence of N-Methyl-D-glucamine dithiocarbamate (MGD). MGD recruits an iron nitrosyl group [Fe(NO)] from the iron-dinitrosyl fragment [Fe(NO)2] of B-DNIC-MS forming stable mononitrosyl complexes of iron with MGD and releasing NO+ cations from a [Fe(NO)2] fragment.

Dinitrosyl iron complexes with glutathione incorporated into a collagen matrix as a base for the design of drugs accelerating skin wound healing.

Composites of a collagen matrix and dinitrosyl iron complexes with glutathione (DNIC-GS) (in a dose of 4.0 µmoles per item) in the form of spongy sheets (DNIC-Col) were prepared and then topically applied in rat excisional full-thickness skin wound model. The effects of DNIC-Col were studied in comparison with spontaneously healing wounds (SpWH) and wounds treated with collagen sponges (Col) without DNIC-GS. The composites induced statistically and clinically significant acceleration of complete wound closure (21±1 day versus 23±1 day and 26±1 day for DNIC-Col, Col and SpWH, respectively). Histological examination of wound tissues on days 4, 14, 18 and 21 after surgery demonstrated that this improvement was supported by enhanced growth, maturation and fibrous transformation of granulation tissue and earlier epithelization of the injured area in rats treated with DNIC-Col composites benchmarked against Col and SpWH. It is suggested that the positive effect of the new pharmaceutical material on wound healing is based on the release of NO from decomposing DNIC. This effect is believed to be potentiated by the synergy of DNIC and collagen.