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9-Azido Analogs of Three Sialic Acid Forms for Metabolic Remodeling Of Supporting Information 9-Azido Analogs of Three Sialic Acid Forms for Metabolic Remodeling of Cell-Surface Sialoglycans Bo Cheng,†,‡ Lu Dong,†,§ Yuntao Zhu,†,‡ Rongbing Huang,†,‡ Yuting Sun,†,‖ Qiancheng You,†,‡ Qitao Song,†,§ James C. Paton, ∇ Adrienne W. Paton,∇ and Xing Chen*,†,‡,§,⊥,# †College of Chemistry and Molecular Engineering, ‡Beijing National Laboratory for Molecular Sciences, §Peking−Tsinghua Center for Life Sciences,‖Academy for Advanced Interdisciplinary Studies, ⊥Synthetic and Functional Biomolecules Center, and #Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Peking University, Beijing 100871, China ∇Research Centre for Infectious Diseases, Department of Molecular and Biomedical Science, University of Adelaide, Adelaide SA 5005, Australia Page S1 Table of Contents: Scheme S1.……………………………………………………….........……………. S3 Figure S1……………………………………………………..………..……………. S3 Figure S2……………………………………………………..………..…………… S4 Figure S3……………………………………………………..………..…………… S4 Figure S4……………………………………………………..………..…………… S5 Figure S5……………………………………………………..………..…………… S6 Figure S6……………………………………………………..………..…………….S7 Figure S7……………………………………………………..………..…………….S8 Figure S8……………………………………………………..………..…………….S9 Experimental Procedures……………………………….…........…………....S10-S27 Table S1………………………………………………..………..…………….S28-S48 Supporting Reference……………………………………………….......………...S49 Page S2 Scheme S1. Synthesis of 9AzNeu5Gc Figure S1: a, b, c, d) Representative scatter plots (FSC vs. SSC) and histograms of flow cytometry analysis on Vero cells treated with 9AzNeu5Gc (a, c) or 9AzNeu5Ac (b, d) at varied concentrations. The scatter plots were gated based on the FSC (forward scatter) and SSC (side scatter) signals for cell debris discrimination. The gated cells were used to analyze cell-surface fluorescence. This figure is related to Figure 1b in the main text. Page S3 Figure S2. Evaluation of metabolic incorporation of 9AzNeu5Gc and 9AzNeu5Ac in CHO (a), HeLa (b), and Jurkat cells (c). The cells were treated with 9AzNeu5Gc or 9AzNeu5Ac at varied concentrations for 24 h, reacted with alkyne-PEG4-biotin and stained with streptavidin−Alexa Fluor 488, followed by flow cytometry analysis. MFI (mean fluorescence intensity) was normalized to that of control cells treated with vehicle. Error bars represent mean ± SD from three independent experiments. Figure S3. HeLa cells were treated with 9AzNeuAc or 9AzNeu5Gc at varied concentrations for 24 h. The treated cells were reacted with DBCO-biotin and stained with streptavidin−Alexa Fluor 647. The nuclei were stained with Hoechst 33342 (blue). The cells were imaged by confocal fluorescence microcopy. DIC, differential interference contrast. Scale bar: 50 m. Page S4 Figure S4. HPLC analysis of metabolic incorporation of 9-azido sialic acid analogs. (a) CHO cells were treated with 9AzNeu5Ac, 9AzNeu5Gc, or 9AzKDN for 24 h. (b) Vero cells were treated with 9AzNeu5Ac at varied concentrations for 24 h. (c) MDCK II cells were cultured with 9AzNeu5Ac at varied concentrations for 24 h. The treated cells were lysed, from which the proteins were precipitated and subjected to acid hydrolysis to release ketosidically bound sialic acids. The released sialic acids were then derivatized with DMB and analyzed by HPLC with fluorescence detection. The symbol (*) indicates the peaks of DMB-azido sialic acid. The dashed box indicates no peak of DMB-9AzNeu5Gc in the 9AzNeu5Ac-treated cells. The quantified incorporation ratios were shown on the right. Page S5 Figure S5. Evaluation of metabolic incorporation of 9AzKDN and 9AzNeu5Ac in A375 (a), CHO (b), BJA-B K20 cultured with FBS in the medium (c), Daudi (d), HeLa (e) and Vero cell (f). The cells were treated with 9AzKDN or 9AzNeu5Ac at varied concentrations for 24 h, reacted with alkyne-PEG4-biotin and stained with streptavidin−Alexa Fluor 488, followed by flow cytometry analysis. The zoomed out bar graph inserted highlights 9AzKDN-labeling. MFI (mean fluorescence intensity) was normalized to that of control cells treated with vehicle. Error bars represent mean ± SD from three independent experiments. Page S6 Figure S6: MS analysis of CMP-9AzKDN from CHO cells treated with 9AzKDN. CHO cell was treated with 4 mM 9AzKDN for 24 h. The lysates were separated using HPAEC-UV and the CMP-9AzKDN fraction corresponding to the peak eluting at 29.4 min in figure 2a was collected and analyzed by MALDI-TOF mass spectrometry using the cationic mode. Page S7 Figure S7. HPAEC-UV/PAD analysis of CMP-9AzKDN from BJA-B K20 cells treated with 4 mM 9AzKDN or vehicle for 24 h in medium containing FBS. Pure CMP-9AzKDN was shown as the standard. Pure CMP-9AzKDN was co-injected with the 9AzKDN-treated sample to further validate the CMP-9AzKDN peak. Page S8 Figure S8. Competitive metabolic incorporation of azido sialic acid analogs with Neu5Ac in CHO and PA-1 cells. (a) CHO cells were treated with 4 mM 9-azido sialic acid analog (9AzKDN, 9AzNeu5Gc or 9AzNeu5Ac, respectively) and Neu5Ac at varied concentration for 24 h. (b) PA-1 cells were treated with 4 mM 9-azido sialic acid analog (9AzKDN or 9AzNeu5Ac, respectively) and Neu5Ac at varied concentration for 24 h. The treated cells were reacted with alkyne-PEG4-biotin via BTTAA-assisted CuAAC and stained with streptavidin−Alexa Fluor 488, followed by flow cytometry analysis. MFI (mean fluorescence intensity) was normalized to that of control cells treated with vehicle. Error bars represent mean ± SD from three independent experiments. Page S9 Experimental Procedures General materials. Reagents for chemical synthesis were obtained from commercial sources and used as directly without further purification unless otherwise noted. KDN1, Neu5Gc2, 9AzKDN3, 9AzNeu5Ac4, CMP-Neu5Ac5, CMP- 9AzNeu5Ac6and CMP-9AzKDN5 were synthesized as previously described. Biotin- PEG4-alkyne, DBCO-biotin and Cy5 Alkyne were purchased from Click Chemistry Tools (Scottsdale, AZ, USA), streptavidin-Alexa Fluor 488 and streptavidin-Alexa Fluor 647 were purchased from Invitrogen (Carlsbad, CA, USA), BCA Protein Assay Kit were obtained from Pierce. EDTA-free protease inhibitor cocktail and Nutridoma SP were purchased from Roche. For metabolic incorporation experiments, azido sialic acids were dissolved in PBS (500 mM) and pH was adjusted to 7.4 using NaOH solution, the stock was kept at 4 oC. CMP-9AzKDN was dissolved in PBS (10 mM) and kept at -80 oC. Oregon-Green-labelled SubAB (OG-SubAB) was prepared according to a reported procedure.7 Cell culture. CHO cells (Chinese hamster ovary cell), HeLa cells, MDCK Ⅱ cells, A375 cell and Vero cells were grown in DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 10% FBS (fetal bovine serum), 100 units/mL penicillin and 100 µg/mL streptomycin. PA-1 cells were incubated in MEM supplemented with 10% FBS (fetal bovine serum), 100 units /mL penicillin and 100 µg/mL streptomycin. BJA-B K20 cells (a gift from Prof. Michael Pawlita), Daudi cells and Jurkat cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 100 units /mL penicillin and 100 µg /mL streptomycin. All the cells were incubated at o 37 C under 5% CO2 in a water-saturated atmosphere. To obtain BJA-B K20 cell in serum-free conditions, the cells were grown carefully for two passages in serum free Page S10 medium (RPMI-1640 with 2 mM L-glutamine containing 1x Nutridoma SP, 50 units/mL penicillin, and 50 μg/mL streptomycin) to deplete endogenous sialic acid. Flow cytometry analysis. After cells were incubated in 6-well plates with azido sialic acids at the indicated concentrations for 24 h. The cells were harvested with trypsin, transferred and distributed into 96-well V-bottomed plate, pelleted (800 g, 6 min, 4 oC), and washed with ice-chilled PBS containing 1% FBS for three times. The pelleted cells were then suspended in PBS containing 0.5% FBS, 50 M alkyne- PEG4-biotin, BTTAA-CuSO4 (50 M CuSO4, BTTAA: CuSO4 = 6:1), and 2.5 mM L- sodium ascorbate at room temperature. After 5 min, the reaction was quenched by adding 1 mM BCS (bathocuproine disulphonate). The cells were washed with cold PBS containing 1% FBS for three times, and incubated with chilled PBS containing 1% FBS and 1 µg/mL Streptavidin-Alexa Fluor 488 for 30 min. After three washes with chilled PBS containing 1% FBS, the cells were suspended in chilled PBS containing 1% FBS and used for FACS (fluorescence-activated cell sorting) analysis on a BD FACSCalibur Flow Cytometer system or BD AccuriTM C6 flow cytometer. In-gel fluorescence scanning. The cells treated with sialic acid probes at the indicated concentrations or vehicle for 24 hours were harvested by trypsin, washed three times with ice-chilled PBS (800 g, 6 min). Then the pelleted cells were lysed in modified RIPA lysis buffer (1% Nonidet P 40, 1% sodium deoxycholate, 0.1% SDS, 50 mM triethanolamine, pH=7.4, 150 mM NaCl, EDTA-free Piercent HaltTM protease inhibitor cocktail, 1 tablet/50 mL). Protein concentrations in the homogenous lysis was normalized to 1 mg/ mL with lysis buffer. The normalized samples were then reacted with 100 µM alkyne-Cy5 in a 60 µL reaction containing premixed BTTAA-CuSO4 complex (50 µM CuSO4, BTTAA: CuSO4 in a 2:1 molar ratio) and 2.5 mM L-sodium ascorbate (freshly prepared). The samples were vortexed for 2 h at 4 °C and resolved on 10% SDS-PAGE gels. The gel was washed in destaining solution (50% methanol, 40% H2O, 10% acetic acid) for 5 min, followed Page S11 by washing in water for another 5 min. After that, the in-gel fluorescence was scanned on a Typhoon FLA 9500 laser scanner (GE Healthcare, USA). Sialylated glycoproteomic identification. For sialylated glycoproteomic identification with three 9-azido sialic acid probes in PA-1 cells, we used a reported procedure.8 Briefly, PA-1 cells treated with azido sialic acid were washed three times in ice-chilled PBS, pelleted and lysed in RIPA buffer. Homogeneous cell lysis was obtained by centrifugation (10,000 × g, 10 min) to remove cell debris. BCA assay was used to determine protein concentration. 5 mg proteins in 5 mL RIPA buffer were incubated with 100 μM alkyne-PEG4-biotin, premixed BTTAA-CuSO4 complex (100 μM CuSO4, BTTAA: CuSO4 at a molar ratio of 2:1), and 2.5 mM L- sodium ascorbate (freshly prepared) for 1 h.
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