N-α-(t-Butoxycarbonyl)-α-methyl-4-fluoro-L-phenylalanine

N(omega)-nitro-L-arginine methyl ester (L-NAME) was labelled with carbon-11 as a potential PET tracer for NO synthase. N(alpha)-t-butoxycarbonyl-N(omega)-nitro-L-arginine was reacted with [11C]diazomethane. After deprotection with trifluoroacetic acid the formed [11C]L-NAME was purified using HPLC. Biodistribution studies in rats and PET studies in monkeys and dogs showed no correlation between radioactivity distribution and NO synthase localization in brain and heart. Substantial amounts of [11C]methanol were detected in dog plasma shortly after injection. These findings preclude the use of [11C]L-NAME as a PET tracer.

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.