Laboratory Synthesis Of Dnic With Lowmolecular Anionic Ligands

For laboratory synthesis of DNIC with low-molecular-weight thiol ligands, two facile pathways exist: First, exposure of solutions with iron and thiols to gaseous NO [69]. Second, by mixing nitrosothiols with iron [46].

As a typical example of the first pathway, a Thunberg vial with two separate containers is prepared. The upper container holds 1 ml distillate water with 25 mM ferrous sulfate, the bottom container holds 4 ml 15 mM HEPES or Tris buffer, pH 7.4 with 100 mM thiol. After evacuation to remove oxygen and addition of gaseous NO, the liquids are mixed and produce a dark-green solution with DNIC of final concentration [Fe] = 5 mM and Fe:thiol ~ 1:20. The complexes are monomeric because the thiolates are present in twentyfold excess over iron. In an alternative synthesis, dimeric DNIC is obtained in a similar way except that a smaller quantity of thiol is added. The addition of 10 mM thiol to the bottom container results in a final ratio Fe:thiol ~ 1:2. In this solution, dimeric DNIC is formed. The solution has yellow-orange color and does not give EPR spectra. The reason is that the dimers are diamagnetic due to the antiferromagnetic coupling between the two constituents. After the exposure to NO, the acidity of the mixture should have remained near pH ~ 7. In particular, acidification should have been avoided as it usually is a sign that the NO gas was contaminated with NO2. The yield of DNIC may be verified from the optical extinction coefficient of the complexes. The monomer has strong absorption band at 392 nm with £392 = 3580 (Mcm)-1. Additionally, it has two weaker d-d absorption bands with £603 = 299 (Mcm)-1 and £772 = 312 (Mcm)-1 [45]. The dimer has £310 = 7200 (Mcm)-1 and £360 = 6800 (Mcm)-1 plus a shoulder around 450 nm. The optical spectra can be found in Refs. [45,47].

After the formation of DNIC, the solutions may be exposed to oxygen from ambient air, and will remain stable only for a limited amount of time due to oxidation of the thiol ligands by the formation of disulfide bridges. The timescale is determined by the type of thiol. Cysteine oxidizes fairly rapidly (within 10-15 min), that results in the transformation of paramagnetic monomeric DNIC to diamagnetic dimeric DNIC with cysteine. The latter can be back transformed into paramagnetic form by the addition of excess cysteine. The dimeric DNIC with cysteine remains stable in air for 1 h [47]. Monomeric glutathione complexes are stable in air for 1-1.5 h [47]. The dimeric DNIC with glutathione keep stability in air for 3-4 h [47]. Small aliquots of freshly prepared DNIC may be snap frozen and will remain stable for weeks when stored in liquid nitrogen.

DNICs with non-thiolic ligands like citrate or phosphate can also be synthesized in this way, but yields also depend on the Fe:ligand ratio and should always be verified via EPR spectroscopy. Additionally, such non-thiolic DNICs are considerably more susceptible to be destroyed by oxygen from ambient air. Paramagnetic DNICs with non-thiolic ligands are formed at high amount of the ligands: at the molar ratio of ligand:Fe > 50. The diamagnetic, probably dimeric DNICs appear at the lower ratio [36]. A structure similar to the structure of Roussin's Red Salt is proposed for these complexes, where non-thiolic ligands function as a bridge between two iron atoms [36].

The exposure to NO gas requires care because the NO gas should be carefully scrubbed to remove contaminating traces of higher oxides, in particular NO2 radicals and N2O. The use of gas from compressed pure NO is not recommended as these contaminants unavoidably form at high pressure from the dismutation reaction (cf Chapter 1).

The common procedure of bubbling the NO gas through an alkaline solution does not remove such traces with adequate precision for reproducible results. Purification by low-temperature sublimation [71] in liquid nitrogen and subsequent evaporation is much preferred.

Generation of NO gas from acidic reduction of nitrite is preferable if oxygen can be excluded from the reaction vessels. A very convenient alternative is the use of compressed inert atmospheres with low NO content. For NO contents below 1%, the above dismutation reaction is kinetically inhibited even under pressure in an inert gas like nitrogen or argon.

Recently, a very different recipe has proven useful for the synthesis of DNIC with thiol- or non-thiol ligands: The addition of ferrous iron to the solution of S-nitrosothiols in the presence of thiolic or non-thiolic anionic ligands, respectively [45,46,71]. The method exploits the chemical equilibrium between the various constituents in any solution containing iron, nitrosothiols and excess thiol ligands [46]. The details of this chemical equilibrium will be discussed in Chapter 11. DNIC solutions obtained in this way always contain significant quantities of free thiol ligands and nitrosothiols. This should be kept in mind when the DNIC yields are tested via optical absorption of the solutions. For laboratory use, this second pathway is very convenient as it completely avoids NO gas with its cumbersome requirement of the first method, namely the scrubbing and removal of oxidized contaminants. Now some groups [45,81] synthesize DNIC with thiol-containing ligands (cysteine or glutathione) from S-nitrosothiols and ferrous ions via this second pathway. Therefore, nitrosothiols will prove very useful for DNIC synthesis in the further studies of physico-chemical and biological properties of these complexes. The recipes for synthesizing DNICs from S-nitrosothiols or mixtures of ferrous iron and thiols are described in detail in Chapter 11 of this book.

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