Synthesis of N-Ac-Ala-Gly-Gly-OMe. To 775 mg (3.8 mmol) of Lalanylglycylglycine (Ala-Gly-Gly, Nutritional Biochemicals Corp., Cleveland, Ohio) slurried in 125 mL of dry methanol at -60 °C was added 10.0 mL (139 mmol) of thionyl chloride in 50 mL of CHC13. The solution was allowed to warm to room temperature with constant stirring, after which the solvents were removed in vacuo, followed by redissolution of the residue in 50 mL each of pyridine and water. At 0 °C, 4.0 mL (116 mmol) of acetic anhydride was added with vigorous stirring, followed by a second 4.0 mL 15 min later. The reaction proceeded 30 min longer, followed by evaporation and chromatography on silica gel (14.5 X 3.8 cm) in 300-mL steps of 5, 10, and 15% methanol-chloroform. The acetylated tripeptide methyl ester, A'-Ac-Ala-Gly-Gly-OMe, eluted in the last two steps: yield 530 mg (54%); mp 133-135 °C; MS parent peak at 259 (A + \ j A = 0.124 ± 0.004; caled., 0.124), fragmentation peaks at m/e 171, 144, 114, and 87 for CH3CONHCHCH3CONHCH2CO+, +NCH2C0NHCH2C02CH3, CH3CONHCHCH3CO+, and +NCH2C02CH3, respectively; 'H NMR (2H20, DSS internal reference) 4.28 (quartet, C"-H of Ala), 4.04 and 3.96 (2s, a-CH2 of each Gly), 3.75 (s, methyl ester CH3), 2.03 (s, CH3 of acetyl group), 1.39 ppm (doublet /3-CH3 of Ala).Procedures. Enzyme alkylation, assay, and purification have been described in the preceding paper.3 p-Nitrophenyl acetate kinetics were measured spectrophotometrically on a Unicam SP 1800 instrument at pH 7.16 (0.09 M phosphate), 25 °C, 6.9% CH3CN (to dissolve the substrate), at 347.8 nm where an isosbestic point for nitrophenol was used: At = 5.06 X 103 M"1 cm'1. The pH-rate profile was measured in phosphate buffers as well.Ac-Tyr-OEt and A'-Ac-Ala-Gly-Gly-OMe kinetics were measured with a Radiometer pH-stat system at pH 8.0, 0.1 M, KC1, 25 °C. All kinetic data were processed on a Data General Nova 1220 minicomputer by either linear least squares or a nonlinear regression analysis written by . H. Klapper based on curfit by Bevington.10
