Challenges in Glycopeptide Synthesis for Vaccine Research - and How to Overcome Them?

Common Pain Points in Glycopeptide Synthesis

(1) Low Yield and Poor Solubility

The issues of low yield and solubility are particularly troublesome during the synthesis of glycopeptides. The failure of coupling reactions in SPPS is a common reason for the low yield of glycopeptides. This might be caused by the carbohydrate moiety itself, which will cause aggregation and precipitation of the peptides during synthesis and purification steps, the efficiency of the peptide elongation steps can be reduced by introducing side reactions and epimerization. Alternatively, a protecting group on the glycosylated building blocks might require harsh conditions for removal which might also lead to undesired destruction of the peptide moiety and glycan moiety as well. This is, in particular, a problem with the poor aqueous solubility of glycopeptides in the presence of complex carbohydrate structures which can lead to precipitation of the peptide product and this problem is also known to be a limitation in purification steps, for example by HPLC. In this case, alternative coupling agents and solvents and more efficient protecting groups that can be removed under milder conditions have been investigated. The application of convergent synthesis methods to the synthesis of glycopeptides in which glycopeptide fragments are prepared separately and coupled together in a modular fashion can also lead to increased yields by reducing the number of synthetic steps and minimizing side reactions.

(2) Complex Glycan Attachment Strategies

N- and O-linked glycosylation: N- and O-linked glycosylation, the natural post-translational modifications which add carbohydrates to the polypeptide chain co-translationally or post-translationally, involve N-glycosidic and O-glycosidic linkage. The former involves the amine group of Asn residue and forms an amide bond; while the latter involves the oxygen atom of side chain of Ser or Thr residues and forms an ether bond with the carbohydrate moiety. Direct and convergent syntheses are two common chemical approaches for the synthesis of N- or O-linked glycopeptides. During direct method synthesis solid phase peptide synthesis (SPPS) sequentially adds pre-synthesised glycosylated amino acids to the growing peptide chain. SPPS utilizes both Fmoc and Boc chemistry methods as its primary strategies. Generally, glycopeptide synthesis is performed by the Fmoc strategy because the strong acidic condition of Boc-chemistry affects the glycosidic linkages in common oligosaccharide. Synthesis of long peptide with more than 50 residues is difficult by stepwise synthesis due to incomplete couplings and epimerisation. As a result, a number of side products are formed and a low yield of final product is obtained. To overcome this problem, convergent (fragment-condensation) methods including on-resin linked glycopeptide and Lansbury aspartylation are also applied. Convergent approach is used for the synthesis of N-linked glycopeptide, as O-glycosylation cannot be made by this method. In these convergent methods, the glycosylamine unit is conjugated to a free Asp residue on a peptide by condensation of amino acid.

Fig. 1 Overview of mammalian N-glycosylation.^1,2

Chemo-enzymatic glycosylation: Chemo-enzymatic approaches take advantage of the synthetic flexibility of chemical synthesis and the high regio- and stereo-selectivity of enzyme-catalysed reactions to reach the highly efficient synthesis of complex carbohydrates. The most used enzymes in the chemoenzymatic toolbox are the Endo-β-N-acetylglucosaminidases (ENGases) and oligosaccharyltransferases (OST). The former stand out because they enable the attachment of complete oligosaccharide structures to GlcNAc-containing peptides or proteins in a single enzymatic reaction. They demonstrate transglycosylation capabilities which enable them to transfer released oligosaccharides to appropriate acceptors to produce new glycopeptides besides breaking glycosidic bonds to cleave chitobiose cores of N-linked glycans between GlcNAc residues. The two most commonly used ENGases, Endo-A from Arthrobacter and Endo-M from Mucor hiemalis function distinctly by processing oxalines as donors and binding them to GlcNAc derivatives as acceptors. Endo-A specifically adds high-mannose N-glycans to a range of acceptors bearing GlcNAc residues, while Endo-M is responsible for the attachment of three major types of N-glycan (high-mannose type, hybrid type, and complex type). Although the transglycosylation activity is different for each enzymes, their hydrolytic activity results generally in product hydrolysis and limits their broad use for chemoenzymatic approaches.

Choosing the Right Linker and Conjugation Strategy

(1) N-Linked vs O-Linked Glycosylation

N-linked glycans are attached to the amide nitrogen of asparagine residues in Asn-X-Ser/Thr sequons, where X is any amino acid except proline. N-linked glycosylation (NLG) is co-translationally added in the endoplasmic reticulum (ER) and further modified in the Golgi apparatus. N-linked glycans typically have a core of two N-acetylglucosamine (GlcNAc) and three mannose residues (GlcNAc2-Man9Glc3), to which other sugars (e.g. galactose, fucose, sialic acid) may be added. NLG is involved in protein folding, stability, and recognition by other molecules or cells. NLG can also affect protein trafficking and secretion as well as modulating immune responses. O-linked glycans develop when oligosaccharide chains bind to serine or threonine residues through their hydroxyl groups. Despite lacking a stringent consensus sequence O-linked glycosylation (OLG) is added to proteins after translation within the Golgi complex. O-linked glycans are more structurally diverse than N-linked glycans as they begin with a GalNAc core to which other sugars (galactose, fucose, sialic acid) can be added. OLG engages in cell-cell recognition activities alongside adhesion and signaling functions. It is particularly important for mucin-type proteins. OLG can also impact protein stability, solubility, and enzymatic activity. NLG can result in more complex and branched glycans than OLG, which can influence the immunogenicity and receptor interactions of the glycoprotein.

(2) Spacer Design for Carrier Proteins (e.g. KLH, CRM197)

Polysaccharide‐based vaccines are unable to elicit T-cell responses and hence only poorly neutralizing IgM antibodies are induced. A ground‐breaking technology that is able to circumvent this limitation is based on conjugating the bacterial polysaccharide to a foreign carrier protein, which provides the missing helper T-epitopes thus enabling T‐cell dependent immune activation. CRM197 is one of the most widely used carrier proteins for both clinically approved as well as investigational poly‐ and oligosaccharide conjugate vaccines and a large body of clinical data in various age groups support favorable immunogenicity, safety and tolerability. CRM197 is a mutant of C. diphtheriae toxin that has a Gly52 to Glu mutation that greatly reduces its toxicity. The protein is made up of two domains that are covalently linked through a disulfide bridge. KLH is a large, multimeric protein that provides a strong scaffold for conjugating glycopeptides. Spacer molecules such as PEG chains can be used to link the glycopeptide and KLH and to make sure that the antigenic sites remain accessible to the immune system. The spacer should be long enough to allow proper presentation of the glycopeptide to the immune system without steric hindrance from the carrier protein. A common spacer length is between 6-12 carbon atoms to provide sufficient flexibility and distance between the glycopeptide and carrier protein. They should also be chemically stable and not prone to degradation under physiological conditions.

Solutions from Our Expert Glycopeptide Team

(1) Site-Specific Synthesis with High Purity

Site-specific synthesis is the key to ensuring both high purity of product and well-defined glycosylation. We have established chemical and chemoenzymatic methods to tightly control site-specific glycosylation, because random glycosylation can change or abolish the biological activity of glycopeptides and influence their immunogenicity. We can accommodate N-linked as well as O-linked glycosylation for site-specific and homogeneous glycopeptides with defined glycan structures. For N-linked glycosylation, we use strategies that incorporate the glycan onto the asparagine residue. This is commonly achieved using glycosyl amine intermediates that incorporate the crucial glycoside-asparagine linkage. For O-linked glycosylation, we can directly synthesize the glycosylated serine or threonine residue building blocks, which can be incorporated into the peptide sequence on standard Fmoc-SPPS conditions. These methods have been developed to high yield and high purity (standard target purity is >90%). We can further modify methods for higher purity if necessary.

(2) Customized Analytical Characterization Services

We offer a full range of customized analytical characterization services to support your glycopeptide product development and validation. LC-MS and HPLC are effective tools to confirm glycan structure and peptide sequence. In addition, we are able to provide detailed structural analysis by other methods. Analytical tests to assess purity, stability and homogeneity of glycopeptides can be done by using state-of-the-art analysis methods available to our analytical team. We also provide glycan profiling for identification and quantification of glycan structures. Glycan profiling is important to evaluate the biological function and immunogenicity of glycopeptides. Our stability testing services evaluate glycopeptide products under different conditions to guarantee their long-term structural integrity. Our analysis services include customization capabilities that cater to your research needs by developing methods for new glycopeptide constructs. Our goal is to deliver robust and reliable analytical support that meets the highest standards of quality and regulatory compliance.

Glycoamino acids we can provide

References

1. Image retrieved from Figure 1 " Overview of mammalian N-glycosylation," Dammen-Brower K.; et al., used under [CC BY 4.0](https://creativecommons.org/licenses/by/4.0/). The original image was not modified.
2. Dammen-Brower K.; et al. " Strategies for glycoengineering therapeutic proteins." Frontiers in chemistry, 2022, 10: 863118.
3. Möginger U, Resemann A, Martin C E, et al. Cross Reactive Material 197 glycoconjugate vaccines contain privileged conjugation sites[J]. Scientific reports, 2016, 6(1): 20488. https://doi.org/10.1038/srep20488.
4. Chen B, Liu W, Li Y, et al. Impact of N-linked glycosylation on therapeutic proteins[J]. Molecules, 2022, 27(24): 8859. https://doi.org/10.3390/molecules27248859.
5. Dozio E, Massaccesi L, Corsi Romanelli M M. Glycation and glycosylation in cardiovascular remodeling: focus on advanced glycation end products and O-linked glycosylations as glucose-related pathogenetic factors and disease markers[J]. Journal of clinical medicine, 2021, 10(20): 4792. https://doi.org/10.3390/jcm10204792.