We herein describe a straightforward approach for the introduction of a solubilizing tag on N-terminal cysteinyl segments used in native chemical ligation-based protein chemical synthesis. Conveniently, the tag is removed during the ligation.

Deciphering the formation of side-products during the synthesis of N-Hnb-Cys crypto-thioesters led to the development of an automatable optimized protocol.

The total synthesis of long proteins requires the assembly of multiple fragments through successive ligations. The need for intermediate purification steps is as trong limitation, particularly in terms of overall yield. One solution to this problem would be solid-supported chemical ligation (SPCL), for which afirst peptide segment must be immobilized on aSPCL-compatible solid support through alinker that can be cleaved under very mild conditions to release the assembled protein. The cleavage of SPCL linkers has previously required chemical conditions sometimes incompatible with sensitive protein targets.H erein, we describe an alternative enzymatic approach to trigger cleavage under extremely mild and selective conditions.O ptimization of the linker structure and use of as mall enzyme able to diffuse into the solid support were key to the success of the strategy.W ed emonstrated its utility by the assembly of three peptide segments on the basis of native chemical ligation to afforda15kDa polypeptide.