Derek Macmillan and colleagues from University College London, UK, have fragmented peptides and proteins sequence-specifically in the presence of 3-mercaptopropionic acid to afford thioesters which can be used in native chemical ligation reactions. 

Native chemical ligation is a technique for constructing a large polypeptide from two or more unprotected peptides. A peptide containing a C-terminal thioester reacts with another peptide containing an N-terminal cysteine, in the presence of an added thiol catalyst. 

New methods which provide simpler access to the components required for native chemical ligation are much sought after. The procedure developed by Macmillan and colleagues to synthesise peptide and protein thioesters is simple and relies on the inherent stabilities of peptides. A one-step thioesterification reaction causes the peptide and protein samples to fragment at X-Cys junctions in the presence of 3-Mercaptopropionic acid. The peptides fragment at these sites to provide thioesters at the C-terminus. 

Macmillan found that the reaction is most efficient at Gly-Cys, His-Cys and Cys-Cys junctions with significant peptide fragmentation occurring at temperatures around 60 ^oC. Peptides also appear to fragment at additional X-Cys sites though only under harsher conditions at 80 ^oC. 

In future Macmillan 'would like to investigate the use microwave irradiation to allow the reaction to be conducted at lower temperature and conduct a comprehensive screen of reagents and potential catalysts. The current process is too harsh and could not be conducted, for example, in live cells.' Macmillan hopes that 'optimisation of the reaction conditions and exploration of alternative experimental protocols. may ultimately yield a direct, small molecule-mediated, method for the production of bacterially expressed protein thioesters to compliment the intein-fusion system.' 

Kathryn Sear