Glycovaccine Design: Optimization of Model and Antitubercular Carrier Glycosylation via Disuccinimidyl Homobifunctional Linker

Abstract

Conjugation via disuccinimidyl homobifunctional linkers is reported in the literature as a convenient approach for the synthesis of glycoconjugate vaccines. However, the high tendency for hydrolysis of disuccinimidyl linkers hampers their extensive purification, which unavoidably results in side-reactions and non-pure glycoconjugates. In this paper, conjugation of 3-aminopropyl saccharides via disuccinimidyl glutarate (DSG) was exploited for the synthesis of glycoconjugates. A model protein, ribonuclease A (RNase A), was first considered to set up the conjugation strategy with mono- to tri- mannose saccharides. Through a detailed characterization of synthetized glycoconjugates, purification protocols and conjugation conditions have been revised and optimized with a dual aim: ensure high sugar-loading and avoid the presence of side reaction products. An alternative purification approach based on hydrophilic interaction liquid chromatography (HILIC) allowed the formation of glutaric acid conjugates to be avoided, and a design of experiment (DoE) approach led to optimal glycan loading. Once its suitability was proven, the developed conjugation strategy was applied to the chemical glycosylation of two recombinant antigens, native Ag85B and its variant Ag85B-dm, that are candidate carriers for the development of a novel antitubercular vaccine. Pure glycoconjugates (≥99.5%) were obtained. Altogether, the results suggest that, with an adequate protocol, conjugation via disuccinimidyl linkers can be a valuable approach to produce high sugar-loaded and well-defined glycovaccines.

Keywords: Ag85B antigen; antitubercular vaccines; chemical glycosylation; disuccinimidyl linkers; glycoconjugate characterization; glycoconjugate vaccines; glycosylation optimization.

Scheme 1

Preparation of monosaccharide 7.

Scheme 2

Preparation of disaccharide 11.

Scheme 3

Preparation of trisaccharide 18.

Scheme 4

(i) DSG (15 equiv.) in DMF/100 mM phosphate buffer, pH 8.0 (4:1 v/v), room temperature, 4 h. (ii) 100 mM phosphate buffer, pH 8, 20 °C, 16 h. Protein graphical representation depicts model protein RNase A and was taken from the Protein data Bank (PDB DOI: 10.2210/pdb2E3W/pdb).

Scheme 5

Side products arising from inadequate linker removal.

Figure 1

HILIC-UV profile of (a) RNase A; (b) RNase A conjugated with 20 purified by precipitation with EtOAc, conjugation performed in non-optimized conditions; (c) RNase A conjugated with 20 purified by HILIC, conjugation performed in non-optimized conditions; (d) RNase A conjugated with 20 purified by HILIC, conjugation performed in optimized conditions. Detection was performed at 214 nm. Numbers indicate the incorporated Man₂ units.

Figure 2

Coefficients and significance obtained in the full factorial design. The numbers on the x-axis refer to the coefficients of the terms of the models X1–X4, as defined in the text and in Table S2. The height of the boxes (y-axis) represents the value of each coefficient. The whiskers represent the confidence interval computed for each coefficient, and the stars refer to the p-value for each coefficient: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Model equation: y = 3.0 + 0.35·X1 + 0.84·X2 + 0.13·X3 − 0.21·X4 − 0.18·X3·X4.

Figure 3

Percentage of relative abundance of each glycosylation site in the conjugation of RNase A with 19, 20, or 21. Site 1 was considered to be the glycosylation site of both the N-terminal amino group and the ε-amino group of the lateral chain of K1.

Figure 4

EIXs for doubly charged ions corresponding to N-terminal peptide/glycopeptide of RNase A. Pink trace: peptide conjugated to glutaric acid; blue trace: unmodified peptide; green trace: peptide conjugated to one mannose units; red trace: peptide conjugated to two mannose units; black trace: peptide conjugated to three mannose units.

Figure 5

Percentage relative abundance of each glycosylation site in the conjugation of Ag85B wt and -dm with 21.