Red blood cells are responsible for sequestering oxygen from the lungs and transporting it to all of the tissues of the human body. Consequently the production and maintenance of red blood cells are critical processes for our survival. Erythropoietin (EPO) is a naturally occurring hormone that is responsible for these tasks. EPO is currently administered as a treatment for individuals suffering from anemia, which is a common side effect of cancer chemotherapy. The introduction of EPO into the body of such patients has been shown to increase red blood cell production and hence has led to the manufacturing of marketed forms of EPO. However, EPO that is isolated from natural sources is not a strictly defined structure. EPO is composed of a protein backbone bearing four pendant carbohydrates. The installment of these carbohydrates is entirely random and their composition is dictated by the environment within the cells during the moment of attachment to the protein core. The result is a pharmaceutical that exists as a mixture of related, but distinct molecules. Moreover, it has been demonstrated that these carbohydrates play key roles in the proper function of EPO and that the incorporation of certain carbohydrates can dramatically alter the efficacy of the molecule. Our goal is to develop a method for the synthesis of EPO as a pure and defined compound exhibiting carbohydrates, which can be specifically chosen and incorporated into the structure. The synthesis we propose would be a general approach to EPO that would allow us to investigate the effects of introducing various carbohydrates at different sites on the protein scaffold. The novelty of our synthetic route rests on the use of the innate reactivity of specific amino acids that compose the natural protein structure. We envision a synthesis that relies on the preparation of three linear fragments of EPO, which can then be united employing Native Chemical Ligation, a powerful chemical reaction which enables the coupling of large complex protein fragments. Our synthesis is also designed such that the carbohydrates are introduced onto the fragments prior to the final coupling sequence. This will allow us to prepare collections of each fragment containing any one of a number of available carbohydrate units. We can then pick and choose which forms of each of the three fragments to couple to one another according to the ligation strategy we will have established. This modular approach would allow for the rapid preparation of various forms of EPO, all of which contain the parent protein core, but present distinct carbohydrate domains. We will then submit these EPO molecules to biological testing. It is our expectation that these studies will reveal the ways in which specific carbohydrates affect the function of EPO in the clinical setting. This knowledge may then lead to the development of a more efficacious treatment for anemia.
This proposal seeks to accomplish a concise and modular total synthesis of the anemia therapeutic Erythropoietin (EPO). EPO is commonly used in the treatment of cancer chemotherapy induced anemia. Our approach to EPO will allow us to evaluate various structural aspects of the molecule in the hopes of developing a more efficacious treatment.
|FernÃ¡ndez-Tejada, Alberto; Vadola, Paul A; Danishefsky, Samuel J (2014) Chemical synthesis of the Î²-subunit of human luteinizing (hLH) and chorionic gonadotropin (hCG) glycoprotein hormones. J Am Chem Soc 136:8450-8|