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Jennifer PooleJennifer Poole, Ph.D., is a postdoctoral associate at Yale University, Department of Chemistry.

Glycosylation is a widespread non-genetically encoded post-translational modification of many naturally occurring proteins. These sugars assist in the transmission of essential cellular information with known involvement in signaling, protein folding, transport, and molecular recognition. Nevertheless, the specific roles of many of these secondary modifications remain a mystery. Given the importance of protein glycosylation in living systems, researchers have worked extensively toward the development of methods to analyze the structural diversity of glycans. These efforts, which define one of the frontiers of glycoprotein science, have demonstrated that variations in the glycan can lead to dramatic differences in activity in vivo, protein stability, and pharmacodynamics. In addition, glycan microheterogenicity is a common feature of many glycoproteins, the nature of which is often dependent on the biochemical environment characteristic of the method of production. With interest in therapeutic glycoproteins growing at a remarkable rate and potential market revenues that reach into the tens of billions of dollars, the ability to control heterogeneity and to make specific changes to glycans has emerged as a defining focus of the glycoprotein field.

Recently, Samuel Danishefsky and coworkers described the first preparation of the glycoprotein erythropoietin in homogenous form (i.e., as a single molecular entity) by total synthesis. The synthetic approach presented by the authors makes use of a convergent strategy where uniquely glycosylated peptide fragments were assembled into the target protein using native chemical ligation and metal-free desulfurization reactions as key steps. The benefit of this modular strategy is that the sugar identity may be easily interchangeable, allowing for the exploration of an almost unlimited number of glycoforms using the same synthetic sequence. This systematic synthetic approach also gives the ability to prepare samples that are unnatural, biologically transient, naturally enzymatically degraded, or unstable to isolation conditions and gives researchers independence from cell-type specific biochemistry.

This method does suffer from several common limitations of large biomolecule total synthesis, including complex route selection, low material throughput, and difficulties with protein folding post-synthesis, which restricts its utility as a large-scale source for biopharmaceutical production. Despite these challenges, the ability to produce small amounts of pure, homogenous glycoprotein presents several opportunities for scientific study. Access to pure homogeneous samples of structural certainty could facilitate a better understanding of the structural relationship between biomolecule glycosylation and functional recognition. These products could also serve as standards for future testing and pharmaceutical quality control purposes, which, considering the difficulties in assessing isolated glycoform mixtures with modern analytical techniques, would be highly useful.

As the field of glycobiology advances and we gain a greater understanding of protein modification in complex cellular processes, it will be interesting to see what contributions result from these efforts. The ultimate test may be in the translation and application of a total synthesis approach to other, less-structurally stable families of proteins. While alternative methods exist for the study of many types of post-translational modifications, the ability to examine a singular glycoprotein species has been lacking. Total synthesis offers a direct way to relate absolute structure to function for glycoproteins.