Alternative chemistries for the synthesis of thrombospondin-1 type 1 repeats

Synthetic protein engineering has benefited from the development of diverse methods for the synthesis of functionalized peptide fragments and approaches for their subsequent assembly into full length polypeptides. Here we describe a series of synthetic approaches for the total chemical synthesis of the second type 1 repeat of thrombospondin-1 (TSR2) that vary in both the location of the ligation site and alpha-amine protecting group strategy (Boc/Fmoc) used for the synthesis of the associated peptide fragments. These syntheses illustrate that challenging peptide sequences can result from the protecting group strategy as well as from sequence-dependent factors. Importantly, we find that such challenges can be overcome by altering the chemistry used for solid phase peptide synthesis, the choice of ligation site, and the resin used as a solid support. From these studies, we have developed a robust synthetic route to the TSR2 polypeptide consisting of native chemical ligation between an N-terminal fragment synthesized by Boc-SPPS and a C-terminal fragment synthesized by Fmoc-SPPS. Finally, the folded TSR2 domain is obtained following an optimized oxidative folding protocol using an excess of oxidized glutathione. This optimized synthesis will enable the use of unnatural amino acids to probe the unusual structure and anti-angiogenic activity of this protein domain.