Oxidation of N hydroxyguanidine by DbH was studied

Oxidation of N-hydroxyguanidine 1 by DbH was studied by HPLC and some oxidation products for 1 could be characterized. The compounds generally observed with iron-containing systems are 4-methoxyphenylurea 11 and 4-methoxyphenylcyanamide 12 (Fig. 2). Oxidation of N-(4-chlorophenyl)-N′-hydroxyguanidine 6 by NOS containing all of its cofactors showed the selective formation of the corresponding urea [16]. Addition of N-hydroxyguanidine 6 to the hemeprotein-model microperoxidase-8 (MP8) in the presence of excess hydrogen peroxide leads to selective formation of the cyanamide derivative [17]. Finally, a reaction performed by a cytochrome c peroxidase active site mutant shows that N-hydroxy-l-arginine yielded a nitrosoguanidine as the main identified product, together with very low amounts of citrulline and N-cyano-ornithine [31]. Apart from urea 11 and cyanamide 12, oxidation of N-hydroxyguanidine 1 by DbH led to the predominant formation of a product that was identified by mass spectroscopy as nitrosoimine 13. Transformation of N-aryl-N′-hydroxyguanidines by DbH is clearly distinct from those previously carried out with iron-containing enzymes. Another point to underline is that nitrosoimine 13 has never been observed as an oxidation product in these studies [15], [16], [17], [18], [28], [29], [31], [32]. Some details about the way N-hydroxyguanidines interact with DbH can be deduced from the activities measured when using different aromatic-ring substituents. First, considering the relative activities observed for compounds 1 and 6–10, the reaction seems not to be governed by electronic effects only. The fact that 10, the ortho-isomer of 1, is not a cosubstrate for DbH supports the meclofenamate australia that the para-position of the aromatic ring of the cosubstrate is involved in the interactions with the active site of DbH. The ability of N-hydroxyguanidine 8 bearing a bulky substituent at the para-position, to act as a cosubstrate, suggests that steric constraints on the ortho-position are more critical than those in the para-position and that the binding site of the N-aryl-N′-hydroxyguanidines displays more hindrance at the ortho- than at the para-position. Taken together, these results show that DbH is selective towards its reducing cosubstrate and that specific interactions do take place between this molecule and the active site of the enzyme. Better understanding of the way N-aryl-N′-hydroxyguanidines interact with the active site of DbH or with other copper-containing enzymes could be provided by the use of model chemical complexes. Such studies are under way in our group.
Introduction Over the last several decades, considerable evidence has accumulated indicating a role of norepinephrine (NE) in the development, expression, and susceptibility to seizures (reviewed by Weinshenker and Szot, 2002). Generally, it has been observed that an activation of the NE system has anticonvulsant effects while an impairment of the NE system has proconvulsant consequences. Multiple animal models exist which lend support to the notion that endogenous NE is anticonvulsant, including the genetically epilepsy-prone rat (Browning et al., 1989, Dailey et al., 1991, Yan et al., 1993, Jobe et al., 1994), the E1 mouse line (Tsuda et al., 1990, Tsuda et al., 1993), and NE-deficient mice (Szot et al., 1999). Of particular interest are findings indicating that an intact NE system is necessary for the action of multiple anticonvulsant therapies. For example, lesions of the locus coeruleus (LC), the major NE nucleus in the brain, attenuate the effects of multiple anticonvulsant drugs, including carbemazepine, phenytoin, and phenobarbital (Quattrone and Samanin, 1977, Quattrone et al., 1978, Crunelli et al., 1981, Waller and Buterbaugh, 1985). An intact noradrenergic system is also required for the efficacy of alternative anticonvulsant therapies, such as vagal nerve stimulation and the ketogenic diet (Krahl et al., 1998, Szot et al., 2001).