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Chemical synthesis can generate homogeneous glycoproteins with well-defined and modifiable glycan structures at designated sites. The precision and flexibility of the chemical synthetic approach provide a solution to the heterogeneity problem of glycopeptides/glycoproteins obtained through biological approaches. In this study, we reported that the conserved N -glycosylation sequon (Asn-Xaa-Ser/Thr) of glycoproteins can serve as a general site for performing Ser/Thr ligation to achieve N -linked glycoprotein synthesis. We developed an N + 2 strategy to prepare the corresponding glycopeptide salicylaldehyde esters for Ser/Thr ligation and demonstrated that Ser/Thr ligation at the sequon was not affected by the steric hindrance brought about by the large-sized glycan structures. The effectiveness of this strategy was showcased by the total synthesis of the glycosylated receptor-binding domain (RBD) of the SARS-CoV-2 spike protein.

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.

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.

Interactions between the tumor-associated carbohydrate antigens of Mucin 1 (MUC1) and the carbohydrate-binding proteins, lectins, often lead to the creation of a pro-tumor microenvironment favoring tumor initiation, progression, metastasis, and immune evasion. Macrophage galactose binding lectin (MGL) is a C-type lectin receptor found on antigen-presenting cells that facilitates the uptake of carbohydrate antigens for antigen presentation, modulating the immune response homeostasis, autoimmunity, and cancer. Considering the crucial role of tumor-associated forms of MUC1 and MGL in tumor immunology, a thorough understanding of their binding interaction is essential for it to be exploited for cancer vaccine strategies. The synthesis of MUC1 glycopeptide models carrying a single or multiple Tn and/or sialyl-Tn antigen(s) is described. A novel approach for the sialyl-Tn threonine building block suitable for the solid phase peptide synthesis was developed. The thermodynamic profile of the binding interaction between the human MGL and MUC1 glycopeptide models was analyzed using isothermal titration calorimetry. The measured dissociation constants for the sialyl-Tn-bearing peptide epitopes were consistently lower compared to the Tn antigen and ranged from 10 μM for mono- to 1 μM for triglycosylated MUC1 peptide, respectively. All studied interactions, regardless of the glycan's site of attachment or density, exhibited enthalpy-driven thermodynamics., (© 2024 Wiley-VCH GmbH.)

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., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

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., (© 2024 Wiley-VCH GmbH.)

Glycosylation is one of the most ubiquitous post-translational modifications. It affects the structure and function of peptides/proteins and consequently has a significant impact on various biological events. However, the structural complexity and heterogeneity of glycopeptides/proteins caused by the diversity of glycan structures and glycosylation sites complicates the detailed elucidation of glycan function and hampers their clinical applications. To address these challenges, chemical and/or enzyme-assisted synthesis methods have been developed to realize glycopeptides/proteins with well-defined glycan morphologies. In particular, N-glycans are expected to be useful for improving the solubility, in vivo half-life and aggregation of bioactive peptides/proteins that have had limited clinical applications so far due to their short duration of action in the blood and unsuitable physicochemical properties. Chemical glycosylation performed in a post-synthetic procedure can be used to facilitate the development of glycopeptide/protein analogues or mimetics that are superior to the original molecules in terms of physicochemical and pharmacokinetic properties. N-glycans are used to modify targets because they are highly biodegradable and biocompatible and have structures that already exist in the human body. On the practical side, from a quality control perspective, close attention should be paid to their structural homogeneity when they are to be applied to pharmaceuticals., (© 2024 Wiley-VCH GmbH.)

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 Lewis X (C2- O -sLe X ). 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 -sLe X -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 -sLe X -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 -sLe X -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.

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 D PPA-1. Subsequent glycosylation resulted in the production of two distinct glycopeptides, D-glu- L PPA-1 and D-gal- L PPA-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 (IC 50s ) 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.

A technically straightforward total synthesis of a new class of vancomycin analogues of reduced synthetic complexity was developed that provided tetrachlorovancomycin ( 1 , LLS = 15 steps, 15% overall yield) and its precursor aglycon 29 (nearly 20% overall yield). The class retains all the intricate vancomycin structural features that contribute to its target binding affinity and selectivity, maintains the antimicrobial activity of vancomycin, and achieves the simplification by an unusual addition, not removal, of benign substituents to the core structure. The modification, accomplished by addition of two aryl chloride substituents to provide 1 , permitted a streamlined total synthesis of the new glycopeptide antibiotic class by removing the challenges associated with CD and DE ring system atropisomer stereochemical control. This also enabled their simultaneous and further-activated S N Ar macrocyclizations that establish the tricyclic skeleton of 1 . Key elements of the approach include catalyst-controlled diastereoselective formation of the AB biaryl axis of chirality (>30:1 dr), an essentially instantaneous macrolactamization of the AB ring system free of competitive epimerization (>30:1 dr), racemization free coupling of the E ring tetrapeptide, room temperature simultaneous CD and DE ring system cyclizations, a highly refined 4-step conversion of the cyclization product to the aglycon, and a protecting-group-free one-pot enzymatic glycosylation for disaccharide introduction. In addition to the antimicrobial evaluation of tetrachlorovancomycin ( 1 ), the preparation of key peripherally modified derivatives, which introduce independent and synergistic mechanisms of action, revealed their exceptional antimicrobial potency and provide the foundation for future use of this new class of synthetic glycopeptide analogues.

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.

With the aging problems increasing, the discovery of anti-aging compounds has become a popular research direction. Accumulation of free radicals and the consequent oxidative stress are the chief culprit of aging. Given this, cultured crocodile meat peptides-Maillard reaction product (CMP-MRP) with remarkable antioxidant activity was obtained via Maillard reaction of cultured crocodile meat hydrolysates and xylose. The antioxidant activity in vitro and anti-aging activity in vivo of CMP-MRP were investigated. Results indicated that the lifespan and the athletic ability of Drosophila were significantly improved after the administration of CMP-MRP in natural aging, H 2 O 2 - and paraquat-induced models. Furthermore, the antioxidant enzyme activities of Drosophila treated with CMP-MRP were enhanced while the levels of malondialdehyde (MDA) and protein carbonyl (PCO) were reduced in three Drosophila models. With the supplement of 5 mg/mL CMP-MRP in natural aging Drosophila model, the maximum lifespan increased from 61 days to 73 days, athletic ability raised by 95.45%, MDA and PCO reduced by 52.72% and 47.43%, respectively. Taken together, CMP-MRP exhibited outstanding antioxidant and anti-aging capacities in Drosophila models, suggesting that CMP-MRP possesses great potential in the health food and biomedicine fields as a food-derived anti-aging agent., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Mucin-type O-glycosylation is among the most complex post-translational modifications. Despite mediating many physiological processes, O-glycosylation remains understudied compared to other modifications, simply because the right analytical tools are lacking. In particular, analysis of intact O-glycopeptides by mass spectrometry is challenging for several reasons; O-glycosylation lacks a consensus motif, glycopeptides have low charge density which impairs ETD fragmentation, and the glycan structures modifying the peptides are unpredictable. Recently, we introduced chemically modified monosaccharide analogues that allowed selective tracking and characterization of mucin-type O-glycans after bioorthogonal derivatization with biotin-based enrichment handles. In doing so, we realized that the chemical modifications used in these studies have additional benefits that allow for improved analysis by tandem mass spectrometry. In this work, we built on this discovery by generating a series of new GalNAc analogue glycopeptides. We characterized the mass spectrometric signatures of these modified glycopeptides and their signature residues left by bioorthogonal reporter reagents. Our data indicate that chemical methods for glycopeptide profiling offer opportunities to optimize attributes such as increased charge state, higher charge density, and predictable fragmentation behavior.

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 β.

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 PACAP 1-23 is capable of inducing neuroprotection in vitro, we were inspired to synthesize shortened glycopeptide analogues of PACAP 1-23 . Herein, we report the synthesis and in vitro characterization of glycosylated PACAP 1-23 analogues that interact strongly with the PAC1 and VPAC1 receptors, while showing reduced activity at the VPAC2 receptor.

C-Glycosyl peptides/proteins are metabolically stable mimics of the native glycopeptides/proteins bearing O/N-glycosidic linkages, and are thus of great therapeutical potential. Herein, we disclose a protocol for the syntheses of vinyl C-glycosyl amino acids and peptides, employing a nickel-catalyzed reductive hydroglycosylation reaction of alkyne derivatives of amino acids and peptides with common glycosyl bromides. It accommodates a wide scope of the coupling partners, including complex oligosaccharide and peptide substrates. The resultant vinyl C-glycosyl amino acids and peptides, which bear common O/N-protecting groups, are amenable to further transformations, including elongation of the peptide and saccharide chains., (© 2021. The Author(s).)

Glycosylation plays important roles in SARS-CoV-2 infection. We describe here a facile chemoenzymatic synthesis of core-fucosylated N-glycopeptides derived from the SARS-CoV-2 Spike protein and their binding with glycan-dependent neutralizing antibody S309 and human lectin CLEC4G. The synthetic glycopeptides provide tools for further functional characterization of viral glycosylation.

Site-selective acylations of α-and β-hydroxyamides in complex polyols are described. The combination of a pyridine aldoxime ester and Zn(OTf) 2 facilitates the acylation of two types of N -glycolyl disaccharides, namely, Gal-GlcNGc and Neu5Gc-Gal, both of which are partial structures of polysaccharides responsible for biological actions, with highly site-selective modifications achieved. Furthermore, biotinylation, one of the most important techniques in chemical biology, is used to site-selectively acylate the β-hydroxyl group in a glycopeptide.

Proteoglycans have important biological activities. To improve the overall synthetic efficiency, a new chemoenzymatic route has been established for the proteoglycan linkage region bearing a galactose-xylose disaccharide. The xylosylated glycopeptides were synthesized via solid phase synthesis, which was followed by the addition of the galactose unit by the galactosyl transferase β4GalT7. This work leads to a better understanding of the acceptor preference of β4GalT7 and opens the door for expeditious synthesis of the proteoglycan linkage region.

Capitalizing on a recently reported iminosugar-based aza-crown (ISAC) accessed by a double Staudinger azaWittig coupling reaction, we have expanded the structural diversity of this new family of sweet cyclam analogs. Replacement of the two secondary amines linking the iminosugar units by two amide bonds obtained a cyclodimerization by with BOP and DIPEA led to a macrocycle that did not demonstrate efficient Zn 2+ chelation unlike the parent ISAC. Introduction of two pyrene moieties on the secondary amines of the parent ISAC yielded a new fluoroionophore that selectively binds Hg 2+ in methanol., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

It is largely unknown how post-translational protein modifications, including glycosylation, impacts recognition of self and non-self T cell epitopes presented by HLA molecules. Data in the literature indicate that O - and N -linked glycosylation can survive epitope processing and influence antigen presentation and T cell recognition. In this perspective, we hypothesize that glycosylation of viral proteins and processed epitopes contribute to the T cell response to HIV. Although there is some evidence for T cell responses to glycosylated epitopes (glyco-epitopes) during viral infections in the literature, this aspect has been largely neglected for HIV. To explore the role of glyco-epitope specific T cell responses in HIV infection we conducted in silico and ex vivo immune studies in individuals with chronic HIV infection. We found that in silico viral protein segments with potentially glycosylable epitopes were less frequently targeted by T cells. Ex vivo synthetically added glycosylation moieties generally masked T cell recognition of HIV derived peptides. Nonetheless, in some cases, addition of simple glycosylation moieties produced neo-epitopes that were recognized by T cells from HIV infected individuals. Herein, we discuss the potential importance of these observations and compare limitations of the employed technology with new methodologies that may have the potential to provide a more accurate assessment of glyco-epitope specific T cell immunity. Overall, this perspective is aimed to support future research on T cells recognizing glycosylated epitopes in order to expand our understanding on how glycosylation of viral proteins could alter host T cell immunity against viral infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Olvera, Cedeño, Llano, Mothe, Sanchez, Arsequell and Brander.)

The synthesis of MUC1 glycopeptides bearing modified tumor-associated carbohydrate antigens (TACAs) represents an effective strategy to develop potential antitumor vaccines that trigger strong immune response. In this context, we present herein the multistep synthesis of the triazole glycosyl amino acid Neu5Ac-α/β2-triazole-6-βGalNAc-ThrOH 1 as STn antigen analog, along with its assembly on the corresponding MUC1 peptide to give NAcProAsp [Neu5Acα/β2-triazole-6-βGalNAc]ThrArgProGlyOH 2. Despite interacting differently with SM3 monoclonal antibody, as shown by molecular dynamic simulations, this unnatural triazole glycopeptide may represent a promising candidate for cancer immunotherapy., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

We report herein an efficient chemical synthesis of homogeneous human E-cadherin N-linked glycopeptides consisting of a heptapeptide sequence adjacent to the Asn-633 N-glycosylation site with representative N-glycan structures, including a conserved trisaccharide, a core-fucosylated tetrasaccharide, and a complex-type biantennary octasaccharide. The key steps are a chemoselective on-resin aspartylation using a pseudoproline-containing peptide and stereoselective glycosylation using glycosyl fluororide as a donor. This synthetic strategy demonstrates potential utility in accessing a wide range of homogeneous N-linked glycopeptides for the examination of their biological function.

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., (© 2020 Wiley-VCH GmbH.)

Glycopeptide antibiotics (GPAs) are essential for the treatment of severe infectious diseases caused by Gram-positive bacteria. The emergence and spread of GPA resistance have propelled the search for more effective GPAs. Given their structural complexity, genetic intractability, and low titer, expansion of GPA chemical diversity using synthetic or medicinal chemistry remains challenging. Here we describe a synthetic biology platform, GPAHex (GPA Heterologous expression), which exploits the genes required for the specialized GPA building blocks, regulation, antibiotic transport, and resistance for the heterologous production of GPAs. Application of the GPAHex platform results in: (1) a 19-fold increase of corbomycin titer compared to the parental strain, (2) the discovery of a teicoplanin-class GPA from an Amycolatopsis isolate, and (3) the overproduction and characterization of a cryptic nonapeptide GPA. GPAHex provides a platform for GPA production and mining of uncharacterized GPAs and provides a blueprint for chassis design for other natural product classes.

Glycan recognition is typically studied using free glycans, but glycopeptide presentations represent more physiological conditions for glycoproteins. To facilitate studies of glycopeptide recognition, we developed Glyco-SPOT synthesis, which enables the parallel production of diverse glycopeptide libraries at microgram scales. The method uses a closed system for prolonged reactions required for coupling Fmoc-protected glycoamino acids, including O-, N-, and S-linked glycosides, and release conditions to prevent side reactions. To optimize reaction conditions and sample reaction progress, we devised a biopsy testing method. We demonstrate the efficient utilization of such microscale glycopeptide libraries to determine the specificity of glycan-recognizing antibodies (e.g., CTD110.6) using microarrays, enzyme specificity on-array and in-solution (e.g., ST6GalNAc1, GCNT1, and T-synthase), and binding kinetics using fluorescence polarization. We demonstrated that the glycosylation on these peptides can be expanded using glycosyltransferases both in-solution and on-array. This technology will promote the discovery of biological functions of peptide modifications by glycans., Competing Interests: Declaration of Interests The authors declare no conflicts of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Glycoproteins are produced by the post-translational modification process of proteins and they play an important role in mediating various biological processes. Our understanding towards biochemical functions of individual glycoproteins has been seriously hampered due to the heterogeneous expression of carbohydrate parts in glycoproteins. Despite the advancement in recombinant expression and chromatographic techniques, the isolation of pure glycoforms remains nearly impossible. To obtain homogenous glycoproteins, tremendous efforts hves been spent in developing various ligation and glycosylation techniques. This minireview discusses selected methods for the preparation and ligation of glycopeptides. The importance of the development of new chemical synthesis method for glycoproteins has also been discussed, which would be one of the next directions in this field., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Peptides are an important class of molecules with diverse biological activities. Many endogenous peptides, especially neuropeptides and peptide hormones, play critical roles in development and regulating homeostasis. Furthermore, as drug candidates their high receptor selectivity and potent binding leads to reduced off-target interactions and potential negative side effects. However, the therapeutic potential of peptides is severely hampered by their poor stability in vivo and low permeability across biological membranes. Several strategies have been successfully employed over the decades to address these concerns, and one of the most promising strategies is glycosylation. It has been demonstrated in numerous cases that glycosylation is an effective synthetic approach to improve the pharmacokinetic profiles and membrane permeability of peptides. The effects of glycosylation on peptide stability and peptide-membrane interactions in the context of blood-brain barrier penetration will be explored. Numerous examples of glycosylated analogues of endogenous peptides targeting class A and B G-protein coupled receptors (GPCRs) with an emphasis on O-linked glycopeptides will be reviewed. Notable examples of N-, S-, and C-linked glycopeptides will also be discussed. A small section is devoted to synthetic methods for the preparation of glycopeptides and requisite amino acid glycoside building blocks., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

A successful synthesis of O -sulfated syndecan-1-like (Q23-E120) glyco-polypeptide was accomplished. The synthesis features the integration of an O -sulfated carbohydrate-bearing glycopeptide cassette with efficient protein ligation strategies, overcoming the acid lability of carbohydrate sulfates as a major hurdle in solid-phase peptide synthesis. Crucial to the synthesis is the microwave-assisted Ag(I) ligation, which afforded the ligation product in improved overall yield. This O -sulfated syndecan-1 (Q23-E120) is the longest O -sulfated glyco-polypeptide prepared to date.

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., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

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, dTAGs 3 , Trim-Away 4 , chaperone-mediated autophagy targeting 5 and SNIPERs 6 ) 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 genes 7 -are key agents in cancer, ageing-related diseases and autoimmune disorders 8 , 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.

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., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

O-Glycopeptides derived from natural bioresources are an attractive material for a variety of purposes. Whey protein products are used as a human dietary supplement and in animal feed and are a readily available resource for the preparation of O-glycopeptides. The protein composition of bovine milk is well-studied, and many glycoproteins carrying N-glycans and O-glycans have been found in commercial whey protein products. In particular, κ-casein glycomacropeptide and lactophorin, which have several O-glycans, are known to exist in whey protein. Here, we report an isolation method of O-glycopeptides bearing disialyl core 1 type and core 2 type glycan moieties from commercially available whey protein products using proteose peptone extraction, enzymatic digestion (with trypsin or thermolysin), and sequential high-performance liquid chromatography purification. We were able to isolate several kinds of O-glycopeptides from lactophorin and κ-casein: six peptide sequences and five kinds of O-glycans. The O-glycopeptides were detected and identified by flow injection analysis combined with electrospray ionization mass spectrometry and tandem mass spectrometry using collision-induced dissociation and electron transfer dissociation. O-Glycopeptides bearing a variety of O-glycans could be used as a substrate for endo-α-N-acetyl galactosaminidase, and their various O-glycan structures were useful for the investigation of enzyme activities., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

A glycal based synthesis of (+)-bulgecinine, 3-hydroxy-2,5-dihydroxymethylpyrrolidine and 2-oxapyrrolizidin-3-ones proceeding through a common intermediate is reported. The key step in the work presented here is a two-step conversion of 4,6-di-O-benzyl-d-glucal to 2,3-dideoxy-2-tosylamido-d-glucose. This manuscript reports the first carbohydrate based approach to the synthesis of (+)-bulgecinine and the whole sequence has been accomplished with complete stereochemical integrity without the formation of mixture of products in any of these steps., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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-Ga 3+ ) 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.

Glycosylation is one of the most common posttranslational modifications of proteins and can exert profound effects on the inherent properties and biological functions of a given protein. Structurally well-defined homogeneous glycopeptides are highly demanded for functional studies and biomedical applications. Various chemical and chemoenzymatic methods have been reported so far for synthesizing different N- and O-glycopeptides. Among them, the chemoenzymatic method based on an endoglycosidase-catalyzed ligation of free N-glycans and GlcNAc-tagged peptides is emerging as a highly efficient method for constructing large complex N-glycopeptides. This chemoenzymatic approach consists of two key steps. The first step is to prepare the GlcNAc peptide through automated solid-phase peptide synthesis (SPPS) by incorporating an Asn-linked GlcNAc moiety at a predetermined glycosylation site; and the second step is to transfer an N-glycan from the corresponding N-glycan oxazoline en bloc to the GlcNAc peptide by an endoglycosidase or its efficient glycosynthase mutant. In this chapter, we provide detailed procedures of this chemoenzymatic method by demonstrating the synthesis of two HIV-1 V3 glycopeptide antigens carrying a high-mannose-type and a complex-type N-glycan, respectively. The described procedures should be generally applicable for the synthesis of other biologically important 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., (© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Anti-mucin1 (MUC1) antibodies have long been used clinically in cancer diagnosis and therapy and specific bindings of some of them are known to be dependent on the differential glycosylation of MUC1. However, a systematic comparison of the binding specificities of anti-MUC1 antibodies was not previously conducted. Here, a total of 20 glycopeptides including the tandem repeat unit of MUC1, APPAHGVTSAPDTRPAPGSTAPPAHGV with GalNAc (Tn-antigen), Galβ1-3GalNAc (T-antigen), NeuAcα2-3Galβ1-3GalNAc (sialyl-T-antigen), or NeuAcα2-6GalNAc (sialyl-Tn-antigen) at each threonine or serine residue were prepared by a combination of chemical glycopeptide synthesis and enzymatic extension of carbohydrate chains. These glycopeptides were tested by the enzyme-linked immunosorbent assay (ELISA) for their capacity to bind 13 monoclonal antibodies (mAbs) known to be specific for MUC1. The results indicated that anti-MUC1 mAbs have diverse specificities but can be classified into a few characteristic groups based on their binding pattern toward glycopeptides in some cases having a specific glycan at unique glycosylation sites. Because the clinical significance of some of these antibodies was already established, the structural features identified by these antibodies as revealed in the present study should provide useful information relevant to their further clinical use and the biological understanding of MUC1.

Natural glycopeptides have been shown to possess interesting biological activities. In this work, we have developed a general solid-phase approach to C-terminal glycopeptides. Taking advantage of oxime resin ester bond nucleophile susceptibility, we optimised the nucleophilic cleavage step with glycosylamines and demonstrated the generality and scope of this method. In addition, this reaction has high functional group tolerance and can be used for the preparation of longer C-terminal glycopeptides, demonstrated with the synthesis of a glycododecapeptide in one single step. The results pave the way to access efficiently novel medically relevant compounds.

Mucin-1 (MUC1) is a highly attractive antigenic target for anticancer vaccines. Naturally existing MUC1 can contain multiple types of O-linked glycans, including the Thomsen-Friedenreich (Tf) antigen and the Sialyl Thomsen-nouveau (STn) antigen. In order to target these antigens as potential anticancer vaccines, MUC1 glycopeptides SAPDT*RPAP (T* is the glycosylation site) bearing the Tf and the STn antigen, respectively, have been synthesized. The bacteriophage Qβ carrier is a powerful carrier for antigen delivery. The conjugates of MUC1-Tf and -STn glycopeptides with Qβ were utilized to immunize immune-tolerant human MUC1 transgenic (MUC1.Tg) mice, which elicited superior levels of anti-MUC1 IgG antibodies with titers reaching over 2 million units. The IgG antibodies recognized a wide range of MUC1 glycopeptides bearing diverse glycans. Antibodies induced by Qβ-MUC1-Tf showed strongest binding, with MUC1-expressing melanoma B16-MUC1 cells, and effectively killed these cells in vitro . Vaccination with Qβ-MUC1-Tf first followed by tumor challenge in a lung metastasis model showed significant reductions of the number of tumor foci in the lungs of immunized mice as compared to those in control mice. This was the first time that a MUC1-Tf-based vaccine has shown in vivo efficacy in a tumor model. As such, Qβ-MUC1 glycopeptide conjugates have great potential as anticancer vaccines.

Antimicrobial resistance in bacteria is frightening, especially resistance in Gram-negative Bacteria (GNB). In 2017, the World Health Organization (WHO) published a list of 12 bacteria that represent a threat to human health, and among these, a majority of GNB. Antibiotic resistance is a complex and relatively old phenomenon that is the consequence of several factors. The first factor is the vertiginous drop in research and development of new antibacterials. In fact, many companies simply stop this R&D activity. The finding is simple: there are enough antibiotics to treat the different types of infection that clinicians face. The second factor is the appearance and spread of resistant or even multidrug-resistant bacteria. For a long time, this situation remained rather confidential, almost anecdotal. It was not until the end of the 1980s that awareness emerged. It was the time of Vancomycin-Resistance Enterococci (VRE), and the threat of Vancomycin-Resistant MRSA (Methicillin-Resistant Staphylococcus aureus ). After this, there has been renewed interest but only in anti-Gram positive antibacterials. Today, the threat is GNB, and we have no new molecules with innovative mechanism of action to fight effectively against these bugs. However, the war against antimicrobial resistance is not lost. We must continue the fight, which requires a better knowledge of the mechanisms of action of anti-infectious agents and concomitantly the mechanisms of resistance of infectious agents.

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., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

Endostatin (ES) has attracted considerable attention for the treatment of anti-angiogenesis-related disorders. An 11-amino-acid peptide (ES2, IVRRADRAAVP) from the amino terminal of ES is of interest because it is the main active fragment of ES. However, both ES and ES2 have a poor stability and a short half-life, and other disadvantages need to be further resolved. Thus, we conjugated ES2 to glycol-split heparin derivatives (GSHPs) to yield the polymer-peptide conjugate, GSHP-ES2. This study showed that GSHP-ES2 exhibited increased stability, a wider pH activity range, better inhibition of endothelial cell proliferation, migration and tube formation in vitro, better anti-angiogenic activity and a longer half-life in vivo compared with ES2. These results also indicate that GSHP-ES2 has good potential for the treatment of angiogenesis-related diseases, either alone or in combination with other chemicals., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

α-Dystroglycan (α-DG) mucins are essential for maintenance of the structural and functional stability of the muscle fiber and, when hypoglycosylated, they are directly involved in pathological processes such as dystroglycanopathies. Thus, this work reports the synthesis of the novel 1,2,3-triazole-derived glycosyl amino acids αGlcNAc-1-O-triazol-2Manα-ThrOH (1) and Gal-β1,4-αGlcNAc-1-O-triazol-2Manα-ThrOH (2), followed by solid-phase assembly to get the corresponding glycopeptides NHAcThrVal[αGlcNAc-1-triazol-2Manα]ThrIleArgGlyOH (3) and NHAcThrVal[Gal-β1,4-αGlcNAc-1-triazol-2Manα]ThrIleArgGlyOH (4) as analogs of α-DG mucins. The glycosyl amino acids 1 (72%) and 2 (35%) were synthesized by Cu(I)-assisted 1,3-dipolar azide-alkyne cycloaddition reactions (CuAAC) between the azide-glycosyl amino acid αManN 3 -FmocThrOBn (5) and the corresponding alkyne-functionalyzed sugars 2'-propynyl-αGlcNAc (6) and 2'-propynyl-Gal-β1,4-αGlcNAc (7), followed by hydrogenation reactions. Subsequently, glycopeptides 3 (23%) and 4 (12%) were obtained by solid phase synthesis, involving sequential couplings of Fmoc-protected amino acids or the glycosyl amino acids 1 and 2, followed by cleavage from resin, N-acetylation and O-deacetylation (NaOMe) reactions. Lastly, enzymatic galactosylation of glycopeptide 3 with bovine β-1,4-GalT showed that it was not a substrate for this enzyme, which could be better elucidated by docking simulations with β-1,4-GalT., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Recent advances have demonstrated the feasibility and robustness of chemical synthesis for the production of homogeneously glycosylated protein forms (glycoforms). By taking advantage of the unmatchable flexibility and precision provided by chemical synthesis, the quantitative effects of glycosylation were obtained using chemical glycobiology approaches. These findings greatly advanced our fundamental knowledge of glycosylation. More importantly, analysis of these findings has led to the development of glycoengineering guidelines for rationally improving the properties of peptides and proteins. In this chapter, we present the key experimental steps for chemical biology studies of protein glycosylation, with the aim of facilitating and promoting research in this important but significantly underexplored area of biology., (© 2019 Elsevier Inc. All rights reserved.)

Access to lipopeptide-based vaccines for immunological studies remains a significant challenge owing to the amphipathic nature of the molecules, which makes them difficult to synthesize and purify to homogeneity. Here, we describe the application of a new peptide ligation technology, the diselenide-selenoester ligation (DSL), to access self-adjuvanting glycolipopeptide vaccines. We show that rapid ligation of glyco- and lipopeptides is possible via DSL in mixed organic solvent-aqueous buffer and, when coupled with deselenization chemistry, affords rapid and efficient access to a vaccine candidate possessing a MUC1 glycopeptide epitope and the lipopeptide adjuvant Pam 2 Cys. This construct was shown to elicit MUC1-specific antibody and cytotoxic T lymphocyte responses in the absence of any other injected lipids or adjuvants. The inclusion of the helper T cell epitope PADRE both boosted the antibody response and resulted in elevated cytokine production.