Andrew G. Roberts, Eric V. Johnston, Jae-Hung Shieh, Joseph P. Sondey, Ronald C. Hendrickson, Malcolm A. S. Moore, Samuel J. Danishefsky

Human granulocyte colony-stimulating factor (G-CSF) is an endogenous glycoprotein involved in hematopoiesis. Natively glycosylated and non-glycosylated recombinant forms, lenograstim and filgrastim, respectively, are used clinically to manage neutropenia in patients undergoing chemotherapeutic treatment. Despite their comparable therapeutic potential, the purpose of O-linked glycosylation at Thr133 remains a subject of controversy. In light of this, we have developed a synthetic platform to prepare G-CSF aglycone with the goal of enabling access to native and designed glycoforms with site-selectivity and glycan homogeneity. To address the synthesis of a relatively large, aggregation-prone sequence, we advanced an isonitrile-mediated ligation method. The chemoselective activation and coupling of C-terminal peptidyl Gly thioacids with the N-terminus of an unprotected peptide provides ligated peptides directly in a manner complementary to conventional native chemical ligation-desulfurization strategies. Herein, we describe the details and application of this method as it enabled the convergent total synthesis of G-CSF aglycone.