Design, Synthesis, and Characterization of Stapled Oligosaccharides

Abstract

Stapling short peptides to lock specific conformations and thereby obtain superior pharmacological properties is well established. However, similar concepts have not been applied to oligosaccharides. Here, we describe the design, synthesis, and characterization of the first stapled oligosaccharides. Automated assembly of β-(1,6)-glucans equipped with two alkenyl side chains was followed by on-resin Grubbs metathesis for efficient ring closure with a variety of cross-linkers of different sizes. Oligosaccharide stapling increases enzymatic stability and cell penetration, therefore opening new opportunities for the use of glycans in medicinal chemistry.

Figure 1

Peptide stapling vs glycan stapling: structural and chemical considerations. (A) α-Helical peptide model consisting of an l-Ala-hexamer. The amino acids commonly used for stapling are highlighted in light blue. (B) Examples of chemical strategies used for peptide stapling. (C) Helical structure of a β-(1–6)-glucose hexamer (minimal energy conformation) highlighting the monosaccharides located at the same face and the multiple combinations available for stapling. (D) Schematic stereochemical representation of the optimal combination of residues proposed for glycan stapling of helical β-(1–6)-glucans.

Figure 2

Synthesis of stapled oligosaccharides employing Grubbs metathesis. (A) Schematic representation of the substrates, AGA, and off-resin methodologies to afford stapled glycans. (B) Summary of BBs, methods, and final yields of all stapled and linear glycans. (C) HPLC traces (after microcleavage) of the glycan with the shortest cross-linkerbefore (5a) and after (6a) RCM. *No RCM was executed to obtain an acyclic glycan bearing hydrocarbon linkages. GI: Grubbs’ first-generation catalyst. GII: Grubbs’ second-generation catalyst.

Figure 3

Cell penetration studies. (A) Reaction of glycans 8d, 9, and 10 with fluorescein-NHS (1.5 equiv) and Et₃N (3 equiv) in DMF (2 mL) for 2 h, in the formation of the fluorescein-labeled glycans 11 (46% yield), 12 (58% yield), and 13 (66% yield). (B) Representative flow cytometry histogram of cell penetration after 3 h of incubation at 37 °C of Jurkat cells with glycans 11, 12, and 13. (C) Quantification of flow cytometry of the cell penetration study of glycans 11, 12, and 13. Values represent mean ± SEM. (D) Confocal fluorescence microscopy images of Jurkat cells incubated with the glycans 11, 12, and 13 for 3 h at 37 °C. Scale bars correspond to 5 μm. (E) Quantification of confocal fluorescence microscopy. Values represent mean ± SEM. Differences were tested for significance using one-way ANOVA followed by Tukey’s post hoc test with (****) p < 0.0001.

Figure 4

Analysis of the enzymatic stability of stapled and linear glycans. (A) Schematic representation of the hydrolysis of 8d, 9, and 10 with a β-endoglucosidase. (B) Optimal experimental conditions for comparative hydrolysis. (C) Enzymatic hydrolysis rates of stapled glycan 8d (purple), alkylated glycan 9 (red), and linear glycan 10 (blue) during enzymatic degradation, highlighting the different half-lives.