Synthesis of the Pentasaccharide Unit of the Pseudomonas aeruginosa Exopolysaccharide Psl Conjugation with CRM197, and Evaluation of Antigenicity in a QS-21/Pam3CSK4-Liposomal Formulation
Abstract
:1. Introduction
2. Results and Discussion
2.1. Synthesis of the Aminooxy Pentasaccharide 1
2.2. Synthesis of Psl Conjugates with CRM197, BSA, and Biotin
2.3. Vaccine Preparation Using Pam3CysSK4-QS-21 Liposomes and Psl-CRM197 Conjugate (16)
2.4. Vaccination Results
3. Experimental Section
3.1. General Methods
3.2. O-Succinimidyl-3-O-(4-Methoxybenzyl)-4,6-O-Benzylidene-2-Benzoyl-β-D-Glucopyranoside (12d)
3.3. O-Succinimidyl-4,6-O-Benzylidene-2-Benzoyl-β-D-Glucopyranoside (4)
3.4. Tolyl-2-O-Benzyl-4,6-O-Benzylidene-3-O-Napthyl-1-Thio-α-D-Mannoyranoside (9)
3.5. Tolyl-[2-O-Benzyl-4,6-O-Benzylidene-3-O-Napthyl-β-D-Mannopyranosyl]-(1→3)-2-O-Benzoyl-4-O-Benzyl-1-Thio-α-L-Rhamnopyranoside (14)
3.6. Tolyl-[2-O-Benzyl-4,6-O-Benzylidene-β-D-Mannopyranosyl]-(1→3)-2-O-Benzoyl-4-O-Benzyl-1-Thio-α-L-Rhamnopyranoside (6)
3.7. Tolyl-(2,3,4,6 Tetra-O-Acetyl)-α-D-Mannopyranosyl)-(1→2)-2-O-Benzyl-4,6-O-Benzylidene 1-Thio-α-D-Mannoyranoside (5)
3.8. Tolyl-(2,3,4,6 Tetra-O-Acetyl)-α-D-Mannopyranosyl)-(1→2)-2-O-Benzyl-4,6-O-Benzylidene (1→3)-[2-O-Benzyl-4,6-O-Benzylidene-β-D-Mannopyranosyl]-(1→3)-2-O-Benzoyl-4-o-Benzyl-1-Thio-α-L-Rhamnopyranoside (3)
3.9. N-Succinimidyl-(2,3,4,6 Tetra-O-Acetyl)-α-D-Mannopyranosyl)-(1→2)-2-O-Benzyl-4,6-O-Benzylidene (1→3)-[2-O-Benzyl-4,6-O-Benzylidene-β-D-Mannopyranosyl]-(1→3)-2-O-Benzoyl-4-O-Benzyl-1-Thio-α-L-Rhamnopyranosyl]-(1→3)-2-O-Benzoyl-4,6-O-Benzylidene-β-D-Glucopyranoside (2)
3.10. Aminooxy-(α-D-Mannopyranosyl)-(1→2)-(β-D-Mannopyranosyl)-(1→3)-(β-D-Mannopyranosyl)-(1→3)-(α-L-Rhamnopyranosyl)-(1→3)-β-D-Glucopyranoside (1)
4. Synthesis of Psl-Conjugates
4.1. Methoxy-Squaramido-(α-D-Mannopyranosyl)-(1→2)-(β-D-Mannopyranosyl)-(1→3)-(β-D-Mannopyranosyl)-(1→3)-(α-L-Rhamnopyranosyl)-(1→3)-β-D-Glucopyranoside (15)
4.2. Synthesis of Psl-CRM197 Conjugate (16)
4.3. Synthesis of Psl-Biotin Conjugate for ELISA Coating (18)
4.4. Mice and Vaccination
4.5. Spleen Cell Isolation
4.6. ELISA
4.7. Whole Cell ELISA Assay
4.8. Opsonophagocytic Killing Assay
4.9. Cell Proliferation Using the MTT Assay
4.10. Single-Color Murine IgG ELISPOT
4.11. Double-Color Murine IFN-γ/IL-17 ELISPOT
5. Statistics
6. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Formulation | DPMG | DMPC | Cholesterol | Pam3CysSK4 | QS-21 |
---|---|---|---|---|---|
mol % | 4.4 | 40 | 54.2 | 1.5 | |
Amount (mg) | 0.909 | 8.135 | 6.288 | 0.733 | 0.010 |
Molecular weight (g/mol) | 688.8 | 677.9 | 386.7 | 1631 | 1990 |
Samples | DPMG (mg) | DMPC (mg) | Cholesterol (mg) | Pam3CysSK4 (mg) | QS-21 (mg) | 16 (mg) |
---|---|---|---|---|---|---|
QS-21 liposome (Control) | 0.909 | 8.135 | 6.288 | 0.733 | 0.01 | - |
QS-21 + Psl-CRM197 liposome | 0.909 | 8.135 | 6.288 | 0.733 | 0.01 | 0.28 |
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Bokolo, U.C.; Dissanayake, R.; Ghosh, S.; Nada, S.; Obadawo, B.S.; Prestwich, E.G.; Wall, K.A.; Sucheck, S.J. Synthesis of the Pentasaccharide Unit of the Pseudomonas aeruginosa Exopolysaccharide Psl Conjugation with CRM197, and Evaluation of Antigenicity in a QS-21/Pam3CSK4-Liposomal Formulation. Molecules 2025, 30, 1720. https://doi.org/10.3390/molecules30081720
Bokolo UC, Dissanayake R, Ghosh S, Nada S, Obadawo BS, Prestwich EG, Wall KA, Sucheck SJ. Synthesis of the Pentasaccharide Unit of the Pseudomonas aeruginosa Exopolysaccharide Psl Conjugation with CRM197, and Evaluation of Antigenicity in a QS-21/Pam3CSK4-Liposomal Formulation. Molecules. 2025; 30(8):1720. https://doi.org/10.3390/molecules30081720
Chicago/Turabian StyleBokolo, Uzoamaka Clara, Ravindika Dissanayake, Samir Ghosh, Shadia Nada, Babatunde S. Obadawo, Erin G. Prestwich, Katherine A. Wall, and Steven J. Sucheck. 2025. "Synthesis of the Pentasaccharide Unit of the Pseudomonas aeruginosa Exopolysaccharide Psl Conjugation with CRM197, and Evaluation of Antigenicity in a QS-21/Pam3CSK4-Liposomal Formulation" Molecules 30, no. 8: 1720. https://doi.org/10.3390/molecules30081720
APA StyleBokolo, U. C., Dissanayake, R., Ghosh, S., Nada, S., Obadawo, B. S., Prestwich, E. G., Wall, K. A., & Sucheck, S. J. (2025). Synthesis of the Pentasaccharide Unit of the Pseudomonas aeruginosa Exopolysaccharide Psl Conjugation with CRM197, and Evaluation of Antigenicity in a QS-21/Pam3CSK4-Liposomal Formulation. Molecules, 30(8), 1720. https://doi.org/10.3390/molecules30081720