Site-Specific Glycosylation Strategies for Peptide-Based Therapeutics

Why Site-Specific Glycosylation Is Essential in Drug Development?

Glycosylation allows for a greater diversity of protein and/or peptide and extends their functional repertoire. Currently four types of glycosylation are well known and studied: N-, O-, C-, and S-glycosylation. The type of the glycosylation is determined based on the nature of the sugar–peptide bond and can be further subdivided on the basis of glycosidic linkage, glycan composition, structure, and length. N-type glycosylation, which is also called N-linked glycosylation, is well studied. The type of glycosylation where glycan is attached to the amino group of asparagine. The oligosaccharides attach covalently upon recognition of Asn-X-Ser/Thr (X is any amino acid) via the nitrogen atom. There are many bacterial glycosyltransferases in use already for controlled in vitro glycosylation and such research has established the field of protein glycoengineering.

(1) Reducing Off-Target Effects via Precise Glycan Placement

Drug development heavily relies on site-specific glycosylation (SSG) for its effectiveness. Because glycans can be placed on a peptide or protein at precise locations, the chances of off-target effects are minimized. Glycans can be attached to a certain area of the agent to direct and optimize the interaction with the target receptor and thus bind to only the desired cells or tissues. By binding only to the targeted area, there should be minimal interaction between the drug and other non-targeted cells, improving its therapeutic index. Chromacin, an antimicrobial peptide from bovine intestine, is an exception among other mammalian antimicrobial peptides as it is both negatively-charged (instead of the usual positive charge), and hydrophilic in nature, as a result, it was expected to have low affinity toward bacterial membranes. Still, the peptide inhibits the growth of Gram-positive bacteria. It was found that O-glycosylation on Ser186 residue is required for activity. The glycosylation targets the peptide to a specific cell surface receptor which overcomes the peptide's naturally low affinity to bacteria by virtue of its chemical properties. SSG results in enhanced binding affinity and specificity for target antigens in monoclonal antibody drug development while minimizing non-specific interactions with other proteins. SSG can be introduced into the antibody by developing an engineered cell line that glycosylates at specific sites, or via enzymatic processes. The glycosylation can then be optimized for improved therapeutic efficacy.

Fig. 1 Current knowledge about the glycosylation roles in the cancer immunotherapy.^1,2

(2) Enhancing Half-Life, Solubility, and Bioavailability

The energetics of Glycosylation stabilization: Peptide folding is speeded up by the inherent chemical characteristics of the N-glycans which direct the folding energy landscape by entropic limitation. The thermodynamic stabilisation that glycosylation brings is also related to kinetic stabilisation. The N-glycan chaperone function is well documented in terms of the stabilising effect that it has on disulphide bond creation as well as proline isomerisation during β-turns or β-sheet production. Peptide stability is a topic which is of particular importance in the pharmaceutical creation of therapeutic peptides, and can be influenced by various things including pH and temperature, as well as storage and delivery conditions. Peptide glycosylation has a huge range of potential in terms of both the degree of glycosylation, and glycan type, structural composition and size. It also has a number of potential attachment sites on the protein which could lead to even more heterogeneity.

Glycosylation and stability: The application of SSG to peptides enables substantial modification of their biological properties in vivo especially through N-linked glycosylation which improves serum half-life and bioavailability of peptides and other compounds. Enfuvirtide is an example of a novel antiretroviral peptide in which glycosylation of sialic acid residues lengthens its half-life by more than ten-fold, without altering its target sensitivity. The pharmacokinetics of such glycosylation by addition of N- or O-glycans has been found to be similar to the addition of polyethylene glycol moieties (PEGs). PEGs increase the rigidity of peptides protecting them from rapid renal clearance. Such modifications are designed to increase the size of a given peptide, as it is known that molecules with a molecular weight of <5 kDa that are not bound to plasma proteins are rapidly excreted via the renal system. Glyco-modification is not associated with the same safety issues as PEG modification through the introduction of a synthetic polymer, and it is also technically less challenging to achieve.

Current Strategies for Site-Specific Glycosylation

(1) Chemical Methods (e.g., SPPS with Glycosylated Amino Acids)

Direct and convergent syntheses are the two general chemical approaches for the synthesis of N- or O-linked glycopeptides. In the direct method, the pre-synthesised glycosylated amino acid is coupled to the elongating peptide one residue at a time using solid phase peptide synthesis (SPPS). Two methods of SPPS, fluorenylmethyloxycarbonyl (Fmoc) and tert-butyloxycarbonyl (Boc) chemistry, are commonly used. In general, glycopeptide synthesis is performed by Fmoc strategy because the strong acidic condition of Boc-chemistry disturbs the glycosidic linkages in common oligosaccharide. Synthesis of long peptides, with over 50 residues, is challenging by stepwise synthesis, due to incomplete couplings and epimerisation. This produces side products and results in a low yield of final product. To address this issue, convergent (fragment-condensation) methods including on-resin linked glycopeptide and Lansbury aspartylation are employed. The convergent approach is mostly used for N-linked glycopeptide synthesis, as O-glycosylation can not be achieved by this method. In these convergent methods, glycosylamine unit is conjugated to a free Asp residue on a peptide through condensation of the amino acid. An on-resin convergent synthesis has been reported in which 2-phenylisopropyl protecting group is used as an orthogonal handle to create glycosylation sites on-resin. This is used for coupling a large high mannose oligosaccharide to peptides to suppress the aspartamide formation.

(2) Enzymatic Approaches (e.g., GalNAc-Ts, EndoS Mutants)

Endo-β-N-acetylglucosaminidases (ENGases) are the most commonly used enzymes for chemoenzymatic approaches. ENGases are able to couple an intact oligosaccharide to the N-acetylglucosamine (GlcNAc)-containing peptide or protein as an efficient acceptor in a single step. In addition to the hydrolysis of the glycosidic bond (cleaving the chitobiose core of N-linked glycans between two GlcNAc residues), ENGases have transglycosylation activity that can attach the released oligosaccharyl moiety to a suitable acceptor and form a new glycopeptide. Endo-A and Endo-M are common ENGases with distinct substrate activity to process oxalines as donors and attach them to GlcNAc derivatives as acceptors. Endo-A specifically adds high-mannose N-glycans to a variety of acceptors bearing GlcNAc residues, whereas Endo-M acts on the attachment of three major types of N-glycan (high-mannose type, hybrid type, and complex type). Glycosyltransferases are able to extend the sugar chain by the attachment of one monosaccharyl residue at a time. β-(1,3)-N-Acetylglucosaminyltransferase is an enzyme isolated from Neisseria meningitides and was used for the conjugation of GlcNAc residue to the lactose moiety of both endomorphin-1 and enkephalin derivatives. Lipopolysaccharyl α-1,4-galactosyltransferase is another glycosyltransferase derived from Neisseria meningitides, which has been used to attach the galactose unit to the terminal lactose residue of lipooligosaccharide.

(3) Bioorthogonal Labeling for In Vivo Applications

Bioorthogonal labeling can also be used for glycan-peptide/protein conjugates. The idea is to take advantage of chemical reactions that can be performed in living systems without perturbing normal biochemistry (bioorthogonal reactions) to introduce chemical handles that allow the attachment of glycans to peptides or proteins in living systems. This is another strategy for site-specific glycosylation and the beauty of these strategies is that one can design chemical probes that do not interfere with the biological activity of interest. For instance, click chemistry has been used to attach glycans to peptides using bioorthogonal reactions, such as azide-alkyne cycloaddition (AZIDE-ALKyne) to introduce glycans at specific sites. The modified peptides in some cases have higher stability and bioavailability.

Our Site-Specific Glycopeptide Solutions

(1) N-linked and O-linked Glycopeptide Synthesis

Site-specific N and O glycopeptides, Our full range of site-specific glycopeptide services includes the synthesis of N- and O-glycopeptides. For N-glycopeptides, glycans can be attached to the amide nitrogen of asparagine, usually through the intermediate of a glycosyl amine. This form of N-glycanation is now a well-established way to site-specifically introduce complex N-glycans to proteins and has been used to dramatically increase the stability and activity of several peptides, for example the group of Ogawa recently synthesized a tripeptide with a core-pentasaccharide N-glycan using a building block approach, with the glycan synthesized first and then coupled to the peptide sequence. O-glycosylation can also be site-specifically introduced to a peptide by using Fmoc-protected glycosylated amino acids as building blocks and then incorporating them into a peptide sequence as part of an Fmoc-SPPS under standard Fmoc-SPPS conditions, with the glycan attached to the hydroxyl group of a serine or threonine.

(2) Glycosylated Amino Acid Building Blocks

We provide an extensive assortment of glycosylated amino acid building blocks to facilitate the synthesis of glycopeptides. The building blocks have been designed to be compatible with standard SPPS protocols. For instance, Fmoc-protected O-glycosylated serine and threonine residues can be prepared by reacting commercially available peracetates and Fmoc amino acids in the presence of a Lewis acid (SnCl4, BF3·Et2O). This straightforward approach enables the fast and efficient generation of glycosylated building blocks that can subsequently be used to synthesize a diverse array of glycopeptides. Our glycosylated amino acid building blocks are designed to minimize side reactions and degradation during peptide synthesis. For example, O-acetylation is often used as a protective group for the sugar hydroxyl groups. This increases the acid stability of the molecule and the acetyl groups can be removed under mild conditions to prevent base-mediated side reactions during deprotection, while maintaining the structural integrity and biological activity of the glycopeptides.

(3) Custom Modification on Specific Residues

We also have custom modification services for the placement of glycans on custom residues within peptides. This can be performed through a variety of chemical and enzymatic methods to place glycans at desired residues, ultimately influencing the pharmacokinetic properties and targeting of the peptide of interest. For example, glycosylation can be added enzymatically through the use of glycosyltransferases to achieve site-specific modification of serine, threonine, or asparagine residues with specific glycans in a highly specific and reproducible fashion. In addition to enzymatic addition, chemical modifications can also be performed using bioorthogonal labeling strategies to label and tag molecules in vivo. Bioorthogonal reactions, such as azide-alkyne cycloaddition, can be used to add glycans to peptides in living systems and thus be studied to gain a better understanding of glycosylation in various processes.

Peptide Modification Services at Creative Peptides

References

1. Image retrieved from Figure 1 " Current knowledge about the glycosylation roles in the cancer immunotherapy," Chiang A W T.; et al., used under [CC BY 4.0](https://creativecommons.org/licenses/by/4.0/). The original image was not modified.
2. Chiang A W T.; et al. " Systems glycobiology for discovering drug targets, biomarkers, and rational designs for glyco-immunotherapy." Journal of biomedical science, 2021, 28: 1-15.
3. Bellavita R, Braccia S, Galdiero S, et al. Glycosylation and lipidation strategies: Approaches for improving antimicrobial peptide efficacy[J]. Pharmaceuticals, 2023, 16(3): 439. https://doi.org/10.3390/ph16030439.
4. Dammen-Brower K, Epler P, Zhu S, et al. Strategies for glycoengineering therapeutic proteins[J]. Frontiers in chemistry, 2022, 10: 863118. https://doi.org/10.3389/fchem.2022.863118.
5. Tvaroška I. The Role of Glycans in Human Immunity—A Sweet Code[J]. Molecules, 2025. https://doi.org/10.3390/molecules30132678.