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Factors affecting aspartimide formation, such as protecting groups, acidity, basicity, and temperature, were studied using the model tetrapeptide, Glu-Asp-Gly-Thr. The aspartyl carboxyl side chain in this tetrapeptide was either free or protected as a benzyl or cyclohexyl ester. Our results showed that the cyclohexyl ester led to far less aspartimide formation during acidic or tertiary amine treatment than the corresponding benzyl ester. The rate constants of aspartimide formation in HF-anisole (9:1, v/v) for the tetrapeptide protected as the benzyl ester were found to be 6.2 x 10(-6) and 73.6 x 10(-6) s-1 at -15 degrees and 0 degrees C respectively. These values were about three times faster than the corresponding free- or cyclohexyl ester-protected tetrapeptide. Little difference was seen when the studies were carried out at room temperature. The cyclohexyl protected tetrapeptide gave only 0.3% aspartimide in diisopropylethylamine treatment in 24 h, a 170-fold reduction of imide formation when compared with the benzyl protected tetrapeptide. Thus, using the cyclohexyl ester for aspartyl protection, our studies showed aspartimide formation could be significantly reduced to less than 2% under standard peptide synthesis conditions. Furthermore, with these model peptides, the mechanism of acid catalyzed aspartimide was studied in a range of HF concentrations. In dilute HF cleavage conditions (HF:dimethylsulfide 1:3, v/v), the mechanism was found to be of the AAC2 type, with the rate of aspartimide formation increasing very slowly with increasing acid concentration. In concentrated HF solutions (HF greater than 70% by volume), the rate of aspartimide formation increased rapidly with the increase in acid concentration. However, from model studies, the mechanism of aspartimide formation in concentrated HF was AAC2 rather than AAC1.