Given the central role of erythrocytes in enabling life, it is not surprising that there has evolved a highly sophisticated system for their production and orchestration. Broadly speaking, the multifaceted erythrocyte management process is encompassed in the term “erythropoiesis.”[1] In the early mammalian fetus, erythropoiesis begins in mesodermal cells in the yolk sac. With further development, the spleen and liver become the venues of erythrocyte production. Ultimately, the bone marrow becomes the “plant site” of erythropoiesis.[2] Upon sensing decreased oxygen in circulation, the kidneys secrete a hormone called erythropoietin (EPO). Contact of EPO with its receptor initiates signaling routines, which trigger erythropoiesis. Thus, EPO is clearly a major participant in erythropoiesis, which is central to life itself. The history of erythropoietin and the intellectual milestones leading to its recognition, demonstration, purification, sequencing, expression, production, and multifaceted medical ramifications (including applications to anemia induced by dialysis, and cancer chemotherapy) are continually being updated in various review forums.[3] Notwithstanding its celebrated status in biology and medicine, the term “erythropoietin” – insofar as it implies a chemically discrete entity – is a misnomer. Erythropoietin (EPO), as encountered by researchers and employed by physicians, is actually a large family of entities. The primary protein structure is highly conserved, as are its sites of glycosylation. Indeed, its sole O-linkage (to glycophorin) at Ser126 is substantially conserved. By contrast, the remaining three oligosaccharide domains (i.e. the N-linkages at asparagines 24, 38 and 83) are not under tight genetic supervision. This uncharacteristically permissive biomanagement leads to a highly complex medley of non-separable EPO glycoforms,[4] which has defied diligent efforts at separation.[5] To our knowledge, erythropoietin as a homogeneous chemical entity, containing a defined unitary array of N-linked carbohydrate domains, was unknown prior to our study. Curiously, our laboratory first became interested in erythropoietin from its presumably less functionally critical carbohydrate sectors. These complicated domains posed seemingly daunting problems from the perspective of organic synthesis. As we developed strategies and methods to deal with assembling suitably complex carbohydrate domains,[6] we began, in ca. 2002, to fantasize about the possibility of generating homogeneous erythropoietin itself, solely through the resources of organic chemistry. This paper describes a major advance in the realization of this goal. A prime reason that the EPO-directed venture gained increasing fascination in our chemistry–centered laboratory was the perception that, powerful as it was, the “state of the art” of protein synthesis was not then up to the task of solving the problem. It seemed that new methods, and conceptual advances would be necessary to synthesize EPO by chemical means. The two vital field resources, then available toward the synthesis of an EPO-sized protein, were step-by-step solid phase peptide synthesis (SPPS)[7] and possible ligations for merging polypeptides. While there were, and still are, no reliable rules limiting the size of a polypeptide which is accessible by linear reiterative SPPS, the size of the EPO protein we were after (166-mer), in the context of its seriously hydrophobic stretches, not to speak of its four carbohydrate domains, seemed to place it out of the range of SPPS, per se. Rather, SPPS, properly employed, could hopefully provide for the synthesis of useful (i.e. combinable) fragments of EPO. It would then be necessary to ligate judiciously selected subunits, bearing N- and O-oligosaccharide domains to reach target EPO, itself. Of the ligation methods then available, the seminal native chemical ligation (NCL) protocols of Kent and associates were certainly the most powerful.[8] However, the NCL method requires an N-terminal cysteine at the N→C ligation site (see Figure 2). Examination of the primary structure of EPO protein reveals that the positioning of its four cysteine residues is such that NCL per se would be of likely value only for the Cys29 (or Cys33) site. Figure 2 New Methods for the synthesis of proteins and glycoproteins. GP = glycopeptide. In Figure 2, we briefly summarize a menu of new methods that were developed for EPO and related protein-based synthesis projects. Of particular interest to this program early on, was the development of the ortho-mercaptoaryl ester rearrangement (OMER) methodology.[9] Thus, an incipient C-terminal thioester is generated from a phenyl ester following TCEP mediated cleavage of an ortho positioned disulfide bond (Figure 2b). The thioester, thus generated, participates as the C-terminus in an NCL ligation, or in HOBT–mediated ligations with other N-terminal protected but non-cysteine containing peptides. Moreover, it was shown that NCL could be applied to the coupling of glycopolypeptide fragments.[10a] This complements an earlier report of a NCL between two O-linked glycopeptides.[10b] Another advance, which was stimulated by EPO and related projects, involved hindered isonitrile mediated N-terminal elongation of a polypeptide chain with an N-protected thioacid (Figure 2c).[11] Finally, and most critically for the purpose at hand, we were able to accomplish major extensions of NCL, occasioned by the discovery of metal-free dethiylation (MFD)[12] (Figure 2d). This finding has had a huge enhancing effect on the reach of NCL. Our first application of MFD was to enable N-terminal alanine ligations.[12] NCL logic has been extended, from our laboratory and others, to embrace ligations at N-terminal valine,[13] leucine,[14] lysine,[15] threonine,[16] proline,[17] and phenylalanine.[18] Cumulatively, these capabilities served to change the landscape of retrosynthetic analysis in the polypeptide field, by building into planning exercise, options for using non-cysteine containing N-terminal fragments, which would reenter the world of proteogenic amino acids through MFD.[12a] The first demonstration of the consequences of this capability in the context of building therapeutic-sized proteins arose from our recently reported synthesis of the human parathyroid hormone (hPTH),[19a] as well as truncated versions thereof.[20] Following this demonstration, the combined NCL/MFD logic for the synthesis of glycoproteins has been reported by other laboratories.[19b,c] As it turned out, our earlier engagements in trying to synthesize erythropoietin preceded some of these enabling discoveries. Our most advanced point previous to this disclosure, using largely OMER technology, as well as applications to glycopeptide synthesis, brought us to three fragments – EPO(1-28),[21] EPO(29-77),[22] and EPO(78-166)[23] – which formally corresponds to erythropoietin in need of two ligations (Figure 3). Unfortunately, the weak acyl donor and acyl acceptor reactivity of the various fragments, in the face of solubility problems with the partially protected substrates, and serious aggregation tendencies, served to frustrate all attempts to join EPO(78–166) with EPO(29–77) in a meaningful yield. Figure 3 Earlier synthetic routes from our laboratory toward EPO. Fortunately, at the time that these major limitations were surfacing, the MFD method, which vastly extended the logic of NCL, came into use. At that difficult juncture, we could undertake a revised plan for the total synthesis of erythropoietin, the success of which we are pleased to present below. A central question that we were addressing in the first instance was the ability to implement the new technologies described above to reach a homogeneous, “wild-type” erythropoietin. Unlike concurrent and illuminating programs in other laboratories, which were also exploiting the possibilities of EPO retrosyntheses based on MFD technology,[24] we set as a non-compromisable condition, that our target would be strictly of the wild-type, rather than contain artificial mutants to simplify handling and isolation. Furthermore, we adopted as a sine qua non that all three wild-type asparagine sites, and the one serine site be glycosylated. In so doing, we were asking whether we could obtain indications for erythropoietic activity from a homogeneous, but more simply, glycosidated EPO, lacking the “high mannose” and sialic acid containing sectors (see Figure 1). We would hope to determine whether a structure of that sort would be foldable and would manifest both activity and stability. Finally, we hoped to compare the properties of such a homogeneous synthetically derived construct with those of wild-type “aglycone protein”. In this fashion, we would be providing the scientific basis need to address the fascinating question as to why nature glycosidates many of its most precious proteins. Figure 1 Ribbon structure of erythropoietin containing a consensus sequence of N-linked carbohydrate domains.