| Abstract
| - The formation of nitric oxide (NO) was followed during the oxidation of 37 N-hydroxyguanidinesor related derivatives, including 18 new N-aryl N‘-hydroxyguanidines, by recombinant induciblenitric oxide synthase (NOS II). Several N-aryl N‘-hydroxyguanidines bearing a relatively small,electron-donating para subtituent, such as H, F, Cl, CH3, OH, OCH3, and NH2, led to NOformation rates between 8 and 41% of that of NO formation from the natural NOS substrate,Nω-hydroxy-l-arginine (NOHA). The characteristics of these reactions were very similar to thosepreviously reported for the oxidation of NOHA by NOS: (i) the strict requirement of NOScontaining (6R)-5,6,7,8-tetrahydro-l-biopterin, reduced nicotinamide adenine dinucleotidephosphate, and O2 for the oxidation to occur, (ii) the formation of NO and the correspondingurea in a 1:1 molar ratio, and (iii) a strong inhibitory effect of the classical NOS inhibitorssuch as Nω-nitro-l-arginine and S-ethyl-iso-thiourea. Structure−activity relationship studiesshowed that two structural factors are crucial for NO formation from compounds containing aCNOH function. The first one is the presence of a monosubstituted N-hydroxyguanidinefunction, since disubstituted N-hydroxyguanidines, amidoximes, ketoximes, and aldoximes failedto produce NO. The second one is the presence of a N-phenyl ring bearing a relatively small,not electron-withdrawing para substituent that could favorably interact with a hydrophobiccavity close to the NOS catalytic site. The kcat value for NOS II-catalyzed oxidation of N-para-fluorophenyl N‘-hydroxyguanidine was 80% of that found for NOHA, and its kcat/Km value wasonly 9-fold lower than that of NOHA. Interestingly, the Km value found for NOS II-catalyzedoxidation of N-(3-thienyl) N‘-hydroxyguanidine was 25 μM, almost identical to that of NOHA.Recombinant NOS I and NOS III also oxidize several N-aryl N‘-hydroxyguanidines with theformation of NO, with a clearly different substrate specificity. The best substrates of the studiedseries for NOS I and NOS III were N-(para-hydroxyphenyl) and N-(meta-aminophenyl) N‘-hydroxyguanidine, respectively. Among the studied compounds, the para-chlorophenyl and para-methylphenyl derivatives were selective substrates of NOS II. These results open the waytoward a new class of selective NO donors after in situ oxidation by each NOS family.
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