The event of aspartimide formation represents a serious challenge to tackle during the synthesis of aspartate-containing peptides as it leads to lowered yields, difficult purifications, or even inaccessible sequences. Aspartimides are formed upon ring-closure between the nitrogen of the alpha-carboxyl amide bond and the beta-carboxyl sidechain and subsequent release of the beta-carboxyl protecting group. The formed aspartimides undergo rapid epimerization followed by ring opening either by hydrolysis or by virtue of base (as used frequently during Fmoc SPPS), thus leading to the formation of up to eight additional potential by-products.

Aspartimide formation is reported to be sequence dependent and often occurs in Asp-Gly containing sequences. For such peptides, the use of Fmoc-Asp(OtBu)-(Dmb)Gly-OH (FDP1380) (see related products) is shown to be highly beneficial. 2,4-Dimethylbenzyl (Dmb) acts as an auxiliary protecting group temporarily masking the amide nitrogen of a peptide bond. Its efficacy and ease of introduction under standard coupling methods, e.g. PyBOP®/DIPEA or DIPCDI/HOBt, make it a valuable building block to prevent aspartimide formation and thus the subsequent formation of undesired side products arising from it. After successful peptide synthesis, the N-Dmb group can be removed by addition of TFA, typically during TFA cleavage of the peptide from the resin. If a differently modified Asp derivative is desired, Iris Biotech is also offering Fmoc-(Dmb)Gly-OH (FAA3390) itself, which can then be introduced ahead of any Asp residue of choice.

Moreover, aspartimide formation also depends on the peptide conformation and can thus be influenced by placing pseudoproline dipeptides, which are reported to be structure disrupting, ahead of an aspartimide-prone Asp residue.

Another practical approach, which is rather sequence-independent, describes the use of sterically demanding, longer alkyl chain aspartic acid esters instead of the commonly used OtBu side chain protecting group. The resulting shielding of the aspartyl beta-carboxyl group allows to minimize the formation of aspartimide-derived by-products. Iris Biotech offers a selection of bulky beta-trialkyl-methyl ester protected aspartic acid building blocks (see related products).

Just recently, the Bode group invented a Fmoc-protected Asp(cyanosulfurylide) (FAA8480) which turned out to completely suppress aspartimide formation during peptide synthesis. In contrast to hydrophobic bulky Asp derivatives, which often suffer from poor solubility and low coupling efficiency, CSY benefits of enhanced solubility. Instead of the C-O bond present in bulky Asp-esters, Asp(CSY) bears a stable C-C bond. The protecting group can be selectively and quantitatively cleaved from protected or unprotected peptides under aqueous conditions with electrophilic halogen species, e.g. N-chlorosuccinimide, to regenerate the carboxylic acid from the ylide, while being stable towards strong reducing agents, transition metals, strong acids and strong bases.

In summary, different options are available to tackle aspartimide formation and the most fitting one needs to be chosen with regard to sequence and synthetic requirements. If you are interested in details on how to avoid aspartimide formation, register for our Workshop Series and benefit from the unique research insights of Prof. Dr. Jeffrey W. Bode (ETH Zurich).

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