Identification, occurrence and prevention of aspartimide-related byproducts in chemical protein synthesis

Formation of a five-membered ring aspartimide through the attack of a backbone amide to the side chain of aspartate and asparagine residues is a long-known side-reaction in solid phase peptide synthesis, and is also associated with in vivo protein ageing and instability of purified proteins. Conversely, its possible occurrence during chemical ligation-based protein synthesis, in particular when using the gold-standard reaction NCL (native chemical ligation), is dubious. We herein report a systematic study which demonstrates that the prevalence of this side-reaction may have been overlooked, due to the difficulty to identify it through standard HPLC analytical methods, but also the in situ conversion of aspartimide into other byproducts, having the same molecular mass as the parent aspartate residue. We show that the formation of aspartimide and derived byproducts can be limited by adopting "good NCL practices", which involve restricting the ligation temperature and reaction times, as well as replacing the commonly used phosphate buffer with HEPES. However, the efficiency of such precautions is expected to vary considerably depending on the sequence of the target protein, and the amount of byproducts is expected to grow with the length of the target protein, as a result of the number of NCL reactions and potential aspartimide hotspots. To overcome such limitations, we developed a novel straightforward and potentially generally applicable methodology based on the temporary protection of the backbone nitrogen by a 2-(4-aminobutanoyloxy)-4-methoxybenzyl (GABA-Hmb) group. This strategy was validated by the byproduct-free synthesis of SUMO-2 and a SUMOylated peptide mimic.