Jessica R. Kramer

Glycopolypeptides via Living Polymerization of Glycosylated-l-lysine N-Carboxyanhydrides

The preparation of new glycosylated-L-lysine-N-carboxyanhydride (glyco-K NCA) monomers is described. These monomers employ C-linked sugars and amide linkages to lysine for improved stability without sacrificing biochemical properties. Three glyco-K NCAs were synthesized, purified, and found to undergo living polymerization using transition metal initiation. These are the first living polymerizations of glycosylated NCAs and were used to prepare well-defined, high molecular weight glycopolypeptides and block and statistical glycocopolypeptides. This methodology solves many long-standing problems in the direct synthesis of glycopolypeptides from N-carboxyanhydrides relating to monomer synthesis, purification, and polymerization and gives polypeptides with 100% glycosylation. These long chain glycopolypeptides have potential to be good mimics of natural high molecular weight glycoproteins.

General Method for Purification of α-Amino acid-N-carboxyanhydrides Using Flash Chromatography

We describe the application of flash column chromatography on silica gel as a rapid and general method to obtain pure α-amino acid-N-carboxyanhydride (NCA) monomers, the widely used precursors for the synthesis of polypeptides, without the need for recrystallization. This technique was effective at removing all common impurities from NCAs and was found to work for a variety of NCAs, including those synthesized using different routes, as well as those bearing either hydrophilic or hydrophobic side chains. All chromatographed NCAs required no further purification and could be used directly to form high molecular weight polypeptides. This procedure is especially useful for the preparation of highly functional and low melting NCAs that are difficult to crystallize and, consequently, to polymerize. This method solves many long-standing problems in NCA purification and provides rapid access to NCAs that were previously inaccessible in satisfactory quality for controlled polymerization. This method is also practical in that it requires less time than recrystallization and often gives NCAs in improved yields.

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.

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.

Reversible chemoselective tagging and functionalization of methionine containing peptides

Reagents were developed to allow chemoselective tagging of methionine residues in peptides and polypeptides, subsequent bioorthogonal functionalization of the tags, and cleavage of the tags when desired. This methodology can be used for triggered release of therapeutic peptides, or release of tagged protein digests from affinity columns.

Recent advances in glycopolypeptide synthesis

Glycosylated peptides and proteins are ubiquitous in nature and display a wide range of biological functions including mediation of recognition events, protection from proteases, and lubrication in eyes and joints. Similarly, synthetic glycopolypeptides are also expected to show great potential as biomedical materials (e.g. scaffolds for tissue repair and drug carriers), as well as serve as valuable tools for probing carbohydrate–protein interactions. Although block copolypeptides and other complex polypeptide architectures have been known for some time, the synthesis of complex and well-defined glycopolypeptide materials, until recently, has been challenging. This article reviews the many advances and accomplishments made in the past few years toward development of strategies and methods for the preparation of synthetic glycopolypeptides via ring opening polymerization.

Glycopolypeptide conformations in bioactive block copolymer assemblies influence their nanoscale morphology

We describe the preparation and assembly of glycosylated amphiphilic diblock copolypeptides, where the hydrophilic glycosylated segments adopt either α-helical or disordered conformations. In this study, glycosylated amphiphilic diblock copolypeptides were prepared using poly(L-leucine), poly(L), as the hydrophobic segment, and poly(α-D-galactopyranosyl-L-lysine), poly(α-gal-K), or poly(α-D-galactopyranosyl-L-cysteine sulfone), poly(α-gal-CO2), as the hydrophilic segment. The poly(α-gal-K) and poly(α-gal-CO2) segments are known to be fully α-helical (>90% at 20 °C) and fully disordered in water, respectively. We found that block copolypeptides containing galactosylated hydrophilic segments of either α-helical or disordered conformation give different assembly morphologies, where the disordered glycopolypeptide segments favor vesicle formation and also present sugar residues that can bind to biological targets.

Chemically tunable mucin chimeras assembled on living cells

Significance Mucins are a diverse and heterogeneous family of glycoproteins that have been implicated in immunity and cancer. This work establishes a rapid and scalable route to synthetic mucin constructs with precisely defined glycan densities and chain lengths. Physical characterization indicates that the constructs both chemically and structurally emulate biological mucins and that dense mucin-type O-glycosylation imparts a remarkable degree of rigidity to the peptide backbone. Dual end-functionalized mucins were covalently conjugated to an engineered membrane protein on live mammalian cells. This strategy allows systematic variation in display of cell surface mucins with numerous applications in understanding the many biological roles of this unusual glycoprotein family. 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.

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.

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.