In the case of the reaction of a with
In the case of the reaction of 1a with Py2SMeCu(II), compound 4a was observed as the major oxidation product suggesting that further oxidation or hydrolysis of 4a was prevented under our conditions. The precise factors that govern the reactivity and stability of nitrosoamidines remain poorly known. In the case of NOHA, an aliphatic NOHG, the corresponding nitrosoamidine and cyanamide (-cyano-l-ornithine) were highly unstable , , , -cyano-l-ornithine being readily hydrolysed to l-citrulline , . During the oxidation of (N-hydroxyamidino)piperidine, the proposed nitroso intermediate has not been identified but the end-product, N-cyano-piperidine, was stable during gas chromatography analysis . One can thus suggest that in the case of 1a and under our experimental conditions, the aromatic substituent stabilizes the nitrosoamidine 4a toward hydrolysis or further oxidation that would give urea 5a or cyanamide 6a, respectively. The presence of a new copper EPR signal in the first seconds of the reactions performed in the presence of excess NOHG 1a or 1b suggests a direct coordination of 1a or 1b to Py2SMeCu(II) (Fig. 4). Moreover, we observed that upon addition of NOHG 1a to Py2SMeCu(II), the intense sulfur-to-copper charge-transfer band centered at 343nm decreased rapidly with a concomitant formation of a new KT 5720 sale band centered at 292.5nm (Fig. 5). During this process, existence of two isobestic points at 270nm and 341nm suggested occurrence of a single phenomenon. These data suggest that one new species was formed during the reaction of 1a with Py2SMeCu(II) and that either 1a or this new species could bind copper. Coordination of copper by 1a–b could involve either N- or O-atoms. Studies with iron(III)-heme proteins NOS and iron(III)-heme models MP-8 have shown that NOHG derivatives predominantly bind iron through their O-atom , . In the case of Py2SMeCu(II), various considerations are in favor of a nitrogen-coordination: (i) Pearson’s HSAB model suggests that affinity of Cu(II) is stronger for nitrogen than for oxygen; (ii) published structures describing the coordination of a copper ion to amidoximes show that nitrogen binds to the metal rather than the terminal oxygen ; (iii) analysis of the EPR parameters for adducts between Py2SMeCu(II) and excess NOHG 1a or 1b (Fig. 4) suggests that no oxygen coordination is involved. Indeed, incorporation of an O-atom in the coordination sphere of Cu(II), with no overall charge modification, would induce a shift of g// to higher values , whereas a small decrease is observed. Coordination of Cu(II)by NOHG 1a should thus occur via its N-atoms. According to these data, we propose that, in the first step, NOHG 1 binds to Py2SMeCu(II) by its NH2 group substituting the CH3S- group to give the Cu(II) species A (Scheme 3). Due to chelate effect, the HO- group of the NOHG could also bind the copper center. An electron transfer from bound NOHG to the copper center and loose of a proton could lead to species B that could be further oxidized by excess of Py2SMeCu(II) complex. In conclusion, use of complex Py2SMeCu(II) helps us to better understand the reactivity of aromatic NOHG as co-substrates for DBH. Direct reduction of the copper ion of Py2SMeCu(II) by NOHG 1a–d has been observed, while in the case of DBH reduction of the copper centers was only deduced from the existence of a catalytic activity . A release of more than one electron by NOHG 1a–d was observed and the EPR experiments suggested that an adduct was formed between Py2SMeCu(II) and N-aryl-N-hydroxyguanidines. The main oxidation product for NOHG 1a in the presence of Py2SMeCu(II) was identified to nitrosoamidine 4a, identical to the main oxidation product of 1a in the presence of DBH, and clearly distinct from any of the other chemical or enzymatic systems previously investigated. Reactivity of NOHG with copper centers could be useful in the study of other copper-containing monooxygenases. However, the physiological relevance of this new reaction of NOHG with DBH remains to be explored. It seems unlikely that under normal conditions, the concentration of the NOS intermediate NOHA could be high enough to compete with the natural reductants ascorbate or glutathione and to act as co-substrate for DBH. However, a distinct situation could prevail under pathological conditions such as septic shock in rats, where the serum concentration of NOHA has been found to be elevated from 3.7 to 15.8μM, and up to 37μM NOHA in the conditioned medium of rat macrophages exposed to bacterial lipopolysaccharide , . Furthermore, the design of new NOHG that could be specifically targeted to DBH could open the way to new useful drugs in the case of neurological disorders.