Solid phase peptide synthesis (SPPS) provides the possibility to chemically synthesize peptides and proteins. Applying the method on hydrophilic structures is usually without major drawbacks but faces extreme complications when it comes to "difficult sequences." These includes the vitally important, ubiquitously present and structurally demanding membrane proteins and their functional parts, such as ion channels, G-protein receptors, and other pore-forming structures. Standard synthetic and ligation protocols are not enough for a successful synthesis of these challenging sequences. In this review we highlight, summarize and evaluate the possibilities for synthetic production of "difficult sequences" by SPPS, native chemical ligation (NCL) and follow-up protocols.

NCL results in the quantitative yield of a membrane protein, where a thioester peptide is formed from an oxo-ester with an in situ cleavable solubilizing tag.

The neat ionic liquid (IL) [Cmim][OAc] is not just capable of dissolving thiol- and disulfide-containing compounds, but is able to chemically react with them without addition of any catalytic reagent. Through the analysis of four small organic molecules and a cysteine-containing peptide we could postulate a general reaction mechanism. Here, the imidazolium-carbenes preferentially react with the disulfide bond, but not thiol group. Moreover, the imidazole moiety was found to abstract the sulfur atom from the cysteine residue, providing an alternative way to transform Cys residues, which were artificially inserted into a peptide sequence in order to perform native chemical ligation (NCL) of two peptide fragments. Finally, the chemical reaction of [Cmim][OAc] with a cysteine-containing biomolecules can be tuned or even suppressed through the addition of at least 30% of water to the reaction mixture.

The chemical synthesis of a highly hydrophobic membrane-associated peptide by native chemical ligation (NCL) in an ionic liquid (IL) [C 2 mim][OAc]/buffer mixture was achieved by employing peptide concentrations up to 11 mM. NCL was studied at different pH and water content and compared to several “gold-standard” ligation protocols. The optimized reaction protocol for the NCL in IL required the addition of 40% water and pH adjustment to 7.0–7.5, resulting in ligation yields of up to 80–95% within 1 to 4 h. This new ligation protocol is generally applicable and outperforms current “gold-standard” NCL methods.