Peptide Glycosylation Research Articles

Site‐selective construction of N‐linked glycopeptides through photoredox catalysis

The glycosylation of peptides and proteins can significantly impact their intrinsic properties, such as conformation, stability, antigenicity, and immunogenicity. Current methods for preparing N‐linked glycopeptides typically rely on amide bond formation, which can be limited by the presence of reactive functional groups like acids and amines. Late‐stage functionalization of peptides offers a promising approach to obtaining N‐linked glycopeptides. In this study, we demonstrate the preparation of N‐linked glycopeptides through a photoredox‐catalyzed site‐selective Giese addition between N‐glycosyl oxamic acid and peptides containing dehydroalanine (Dha) under visible light conditions. Unlike traditional methods that rely on the coupling of aspartic acid and glycosylamine, this approach utilizes the conjugation of N‐glycosylated carbamoyl radicals with Dha, facilitating the straightforward modification of complex peptides.

Site-selective construction of N-linked glycopeptides through photoredox catalysis.

The glycosylation of peptides and proteins can significantly impact their intrinsic properties, such as conformation, stability, antigenicity, and immunogenicity. Current methods for preparing N-linked glycopeptides typically rely on amide bond formation, which can be limited by the presence of reactive functional groups like acids and amines. Late-stage functionalization of peptides offers a promising approach to obtaining N-linked glycopeptides. In this study, we demonstrate the preparation of N-linked glycopeptides through a photoredox-catalyzed site-selective Giese addition between N-glycosyl oxamic acid and peptides containing dehydroalanine (Dha) under visible light conditions. Unlike traditional methods that rely on the coupling of aspartic acid and glycosylamine, this approach utilizes the conjugation of N-glycosylated carbamoyl radicals with Dha, facilitating the straightforward modification of complex peptides.

Methionine-Directed C–H Glycosylation of Peptides Catalyzed by Pd(OAc)2

Methionine-Directed C–H Glycosylation of Peptides Catalyzed by Pd(OAc)2

Late-Stage Glycosylation of Peptides by Methionine-Directed β-C(sp3)-H Functionalization with 1-Iodoglycals.

Using l-methionine (Met) as the endogenous directing group, we developed Pd-catalyzed β-C(sp3)-H glycosylation of peptides with 1-iodoglycals. A wide range of tri- to hexapeptides containing the Ala-Met motifs underwent Ala C-H glycosylation under the standard conditions to give the glycopeptides smoothly. 15 proteinogenic amino acids (with easily removable protecting groups) were well tolerated. Control experiments indicated that Met acted as a N,S-bidentate directing group and exhibited an effect superior to other amino acid residues such as l-aspartic acid (Asp), l-asparagine (Asn), and S-protected l-cysteine (Cys). In addition, further transformation by HFIP-promoted 1,4-elimination furnished another type of glycopeptide with the 1,3-diene motif, which provides a handle for further derivatization.

Photoredox and Nickel-Catalyzed Glycosylation of Amino Acids and Peptides

Photoredox and Nickel-Catalyzed Glycosylation of Amino Acids and Peptides

Direct and Detailed Site-Specific Glycopeptide Characterization by Higher-Energy Electron-Activated Dissociation Tandem Mass Spectrometry.

Glycosylation is widely recognized as the most complex post-translational modification due to the widespread presence of macro- and microheterogeneities, wherein its biological consequence is closely related to both the glycosylation sites and the glycan fine structures. Yet, efficient site-specific detailed glycan characterization remains a significant analytical challenge. Here, utilizing an Orbitrap-Omnitrap platform, higher-energy electron-activated dissociation (heExD) tandem mass spectrometry (MS/MS) revealed extraordinary efficacy for the structural characterization of intact glycopeptides. HeExD produced extensive fragmentation within both the glycan and the peptide, including A-/B-/C-/Y-/Z-/X-ions from the glycan motif and a-/b-/c-/x-/y-/z-type peptide fragments (with or without the glycan). The intensity of cross-ring cleavage and backbone fragments retaining the intact glycan was highly dependent on the electron energy. Among the four electron energy levels investigated, electronic excitation dissociation (EED) provided the most comprehensive structural information, yielding a complete series of glycosidic fragments for accurate glycan topology determination, a wealth of cross-ring fragments for linkage definition, and the most extensive peptide backbone fragments for accurate peptide sequencing and glycosylation site localization. The glycan fragments observed in the EED spectrum correlated well with the fragmentation patterns observed in EED MS/MS of the released glycans. The advantages of EED over higher-energy collisional dissociation (HCD), stepped collision energy HCD (sceHCD), and electron-transfer/higher-energy collisional dissociation (EThcD) were demonstrated for the characterization of a glycopeptide bearing a biantennary disialylated glycan. EED can produce a complete peptide backbone and glycan sequence coverage even for doubly protonated precursors. The exceptional performance of heExD MS/MS, particularly EED MS/MS, in site-specific detailed glycan characterization on an Orbitrap-Omnitrap hybrid instrument presents a novel option for in-depth glycosylation analysis.

Electrochemical aptamer-based biosensing of glycan-conjugated targets

As the most complex post-translational modification, glycosylation involves the covalent linking of glycan(s) to a biomolecule. The glycosylation of proteins, peptides, lipids, and nucleic acids results in the formation of diverse glycoconjugates, like glycoproteins, peptidoglycans, glycopeptides, glycolipids, lipopolysaccharides, and glycoRNAs, and glycoconjugates are also ubiquitous on the surface of extracellular vesicles (EVs), cells, bacteria, and viruses. The conjugated glycans are integral to cell recognition (e.g., adhesion and homing), cell trafficking, immune recognition, hormone-receptor binding, protein folding and stabilization, and many other biological processes. Currently, the glycan-conjugated biological objects have found widespread applications in disease diagnosis (e.g., glycoproteins, exosomes, and circulating tumor cells) and therapy (e.g., therapeutic monoclonal antibodies). Over the past decades, a variety of methods have been established for the quantitative assay of the glycan-conjugated biological objects, in light of their significant diagnostic and therapeutic values, using antibodies, aptamers, or affinity peptides as recognition ligands. Relative to other methods, the aptamer-based electrochemical biosensors (i.e., electrochemical aptasensors) have attracted much attention in recent years in the detection of the glycan-conjugated targets, by virtue of their intrinsic advantages such as low cost, good portability, simple instrument, superior stability and anti-interference capability, and high sensitivity and selectivity. This review summarizes the biological, diagnostic, and therapeutic significance of the glycan-conjugated biological objects, and then presents the progress in the electrochemical aptasensing of the glycan-conjugated biological objects, including glycoproteins, lipopolysaccharides (LPSs), exosomes, circulating tumor cells (CTCs), and pathogenic microbes (e.g., bacteria and viruses). Moreover, the challenges and future prospects of the electrochemical aptasensing of the glycan-conjugated biological objects are discussed.

Pd-catalyzed S-glycosylation of cysteine-containing peptides at room temperature

This study reports the synthesis of thioglycopeptides via a Pd-catalyzed coupling of cysteine-containing peptides with iodoglycals. A variety of cysteine-containing peptides and other thiol nucleophiles including thiosugars and thiophenols were used.

Recent Advances in Glycopeptide and Glycoprotein Synthesis: A Refined Synthetic Probe towards the Biological World

Comprehensive SummaryProtein glycosylation is the most complex and diverse form of post‐translational modification in human body. Meanwhile, glycosylation of peptides and proteins emerges as a promising strategy to improve the pharmacokinetic profile of peptide‐ and protein‐based therapeutics. Owing to the importance of protein glycosylation, a rigorous evaluation of the relationship between the precise structure and biological function of glycoproteins has to be performed. Recently, chemical synthesis, chemoenzymatic synthesis and semisynthesis strategies have attracted extensive attentions towards the preparation of structurally defined glycopeptides and glycoproteins; the obtained synthetic glycoforms thus enable the thorough investigation of specific effects of protein glycosylation. This review highlights the recent progress in the development of novel strategies, preparation of homogeneous glycoproteins and exploration of structure‐activity relationships. On this basis, the challenges and prospects are discussed.

Glycosylation and Lipidation Strategies: Approaches for Improving Antimicrobial Peptide Efficacy.

Antimicrobial peptides (AMPs) have recently gained attention as a viable solution for combatting antibiotic resistance due to their numerous advantages, including their broad-spectrum activity, low propensity for inducing resistance, and low cytotoxicity. Unfortunately, their clinical application is limited due to their short half-life and susceptibility to proteolytic cleavage by serum proteases. Indeed, several chemical strategies, such as peptide cyclization, N-methylation, PEGylation, glycosylation, and lipidation, are widely used for overcoming these issues. This review describes how lipidation and glycosylation are commonly used to increase AMPs' efficacy and engineer novel AMP-based delivery systems. The glycosylation of AMPs, which involves the conjugation of sugar moieties such as glucose and N-acetyl galactosamine, modulates their pharmacokinetic and pharmacodynamic properties, improves their antimicrobial activity, and reduces their interaction with mammalian cells, thereby increasing selectivity toward bacterial membranes. In the same way, lipidation of AMPs, which involves the covalent addition of fatty acids, has a significant impact on their therapeutic index by influencing their physicochemical properties and interaction with bacterial and mammalian membranes. This review highlights the possibility of using glycosylation and lipidation strategies to increase the efficacy and activity of conventional AMPs.

Glycosylated Lipopeptides—Synthesis and Evaluation of Antimicrobial Activity and Cytotoxicity

Ultrashort cationic lipopeptides (USCLs) are promising antimicrobial agents that may be used to combat pathogens such as bacteria and fungi. USCLs consist of a few basic amino acid residues and at least one lipid moiety, usually a fatty acid chain. Generally, USCLs are potent antimicrobials but their major shortcoming is a relatively high cytotoxicity and hemolytic activity. Glycopeptide antibiotics (e.g. vancomycin) are essential in combating bacterial infections and are popular in medicinal practice. However, literature concerning the effect of glycosylation of peptides on their antimicrobial activity is rather scarce. For the first time, this study highlights the effect of USCLs glycosylation on in vitro biological activity. The aim of this study was to evaluate the impact of glycosylation of a series of USCLs on antimicrobial activity, cytotoxicity and hemolytic activity. Straight-chain fatty acids (C14, C16, C18) were attached to the N-terminal amino group of tripeptides-SRR-NH2, RSR-NH2 and RRS-NH2. Two groups of the lipopeptides were synthetized, the first with unmodified L-serine (USCLs) and the other with L-serine O-glycosylated by N-acetyl-β-d-glucosamine to produce new class of glycosylated ultrashort cationic lipopeptide (gUSCLs). Both USCLs and gUSCLs were tested against planktonic and biofilm cultures of ESKAPE strains (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp.) and Candida glabrata, and hemolytic activity on human erythrocytes and cytotoxicity against the HaCaT cell line was examined. Generally, USCLs and gUSCLs proved to be active against all the tested strains. The highest activity displayed was by lipopeptides containing the C18 fatty acid. Antimicrobial, hemolytic and cytotoxic activities were mainly correlated with amino acid sequence (position of serine/glycosylated serine) and hydrophobicity of molecule and were found to be highly strain-dependent. In general, glycosylation did not guarantee an increased antimicrobial activity or a decreased hemolytic and cytotoxic activities. However, in some cases, gUSCLs proved to be superior to their USCLs analogs. The most pronounced differences were found for peptides with C18 fatty acid and serine at the first and second position against both planktonic cells and biofilm of C. glabrata, as well as the second and third position against S. aureus. It is noteworthy that gUSCLs were also more active against biofilm than were USCLs.

Chemoselective Solution- and Solid-Phase Synthesis of Disulfide-Linked Glycopeptides.

Glycosylation of peptides and proteins is a widely employed strategy to mimic important post-translational modifications or to modulate the physicochemical properties of peptides to enhance their delivery. Furthermore, glycosylation via a sulfur atom imparts increased chemical and metabolic stability to the resulting glycoconjugates. Herein, we report a simple and chemoselective procedure to prepare disulfide-linked glycopeptides. Acetate-protected glycosylsulfenyl hydrazines are shown to be highly reactive with the thiol group of cysteine residues within peptides, both in solution and as part of conventional solid-phase peptide synthesis protocols. The efficiency of this glycosylation methodology with unprotected carbohydrates is also demonstrated, which avoids the need for deprotection steps and further extends its utility, with disulfide-linked glycopeptides produced in excellent yields. Given the importance of glycosylated peptides in structural glycobiology, pharmacology, and therapeutics, the methodology outlined provides easy access to disulfide-linked glycopeptides as molecules with multiple biological applications.

Design and Synthesis of Brain Penetrant Glycopeptide Analogues of PACAP With Neuroprotective Potential for Traumatic Brain Injury and Parkinsonism.

There is an unmet clinical need for curative therapies to treat neurodegenerative disorders. Most mainstay treatments currently on the market only alleviate specific symptoms and do not reverse disease progression. The Pituitary adenylate cyclase-activating polypeptide (PACAP), an endogenous neuropeptide hormone, has been extensively studied as a potential regenerative therapeutic. PACAP is widely distributed in the central nervous system (CNS) and exerts its neuroprotective and neurotrophic effects via the related Class B GPCRs PAC1, VPAC1, and VPAC2, at which the hormone shows roughly equal activity. Vasoactive intestinal peptide (VIP) also activates these receptors, and this close analogue of PACAP has also shown to promote neuronal survival in various animal models of acute and progressive neurodegenerative diseases. However, PACAP’s poor pharmacokinetic profile (non-linear PK/PD), and more importantly its limited blood-brain barrier (BBB) permeability has hampered development of this peptide as a therapeutic. We have demonstrated that glycosylation of PACAP and related peptides promotes penetration of the BBB and improves PK properties while retaining efficacy and potency in the low nanomolar range at its target receptors. Furthermore, judicious structure-activity relationship (SAR) studies revealed key motifs that can be modulated to afford compounds with diverse selectivity profiles. Most importantly, we have demonstrated that select PACAP glycopeptide analogues (2LS80Mel and 2LS98Lac) exert potent neuroprotective effects and anti-inflammatory activity in animal models of traumatic brain injury and in a mild-toxin lesion model of Parkinson’s disease, highlighting glycosylation as a viable strategy for converting endogenous peptides into robust and efficacious drug candidates.

Identification of the effect of N-glycan modification and its sialylation on proteolytic stability and glucose-stabilizing activity of glucagon-like peptide 1 by site-directed enzymatic glycosylation.

In this study, an approach to prepare long-acting glucagon-like peptide 1 (GLP-1) by site-directed enzymatic glycosylation with homogeneous biantennary complex-type N-glycan has been developed. All the N-glycan-modified GLP-1 analogues preserved an unchanged secondary structure. The glycosylated GLP-1 analogues with sialyl complex-type N-glycan modified at Asn26 and Asn34 exhibited a 36.7- and 24.0-fold in vitro half-life respectively when incubated with dipeptidyl peptidase-IV (DPP-IV), and 25.0- and 13.9-fold respectively when incubated with mouse serum. Compared to native GLP-1, both glycosylated GLP-1 analogues modified at Asn34 by asialyl and sialyl N-glycan demonstrated lower maximum blood glucose levels, as well as more rapid and more persistent glucose-stabilizing capability in type 2 diabetic db/db mice. Our results indicated that the selection of an appropriate position (to avoid hindering the peptide-receptor binding) is crucial for N-glycan modification and its sialylation to improve the therapeutic properties of the modified peptides. The information learned would facilitate future design of therapeutic glycopeptides/glycoproteins with N-glycan to achieve enhanced pharmacological properties.

Dietary soy, pork and chicken proteins induce distinct nitrogen metabolism in rat liver

Diets have been shown to alter metabolism and gene expression. However, few data are available about changes in gene expression in liver after intake of different meat protein diets. This work aimed to explore the long-term effects of protein source on liver metabolic enzymes. Rats were fed protein diets for 90 days to study whether intake of chicken and pork protein diets promoted gene expression involved in hepatic metabolism. Liver proteome profiles were measured by iTRAQ labeling and LC–ESI–MS/MS. Chicken protein diet induced higher level of serum amino acids in rats than soy protein. Amino acid metabolizing enzymes were downregulated by pork and chicken protein diets compared with soy protein diet. Intake of meat protein diets downregulated enzymes involved in protein synthesis, disulfide bond formation, signal peptide addition, transport, localization, degradation and glycosylation modification, but upregulated enzymes involved in prolyl cis–trans isomerization for protein synthesis. Protein diets from different sources affected the amino acid supply, and further influenced ribosome assembly and protein synthesis through mTOR signaling pathway.

Nickel-Catalyzed Glycosylation of Amino Acids or Peptides

Nickel-Catalyzed Glycosylation of Amino Acids or Peptides

Protection-Free Site-Directed Peptide or Protein S -Glycosylation and Its Application in the Glycosylation of Glucagon-like Peptide 1

The development of a facile cysteine-directed S-glycosylation strategy would facilitate the intensive investigation of the effect of glycosylation on protein translational modification. Herein, we ...

Can Glycosylation Mask the Detection of MHC Expressing p53 Peptides by T Cell Receptors?

Proteins of the major histocompatibility complex (MHC) class I, or human leukocyte antigen (HLA) in humans interact with endogenous peptides and present them to T cell receptors (TCR), which in turn tune the immune system to recognize and discriminate between self and foreign (non-self) peptides. Of especial importance are peptides derived from tumor-associated antigens. T cells recognizing these peptides are found in cancer patients, but not in cancer-free individuals. What stimulates this recognition, which is vital for the success of checkpoint based therapy? A peptide derived from the protein p53 (residues 161–169 or p161) was reported to show this behavior. T cells recognizing this unmodified peptide could be further stimulated in vitro to create effective cancer killing CTLs (cytotoxic T lymphocytes). We hypothesize that the underlying difference may arise from post-translational glycosylation of p161 in normal individuals, likely masking it against recognition by TCR. Defects in glycosylation in cancer cells may allow the presentation of the native peptide. We investigate the structural consequences of such peptide glycosylation by investigating the associated structural dynamics.

Protease-protection strategy combined with the SERS tags for detection of O-GlcNAc transferase activity

Protein glycosylation plays a key role in the process of life, and the related O-GlcNAc transferase (OGT) has become a sweet new target for cancer diagnosis and therapy. Herein, based on protease-protection strategy combining with the well-designed surface-enhanced Raman scattering (SERS) tags, a sensitive SERS assay was developed for specifically OGT activity determination. In the assay, a peptide containing both glycosylation and protease sites was designed. The two ends of the peptide are biotin and cysteine residues. Therefore, the SERS tags and the magnetic bead can be connected to the two ends of the peptide through Ag-S bond and affinity interaction, respectively. Peptide glycosylation occurs in the presence of OGT, which will protect the peptide from the next cleavage by proteinase K, and more SERS tags left in the surface of magnetic beads will contribute a high SERS signal. The SERS intensity was identified to be correlate with the activity of OGT during the measurement, and the minimum detection concentration of OGT down to 0.1 nmol/L was achieved with a wide linearity range from 10−10 to 10-7 mol/L. The inhibition assay and serum sample detection confirmed the feasibility of the SERS assay in practical application, which is essential for drug development and clinical diagnosis.

Genetic hybridization of highly active exogenous functional proteins into silk-based materials using “light-clothing” strategy

Genetic hybridization of highly active exogenous functional proteins into silk-based materials using “light-clothing” strategy