1. Custom Glycosylation of Cells and Proteins Using Cyclic Carbamate-Derivatized Oligosaccharides 
Marek W.J. Whitehead, Nikolay Khanzhin, Lubor Borsig, Thierry Hennet 
Cell Chemical Biology 
Volume 24, Issue 11, Pages e3 (November 2017)
DOI: /j.chembiol
Copyright © 2017 Elsevier Ltd Terms and Conditions

2. Cell Chemical Biology 2017 24, 1336-1346. e3DOI: (10. 1016/j. chembiol
Copyright © 2017 Elsevier Ltd Terms and Conditions

3. Figure 1 Synthesis of Oligosaccharide-Cyclic Carbamates
The oligosaccharides 2-fucosyllactose, 3-fucosyllactose, 3-sialyllactose, and 6-sialyllactose were peracetylated and then anomerically brominated. The resulting peracetylated α-bromides were then inverted into β-azides, which could then be converted into β-cyclic carbamate derivatives. 3-Sialyl-3-fucosyllactose-cyclic carbamate was synthesized by trans-sialydating the unprotected 3-fucosyllactose-azide with the trans-sialidase from Trypanosoma cruzi before converting it to the β-cyclic carbamate derivative.
Cell Chemical Biology  , e3DOI: ( /j.chembiol )
Copyright © 2017 Elsevier Ltd Terms and Conditions

4. Figure 2 Coating Cells with Oligosaccharide-Cyclic Carbamates
(A and B) MC-38 cells were coated with 2FL- or 3FL-cyclic carbamate or left uncoated, then stained with fluorescein-isothiocyanate-labeled UEA (A) or AAL (B) before being analyzed by flow cytometry. Unstained cells (red lines), uncoated cells (blue lines), 2FL-coated cells (orange lines), 3FL-coated cells (green lines).
(C, D, F, and G) MC-38 (C, F, G) and B16 melanoma cells (D) were coated with fucosyllactose-cyclic carbamates or left uncoated. The cells were then labeled with an E-selectin (C, D), L-selectin (F), or P-selectin (G) probe with a human Fc domain, which was detected with a biotin-labeled antibody and streptavidin PE-Cy5 in flow cytometry. Negative control MC-38 cells were not coated or stained with the respective selectin probe. Instead they were stained directly with the Fc domain targeting biotinylated antibody and PE-Cy5-labeled streptavidin. Uncoated cells (blue lines), biotin anti-IgG + streptavidin PE-Cy5 (black lines), 2FL-coated cells (orange lines), 3FL-coated cells (green lines), FSL-coated cells (grey lines).
(E) E-selectin mediated adhesion of MC-38 cells coated with 2FL, 3FL, or FSL after 2 h agitation at 80 rpm. Data are shown as average and SD of three samples analyzed.
Cell Chemical Biology  , e3DOI: ( /j.chembiol )
Copyright © 2017 Elsevier Ltd Terms and Conditions

5. Figure 3
Oligosaccharide Coating of E. coli K-12 and Complement Factor H Recruitment by 3SL
(A) Fixed E. coli K-12 coated with 3FL, 2FL, 3SL, and 6SL or left uncoated were stained with fluorescein-isothiocyanate-labeled AAL, UEA, MALI, and SNA and examined by fluorescence microscopy. Scale bar, 10 μm.
(B) Binding of complement factor H to fixed E. coli K-12 coated with 2FL, 3FL, 3SL, or 6SL detected by anti-factor H IgG and anti-heavy chain binding antibody coupled to Alexa 488. Uncoated (red lines), 2FL-coated cells (orange lines), 3FL-coated cells (green lines), 3SL-coated cells (black lines), 6SL-coated cells (grey lines).
(C) Titration of E. coli K-12 cells coated with increasing amounts of 3SL (open circles) or 6SL (closed circles) and detection of complement factor H binding. MFI, median fluorescence intensity. Data are shown as averages and SD of three values.
Cell Chemical Biology  , e3DOI: ( /j.chembiol )
Copyright © 2017 Elsevier Ltd Terms and Conditions

6. Figure 4
Cytokine Response of Human Dendritic Cells to Oligosaccharide-Coated E. coli
Cytokine response of human PBMC-derived dendritic cells to oligosaccharide-cyclic carbamate-coated E. coli K-12 at a ratio of 1:1. (A) IFN-γ; (B) IL-1b; (C) IL-12p70; (D) IL-18; (E) IL-6; (F) TNF-α; (G) IL-10; (H) IL-23. Cytokines were measured after 24 h. Unc, uncoated. Data represent averages and SD of three experiments. *p < 0.05.
Cell Chemical Biology  , e3DOI: ( /j.chembiol )
Copyright © 2017 Elsevier Ltd Terms and Conditions

7. Figure 5
Gel Electrophoresis of Oligosaccharide-Coated Proteins and Selectin Binding
(A and B) The oligosaccharides 2FL, 3FL, 3SL, 6SL were coupled to (A) BSA and (B) Staphylococcus aureus protein A. Custom-glycosylated proteins were stained using Coomassie blue. Molecular weight markers are indicated in kDa at the left of each panel.
(C–E) BSA coated with 2FL, 3FL, 3SL, 6SL, FSL was dotted onto nitrocellulose membranes at the indicated amounts (ng). (C) E-selectin binding, (D) P-selectin binding, and (E) L-selectin binding was assessed using selectin probes fused to a human Fc domain, detected by a biotinylated anti-Fc antibody and streptavidin-linked horseradish peroxidase.
Cell Chemical Biology  , e3DOI: ( /j.chembiol )
Copyright © 2017 Elsevier Ltd Terms and Conditions

8. Figure 6
Effect of PI3K Inhibition on Cytokine Production in LPS Stimulated Dendritic Cells Pre-treated with Glycosylated BSA
FSL-modified BSA was added to PBMC-derived human dendritic cells at the indicated concentrations (μg/mL). Some cells were treated with wortmannin (labeled with W) before the addition of FSL-modified BSA. Finally the cells were stimulated with LPS. The medium was collected and (A) TNF-α, (B) IL-12p70, (C) IL-1b, (D) IL-6, and (E) IL-10 levels were determined. W, wortmannin; -, untreated; +, stimulated with 500 ng/mL LPS. Data represent averages and SD of three experiments. *p < 0.05.
Cell Chemical Biology  , e3DOI: ( /j.chembiol )
Copyright © 2017 Elsevier Ltd Terms and Conditions

9. Figure 7
Effect of PI3K Inhibition on Cytokine Production Depends on Glycan Chains
Native and glycosylated BSA was added to PBMC-derived human dendritic cells at 250 μg/mL with or without pre-treatment with wortmannin. (A) IL-12p70, (B) IL-6 levels in cell supernatants after 24 h incubation. -, untreated; +, stimulated with 500 ng/mL LPS. Data represent averages and SD of three experiments. *p < 0.05.
Cell Chemical Biology  , e3DOI: ( /j.chembiol )
Copyright © 2017 Elsevier Ltd Terms and Conditions