Investigation of novel cyclic structure in glycoconjugate using a simple model system
Introduction
Capsular polysaccharides, presenting in bacteria or parasite cell wall surfaces, provide a means to activate the immune response in humans. However, capsular polysaccharides, while offering vaccine protection to young adults through the innate immune response, fail to generate the adaptive immune response to protect infants under the age of 2 [1] or seniors over the age of 65 [2]. In these cases, the conjugation of the capsular polysaccharides to a carrier protein is necessary to induce T cell dependent immunity [3]. Several reports suggest that glycopeptide fragments may be the key to the activation of the CD4+ T-cell [[4], [5], [6]]. These reports also suggest that the conjugation linkages could have an impact on the immune response activation. Understanding the conjugation linkages will lead to a better understanding of the glycoconjugate vaccine structure and its relationship to the vaccine potency which eventually benefits the vaccine design and production.
Reductive amination is one of the most well-adopted polysaccharide-protein conjugation strategies [[7], [8], [9], [10], [11], [12]]. The capsular polysaccharide is first treated with a mild oxidation reagent such as sodium periodate (NaIO4) to activate the carbohydrate or open individual sugar rings (Fig. 1). During the reaction, NaIO4 binds 1,2-diols and transforms them into aldehydes with cleavage of the C–C bond. Next, the carrier protein is mixed with the oxidized polysaccharide and conjugated via reversible imine linkages. Upon the addition of a reducing reagent such as sodium cyanoborohydride (NaCNBH3), the imines are reduced to secondary amines and an irreversible polysaccharide-protein covalent linkage is established. The amine can react with either of the two aldehydes in the sugar ring and generate a heterogeneous mixture. The remaining unreacted aldehydes on the polysaccharide are reduced to the hydroxyl group upon quenching by sodium borohydride (NaBH4) to avoid potential side reactions (Fig. 1) [[13], [14], [15], [16]]. Although the mechanism of the reductive amination conjugation has been established for decades, there is still debate regarding the structural identity of conjugation linkage generated in the reaction. For example, Schuerman et al. proposed a cyclic ring structure may be present in the glycoconjugate instead of an open ring structure. It is also proposed that the conjugate linkage can impact the level and function of the glycoconjugate induced antibody [6]. Meanwhile, Small molecules which use amino acids to build up the oxazepane and morpholine structures were also reported by different research groups [17,18]. However, direct experimental evidence demonstrating such cyclic linkages present in peptide or protein glycoconjugates using reductive amination has not been reported. Here we report results from an investigation of the reductive amination conjugation linkage structure using a simple model system.
Section snippets
Results and discussion
To gain a better understanding of the reductive amination process, a small peptide 1, α-factor mating pheromone, containing one lysine was conjugated to a monosaccharide-aldehyde. Compound 2, methyl glucopyranoside, was treated with NaIO4 to produce the mixture of aldehyde products 3, 4, 5 [18], which were further conjugated to the model peptide as an approach to mimic a dialdehyde conjugation system. The conjugation reactions were carried out under aqueous conditions (PBS buffer, pH = 6.8).
Conclusion
In summary, we report a model study of reductive amination chemistry using various monosaccharides and a model peptide coupled with state-of-the-art LC-MS, LC-MS/MS and NMR analyses. Three different kinds of dialdehydes were investigated here using NaIO4 oxidation and a glucose model. We report that the primary conjugation linkage in the peptide model study for sugars containing two aldehydes in the same ring (i.e. oxidation products of vicinal diols using NaIO4) is a cyclic tertiary amine
General procedure for oxidizing monosaccharides using sodium periodate
To a water solution of 0.26 mmol/mL monosaccharide 1-O-methyl-glucoside (2) or 1-O-methyl-galactoside, 1.2 eq. of NaIO4 was added. The reaction mixture was allowed to incubate at room temperature for 4 h. Then 0.1 mL of MeOH was added to quench the reaction. The mixture was dried under a nitrogen evaporator for 2 h at 37 °C. It was then re-dissolved in MeOH and all salts were precipitated. The salts were then centrifuged, and the supernatant was transferred to another tube and dried under
Declaration of competing interest
The authors declare the following competing financial interest(s): the authors are employees of Pfizer Inc.
Acknowledgements
This work was supported by Biotherapeutics Pharmaceutical Sciences, in Pfizer, Inc. We thank Margaret Ruesch, Heidi Holovics, Kelly Sackett, and Elizabeth Rainbolt for their critical reviews, and Qi Liu and Yuting Huang for their support. We also thank Mark Ruppen, Lynn Phelan, and David Ricol from Pfizer Vaccine Research and Development for their helpful discussions.
References (25)
- et al.
Predictors of pneumococcal conjugate vaccine immunogenicity among infants and toddlers in an American Indian PnCRM7 efficacy trial
J. Infect. Dis.
(2007) - et al.
Pneumococcal vaccination of elderly adults: new paradigms for protection
Clin. Infect. Dis.
(2008) - et al.
How bacterial carbohydrates influence the adaptive immune system
Annu. Rev. Immunol.
(2009) - et al.
Peptide adjacent to glycosylation sites impacts immunogenicity of glycoconjugate vaccine
Oncotarget
(2018) - et al.
A mechanism for glycoconjugate vaccine activation of the adaptive immune system and its implications for vaccine design
Nat. Med.
(2011) - et al.
Impact of the conjugation method on the immunogenicity of Streptococcus pneumoniae serotype 19F polysaccharide in conjugate vaccines
Clin. Vaccine Immunol.
(2011) - et al.
Immunochemistry of groups A, B, and C meningococcal polysaccharide-tetanus toxoid conjugates
J. Immunol.
(1981) - et al.
Protein-polysaccharide conjugation
- et al.
Development of a glycoconjugate vaccine to prevent meningitis in Africa caused by meningococcal serogroup X
Proc. Natl. Acad. Sci. Unit. States Am.
(2013) - et al.
Enzymatic depolymerization of streptococcus pneumoniae type 8 polysaccharide
Carbohydr. Res.
(2020)
Synthesis and characterization of periodate-oxidized polysaccharides: dialdehyde xylan (DAX)
Biomacromolecules
Periodate oxidation of polysaccharides for modification of chemical and physical properties
Carbohydr. Res.
Cited by (1)
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2022, Frontiers in Chemistry
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These authors contributed equally to the work.