Clickable Polyprolines from Azido-proline N-Carboxyanhydride.

Polyproline is a material of great interest in biomedicine due to its helical scaffold of structural importance in collagen and mucins and its ability to gel and to change conformations in response to temperature. Appending of function-modulating chemical groups to such a material is desirable to diversify potential applications. Here, we describe the synthesis of high-molecular-weight homo, block, and statistical polymers of azide-functionalized proline. The azide groups served as moieties for highly efficient click-grafting, as stabilizers of the polyproline PPII helix, and as modulators of thermoresponsiveness. Saccharides and ethylene glycol were utilized to explore small-molecule grafting, and glutamate polymers were utilized to form polyelectrolyte bottlebrush architectures. Secondary structure effects of both the azide and click modifications, as well as lower critical solution temperature behavior, were characterized. The polyazidoprolines and click products were well tolerated by live human cells and are expected to find use in diverse biomedical applications.

Mirror Image Mucins and Thio Mucins with Tunable Biodegradation.

Mucin glycoproteins are the major component of mucus and are integral to the cellular glycocalyx. Mucins play diverse roles in health and disease, are an important element in epithelial tissue models, and have broad therapeutic potential. All mucin applications are currently challenged by their inherent structural heterogeneity and degradation by proteases. In this study, we describe the synthesis and study of chemically defined mucin analogues bearing native glycans. We utilized combinations of enantiomer amino acids and glycan thioether linkages to achieve tunable proteolysis while maintaining cytocompatibility and binding activity. Structural characterization revealed a previously unknown mirror-image helix and sheds light on the molecular drivers of glycoprotein conformation. This work represents an important step toward the development of artificial mucins for biomedical applications.

Supramolecular Protein Stabilization with Zwitterionic Polypeptide-Cucurbit[7]uril Conjugates.

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.

Principles of glycocalyx engineering with hydrophobic-anchored synthetic mucins.

The cellular glycocalyx is involved in diverse biological phenomena in health and disease. Yet, molecular level studies have been challenged by a lack of tools to precisely manipulate this heterogeneous structure. Engineering of the cell surface using insertion of hydrophobic-terminal materials has emerged as a simple and efficient method with great promise for glycocalyx studies. However, there is a dearth of information about how the structure of the material affects membrane insertion efficiency and resulting density, the residence time of the material, or what types of cells can be utilized. Here, we examine a panel of synthetic mucin structures terminated in highly efficient cholesterylamide membrane anchors for their ability to engineer the glycocalyx of five different cell lines. We examined surface density, residence time and half-life, cytotoxicity, and the ability be passed to daughter cells. We report that this method is robust for a variety of polymeric structures, long-lasting, and well-tolerated by a variety of cell lines.

Synthesis and biomedical applications of mucin mimic materials.

Mucin glycoproteins are the major component of mucus and coat epithelial cell surfaces forming the glycocalyx. The glycocalyx and mucus are involved in the transport of nutrients, drugs, gases, and pathogens toward the cell surface. Mucins are also involved in diverse diseases such as cystic fibrosis and cancer. Due to inherent heterogeneity in native mucin structure, many synthetic materials have been designed to probe mucin chemistry, biology, and physics. Such materials include various glycopolymers, low molecular weight glycopeptides, glycopolypeptides, polysaccharides, and polysaccharide-protein conjugates. This review highlights advances in the area of design and synthesis of mucin mimic materials, and their biomedical applications in glycan binding, epithelial models of infection, therapeutic delivery, vaccine formulation, and beyond.

Mucins Inhibit Coronavirus Infection in a Glycan-Dependent Manner.

Mucins are a diverse and heterogeneous family of glycoproteins that comprise the bulk of mucus and the epithelial glycocalyx. Mucins are intimately involved in viral transmission. Mucin and virus laden particles can be expelled from the mouth and nose to later infect others. Viruses must also penetrate the mucus layer before cell entry and replication. The role of mucins and their molecular structure have not been well-characterized in coronavirus transmission studies. Laboratory studies predicting high rates of fomite transmission have not translated to real-world infections, and mucins may be one culprit. Here, we probed both surface and direct contact transmission scenarios for their dependence on mucins and their structure. We utilized disease-causing, bovine-derived, human coronavirus OC43. We found that bovine mucins could inhibit the infection of live cells in a concentration- and glycan-dependent manner. The effects were observed in both mock fomite and direct contact transmission experiments and were not dependent upon surface material or time-on-surface. However, the effects were abrogated by removal of the glycans or in a cross-species infection scenario where bovine mucin could not inhibit the infection of a murine coronavirus. Together, our data indicate that the mucin molecular structure plays a complex and important role in host defense.

Biomimetic Glycosylated Polythreonines by N-Carboxyanhydride Polymerization.

Glycosylated threonine (Thr) is a structural motif found in seemingly disparate natural proteins from deep-sea collagen to mucins. Synthetic mimics of these important proteins are of great interest in biomedicine. Such materials also provide ready access to probe the contributions of individual amino acids to protein structure in a controlled and tunable manner. N-Carboxyanhydride (NCA) polymerization is one major route to such biomimetic polypeptides. However, challenges in the preparation and polymerization of Thr NCAs have impeded obtaining such structures. Here, we present optimized routes to several glycosylated and acetylated Thr NCAs of high analytical purity. Transition metal catalysis produced tunable homo-, statistical, and block-polypeptides with predictable chain lengths and low dispersities. We conducted structural work to examine their aqueous conformations and found that a high content of free OH Thr induces the formation of water-insoluble ß-sheets. However, glycosylation appears to induce a polyproline II-type helical conformation, which sheds light on the role of glyco-Thr in rigid proteins such as mucins and collagen.

Preparation and applications of artificial mucins in biomedicine.

Mucus is an essential barrier material that separates organisms from the outside world. This slippery material regulates the transport of nutrients, drugs, gases, and pathogens toward the cell surface. The surface of the cell itself is coated in a mucus-like barrier of glycoproteins and glycolipids. Mucin glycoproteins are the primary component of mucus and the epithelial glycocalyx. Aberrant mucin production is implicated in diverse disease states from cancer and inflammation to pre-term birth and infection. Biological mucins are inherently heterogenous in structure, which has challenged understanding their molecular functions as a barrier and as biochemically active proteins. Therefore, many synthetic materials have been developed as artificial mucins with precisely tunable structures. This review highlights advances in design and synthesis of artificial mucins and their application in biomedical studies of mucin chemistry, biology, and physics.

Polypeptoids and Peptoid-Peptide Hybrids by Transition Metal Catalysis.

Peptoids have attracted attention for application in biomedicine due to their advantageous properties as compared to peptides. The structural analogues are typically resistant to protease degradation and offer improved biocompatibility. Chemical routes to an impressive variety of short-chain, low-molecular-weight peptoids are well-established. However, synthetic methods for well-defined, high-molecular-weight polypeptoids with side chain diversity are still in their infancy. Here, we report a facile method for synthesis of polypeptoids via transition-metal-catalyzed controlled, living polymerization of N-substituted N-carboxyanhydrides. Our method is amenable to hydrophilic and hydrophobic side chains and yields high-molecular-weight linear polypeptoids of predictable length and low dispersity. Further, the polymer end groups can be tuned for biological targeting, and polypeptide-polypeptoid hybrids are readily prepared in one pot. Our materials are indeed resistant to common proteases and are well-tolerated by human cells. Overall, this work represents a significant stride toward access to tunable polypeptoids.

Tunable, biodegradable grafting-from glycopolypeptide bottlebrush polymers.

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.

Rethinking Transition Metal Catalyzed N-Carboxyanhydride Polymerization: Polymerization of Pro and AcOPro N-Carboxyanhydrides.

Polyproline (PP) based polypeptides have broad applications as protein mimics, ordered materials, hydrogels, and surface coatings. However, a lack of rapid and efficient preparatory methods has challenged synthesis of well-defined high molecular weight materials. Here, we report facile and high-yielding methods for preparation and polymerization of Pro and trans-4-acetoxy-Pro N-carboxyanhdrides (NCAs). For decades, transition metal initiators of NCA polymerization were assumed to be nonstarters with Pro due to the lack of an amide NH proton. We carefully considered the known steps in the initiation mechanism and applied a Ni initiator that intercepts an intermediate and does not require an NH group. This initiator efficiently catalyzes controlled, living polymerization of Pro NCAs, revealing that routes alternate to the previously proposed mechanism must be at play. We also found Co species can catalyze Pro NCA polymerization, and we improved the synthetic methods to prepare the NCA monomers. Our methods are high-yielding and rapid and give tunable, end-functional PP-based homo, statistical, and block polypeptides. We characterized the conformation of PP and trans-4-hydroxy-PP by CD and confirmed the time scale for quantitative conversion from PPI to PPII helices. Overall, our data shed light on the general propagation mechanism of transition metal catalyzed NCA polymerization and have opened the door for efficient preparation of a desirable class of biomaterials.

Design, synthesis and biological applications of glycopolypeptides.

Carbohydrates play essential structural and biochemical roles in all living organisms. Glycopolymers are attractive as well-defined biomimetic analogs to study carbohydrate-dependent processes, and are widely applicable biocompatible materials in their own right. Glycopolypeptides have shown great promise in this area since they are closer structural mimics of natural glycoproteins than other synthetic glycopolymers and can serve as carriers for biologically active carbohydrates. This review highlights advances in the area of design and synthesis of such materials, and their biomedical applications in therapeutic delivery, tissue engineering, and beyond.

The mucin-selective protease StcE enables molecular and functional analysis of human cancer-associated mucins.

Mucin domains are densely O-glycosylated modular protein domains that are found in a wide variety of cell surface and secreted proteins. Mucin-domain glycoproteins are known to be key players in a host of human diseases, especially cancer, wherein mucin expression and glycosylation patterns are altered. Mucin biology has been difficult to study at the molecular level, in part, because methods to manipulate and structurally characterize mucin domains are lacking. Here, we demonstrate that secreted protease of C1 esterase inhibitor (StcE), a bacterial protease from Escherichia coli, cleaves mucin domains by recognizing a discrete peptide- and glycan-based motif. We exploited StcE's unique properties to improve sequence coverage, glycosite mapping, and glycoform analysis of recombinant human mucins by mass spectrometry. We also found that StcE digests cancer-associated mucins from cultured cells and from ascites fluid derived from patients with ovarian cancer. Finally, using StcE, we discovered that sialic acid-binding Ig-type lectin-7 (Siglec-7), a glycoimmune checkpoint receptor, selectively binds sialomucins as biological ligands, whereas the related receptor Siglec-9 does not. Mucin-selective proteolysis, as exemplified by StcE, is therefore a powerful tool for the study of mucin domain structure and function.

N-Carboxyanhydride Polymerization of Glycopolypeptides That Activate Antigen-Presenting Cells through Dectin-1 and Dectin-2.

The C-type lectins dectin-1 and dectin-2 contribute to innate immunity against microbial pathogens by recognizing their foreign glycan structures. These receptors are promising targets for vaccine development and cancer immunotherapy. However, currently available agonists are heterogeneous glycoconjugates and polysaccharides from natural sources. Herein, we designed and synthesized the first chemically defined ligands for dectin-1 and dectin-2. They comprised glycopolypeptides bearing mono-, di-, and trisaccharides and were built through polymerization of glycosylated N-carboxyanhydrides. Through this approach, we achieved glycopolypeptides with high molecular weights and low dispersities. We identified structures that elicit a pro-inflammatory response through dectin-1 or dectin-2 in antigen-presenting cells. With their native proteinaceous backbones and natural glycosidic linkages, these agonists are attractive for translational applications.

Chemically tunable mucin chimeras assembled on living cells.

Mucins are a family of secreted and transmembrane glycoproteins characterized by a massive domain of dense O-glycosylation on serine and threonine residues. Mucins are intimately involved in immunity and cancer, yet elucidation of the biological roles of their glycodomains has been complicated by their massive size, domain polymorphisms, and variable glycosylation patterns. Here we developed a synthetic route to a library of compositionally defined, high-molecular weight, dual end-functionalized mucin glycodomain constructs via N-carboxyanhydride polymerization. These glycopolypeptides are the first synthetic analogs to our knowledge to feature the native α-GalNAc linkage to serine with molecular weights similar to native mucins, solving a nearly 50-year synthetic challenge. Physical characterization of the mimics revealed insights into the structure and properties of mucins. The synthetic glycodomains were end-functionalized with an optical probe and a tetrazine moiety, which allowed site-specific bioorthogonal conjugation to an engineered membrane protein on live mammalian cells. This strategy in protein engineering will open avenues to explore the biological roles of cell surface mucins.

Quantitative Side-Chain Modifications of Methionine-Containing Elastin-Like Polypeptides as a Versatile Tool to Tune Their Properties.

Tuning the lower critical solution temperature (LCST) of temperature-responsive recombinant elastin-like polypeptides has usually been achieved by designing different protein sequences, in terms of amino acid composition and length, implying tedious molecular cloning steps. In the present work, we have explored the chemoselective alkylation of methionine as an easy means to modify elastin repeat side chains and easily modulate the LCST of the polypeptides. Such a versatile synthetic method shall practically be exploited to modulate any properties of recombinant polymers.

Reinventing Cell Penetrating Peptides Using Glycosylated Methionine Sulfonium Ion Sequences.

Cell penetrating peptides (CPPs) are intriguing molecules that have received much attention, both in terms of mechanistic analysis and as transporters for intracellular therapeutic delivery. Most CPPs contain an abundance of cationic charged residues, typically arginine, where the amino acid compositions, rather than specific sequences, tend to determine their ability to enter cells. Hydrophobic residues are often added to cationic sequences to create efficient CPPs, but typically at the penalty of increased cytotoxicity. Here, we examined polypeptides containing glycosylated, cationic derivatives of methionine, where we found these hydrophilic polypeptides to be surprisingly effective as CPPs and to also possess low cytotoxicity. X-ray analysis of how these new polypeptides interact with lipid membranes revealed that the incorporation of sterically demanding hydrophilic cationic groups in polypeptides is an unprecedented new concept for design of potent CPPs.

Multimodal switching of conformation and solubility in homocysteine derived polypeptides.

We report the design and synthesis of poly(S-alkyl-L-homocysteine)s, which were found to be a new class of readily prepared, multiresponsive polymers that possess the unprecedented ability to respond in different ways to different stimuli, either through a change in chain conformation or in water solubility. The responsive properties of these materials are also effected under mild conditions and are completely reversible for all pathways. The key components of these polymers are the incorporation of water solubilizing alkyl functional groups that are integrated with precisely positioned, multiresponsive thioether linkages. This promising system allows multimodal switching of polypeptide properties to obtain desirable features, such as coupled responses to multiple external inputs.

Enzyme-triggered cargo release from methionine sulfoxide containing copolypeptide vesicles.

We have developed a facile, scalable method for preparation of enzyme-responsive copolypeptide vesicles that requires no protecting groups or expensive components. We designed amphiphilic copolypeptides containing segments of water-soluble methionine sulfoxide, M(O), residues that were prepared by synthesis of a fully hydrophobic precursor diblock copolypeptide, poly(l-methionine)65-b-poly(L-leucine0.5-stat-L-phenylalanine0.5)20, M65(L0.5/F0.5)20, followed by its direct oxidation in water to give the amphiphilic M(O) derivative, M(O)65(L0.5/F0.5)20. Assembly of M(O)65(L0.5/F0.5)20 in water gave vesicles with average diameters of a few micrometers that could then be extruded to nanoscale diameters. The M(O) segments in the vesicles were found to be substrates for reductase enzymes, which regenerated hydrophobic M segments and resulted in a change in supramolecular morphology that caused vesicle disruption and release of cargos.