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  • In the present work we have


    In the present work, we have investigated the reactivity of N-aryl-N′-hydroxyguanidines 1a–d (see Scheme 1 for structures) with the water-soluble Cu(II) complex 8. Using EPR and UV–Visible spectroscopy, we have shown that the studied N-aryl-N′-hydroxyguanidines 1a–d can bind and transfer electrons to the Cu(II) ion of complex 8. The main organic product formed during the oxidation of NOHG 1a by 8 was identified by HPLC/MS as the nitrosoamidine 4a. Based on the stoichiometry of the reaction and its spectroscopic features, a mechanism for the oxidation of NOHG 1a–d by complex 8 is proposed.
    Experimental procedures
    Discussion The present study describes for the first time the interaction between a water-soluble Cu(II) complex and an aryl-NOHG. In a previous paper [15], we have established that some N-aryl-N′-hydroxyguanidines are reducing co-substrates for DBH, a copper-containing enzyme involved in the metabolism of the crucial neuromediator dopamine. The present study describes for the first time an interaction between N-aryl-N′-hydroxyguanidines and a water-soluble copper complex mimicking the CuB site of DBH. Use of this complex leads to conclusions that could not be obtained from experiments performed with purified DBH. First, a direct Actinonin transfer can take place between Cu(II) and the NOHG moiety, and two electrons are transferred from 1a to Cu(II) ions. The oxidations of 1a by DBH or by the water soluble complex Py2SMeCu(II) were found to predominantly form the nitrosoamidine 4a, and to absolutely require the presence of a free hydroxyl group. However, reduction of Py2SMeCu(II) by NOHG was less specific than that of DBH, as acetamidoxime 3a can always reduce Cu(II) of Py2SMeCu(II) whereas 3a was unable to react with DBH [15]. Previous works have shown that oxidation of NOHG 1b by NOS containing all its cofactors leads to urea 5b[13], [14], whereas NOS depleted in its cofactor tetrahydrobiopterine mainly forms cyanamide 6b from 1b[37], and -cyano-ornithine from NOHA [38]. Oxidation of NOHG 1b by excess H2O2 in the presence of the heme protein model microperoxidase-8 (MP-8) also predominantly yields cyanamide 6b[39]. Using an engineered heme-peroxidase that binds NOHA, Hirst and Goodin have identified -nitroso-l-arginine as an oxidation product for NOHA, as well as traces of -cyano-ornithine, the nitrosoamidine derivative being proposed as an unstable intermediate in the course of this reaction [40]. Finally, the reaction of (N-hydroxyamidino)piperidine with monoelectronic oxidants resulted in the formation of N-cyanopiperidine, a nitrosoamidine being also proposed as an intermediate during the course of these reactions [26]. Using the water-soluble complex Py2SMeCu(II), we have identified the main oxidation product of NOHG 1a to N-(4-methoxyphenyl)-nitrosoamidine 4a, a compound previously identified as the main metabolite of 1a incubated in the presence of DBH [15]. Although the mononuclear complex Py2SMeCu(II) is a model of the CuB center of DBH only, our results indicate that it is a good functional model to study the interaction of DBH with reducing co-substrates. A general mechanism for the oxidation of NOHG can be proposed on the basis of previously published mechanisms. One-electron oxidation of the NOHG moiety by Cu(II) generates an iminoxyl radical (Scheme 2) that can undergo dimerisation, either through a N–N coupling (Scheme 2, pathway (a) forming a N-nitrosoguanidine 7 as end-product, as proposed by Cho et al. [28], or upon formation of a C–C bond (Scheme 2, pathway (b) as proposed by Boucher et al. [41]. A further one-electron oxidation of the iminoxyl radical can produce the nitrosoamidine 4 (Scheme 2, pathway (c). Compound 4 could then evolve to urea 5 upon hydrolysis, or to cyanamide 6 either by direct loss of HNO or after a third one-electron oxidation giving NO. Such a mechanism should predominantly form HNO rather than NO. The metastable species HNO is known to rapidly dimerize to hyponitrous acid and to decompose to give N2O and water. We were not equipped to detect N2O by the specific gas chromatographic system [26]. We have detected very low amounts of and ions, stable end-products of NO, in our reaction mixtures. This comforts the proposed mechanism that would generate minor amounts of NO. These low amounts of NO could also arise from oxidation of HNO under our experimental conditions.