In recent years, the infusion of therapeutic proteins (i.e. insulin, growth factors, cytokines, etc.) into the medical field has opened new avenues for researchers to explore treatments for many of the ailments that currently accost society. Attesting to their importance and enormous potential is the fact that therapeutic proteins have become the fastest growing area of pharmaceuticals. Despite the ascribed benefits of these biopharmaceuticals, following administration, they are often recognized as foreign by the body and consequently removed from circulation, preventing them from carrying out their designed function for prolonged periods of time.
The work presented herein examines the glycosylation of biopharmaceutical proteins as means of improving their therapeutic effectiveness. Specifically, this work describes two novel approaches for the site-selective and indiscriminate glycosylation of proteins, procedures that are unique in the fact that they allow the conjugation of high molecular weight carbohydrates—up to 10,000 Daltons and beyond—to the surface of proteins. To highlight the usefulness of the developed methods, three biopharmaceuticals were selected for glycosylation and their effects subsequently studied.
The developed methodology was initially applied to allow for the chemical glycosylation of hemoglobin, a protein widely researched for its potential to serve as a blood substitute in transfusion medicine. In summary, hemoglobin was successfully site-selectively glycosylated and the impact of glycosylation on its various biophysical properties subsequently investigated. The second protein selected for chemical glycosylation was the catalytic enzyme paraoxonase-1 (PON1), a protein valued for its potential to provide prophylactic protection from exposure to a multitude of highly toxic organophosphate compounds—many of which are currently stockpiled around the world for deployment in chemical warfare. Following indiscriminate glycosylation, the enzyme’s catalytic efficiency was investigated and its potential to serve as a viable prophylactic treatment subsequently studied. Lastly, the methods developed for the site-selective glycosylation of hemoglobin and the indiscriminate glycosylation of PON1 were employed for the modification of human serum albumin (HSA)—a protein currently used as a plasma expander in transfusion medicine.
Additionally, described herein is a procedure for the synthesis of glycopolymers capable of self-assembling to form glyconanoparticles in aqueous environments at physiological temperatures. In summary, the GNPs were designed and synthesized to serve as anti-adhesion agents capable of removing Shiga toxin (Stx)—the lethal toxin produced by Escherichia Coli—from the body.
In a separate area of research discussed herein, glycosyltransferases—the superfamily of enzymes responsible for the construction of complex carbohydrates and glycoconjugates—were investigated for their usefulness as powerful synthetic tools. Specifically, detailed in this work is a novel method for analyzing the efficiency and ability of glycosyltransferases to accept non-natural substrates for the synthesis of carbohydrate derivatives. To demonstrate the applicability of the described methodology, a panel of 1,2-fucosyltransferases was investigated for their ability to synthesize the H(O) blood group trisaccharide and related analogs from a variety of chemoenzymatically synthesized GDP-fucose derivatives.
Lastly, reported in this work is an endeavor undertaken to better understand the reversibility of glycosylation reactions catalyzed by glycosyltransferases.