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A facile approach to the synthesis of some side-chain-protected amino acids via oxalic acid dihydrate as the copper sequestering reagent is presented. The copper in the amino acid complex reacted with oxalic acid dihydrate to form insoluble cupric oxalate, with the free amino acid released. Compared with conventional methods, this method is convenient, inexpensive, and environmentally friendly.

Sodium sulfide very efficiently removes copper from protected amino acid-copper complexes. The copper in the amino acid complex was reduced to insoluble cuprous sulfide and the free amino acid was released in pure form. This method is very convenient and rapid, requiring only 5-10 min and 0.55-0.75 equiv of sodium sulfide.

Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to L- citrulline and nitric oxide. N5-(1-iminoethyl)-L-ornithine (L-NIO, 5) is a natural product known to inactivate NOS, but the mechanism of inactivation is unknown. Upon incubation of iNOS with L-NIO a type I binding difference spectrum is observed, indicating that binding at the substrate binding site occurs. L-NIO is shown to be a time-dependent, concentration-dependent, and NADPH-dependent irreversible inhibitor of iNOS with K(I) and k(inact) values of 13.7 ± 1.6 μM and 0.073 ± 0.003 min-1, respectively. During inactivation the heme chromophore is partially lost (Figure 1); HPLC shows that the loss corresponds to about 50% of the heine. Inclusion of catalase during incubation does not prevent heine loss. N5-(1-Imino-2-[14C]ethyl)- L-ornithine (11) inactivates iNOS, but upon dialysis or gel filtration, no radioactivity remains bound to the protein or to a cofactor. The only radioactive product detected after enzyme inactivation is N(ω)-hydroxy-L- NIO (12); no C(ω)-hydroxy-L-NIO (13) or N(δ)-acetyl-L-ornithine (14) is observed (Figure 2). The amount of 12 produced during the inactivation process is 7.7 ± 0.2 equiv per inactivation event. Incubations of 12 with iNOS show time-, concentration-, and NADPH-dependent inactivation that is not reversible upon dilution into the assay solution. Incubations that include an excess of L-arginine or with substitution of NADP+ for NADPH result in no significant loss of enzyme activity. The K(I) and k(inact) values for 12 are 830 ± 160 μM and 0.0073 ± 0.0007 min-1, respectively. The magnitude of these kinetic constants (compared with those of 5) suggest that 12 is not an intermediate of L-NIO inactivation of iNOS. Compound 12 also is a substrate for iNOS, exhibiting saturation kinetics with K(m) and k(cat) values of 800 ± 85 μM and 2.22 min-1, respectively; the product is shown to be N(δ)- acetyl-L-ornithine (14) (Figure 3). The k(cat) and k(inact) values for 12 can be compared directly to give a partition ratio (k(cat)/k(inact)) for inactivation of 304; i.e., there are 304 turnovers to give NO per inactivation event. This high partition ratio further supports the notion that 12 is not involved in L-NIO inactivation of iNOS. C(ω)-Hydroxy-L-NIO (13) is not an inactivator of iNOS. These results suggest that L-NIO inactivation occurs after an oxidation step (NADPH is required for inactivation) but prior to a hydroxylation step (12 and 13 are not involved). Inactivation of iNOS by N5-(1-imino-2-[2H3]-ethyl)-L-ornithine (15) exhibits 3 kinetic isotope effect (H)k(inact)/(D)k(inact) of 1.35 ± 0.08 and on (H)(k(inact)/k(I)/(D)-(k(inact)/K(I) of 1.51 ± 0.3, suggesting that the methyl C-H bond is cleaved in a partially rate-determining step prior to hydroxylation, and that leads to inactivation. A new NADPH-dependent 400 nm peak in the HPLC of L-NIO-inactivated iNOS is produced (Figure 4). LC- electrospray mass spectrometry (Figure 5) demonstrates the m/z of the new metabolite to be 583, which is shown to correspond to biliverdin (23) (Figures 6 and 7). Two possible mechanisms for the formation of biliverdin during inactivation are proposed (Schemes 10 and 11). When 14 is incubated with iNOS, time-, concentration-, and NADPH-dependent loss of enzyme activity is observed (K(I) and k(inact) values are 490 mM and 0.24 min-1, respectively); iNOS inactivation by 14 can be prevented by inclusion of L- arginine, but not D-arginine, in the inactivation mixtures, suggesting that the inactivator acts at the arginine binding site. However, 14 is not produced from L-NIO (Figure 2) and, therefore, is not involved in L-NIO inactivation.

It has been reported elsewerw that Vigna radiata seeds contain a high concentration of γ-glutamyl-S-methylcysteine and its sulphoxide and that these compounds serve as index compounds for the chemotaxonomy of V. radiata.Beside these two γ-glutamyl peptides, nine other γ-glutamyl derivatives, of which three are new compounds, have been isolated and identified from V. radiata seeds.The structures of the new peptides, γ-glutamyl-S-methylcysteinyl-β-alanine (S-methylhomoglutathione), γ-glutamyl-Nδ-acetylornithine and γ-glutamyl-γ-glutamyl-S-methylcysteine, were confirmed by direct comparison with synthetic specimens.The other six γ-glutamyl derivatives were homoglutathione, γ-glutamyl glutamic acid, -aspartic acid, -phenylalanine, -leucine and -isoleucine.Key Word Index- Vigna radiata; Leguminosae; seeds; γ-glutamyl peptides; γ-glutamyl-S-methylcystenyl-β-alanine (S-methylhomoglutathione); γ-glutamyl-Nδ-acetylornithine; γ-glutamyl-γ-glutamyl-S-methylcysteine; homoglutathione; γ-glutamyl derivatives.

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