From EPOthilone to EPO: Aa challenge for natural product synthesis.

A ctivate. Couple. Deprotect. Repeat. So goes the cycle of peptide synthesis, whereby amino acids are iteratively connected to produce oligopeptides. However, only peptides that are fewer than approximately 50 residues can typically be accessed through solid-phase peptide synthesis. Thus, a defining moment in the field of peptide synthesis came about with the advent of native chemical ligation (NCL), a mild and selective reaction that allows two unprotected polypeptides to be joined together via a peptide bond (1). This convergent synthesis approach significantly expanded the accessible chemical space of peptide chemistry, and advances have continued to push the field forward to access even larger protein constructs. In PNAS, Brailsford and Danishefsky (2) use a number of these methodological developments and detail their synthetic adventures to produce a nonglycosylated form of erythropoietin (EPO). The protein of interest is EPO, a 166-aa glycoprotein that serves as the main hormonal regulator of red blood cell production. EPO contains three N-linked glycans (attached to asparagine residues 24, 38, and 83) and a single O-linked glycan at serine 126. The hormone is therapeutically administered to treat anemia that can result from kidney failure, cancer, and chemotherapy. Commercial overexpression of EPO results in a heterogeneous mixture of glycoforms (various sugar appendages that are not necessarily the canonical modifications), and the individual therapeutic contributions of each form are not known. Indeed, one study showed that enzymatic removal of N-linked glycans diminished in vivo activity but not in vitro activity (3). Other studies have found that increasing the sialic acid content through cell engineering techniques leads to advantageous in vivo therapeutic properties (4). Access to homogeneous samples of glycoproteins would lead to a better understanding of the individual roles of the glycans in tuning the folding and function of EPO, perhaps opening the way to more potent analogs. In principle, protein synthesis through NCL is perfectly suited to this problem. However, the synthetic challenges associated with the synthesis of a complex glycoprotein such as EPO are manifold, even with the use of state-of-the-art NCL methodology. Nonetheless, Brailsford and Danishefsky (2) embarked on this ambitious synthetic expedition several years ago, and, in this installment of the journey, report on the successful assembly of the full-length unmodified protein (i.e., without the appended glycosyl groups). With this, they have passed an important milestone. Brailsford and Danishefsky (2) exploit a number of synthetic advances toward the creation of synthetic proteins, highlighting how far the field has come since the introduction of NCL some 18 years ago (5). The methodology they use demonstrates how a toolbox of useful reactions for the generation, manipulation, and elaboration of large peptides has been developed. These include the use of semipermanent protecting groups and active esters, tactics that are routinely used Fig. 1. The logical flow of peptide synthesis (Left) compared with Iterative NCL for the synthesis of EPO (Right). The sequential steps of activation of the C terminus of one building block, coupling of the two fragments, and removing the next protecting group are repeated to assemble peptides (Left) or proteins (Right).