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.

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.

Facile access to C-glycosyl amino acids and peptides via Ni-catalyzed reductive hydroglycosylation of alkynes.

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.

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.

A facile chemoenzymatic synthesis of SARS-CoV-2 glycopeptides for probing glycosylation functions.

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.

Glycated peptides obtained from cultured crocodile meat hydrolysates via Maillard reaction and the anti-aging effects on Drosophila in vivo.

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, H2O2- 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.

Benefits of Chemical Sugar Modifications Introduced by Click Chemistry for Glycoproteomic Analyses.

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.

Site-Selective Acylations of α- and ß-Hydroxyamides in Complex Molecules: Application of Template-Driven Acylation to Disaccharides and a Glycopeptide.

Site-selective acylations of α-and ß-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 ß-hydroxyl group in a glycopeptide.

Synthesis and chelation study of a fluoroionophore and a glycopeptide based on an aza crown iminosugar structure.

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 Zn2+ 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 Hg2+ in methanol.

Chemoenzymatic Synthesis of Glycopeptides Bearing Galactose-Xylose Disaccharide from the Proteoglycan Linkage Region.

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 ß4GalT7. This work leads to a better understanding of the acceptor preference of ß4GalT7 and opens the door for expeditious synthesis of the proteoglycan linkage region.

GPAHex-A synthetic biology platform for Type IV-V glycopeptide antibiotic production and discovery.

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.

Chemical Synthesis of Homogeneous Human E-Cadherin N-Linked Glycopeptides: Stereoselective Convergent Glycosylation and Chemoselective Solid-Phase Aspartylation.

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.

Synthesis of MUC1-derived glycopeptide bearing a novel triazole STn analog.

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/ß2-triazole-6-ßGalNAc-ThrOH 1 as STn antigen analog, along with its assembly on the corresponding MUC1 peptide to give NAcProAsp [Neu5Acα/ß2-triazole-6-ßGalNAc]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.

Synthesis of O-Sulfated Human Syndecan-1-like Glyco-polypeptides by Incorporating Peptide Ligation and O-Sulfated Glycopeptide Cassette Strategies.

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.

Recent Development in Ligation Methods for Glycopeptide and Glycoprotein Synthesis.

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.

Glycopeptide drugs: A pharmacological dimension between "Small Molecules" and "Biologics".

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.

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.

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.

Parallel Glyco-SPOT Synthesis of Glycopeptide Libraries.

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.