SCIENCE & TECHNOLOGY SYNTHETIC TARGET EPO has four major α-helices and several attached oligosaccharide groups.
IN PURSUIT OF SYNTHETIC EPO Complex erythropoietin-like glycopeptides are made from scratch STU BORMAN, C&EN WASHINGTON
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ESEARCHERS ARE BEGINNING TO
close in on the goal of synthesizing erythropoietin-like glycoproteins having defined carbohydrate composition. The antianemia medication erythro poietin (EPO) had 2005 sales of nearly $9 billion, according to IMS Health. The 165residue glycoprotein contains several carbo hydrate groups, some ofwhich are oligosac charides of substantial size and complexity. It's produced commercially by fermentation as a mixture of glycoproteins with variable carbohydrate composition. Homogeneous EPO, in which the carbo hydrate composition is constant, has been difficult to isolate in significant quantities. Obtaining homogeneous versions of the drug in substantial amounts is a long-sought goal, as it could ease studies of structure-ac tivity relationships and lead to more-predict able therapeutic formulations. Chemistry professor Samuel J. Danishefsky of Sloan-Kettering Institute for Cancer Research and Columbia University and coworkers hope to create homoge neous EPO synthetically. They haven't suc ceeded yet, but in a recent paper they re port significant progress toward that goal {Angew. Cbem. Int. Ed., published online May 64
C & E N / J U N E 5, 2006
19, dx.doi.org/10.1002/anie.200600538). In the study, the group developed a revised route to convergent glycopeptide synthesis and demonstrated it by creating some of the largest and most complex homo geneous EPO-like glycopeptides ever made by chemical synthesis. The undertaking has already led to advances in carbohydrate and peptide-ligation chemistry, and Danishefsky believes it will also eventually lead to homogeneous EPO. EPO-like proteins have been synthe sized twice before. In 1999, Nicola Rob ertson and Robert Ramage of Albachem Ltd. (now Almac Sciences), Edinburgh, Scotland, and the University of Edinburgh reported having synthesized an EPO-like protein by solid-phase synthesis. But it was deglycosylated, lacking carbohydrate groups. In 2003, R&D Director Gerd G. Kochendoerfer of Gryphon Therapeutics, in South San Francisco, and coworkers, in cluding researchers at the Blood Research Institute, Milwaukee, made four peptides on a synthesizer, added a carbohydrate-mi metic polymer to two of them, and joined the peptides by a technique called native chemical ligation (NCL). The polypeptide chain was then folded and two disulfide
bonds were added to produce a bioactive EPO-like protein with enhanced in vivo lifetime. Synthesizing EPO with its carbohydrate groups, however, has so far proved elusive. One restraint is that conventional NCL re quires that a terminal cysteine be present on one of the peptide pieces to be joined, and EPO has few cysteines. Associate profes sor of cell biology and chemistry Philip E. Dawson of Scripps Research Institute and coworkers had earlier developed a more versatile NCL reaction that doesn't require terminal cysteines, and Danishefsky and coworkers modified this procedure. Their cysteine-free NCL requires the presence of a terminal auxiliary group on one of the peptides, and this is then removed from the product after the ligation. IN THEIRSTUDYp Danishefsky and cowork ers used one cysteine-based and one noncysteine NCL to join three glycopeptides that they had preassembled. Their most EPO-like product was a glycopeptide with 24 amino acids and three carbohydrate groups, only one ofwhich even approaches the size and complexity of EPO's largest oligosaccharides. But in a yet-unpublished study, they created a biantennary (twobranch) oligosaccharide that more closely resembles EPO's. And in another study (J. Am. Chem. Soc, 2006,128, 7460), they de termined the molecular mechanism of their version of NCL. 'The glycopeptide synthesis is an impres sive piece of work," says chemistry profes sor Carolyn R. Bertozzi of the University of California, Berkeley, and "a major step toward the ultimate goal ofputting together EPO, the most complex natural product to be targeted for total synthesis, as far as I know." Combining glycopeptides "elevates the challenge by orders of magnitude" over peptide ligation alone, she says, "because of the sheer number of functional groups and the demands on chemoselectivity." The noncysteine ligation they use "apparently stands out in that its conditions, yields, and rates are amenable to use with large, glyco sylated fragments." Chemistry professor Stephen Β. Η. Kent of the University of Chicago is more skep tical. "Where we're going to end up when Danishefsky and coworkers have succeed ed—which they undoubtedly will—is with a chemical synthesis of a native glycosylated EPO that will never be reproduced by other groups. It will be one of these heroic syntheWWW.CEN-0NLINE.ORG
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EPO-LIKE The most complex glycopeptide synthesized by Danishefsky and coworkers so far is this 24-amino acid structure with one biantennary oligosaccharide (green) and two simple ones (blue). The Gly-Cys and Gly-Gln pairs (red), respectively, are connection points for the cysteine-based and noncysteine ligations the researchers used. Ac is acetyl, and three-letter codes are amino acids. Aux, an auxiliary chemical group required for noncysteine ligation, can be removed from the product. ses that will be a milestone in the field and will contribute all sorts ofuseful methodol ogy, but it's unlikely that it will be a practical way for others to make analogs. It's closer to traditional synthetic organic chemistry than to the types of new approaches that will be required to make total chemical pro tein synthesis routine, useful, and practical for ordinary mortals." Danishefsky replies that "at present, the effort is really an exciting academic enter prise. However, I am hopeful it will find a practical consequence by allowing medici
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nal chemistry to be done in the EPO field on pure compounds. The practicality of the technique could change radically when all the building blocks are available in a modu lar form and can be shuffled around." Dawson notes that "a major challenge for the future of this synthesis will be to maintain high synthetic yields and optical purity as the peptide fragments get larger and less soluble. Continued refinement of ligation methods will benefit the entire field of synthetic protein chemistry." Although major challenges remain in the
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assembly of large glycoproteins, says asso ciate professor of chemistry and chemical engineering Linda Hsieh-Wilson of Cali fornia Institute of Technology, the stud ies by Danishefsky's group "significantly extend the reach of NCL and take us one step closer to tailoring glycoproteins with defined oligosaccharides at specific sites. This work has the potential to significantly advance our understanding of the roles of protein glycosylation and has exciting im plications for the development of protein and antibody therapeutics." •
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