The synthesis and biological evaluation of a peptide library containing carbon-linked glycosylated serine analogs

Protein-carbohydrate interactions control a variety of cellular communication and disease processes. Remarkably, association constants for these interactions are very weak, typically showing association constants of 103 to 106 M-1. With such weak affinities, therapeutic intervention by competitive inhibition is unfeasible. Most carbohydrate binding proteins, or lectins, assemble into higher order oligomers capable of binding more than one carbohydrate and the suggestion has been made that Nature circumvents weak native affinity through multivalency.; Infection by Shiga-like toxin (SLT)-secreting E. coli O157—a pathogen from bovine fecal material that frequently contaminates a range of foodstuffs—produces a set of symptoms equivalent to shigellosis, including hemorrhagic colitis and, less frequently, fatal hemolytic uremic syndrome. One such protein is the Shiga-like toxin which assembles into a pentamer and binds as many as fifteen copies of the cell surface glycolipid globosyltriosylceramide ($a$Gal(1→4)$$Gal(1→4)$b$Glu(1→1)ceramide). This structure suggests that multivalent ligands might act as effective high affinity ligands for medically relevant multivalent lectins.; In an effort to better understand the energetics of multivalent ligand binding, a library of novel, bivalent glycopeptide ligands for SLT were synthesized using solid phase combinatorial chemistry. The library incorporates a variable peptide-based tether linking carbohydrate recognition elements. Before the library was assayed for binding to the SLT, the possibility of intermolecular interactions between adjacent ligands and the lectin was eliminated through a shaving/capping procedure. High affinity ligands were identified and re-synthesized for solution phase binding studies using titration microcalorimetry. Four glycopeptides, one containing a hydrophilic sequence (YDSK) and three containing a hydrophilic sequence (AYAI) were prepared and evaluated by isothermal titration microcalorimetry for binding to the Shiga-like toxin. The bivalent hydrophobic peptide bound with a 12-fold enhancement in affinity over the trisaccharide portion of the in vivo receptor and a 3 -fold enhancement over the bivalent, hydrophilic peptide. Binding information from these ligands suggest that although multivalent carbohydrate interactions are important for the design of tight binding ligands, adventitious contacts between the ligand and the lectin also contribute favorably to the free energy of binding. In the course of these studies, convenient methodology for the preparation of diastereomerically pure carbon-linked glycosylated serine analogs using DuPHOS-mediated catalytic asymmetric hydrogenation of glycosylated enamide esters was developed. Additionally, the capping requirements for biological assays of protein-carbohydrate interactions on solid support were elucidated.