Nα,Nim-Bis-Boc-L-histidine methyl ester

Nitration of N(alpha),N(1)-bis(trifluoroacetyl)-l-tryptophan methyl ester with HNO(3) in acetic anhydride at 0 degrees C provides N(alpha)-trifluoroacetyl-2-nitro-l-tryptophan methyl ester in 67% yield, whereas nitration in trifluoroacetic acid at 0 degrees C gives N(alpha)-trifluoroacetyl-6-nitro-l-tryptophan methyl ester in 69% yield.

Extracts of Fusarium roseum (ATCC 12822) contain an enzyme which hydrolyzes the ornithine ester bonds of fusarinine C, a cyclic trihydroxamic acid produced by this organism. The methyl ester of Ndelta-dinitrophenyl-L-ornithine is also a substrate for the enzyme, and an assay was devised using this substrate. The enzyme exhibits a sharp maximum of activity at pH 7.5 and is extremely temperature sensitive. It is strongly inhibited by HgCl2 and p-chloromercuribenzoate, and it is competitively inhibited by Ndelta-dinitrophenyl-D-ornithine methyl ester (Ki = 0.3mM). Methyl esters of glycine, L-alanine, dinitrophenyl-L-alanine, dinitrophenyl-beta-alanine, and Ndelta-dinitrophenyl-Nalpha-acetyl-L-ornithine are not substrates, although Nepsilon-dinitrophenyl-L-lysine methyl ester is as effective as the ornithine derivative. Nonspecific lipases do not hydrolyze ornithine esters, nor does trypsin. The three ester bonds of fusarinine C are progressively hydrolyzed by the enzyme to eventually yield the monomer, fusarinine. The ferric chelate of fusarinine C is not hydrolyzed. An enzyme from Penicillium sp. was isolated with identical properties toward Nbeta-dinitro-phenyl-L-ornithine methyl ester as substrate. It also hydrolyzes N,N',N"-triacetylfusarinine C, a cyclic trihydroxamate containing Nalpha-acetylornithine ester bonds, which is produced by this organism. This substrate is hydrolyzed to Nalpha-acetylfusarine. In contrast to the Fusarium enzyme, this enzyme is fully active toward the ferric trihydroxamate chelate. However, replacement of iron by aluminum leads to a completely inactive substrate. Production of the enzyme is severely suppressed by iron in the growth medium. It is proposed that these specific ornithylesterases provide a mechanism of cellular iron release by hydrolysis of the ferric ionophores, and that an iron-exchange step occurs prior to, and is a prerequisite for, hydrolysis of the ester bonds.

The amino acid histidine is an excellent buffer and is therefore included in several organ preservation solutions used in transplantation medicine. However, when used at concentrations as in these solutions, histidine has a marked injurious potential. Therefore, we here assessed the mechanism of histidine-induced cell injury and searched for ways to use the buffering power of histidine but avoid histidine toxicity. When cultured hepatocytes were incubated in HTK solution or in modified Krebs-Henseleit buffer containing 198 mM L-histidine at 37 degrees C, most cells lost viability within 3 h (LDH release 86 +/- 7% and 89 +/- 5%, respectively). This injury was accompanied by marked lipid peroxidation, and was strongly inhibited by hypoxia, by the antioxidants trolox, butylated hydroxytoluene and N-acetylcysteine and by the membrane-permeable iron chelators 2,2'-dipyridyl, 1,10-phenanthroline, LK 614, LK 616 and deferoxamine. Thus, histidine-induced cell injury appears to be mediated by an iron-dependent formation of reactive oxygen species. D-Histidine, imidazol and L-histidine methyl ester also elicited marked injury, while the N-substituted derivatives Nalpha-acetyl-L-histidine and tert-butyl-oxycarbonylhistidine and histidine-containing dipeptides showed almost no toxicity. Histidine toxicity, its iron dependence and the superiority of Nalpha-acetyl-L-histidine were also evident during/after cold (4 degrees C) incubations. Therefore, we suggest the addition of iron chelators to histidine-containing solutions, and/or replacing histidine with Nalpha-acetyl-L-histidine in organ preservation solutions.