Boc-L-aspartic acid β-cyclohexyl ester 4-oxymethylphenylacetamidomethyl resin

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.

The undesirable reaction of aspartimide formation has been proved to occur under both acid and base conditions in solid-phase peptide synthesis and is dependent on the beta-carboxyl protecting group, the acid or base used during the synthesis, as well as the peptide sequence. The hydrolysis of aspartimide-containing peptides, especially during HPLC purification, yields a mixture of alpha- and beta-aspartyl peptides that can not be purified easily. A previous study demonstrated that treatment of aspartimide-containing peptides with methanol in the presence of 2% diisopropylethylamine in solution leads to alpha- and beta-aspartyl peptide methyl esters. Taking advantage of these results and aiming at elucidating the optimal conditions for aspartimide ring opening, the effect of different types and concentrations of alcohols (primary and secondary) and bases (diisopropylethylamine, collidine, 4-pyrrolidinopyridine, 1-methyl-2-pyrrolidone, piperidine and KCN) was tested at various temperatures and reaction times. The best results were obtained with a combination of a primary alcohol and diisopropylethylamine, while aspartimide ring opening by secondary alcohols occurred only at high temperatures. The optimal conditions were also applied to solid-phase peptide synthesis.

Obtaining homogenous aspartyl-containing peptides via Fmoc/tBu chemistry is often an insurmountable obstacle. A generic solution for this issue utilising an optimised side-chain protection strategy that minimises aspartimide formation would therefore be most desirable. To this end, we developed the following new derivatives: Fmoc-Asp(OEpe)-OH (Epe = 3-ethyl-3-pentyl), Fmoc-Asp(OPhp)-OH (Php = 4-n-propyl-4-heptyl) and Fmoc-Asp(OBno)-OH (Bno = 5-n-butyl-5-nonyl). We have compared their effectiveness against that of Fmoc-Asp(OtBu)-OH and Fmoc-Asp(OMpe)-OH in the well-established scorpion toxin II model peptide variants H-Val-Lys-Asp-Asn/Arg-Tyr-Ile-OH by treatments of the peptidyl resins with the Fmoc removal reagents containing piperidine and DBU at both room and elevated temperatures. The new derivatives proved to be extremely effective in minimising aspartimide by-products in each application.