Myristoic acid (Myr-OH) and other long-chain fatty acids are found in amide linkages with the N-terminal Gly residues of proteins. The fatty acid moiety has been identified in electron density maps obtained by high-resolution X-ray crystallographic studies. 3) The acid moiety has also been elucidated by gas chromatography of Myr-peptide and confirmed by FAB-MS. 4) The fatty acids released by hydrolysis of the protein were identified as fatty acid phenacyl esters by HPLC. 5) The N-terminal

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... ng groups prevented straightforward application of amino acid sequence determination such as Edman degradation on the intact protein and peptides. Enzymatic deblocking was developed for removal of Myr-OH from peptides, 6) but no simple chemical means have been reported. To develop a simple chemical method for deblocking of Myr-Gly-peptide, we investigated the susceptibility and stability of the internal peptide linkages of model peptides, Myr-Gly-X-Phe-OH (XϭGly, Ala, Val, Lys, His, Arg, Ile, Glu, Gln, Asn, Asp), to HCl, H 2 SO 4 and MSA at 25 and 60°C, and analyzed the hydrolysates by HPLC. Here, we describe a simple method for regioselective cleavage of the Gly-X bond of Myr-Gly-X-Phe-OH by acids and determination of the decomposition products by RP-HPLC. Results and Discussion Hydrolysis of Myr-Gly-Ala-Phe-OH in HCl To examine whether the regioselective cleavage of a peptide linkage of Myr-Gly-X-Phe-OH is possible, the susceptibilities of the Myr-Gly, Gly-Ala and Ala-Phe bonds of My-Gly-Ala-Phe-OH in dilute acid were examined. When a solution of Myr-Gly-Ala-Phe-OH [4ϫ10 Ϫ4 mol/l in 6% HCl : dioxane (1 : 1)] was incubated at 60°C for 6 h, the hydrolysis resulted in the formation of three hydrolysates. The three peaks and starting material peak in the HPLC were quantified and isolated by HPLC, and assigned on the basis of amino acid analysis and direct comparison with authentic samples. The main reactions were cleavage of the Myr-Gly and Gly-Ala linkages resulting in the production of H-Gly-Ala-Phe-OH and H-Ala-Phe-OH, with H-Phe-OH as a minor product (Fig. 1) . The yield of H-Ala-Phe-OH was higher than that of H-Gly-Ala-Phe-OH. Thus, the Gly-Ala linkage was more susceptible than the Myr-Gly linkage in dilute acid. Next, the susceptibilities of the Gly-Ala and Ala-Phe bonds of N-terminal free peptide H-Gly-Ala-Phe-OH were examined under the same conditions to test the fur-ther decomposition of the hydrolysate. H-Gly-Ala-Phe-OH was quite stable under these conditions, that is, 90% of the starting material remained intact after 6 h incubation. The yields of H-Ala-Phe-OH and H-Phe-OH were less than 4% (Fig. 2) . These results indicated that the hydrolysates of Myr-Gly-Ala-Phe-OH were derived from the direct decomposition of the starting material. Next, to examine whether more selective cleavage of the three amide linkages of Myr-Gly-X-Phe-OH is possible, the susceptibilities of the Myr-Gly, Gly-Ala and Ala-Phe bonds of Myr-Gly-Ala-Phe-OH to several concentrations of 6-36% HCl : dioxane (1 : 1) were examined at 25°C. Our earlier study revealed that acid hydrolysis at low temperature To develop a simple chemical deblocking method for Myr-Gly-peptide, we investigated the susceptibility and stability of the internal peptide linkages of model peptides, Myr-Gly-X-Phe-OH (X‫؍‬Gly, Ala, Val, Lys, His, Arg, Ile, Glu, Gln, Asp, Asn), to HCl, H 2 SO 4 and methanesulfonic acid (MSA) at 25 and 60°C, and analyzed the hydrolysates by HPLC. The results indicated that acid hydrolysis in concentrated acids was superior for selective cleavage of the Gly-X linkage. Here, we describe a simple method for regioselective cleavage of the Gly-X bond of Myr-Gly-X-Phe-OH by acids and determination of the decomposition products by RP-HPLC.