Lysosome-targeting chimaeras for degradation of extracellular proteins.

The majority of therapies that target individual proteins rely on specific activity-modulating interactions with the target protein-for example, enzyme inhibition or ligand blocking. However, several major classes of therapeutically relevant proteins have unknown or inaccessible activity profiles and so cannot be targeted by such strategies. Protein-degradation platforms such as proteolysis-targeting chimaeras (PROTACs)1,2 and others (for example, dTAGs3, Trim-Away4, chaperone-mediated autophagy targeting5 and SNIPERs6) have been developed for proteins that are typically difficult to target; however, these methods involve the manipulation of intracellular protein degradation machinery and are therefore fundamentally limited to proteins that contain cytosolic domains to which ligands can bind and recruit the requisite cellular components. Extracellular and membrane-associated proteins-the products of 40% of all protein-encoding genes7-are key agents in cancer, ageing-related diseases and autoimmune disorders8, and so a general strategy to selectively degrade these proteins has the potential to improve human health. Here we establish the targeted degradation of extracellular and membrane-associated proteins using conjugates that bind both a cell-surface lysosome-shuttling receptor and the extracellular domain of a target protein. These initial lysosome-targeting chimaeras, which we term LYTACs, consist of a small molecule or antibody fused to chemically synthesized glycopeptide ligands that are agonists of the cation-independent mannose-6-phosphate receptor (CI-M6PR). We use LYTACs to develop a CRISPR interference screen that reveals the biochemical pathway for CI-M6PR-mediated cargo internalization in cell lines, and uncover the exocyst complex as a previously unidentified-but essential-component of this pathway. We demonstrate the scope of this platform through the degradation of therapeutically relevant proteins, including apolipoprotein E4, epidermal growth factor receptor, CD71 and programmed death-ligand 1. Our results establish a modular strategy for directing secreted and membrane proteins for lysosomal degradation, with broad implications for biochemical research and for therapeutics.

Total Synthesis of a PSGL-1 Glycopeptide Analogue for Targeted Inhibition of P-Selectin.

The high affinity interaction between P-selectin glycoprotein ligand-1 (PSGL-1) and P-selectin is mediated by a multimotif glycosulfopeptide (GSP) recognition domain consisting of clustered tyrosine sulfates and a Core 2 O-glycan terminated with sialyl LewisX (C2-O-sLeX). These distinct GSP motifs are much more common than previously appreciated within a wide variety of functionally important domains involved in protein-protein interactions. However, despite the potential of GSPs to serve as tools for fundamental studies and prospects for drug discovery, their utility has been limited by the absence of chemical schemes for synthesis on scale. Herein, we report the total synthesis of GSnP-6, an analogue of the N-terminal domain of PSGL-1, and potent inhibitor of P-selectin. An efficient, scalable, hydrogenolysis-free synthesis of C2-O-sLeX-Thr-COOH was identified by both convergent and orthogonal one-pot assembly, which afforded this crucial building block, ready for direct use in solid phase peptide synthesis (SPPS). C2-O-sLeX-Thr-COOH was synthesized in 10 steps with an overall yield of 23% from the 4-O,5-N oxazolidinone thiosialoside donor. This synthesis represents an 80-fold improvement in reaction yield as compared to prior reports, achieving the first gram scale synthesis of SPPS ready C2-O-sLeX-Thr-COOH and enabling the scalable synthesis of GSnP-6 for preclinical evaluation. Significantly, we established that GSnP-6 displays dose-dependent inhibition of venous thrombosis in vivo and inhibits vaso-occlusive events in a human sickle cell disease equivalent microvasculature-on-a-chip system. The insights gained in formulating this design strategy can be broadly applied to the synthesis of a wide variety of biologically important oligosaccharides and O-glycan bearing glycopeptides.

Chemical synthesis and functional evaluation of glycopeptides and glycoproteins containing rare glycosyl amino acid linkages.

Covering: 1987 to 2023Naturally existing glycoproteins through post-translational protein glycosylation are highly heterogeneous, which not only impedes the structure-function studies, but also hinders the development of their potential medical usage. Chemical synthesis represents one of the most powerful tools to provide the structurally well-defined glycoforms. Being the key step of glycoprotein synthesis, glycosylation usually takes place at serine, threonine, and asparagine residues, leading to the predominant formation of the O- and N-glycans, respectively. However, other amino acid residues containing oxygen, nitrogen, sulfur, and nucleophilic carbon atoms have also been found to be glycosylated. These diverse glycoprotein linkages, occurring from microorganisms to plants and animals, play also pivotal biological roles, such as in cell-cell recognition and communication. The availability of these homogenous rare glycopeptides and glycoproteins can help decipher the glyco-code for developing therapeutic agents. This review highlights the chemical approaches for assembly of the functional glycopeptides and glycoproteins bearing these "rare" carbohydrate-amino acid linkages between saccharide and canonical amino acid residues and their derivatives.

HPLC-Based Automated Synthesis and Purification of Carbohydrates.

Reported herein is a new HPLC-based automated synthesizer (HPLC-A) capable of a temperature-controlled synthesis and purification of carbohydrates. The developed platform allows to perform various protecting group manipulations as well as the synthesis of O- and N-glycosides. A fully automated synthesis and purification was showcased in application to different carbohydrate derivatives including glycosides, oligosaccharides, glycopeptides, glycolipids, and nucleosides.

Supramolecular materials constructed from synthetic glycopeptides via aqueous self-assembly and their bioapplications in immunotherapy.

Synthetic glycopeptides capable of self-assembly in aqueous environments form a range of supramolecular nanostructures, such as nanoparticles and nanofibers, owing to their amphiphilic nature and the diverse structures of the saccharides introduced. These glycopeptide-based supramolecular materials are promising for immunotherapy applications because of their biocompatibility and multivalent saccharide display, which enhances lectin-saccharide interactions. This review highlights recent advances in the molecular design of synthetic glycopeptide-based supramolecular materials and their use as immunomodulatory agents.

Development of Glycosylation-Modified DPPA-1 Compounds as Innovative PD-1/PD-L1 Blockers: Design, Synthesis, and Biological Evaluation.

In the context of peptide drug development, glycosylation plays a pivotal role. Accordingly, L-type peptides were synthesized predicated upon the PD-1/PD-L1 blocker DPPA-1. Subsequent glycosylation resulted in the production of two distinct glycopeptides, D-glu-LPPA-1 and D-gal-LPPA-1, by using D-glucose (D-glu) and D-galactose (D-gal), respectively, during glycosylation. Both glycopeptides significantly inhibited the interaction between PD-1 and PD-L1, and the measured half maximal inhibitory concentrations (IC50s) were 75.5 µM and 101.9 µM for D-glu-LPPA-1 and D-gal-LPPA-1, respectively. Furthermore, D-gal-LPPA-1 displayed a pronounced ability to restore T-cell functionality. In an MC38 tumor-bearing mouse model, D-gal-LPPA-1 demonstrated a significant inhibitory effect. Notably, D-gal-LPPA-1 substantially augmented the abundance and functionality of CD8+ T cells in the tumor microenvironment. Additionally, in the lymph nodes and spleens, D-gal-LPPA-1 significantly increased the proportion of CD8+ T cells secreting interferon-gamma (IFN-γ). These strong findings position D-gal-LPPA-1 as a potent enhancer of the antitumor immune response in MC38 tumor-bearing mice, underscoring its potential as a formidable PD-1/PD-L1 blocking agent.

Solid-Phase-Supported Chemoenzymatic Synthesis and Analysis of Chondroitin Sulfate Proteoglycan Glycopeptides.

Proteoglycans (PGs), consisting of glycosaminoglycans (GAGs) linked with the core protein through a tetrasaccharide linkage region, play roles in many important biological events. The chemical synthesis of PG glycopeptides is extremely challenging. In this work, the enzymes required for synthesis of chondroitin sulfate (CS) PG (CSPG) have been expressed and the suitable sequence of enzymatic reactions has been established. To expedite CSPG synthesis, the peptide acceptor was immobilized on solid phase and the glycan units were directly installed enzymatically onto the peptide. Subsequent enzymatic chain elongation and sulfation led to the successful synthesis of CSPG glycopeptides. The CS dodecasaccharide glycopeptide was the longest homogeneous CS glycopeptide synthesized to date. The enzymatic synthesis was much more efficient than the chemical synthesis of the corresponding CS glycopeptides, which could reduce the total number of synthetic steps by 80 %. The structures of the CS glycopeptides were confirmed by mass spectrometry analysis and NMR studies. In addition, the interactions between the CS glycopeptides and cathepsin G were studied. The sulfation of glycan chain was found to be important for binding with cathepsin G. This efficient chemoenzymatic strategy opens new avenues to investigate the structures and functions of PGs.

Integrated Chemoenzymatic Approach to Streamline the Assembly of Complex Glycopeptides in the Liquid Phase.

Glycosylation of proteins is a complicated post-translational modification. Despite the significant progress in glycoproteomics, accurate functions of glycoproteins are still ambiguous owing to the difficulty in obtaining homogeneous glycopeptides or glycoproteins. Here, we describe a streamlined chemoenzymatic method to prepare complex glycopeptides by integrating hydrophobic tag-supported chemical synthesis and enzymatic glycosylations. The hydrophobic tag is utilized to facilitate peptide chain elongation in the liquid phase and expeditious product separation. After removal of the tag, a series of glycans are installed on the peptides via efficient glycosyltransferase-catalyzed reactions. The general applicability and robustness of this approach are exemplified by efficient preparation of 16 well-defined SARS-CoV-2 O-glycopeptides, 4 complex MUC1 glycopeptides, and a 31-mer glycosylated glucagon-like peptide-1. Our developed approach will open up a new range of easy access to various complex glycopeptides of biological importance.

Total Synthesis of Mannopeptimycin ß via ß-Hydroxyenduracididine Ligation.

Nonribosomal peptide synthesis in bacteria has endowed cyclic peptides with fascinating structural complexity via incorporating nonproteinogenic amino acids. These bioactive cyclic peptides provide interesting structural motifs for exploring total synthesis and medicinal chemistry studies. Cyclic glycopeptide mannopeptimycins exhibit antibacterial activity against antibiotic-resistant Gram-positive pathogens and act as the lipid II binder to stop bacterial cell wall biosynthesis. Here, we report a strategy streamlining solution phase-solid phase synthesis and chemical ligation-mediated peptide cyclization for the total synthesis of mannopeptimycin ß.

Synthesis and In Vitro Characterization of Glycopeptide Drug Candidates Related to PACAP1-23.

The search for efficacious treatment of neurodegenerative and progressive neuroinflammatory diseases continues, as current therapies are unable to halt or reverse disease progression. PACAP represents one potential therapeutic that provides neuroprotection effects on neurons, and also modulates inflammatory responses and circulation within the brain. However, PACAP is a relatively long peptide hormone that is not trivial to synthesize. Based on previous observations that the shortened isoform PACAP1-23 is capable of inducing neuroprotection in vitro, we were inspired to synthesize shortened glycopeptide analogues of PACAP1-23. Herein, we report the synthesis and in vitro characterization of glycosylated PACAP1-23 analogues that interact strongly with the PAC1 and VPAC1 receptors, while showing reduced activity at the VPAC2 receptor.

Low Collision Energy Fragmentation in Structure-Specific Glycoproteomics Analysis.

Glycosylation is a major post-translational modification of proteins that regulates many biological processes including protein folding, structure stability, receptor activation, and immune responses. The glycans attached to proteins represent an important determinant of the protein interaction-specificity and maintain the 3D structure of proteins. Mass spectrometry (MS) is one of the most efficient tools used in the current studies of glycoproteins and structure of their glycoforms. Collision energy (CE) is a crucial instrument parameter that can be exploited to improve structural resolution because different linkages of glycan units show different stabilities under CID/HCD fragmentation. Here we report the utility of CE modulation for qualitative and quantitative analysis of site- and structure-specific glycoforms of proteins. Using CE modulation, we were able to break selectively specific glycan linkages on intact glycopeptides and get, to some degree, structure-specific mass spectrometric signals. Structure- and CE-specific oxonium ions provide sufficient information for the resolution of outer arm structure motifs with recognized biological functions. The complementary Y-ions, generated under optimized low CE (soft) conditions, provide additional structural information including features specific to the chitobiose core. This methodology of multiple CE fragmentation without merging spectral information can significantly improve confidence of glycopeptide identification and structural resolution by providing additional information to the established glycopeptide-search algorithms and tools.

Bifunctional Magnetic Supramolecular-Organic Framework: A Nanoprobe for Simultaneous Enrichment of Glycosylated and Phosphorylated Peptides.

Protein glycosylation and phosphorylation are two important protein post-translational modifications. Mass spectrometry (MS) has been proved to be a powerful technique in comprehensive characterization of protein glycosylation and phosphorylation; however, the complexity of biological matrices and weak ionization efficiency bring a big challenge. Capturing glycopeptides and phosphopeptides from complicated biological samples is indispensable before MS determinations. In this study, a bifunctional gallium ion immobilized magnetic pertriflated pillar[5]arene supramolecular-organic framework (magOTfP5SOF-Ga3+) was designed for the one-step simultaneous enrichment of glycopeptides and phosphopeptides. Thanks to the abundant sulfonic acid groups, the material owns strong hydrophilicity and leads to hydrophilic interaction chromatography for glycopeptides enrichment. Simultaneously, the high loading amount of gallium ion provides immobilized metal ion affinity for phosphopeptides enrichment. The established platform possesses quick magnetic response performance, high sensitivity (detection limits as low as 0.1 fmol and 0.05 fmol for glycopeptides and phosphopeptides, respectively), and good reusability. In addition, the method was applied to the determination of glycopeptides and phosphopeptides in clinical specimens, cell lysates, and mouse liver tissue samples, demonstrating its highly sensitive and specific glycoproteomics and phosphorproteomics analysis in complex biosamples.

On-Chip Neo-Glycopeptide Synthesis for Multivalent Glycan Presentation.

Single glycan-protein interactions are often weak, such that glycan binding partners commonly utilize multiple, spatially defined binding sites to enhance binding avidity and specificity. Current array technologies usually neglect defined multivalent display. Laser-based array synthesis technology allows for flexible and rapid on-surface synthesis of different peptides. By combining this technique with click chemistry, neo-glycopeptides were produced directly on a functionalized glass slide in the microarray format. Density and spatial distribution of carbohydrates can be tuned, resulting in well-defined glycan structures for multivalent display. The two lectins concanavalin A and langerin were probed with different glycans on multivalent scaffolds, revealing strong spacing-, density-, and ligand-dependent binding. In addition, we could also measure the surface dissociation constant. This approach allows for a rapid generation, screening, and optimization of a multitude of multivalent scaffolds for glycan binding.

Machine-Driven Chemoenzymatic Synthesis of Glycopeptide.

Historically, researchers have put considerable effort into developing automation systems to prepare natural biopolymers such as peptides and oligonucleotides. The availability of such mature systems has significantly advanced the development of natural science. Over the past twenty years, breakthroughs in automated synthesis of oligosaccharides have also been achieved. A machine-driven platform for glycopeptide synthesis by a reconstructed peptide synthesizer is described. The designed platform is based on the use of an amine-functionalized silica resin to facilitate the chemical synthesis of peptides in organic solvent as well as the enzymatic synthesis of glycan epitopes in the aqueous phase in a single reaction vessel. Both syntheses were performed by a peptide synthesizer in a semiautomated manner.

A Chemoenzymatic Approach to the Synthesis of Glycopeptide Antibiotic Analogues.

Glycopeptide antibiotics (GPAs) are important antibiotics that are highly challenging to synthesise due to their unique and heavily crosslinked structure. Given this, the synthetic production and diversification of this key compound class remains impractical. Furthermore, the possibility of biosynthetic reengineering of GPAs is not yet feasible since the selectivity of the biosynthetic crosslinking enzymes for altered substrates is largely unknown. We show that combining peptide synthesis with enzymatic cyclisation enables the formation of novel examples of GPAs and provides an indication of the utility of these crucial enzymes. By accessing the biosynthetic process in vitro, we identified peptide modifications that are enzymatically tolerated and can also reveal the mechanistic basis for substrate intolerance where present. Using this approach, we next specifically activated modified residues within GPAs for functionalisation at previously inaccessible positions, thereby offering the possibility of late-stage chemical functionalisation after GPA cyclisation is complete.

Glycosylated Cell-Penetrating Peptides (GCPPs).

The cell membrane regulates the exchange of molecules and information with the external environment. However, this control barrier hinders the delivery of exogenous bioactive molecules that can be applied to correct cellular malfunctions. Therefore, the traffic of macromolecules across the cell membrane represents a great challenge for the development of the next generation of therapies and diagnostic methods. Cell-penetrating peptides are short peptide sequences capable of delivering a broad range of biomacromolecules across the cellular membrane. However, penetrating peptides still suffer from limitations, mainly related to their lack of specificity and potential toxicity. Glycosylation has emerged as a potential promising strategy for the biological improvement of synthetic materials. In this work we have developed a new convergent strategy for the synthesis of penetrating peptides functionalized with glycan residues by an oxime bond connection. The uptake efficiency and intracellular distribution of these glycopeptides have been systematically characterized by means of flow cytometry and confocal microscopy and in zebrafish animal models. The incorporation of these glycan residues into the peptide structure influenced the internalization efficiency and cellular toxicity of the resulting glycopeptide hybrids in the different cell lines tested. The results reported herein highlight the potential of the glycosylation of penetrating peptides to modulate their activity.

Glycopeptides by Linch-Pin C-H Activations for Peptide-Carbohydrate Conjugation by Manganese(I)-Catalysis.

Late-stage C-H glycosylations of structurally complex amino acids and peptides were accomplished by means of racemization-free manganese(I)-catalyzed C-H activation. Thus, glycosylative modifications proved to be viable by a linch-pin approach, featuring chemo- and site-selective C-H transformations. The peptide-saccharide conjugation provided modular access to structurally complex glycopeptides, likewise enabling the assembly of fluorescent-labelled glycopeptides.

A Tripeptide Approach to the Solid-Phase Synthesis of Peptide Thioacids and N-Glycopeptides.

A general and robust method for the incorporation of aspartates with a thioacid side chain into peptides has been developed. Pseudoproline tripeptides served as building blocks for the efficient fluorenylmethyloxycarbonyl (Fmoc) solid-phase synthesis of thioacid-containing peptides. These peptides were readily converted to complex N-glycopeptides by using a fast and chemoselective one-pot deprotection/ligation procedure. Furthermore, a novel side reaction that can lead to site-selective peptide cleavage using thioacids (CUT) was discovered and studied in detail.

Synthesis and evaluation of biological activity for dual-acting antibiotics on the basis of azithromycin and glycopeptides.

One of the promising directions of the combined approach is the design of dual-acting antibiotics - heterodimeric structures on the basis of antimicrobial agents of different classes. In this study a novel series of azithromycin-glycopeptide conjugates were designed and synthesized. The structures of the obtained compounds were confirmed using NMR spectroscopy and mass spectrometry data including MS/MS analysis. All novel hybrid antibiotics were found to be either as active as azithromycin and vancomycin against Gram-positive bacterial strains or have superior activity in comparison with their parent antibiotics. One compound, eremomycin-azithromycin conjugate 16, demonstrated moderate activity against Enterococcus faecium and Enterococcus faecalis strains resistant to vancomycin, and equal to vancomycin's activity for the treatment of mice with Staphylococcus aureus sepsis.

Glycoconjugate synthesis using chemoselective ligation.

Chemoselective ligation of carbohydrates and polypeptides was achieved using an adipic acid dihydrazide cross-linker. The reducing end of a carbohydrate is efficiently attached to peptides in two steps, constructing a glycoconjugate in high yield and with high regioselectivity, enabling the production of homogeneous glycoconjugates.