N-α-(t-Butoxycarbonyl)-O-benzyl-3-nitro-D-tyrosine

In order to trace the loss of N tau-methylhistamine, a principal metabolite of histamine, during extraction and purification from human plasma and urine samples, N tau-[3H]methylhistamine was prepared in two steps from N alpha t-butoxycarbonylhistamine (II). In the first step, compound II was deprotonated with NaH in an aprotic solvent and treated with [3H]methyl iodide. The products, N alpha t-butoxycarbonyl-N tau-[3H]methylhistamine (III) and N alpha t-butoxycarbonyl-N pi-[3H]methylhistamine (IV), were then hydrolysed with iodotrimethylsilane under mild and short reaction conditions. Facile purification with Sep-Pak silica cartridges gave the combined two isomers of N tau-[3H]methylhistamine and N pi-[3H]methylhistamine in 10.7% radiochemical yield with a radiochemical purity of > 94% and a ratio of approximately 2:1. Improvements in the extraction of methylhistamine using chromatography on Sep-Pak silica cartridges led to an overall recovery of 82.5 +/- 0.3% (n = 3) based upon total [3H]methylhistamine from normal human plasma.

Cholinesterases are involved in neuronal signal transduction, and perturbation of function has been implicated in diseases, such as Alzheimer's and Huntington's disease. For the two major classes of cholinesterases, such as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), previous studies reported BChE activity is elevated in patients with Alzheimer's disease, while AChE levels remain the same or decrease. Thus, the development of potent and specific inhibitors of BChE have received much attention as a potential therapeutic in the alleviation of neurodegenerative diseases. In this study, we evaluated amino acid analogs as selective inhibitors of BChE. Amino acid analogs bearing a 9-fluorenylmethyloxycarbonyl (Fmoc) group were tested, as the Fmoc group has structural resemblance to previously described inhibitors. We identified leucine, lysine, and tryptophan analogs bearing the Fmoc group as selective inhibitors of BChE. The Fmoc group contributed to inhibition, as analogs bearing a carboxybenzyl group showed ~tenfold higher values for the inhibition constant (K I value). Inclusion of a t-butoxycarbonyl on the side chain of Fmoc tryptophan led to an eightfold lower K I value compared to Fmoc tryptophan alone suggesting that modifications of the amino acid side chains may be designed to create inhibitors with higher affinity. Our results identify Fmoc-amino acids as a scaffold upon which to design BChE-specific inhibitors and provide the foundation for further experimental and computational studies to dissect the interactions that contribute to inhibitor binding.

The minimum primary structural requirement for N-glycosylation of proteins is the sequence -Asn-X-Thr/Ser-. In the present study, NH2-terminal derivatives of Asn-Leu-Thr-NH2 and peptides with asparagine replacements have been tested as substrates or inhibitors of N-glycosylation. The glycosylation of a known acceptor, N alpha-[3H]Ac-Asn-Leu-Thr-NHCH3, was optimized in chicken oviduct microsomes. The reaction was shown to be dependent upon Mn2+ and linear for 10 min at 30 degrees C; the apparent Km for the peptide was found to be 10 microM. N alpha-Acyl derivatives of Asn-Leu-Thr-NH2 (N-acetyl, N-benzoyl, N-octanoyl, or N-t-butoxycarbonyl) inhibited the glycosylation of N alpha-[3H] Ac-Asn-Leu-Thr-NHCH3 in a dose-dependent manner; additional experiments demonstrated that these compounds were alternative substrates rather than true inhibitors. The benzoyl and octanoyl derivatives were 10 times as effective as N alpha-Ac-Asn-Leu-Thr-NH2 in inhibiting glycosylation. In contrast, peptides containing asparagine modifications or substitutions were neither substrates nor inhibitors of N-glycosylation. They did not compete for glycosylation of 3H-peptide at 100-fold greater concentrations, and did not deplete endogenous pools of oligosaccharide-lipid. Thus, the asparagine side chain is an absolute requirement for recognition by the transferase. The majority of the glycosylated product (61%), but only 1% of the unglycosylated peptide, remained associated with the microsomes after high speed centrifugation. A large 41-amino acid residue acceptor peptide, alpha-lac17-58, was a poor substitute for glycosylation unless detergent was added to the microsomes. In contrast, glycosylation of tripeptide acceptors was not stimulated by detergent. Both of these findings suggest that the tripeptides are freely permeable to the microsomal membrane and support the earlier conclusion that glycosylation of proteins occurs at the luminal face of the microsomes.