ABSTRACT
Antifreeze glycoproteins (AFGPs) are produced by extremophiles to defend against tissue damage in freezing climates. Cumbersome isolation from polar fish has limited probing AFGP molecular mechanisms of action and limited development of bioinspired cryoprotectants for application in agriculture, foods, coatings, and biomedicine. Here, we present a rapid, scalable, and tunable route to synthetic AFGPs (sAFGPs) using N-carboxyanhydride polymerization. Our materials are the first mimics to harness the molecular size, chemical motifs, and long-range conformation of native AFGPs. We found that ice-binding activity increases with chain length, Ala is a key residue, and the native protein sequence is not required. The glycan structure had only minor effects, and all glycans examined displayed antifreeze activity. The sAFGPs are biodegradable, nontoxic, internalized into endocytosing cells, and bystanders in cryopreservation of human red blood cells. Overall, our sAFGPs functioned as surrogates for bona fide AFGPs, solving a long-standing challenge in accessing natural antifreeze materials.
ABSTRACT
Protein aggregation is an obstacle for the development of new biopharmaceuticals, presenting challenges in shipping and storage of vital therapies. Though a variety of materials and methods have been explored, the need remains for a simple material that is biodegradable, nontoxic, and highly efficient at stabilizing protein therapeutics. In this work, we investigated zwitterionic polypeptides prepared using a rapid and scalable polymerization technique and conjugated to a supramolecular macrocycle host, cucurbit[7]uril, for the ability to inhibit aggregation of model protein therapeutics insulin and calcitonin. The polypeptides are based on the natural amino acid methionine, and zwitterion sulfonium modifications were compared to analogous cationic and neutral structures. Each polymer was end-modified with a single cucurbit[7]uril macrocycle to afford supramolecular recognition and binding to terminal aromatic amino acids on proteins. Only conjugates prepared from zwitterionic structures of sufficient chain lengths were efficient inhibitors of insulin aggregation and could also inhibit aggregation of calcitonin. This polypeptide exhibited no cytotoxicity in human cells even at concentrations that were five-fold of the intended therapeutic regime. We explored treatment of the zwitterionic polypeptides with a panel of natural proteases and found steady biodegradation as expected, supporting eventual clearance when used as a protein formulation additive.
Subject(s)
Bridged-Ring Compounds , Protein Stability , Humans , Bridged-Ring Compounds/chemistry , Calcitonin/chemistry , Insulins/chemistry , Peptides/chemistryABSTRACT
The cellular glycocalyx and extracellular matrix are rich in glycoproteins and proteoglycans that play essential physical and biochemical roles in all life. Synthetic mimics of these natural bottlebrush polymers have wide applications in biomedicine, yet preparation has been challenged by their high grafting and glycosylation densities. Using one-pot dual-catalysis polymerization of glycan-bearing α-amino acid N-carboxyanhydrides, we report grafting-from glycopolypeptide brushes. The materials are chemically and conformationally tunable where backbone and sidechain lengths were precisely altered, grafting density modulated up to 100%, and glycan density and identity tuned by monomer feed ratios. The glycobrushes are composed entirely of sugars and amino acids, are non-toxic to cells, and are degradable by natural proteases. Inspired by native lipid-anchored proteoglycans, cholesterol-modified glycobrushes were displayed on the surface of live human cells. Our materials overcome long-standing challenges in glycobrush polymer synthesis and offer new opportunities to examine glycan presentation and multivalency from chemically defined scaffolds.