Xyloside Primed Glycosaminoglycans Co ... - ACS Publications

Article Cite This: J. Med. Chem. 2018, 61, 2937−2948

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Aglycone Ebselen and β‑D‑Xyloside Primed Glycosaminoglycans Cocontribute to Ebselen β‑D‑Xyloside-Induced Cytotoxicity Yang Tang,†,‡,⊥ Siqi Zhang,†,⊥ Yajing Chang,†,‡ Dacheng Fan,† Ariane De Agostini,§ Lijuan Zhang,*,‡ and Tao Jiang*,†,∥ †

Key Laboratory of Marine Drugs, Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266071, P. R. China ‡ Medical Systems Biology Center for Complex Diseases, Affiliated Hospital of Qingdao University, Qingdao 266003, P. R. China § Department of Gynecology and Obstetrics, Geneva University Hospitals and University of Geneva, Geneva 14, Switzerland ∥ Laboratory for Marine Drugs and Bioproducts of Qingdao National of Laboratory for Marine Science and Technology, Qingdao 266003, P. R. China S Supporting Information *

ABSTRACT: Most β-D-xylosides with hydrophobic aglycones are nontoxic primers for glycosaminoglycan assembly in animal cells. However, when Ebselen was conjugated to D-xylose, D-glucose, Dgalactose, and D-lactose (8A−D), only Ebselen β-D-xyloside (8A) showed significant cytotoxicity in human cancer cells. The following facts indicated that the aglycone Ebselen and β-Dxyloside primed glycosaminoglycans co-contributed to the observed cytotoxicity: 1. Ebselen induced S phase cell cycle arrest, whereas 8A induced G2/M cell cycle arrest; 2. 8A augmented early and late phase cancer cell apoptosis significantly compared to that of Ebselen and 8B−D; 3. Both 8A and phenyl-β-D-xyloside primed glycosaminoglycans with similar disaccharide compositions in CHO-pgsA745 cells; 4. Glycosaminoglycans could be detected inside of cells only when treated with 8A, indicating Ebselen contributed to the unique property of intracellular localization of the primed glycosaminoglycans. Thus, 8A represents a lead compound for the development of novel antitumor strategy by targeting glycosaminoglycans.

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INTRODUCTION Glycosaminoglycans (GAGs) are hexosamine-containing linear polysaccharides. Two major types of GAGs are heparan sulfate (HS) and chondroitin sulfate (CS) consisting of repeating disaccharide of hexosamine (GlcN/GlcNAc for HS and GalNAc for CS) and hexuronic acid (GlcA and IdoA for both HS and CS). HS and CS share the same initiating tetrasaccharide sequence (GlcA-Gal-Gal-Xyl) where the xylose links to the core proteins of proteoglycans. Biosynthesis of GAG chains can take place independently of proteoglycan core proteins by priming on β-D-xylosides, where the hydrophobic aglycones of β-D-xylosides penetrate cell membranes to initiate the GAG assemble on xylosides in animal cells.1 Because of the immense structural diversity of GAG chains, GAGs play important roles in organizing the extracellular matrix, in activating multiple signaling pathways by serving as scaffolds for the interactions between growth factors/cytokines and their receptors in cytokinesis and in cell division.2−7 Particularly, it is well-documented that GAG biosynthesis dysfunctions are common in many pathological conditions, including tumor progression and inflammation.8−11 Considering that the dysfunctions of GAG biosynthesis are common in many © 2018 American Chemical Society

pathological conditions, the exploitation of xylosides for drug development are reasonable and promising. Some xylosides have reported antithrombosis,12 cell morphology,13,14 angiogenesis,15 and antitumor effects.16 Most β-D-xylosides with hydrophobic aglycones are not toxic to animal cells up to 100 μM concentrations.17,18 Mani et al. reported that the xyloside 2-(6-hydroxynaphthyl)-D-xyloside (XylNapOH), in contrast to 2-naphthyl-D-xyloside (XylNap), specifically reduces tumor growth both in vitro and in vivo,19−21 indicating the subtle differences in aglycone structures lead to significant difference in tumor biology. The same research group recently reported that the CS chains primed on both XylNapOH and XylNap from HCC70 cells have similar structure and similar cytotoxicity, indicating the CS chains but not the difference in aglycones are responsible for the observed cytotoxicity. In contrast, despite the HS chain primed on both XylNapOH and XylNap from HCC70, cells have similar disaccharide compositions; the XylNap-primed HS chains inhibit the cytotoxic effect of the XylNapOH- and Received: December 14, 2017 Published: March 27, 2018 2937

DOI: 10.1021/acs.jmedchem.7b01835 J. Med. Chem. 2018, 61, 2937−2948

Journal of Medicinal Chemistry

Article

Scheme 1. Synthesis of Compound 8A−D

have anticancer properties by inhibiting focal adhesion kinase (FAK), AKT-1, and protein kinase C-a (PKC-a).32 In current study, we conjugated the known cytotoxic compound Ebselen to D-xylose and three other monosaccharides to synthesize Ebselen β-D-glycosides (8A, 8B, 8C, and 8D). We found only Ebselen β-D-xyloside (8A) showed significant cytotoxicity with IC50 ranging from 6.4 to 7.3 μM in four human cancer cell lines tested. Most importantly, we discovered for the first time that the aglycone and β-D-xyloside primed glycosaminoglycan chains co-contributed to the Ebselen β-D-xyloside-induced cytotoxicity.

XylNap-primed CS chains on HCC70 cells, whereas the XylNapOH-primed HS is inactive, which demonstrates that the difference in the aglycones XylNapOH and XylNap are responsible for the observed biological activities.22 However, the underlying molecular mechanisms are largely unknown. Thus, our goal is to develop novel β-D-xylosides by conjugating known cytotoxic compound to xylose in order to understand the role of the aglycone and primed GAGs in contributing to βD-xyloside-dependent cytotoxicity in cancer cells. In the current study, we chose cytotoxic Ebselen as an aglycone. Ebselen is a hydrophobic selenoorganic compound, potently inhibits lipid peroxidation through a glutathione peroxidase-like action,23,24 and exerts a wide spectrum of biological activities, ranging from antioxidant, cytoprotective, neuroprotective, and anti-inflammatory. Moreover, Ebselen also inhibits other enzymes such as cyclooxygenases, lipoxygenases, and indoleamine 2,3-dioxygenase, which play a broad functional role in cancer signaling, as well as the regulation of the immune response.25−30 Furthermore, it has been reported that Ebselen sensitizes glioblastoma cells to Tumor Necrosis Factor (TNFa)-induced apoptosis through NF-kB downregulation.31 In prior studies, we have observed that the benzoisoselenazolone-fragment containing compounds derived from Ebselen

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RESULTS Chemistry. The general method for the synthesis of the four glycosyl conjugated Ebselen is shown in Scheme 1. We used peracetylated xylose, glucose, galactose, and lactose as glycosyl donors and coupled them to 2-chloroselenobenzoyl chloride to generate the glycosyl conjugated Ebselen compounds. Cytotoxicity. Compound 8A Inhibited the Growth of Human Cancer Cells. The cytotoxicity of the Ebselen, 8A, 8B, 8C, and 8D was studied with a negative control condition of 0.1% DMSO in four human cancer cell lines including two lung 2938



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To study how 8A affected the cell cycle, colon cancer cells HCT 116 were treated with 8A, 8B, 8C, 8D, or Ebselen, respectively, for 24 h (Figure S2A). Ebselen (40 μM) caused cell cycle arrest at S phase, as expected. In contrast, 8A at 12.5 μM concentration increased G2/M cell population to 45.2%, 1.3-fold increase of that of the control cells, and 8A at 15 μM concentrations further increased G2/M cell population to 51.9%. At 20 μM concentration, 8B, 8C, and 8D had similar effect on cell cycle as 8A at 12.5 μM (Figure 2A). For the apoptosis analysis, human colon cancer cells HCT 116 were stained with Annexin V/propidium iodide and analyzed by flow cytometry. Viable cells (FITC-negative) and early apoptotic cells (FITC-positive) were PI-negative, whereas late apoptotic and necrotic cells were PI-positive and FITCpositive. As shown in Figure S2B, only viable cells were observed in the untreated control cells (K3), whereas the 8A treatment increased the percentage of both early and late apoptotic cells significantly. The data in Figure 2B showed that 8A at 7.5 μM increased early apoptotic cell population by 27.7fold compared to that of control cells in HCT 116 cells. When the concentration of the 8A was increased to 12.5 μM, the populations of early and late apoptotic cells were further increased to 10.97% and 13.18%, respectively. In contrast, 40 μM Ebselen, 8B, 8C, or 8D induced less early and late apoptotic cells compared to that of 8A at 7.5 μM (Figure 2B). Glycochemistry. Compound 8A Primed Both CS and HS Chains. Compound 8A was more toxic (Figure 1) with much lower IC50 values (Table 1) in four different human cancer cell lines compared to that of Ebselen, 8B, 8C, or 8D. Compound 8A also affected cell cycle and induced apoptosis in a way different from that of Ebselen, 8B, 8C, or 8D (Figure 2). Theoretically, 8A could prime for GAG chains whereas Ebselen, 8B, 8C, or 8D could not, which suggested that the GAG priming ability of 8A might contribute to the observed cytotoxicity. This was tested using a mutant Chinese hamster ovary (CHO) cell line pgsA-745 that lacks active xylosyltransferase.33 As a consequence, CHO pgsA-745 cells cannot substitute xylose on the hydroxyl group of serine on proteoglycan core proteins, thus defective in GAG biosynthesis. However, exogenous supply of β-xylosides in cell culture media could bypass the xylosyltransferase deficiency and stimulate a 3to 5-fold increase in GAG biosynthesis in CHO pgsA-745 cells compared to wild-type CHO K1 cells. In our experiment, the CHO pgsA-745 cells were incubated with 0.1% DMSO (negative control) or with 10 μM of either 8A or phenyl-β-Dxyloside (positive control) with a final 0.1% DMSO in cultural medium supplemented with additional 100 mg/L sodium sulfate. After 48 h of incubation, the GAG chains were isolated by anion exchange resins from cells and their culture media. After complete hydrolysis, the amount of GlcN and GalN in each GAG sample was quantified. The results in Figure 3A showed that the amount of GlcN and GalN detected in the GAGs extracted from the negative control cells (Figure S3A, blue tracer) were barely detectable, which was consistent with the original report about the CHO pgsA-745 cells.33 The amount of GlcN and GalN detected in the GAGs extracted from the 8A-treated CHO pgsA-745 cells had 12.2-fold increase in GlcN and 27.6-fold increase in GalN compared to that of the negative control, indicating 8A stimulated GAG biosynthesis in CHO pgsA-745 cells (Figure S3A, green tracer). However, the positive control, i.e., phenyl-β-D-xyloside-treated CHO pgsA745 cells, stimulated more GAG biosynthesis based on the amount of GlcN and GalN detected (Figure S3A, red tracer).

cancer cell lines A549 and H1299 and two colon cancer cell lines HCT116 and HT29, and their IC50 values are shown in Table 1. Compared to the cytotoxic effect of Ebselen, all four Table 1. IC50 of Ebselen (Ebs) and 8A−D in Four Different Types of Human Cancer Cells IC50 (μM) compd Ebs 8A 8B 8C 8D

A549 16.8 7.0 20.4 25.2 36.5

± ± ± ± ±

0.45 0.37 1.41 1.41 1.35

H1299 27.7 7.0 19.0 21.6 34.0

± ± ± ± ±

2.24 0.49 0.15 0.26 1.33

HCT116 50.0 6.4 18.0 17.8 24.2

± ± ± ± ±

0.90 0.05 0.15 0.25 0.58

HT29 50.4 7.3 11.9 19.3 32.3

± ± ± ± ±

0.42 0.15 0.58 0.86 0.96

Ebselen-β-D-glycosides were more toxic to colon cancer cells. Most importantly, Ebselen β-D-xyloside (8A) had much higher increased cytotoxicity toward all four human cancer cell lines tested compared to Ebselen and other Ebselen-β-D-glycosides (8B, 8C, and 8D). Cancer cell proliferation was significantly suppressed in the presence of 8A in a concentration-dependent manner (Figure 1). Compound 8A had the same IC50 values of

Figure 1. Growth inhibitory effect of 8A on human cancer cells A549, H1299, HCT116, and HT29. Human cancer cells were seeded in 96well plates, and after 24 h, cells were treated with serial concentrations of 8A for 48 h of treatments, followed by adding resazurin to the media for 16 h. The fluorescent signal is monitored using 544 nm excitation wavelength and 595 nm emission wavelength by Spectramax M5 plate reader (Molecular Devices). The relative fluorescence unit (RFU) generated from the assay is proportional to the number of living cells in each well. Data are mean ± range of duplicates. The results are representative of at least three independent experiments (p < 0.01).

7.0 μM in both lung cancer cell lines A549 and H1299. The IC50 values for 8A were 6.4 and 7.3 μM in two colon cancer cell lines HCT116 and HT29 cells, respectively (Table 1). Effects of 8A on Cell Cycle and Apoptosis. To test if the cytotoxicity of 8A was due to the presence of the cytotoxic Ebselen in the xyloside, we compared the extent of apoptosis and cell cycle distribution by flow cytometry analysis among 8A, Ebselen, 8B, 8C, and 8D. 2939



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Figure 2. Effect of Ebselen and its glycosyl conjugates on cell death and cell cycle distribution of HCT116 cells. (A) Cells were seeded in 6-well plates for 24 h, and then the cells were treated with 0.1% DMSO, 40 μM Ebselen, 7.5 μM/12.5 μM 8A, or 20 μM 8B−D. After 24 h of treatments, cells were harvested and subject to cell cycle analyses as described in the Experimental Section. Statistical analysis was conducted among control and treated groups in G0/G1, S, and G2/M phases, separately. (B) HCT116 cells were seeded in 6-well plates for 24 h, and then the cells were treated with 0.1% DMSO, 40 μM Ebselen, 7.5 μM or 12.5 μM 8A, or 20 μM 8B−D. After 48 h of treatments, cells were harvested and subject to apoptosis analyses as described in the Experimental Section. Statistical analysis was conducted among control and treated groups in early apoptosis and late apoptosis, separately.

Since fetal bovine serum used in cell culture media contains a significant amount of GAGs,34 both GlcN and GalN were detected in the GAGs extracted from the media of the negative control (Figure S3B, blue tracer). Compound 8A treatment did not make a big increase in the amount of both GlcN and GalN in GAGs extracted from the culture media compared to the negative control (Figure S3B, red tracer vs blue tracer). However, there was significant increase in the amount of GalN in GAGs extracted from the culture media of phenyl-β-Dxyloside-treated CHO pgsA-745 cells. Based on the GlcN and GalN quantification data, phenyl-β-D-xyloside was more efficient at priming GAG chains, and most of them were secreted into the cell culture media compared to 8A. In contrast, only a limited fraction of the GAG chains primed by 8A was secreted into the culture media. The data shown in Figures 1 and 3 indicated that 8A was different from most of the reported β-D-xylosides in three aspects: 1. potent cytotoxicity; 2. limited amount of primed GAGs in cell culture media; and 3. significant amount of cellassociated GAGs. In order to investigate whether the cellassociated GAG chains primed on 8A were located inside of the CHO pgsA-745 cells, we fixed the β-D-xyloside-treated cells by 10% methanol to permeate the cell membrane and make the staining of intracellular GAGs possible. The stained cells were then analyzed by flow cytometry and confocal microscope independently. Figure 4A is the images under the confocal microscope illustrating the treated CHO pgsA-745 cells doublestained with antibodies against chondroitinase ABC predigested monosulfated nonreducing terminal of CS chains by monoantibody 2B6 and nucleus by DAPI. Theoretically, fluorescence staining intensity on cells with mAb 2B6 should be increased after chondroitinase ABC digestion. In the experiment, we adjusted the exciting laser to an intensity under which the cells treated in control conditions had no visible stain and then used the identical exciting laser to stain the β-D-xyloside-treated cells. Under this condition, cells treated with phenyl-β-D-xyloside

Figure 3. GlcN and GalN quantification of extracted GAG samples. The GAGs samples extracted from the cells (A) or culture medium (B) of the CHO-pgsA745 treated with phenyl-β-D-xyloside or 8A were digested sufficiently in hydrochloric acid to generate respective monosaccharides. The acid hydrolyzed products were labeled with PMP and analyzed by HPLC as described in the Experimental Section. 2940



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Figure 4. Expression of GAGs in CHO pgsA-745 cells. (A,B) Cells were cultured on a glass slide and treated with phenyl-β-D-xyloside or 8A for 24 h. Cells were fixed using 10% ethanol and incubated in PBS (A,B, top panel) with 1 mU chondroitinase ABC (A) or 1 mU heparinase (B), respectively, at 37 °C. The cells were incubated with monoclonal antibody 2B6 of mouse origin that recognizes chondroitinase ABC-treatment generated unsaturated CS structural motif or monoclonal antibody 3G10 of mouse origin that recognizes heparinase-treatment generated unsaturated HS structural motif overnight at 4 °C. For fluorescence labeling, cells were then incubated with Alexa Fluor 488-conjugated antimouse antibody. Cells were counterstained with DAPI for localizing nuclei of the cells. The bar represents 5 μm. (C) Flow cytometry analysis of the stained cells. The DMSO- (black tracer), phenyl-β-D-xyloside- (green tracer), or 8A- (red tracer) treated cells were harvested and fixed, followed by the same staining process with mAb 2B6 or mAb 3G10 followed by staining with Alexa Fluor 488-conjugated antimouse antibody and flow cytometry analysis. The gray shadow represents negative stain.

exhibited little green fluorescence around the nucleus when stained with mAb 2B6 (Figure 4A). In contrast, the green fluorescence with direct mAb 2B6 stain in 8A treated cells was obvious and stronger than that in the phenyl-β-D-xyloside treated cells. After treatment with chondroitinase ABC, the green fluorescence became stronger, as expected, indicating specific CS staining. Similar results were obtained when the antibody against heparin lyase digested HS chains (Mab 3G10) was used for immunostaining the xyloside-treated and untreated CHO pgsA745 cells (Figures 4B) where the fluorescence intensity was strongest in the cells treated with 8A after heparin lyase digestion. To quantify the fluorescent intensity in the xyloside-treated and -untreated CHO pgsA-745 cells after 2B6 and 3G10 plus Alexa 488 labeled secondary antibody staining, we performed flow cytometry analysis on each stained sample (Figure 4C).

Compound 8A treated cells showed the strongest fluorescent intensity followed by that of phenyl-β-D-xyloside-treated and 0.1% DMSO treated cells (control). Overall, results from Figure 4 indicated that 8A-primed GAG chains were more abundant inside CHO pgsA-745 cells. The primed GAG chains could be detected using antibodies that recognize chondroitinase ABC or heparinase generated novel structural motifs after disrupting the cell membrane with 4% formaldehyde. To further confirm the observation that GAGs were localized in the cellular compartment of cells after 8A treatment, we decided to use the cell surface FGF/FGFR/GAG ternary complex formation assay previously used on CHO wild-type and mutant cells35 to detect the 8A primed GAGs inside of CHO pgsA-745 cells. To this end, we chose FGF7 and FGFR2B-Fc fusion protein. We have previously shown that both HS and CS could facilitate FGF7- and FGFR2Bdependent signaling in BaF3 cells. 34 The binding of 2941



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Figure 5. FGF7/GAGs/FGFR2B ternary complex formation on treated CHO cells. Cells were cultured on a glass slide and treated with phenyl-β-Dxyloside or 8A for 24 h. Cells were fixed using 10% ethanol and incubated in PBS or sequentially in FGFR2B-Fc, FGF7, and Protein A-Alexa Fluor 647. The protein A-Alexa Fluor 647-tagged FGFR2B-Fc was monitored under confocal microscope. The arrows mark the observed fluorescence.

Table 2. Quantitative Analysis of Changes in CS Disaccharide Compositions after GAGs from Cell Culture Media of Control, XylPh-, or 8A-Treated CHO pgsA745 Cells Were Treated with Chondroitinase ABC and Labeled with Either 5D-PMP (Control) or 5H-PMP (XylPh or 8A) Followed by LC−MS Analysis ΔUA-GalNAc

control + XylPh control + 8A

Ctrl (5D-PMP) XylPh (5H-PMP) Ctrl (5D-PMP) 8A (5H-PMP)

ΔUA-GalNAc6S

m/z

intensity

relative intensity

718.29 708.23 718.29 708.23

1953.5 21718.1 2643.7 5908.4

8.99 100 44.74 100

FGFR2B-Fc to CHO pgsA-745 cells was monitored by confocal microscopy (Figure 5). The control CHO pgsA-745 cells showed no binding and normal cellular morphology. The cells treated with phenyl-β-D-xyloside also showed no binding. However, under the same detecting condition, we observed the fluorescence signal within the cells treated with 8A. Interestingly, the dividing cells showed a much stronger fluorescent signal, indicating the enrichment of the 8A-primed GAGs in these cells (Figure 5), suggesting the intracellular GAGs might be responsible for the observed cytotoxicity. LC−MS Analysis of PMP-Labeled HS and CS Disaccharides. Quantitative disaccharide compositional analysis of the primed GAG chains is extremely important in understanding the 8A-induced cytotoxicity. However, there is great need for a sensitive, reliable, and quantifiable method for the analysis of the GAG-derived disaccharides from CHO pgsA-745 cells that have grown in different culture conditions. Our laboratory has established LC−MS analytic methods for the quantitative

m/z 798.25 788.18 798.25 788.18

ΔUA-GalNAc4S

intensity

relative intensity

m/z

intensity

relative intensity

490.4 5149.7 555.2 1902.3

9.52 100 29.19 100

798.25 788.18 798.25 788.18

96.9 9631.8 93.9 3231.5

1.01 100 2.9 100

disaccharide compositional analysis of GAGs from cells, tissue, and blood that rely on application of stable isotope labeling.36 Briefly, the 1-phenyl-3-methyl-5-pyrazolone (PMP) can be conveniently introduced into the reducing end of GAG-derived disaccharides in alkaline condition. PMP-labeling offers chromatographic separation of GAG-derived disaccharides and also a strong UV absorbance at 245 nm. We confirmed the identity of each disaccharide qualitatively based on its retention time detected by UV and molecular weights (Mw) detected using MS by injecting PMP-labeled commercial disaccharide standard one at a time. The Mw of PMP-labeled disaccharides was calculated from the sum of the disaccharide and PMP molecular weights, minus water and proton and are listed in Table S1. The mass spectra of the five kinds of PMP-labeled HS disaccharides showed that nonsulfated disaccharide had only an [M − H]− molecular-ion peak, whereas monosulfated disaccharides had both z1 [M − H]− and z2 [M − 2H]2− molecular-ion peaks. All five PMP2942



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labeled HS disaccharides were completely separated using gradient ratio of 10 mM ammonium acetate and acetonitrile as elution solution as described in the Experimental Section. Pair-wised Comparison of the Quantitative Changes in Each CS and HS Disaccharide in 8A- or Phenyl-β-D-xylosideTreated vs 0.1% DMSO-Treated CHO pgsA-745 Cells. In order to quantify GAG disaccharides extracted from different sources, GAGs from the same amount of cellular proteins were digested with either chondroitinase ABC to obtain CS disaccharides or heparinase I, II, III to obtain HS disaccharides. We then used the regular PMP (5H-PMP) to label disaccharides derived from GAGs of 8A- or phenyl-β-D-xyloside-treated CHO pgsA-745 cells and deuterium substituted PMP (5D-PMP) to label the disaccharides derived from GAGs of 0.1% DMSO treated control CHO pgsA-745 cells. We then coinjected the disaccharides with different PMP tagging for the LC−MS analysis. This is an accurate disaccharide quantitative method because the same disaccharide is eluted at the same position after LC separation but with different molecular ion intensities detected by MS that can be quantified pairwise.36,37 All the disaccharides identified by the LC−MS analysis were summarized in Table 2. As shown in Figure 6A, the left peak (708.23) presented the nonsulfated ΔUA-GalNAc labeled by 5H-PMP derived from cells treated with 8A (A), and the right peak (z1 718.29) presented the same nonsulfated ΔUA-GalNAc labeled by 5DPMP from the control cells. Similar increase was observed between phenyl-β-D-xyloside-treated cells and the control cells (Figure S2A). These results confirmed that both 8A and phenyl-β-D-xyloside could effectively prime GAG chains. In contrast, the CHO pgsA-745 cells were unable to synthesize significant amounts of GAGs in the absent of xylosides, which was consistent with a published result.33 In addition, we detected a comparable amount of nonsulfated ΔUA-GalNAc disaccharides from the cell cultural media of both 8A- and 0.1% DMSO-treated cells, which was consistent with our previous published results that fetal bovine serum contains a significant amount of both HS and CS.38 However, the amount of nonsulfated ΔUA-GalNAc from the cell culture media of phenyl-β-D-xyloside-treated cells was 11 times that from the control cells (Figure S2B). The amount of nonsulfated ΔUAGalNAc from the cell culture media of 8A-treated cells was also 2.24 times the control (Figure 6B). As shown in Figure 6C, the peak on the left (z1 788.18) represented the 4-O-sulfated ΔUAGalNAc (ΔUA-GalNAc 4S) from the cells treated with 8A, and the peak on the right (z1 798.25) represented ΔUA-GalNAc4S from the control cells. Based on the MS analysis, 8A-treatment increased the levels of monosulfated disaccharides and 4-O- and 6-O-sulfated ΔUA-GalNAc similar to that of phenyl-β-Dxyloside (Figure S2C). To be precise, we detected 10.5 times of ΔUA-GalNAc6S in phenyl-β-D-xyloside-treated cells and 3.4 times of ΔUA-GalNAc6S in 8A-treated cells compared to that of the control. Furthermore, due to the limited amount of 4-Osulfated ΔUA-GalNAc detected in the 0.1% DMSO-treated control cells, the amount of ΔUA-GalNAc4S was up to 99 times and 34.5 times in phenyl-β-D-xyloside- and 8A-treated cells, respectively. We also digested each GAG sample with heparinase I, II, III and analyzed the HS disaccharides by the LC−MS. As shown in Figure 6D, the most abundant HS disaccharide, nonsulfated ΔUA-GlcNAc tagged by two 5H-PMPs (z1 708.23), was detected in both phenyl-β-D-xyloside- (Figure S2D) and 8Atreated cells. In contrast, the peak on the right (z1 718.29) that

Figure 6. Extracted ion chromatogram and mass spectra of HS- or CSderived disaccharides. (A) ΔUA-GalNAc labeled by 5H-PMP (m/z = 708.23) derived from cells treated with 8A vs ΔUA-GalNAc labeled by 5D-PMP from the control cells (m/z = 718.29). (B) ΔUA-GalNAc labeled by 5H-PMP (m/z = 708.23) derived from culture medium of the cells treated with 8A vs ΔUA-GalNAc labeled by 5D-PMP from the culture medium of control cells (m/z = 718.29). (C) Monosulfated ΔUA-GalNAc labeled by 5H-PMP (m/z = 788.18) derived from culture medium of the cells treated with 8A vs monosulfated ΔUAGalNAc labeled by 5D-PMP from culture medium of the control cells (m/z = 798.25). (D) ΔUA-GlcNAc labeled by 5H-PMP (m/z = 708.23) derived from cells treated with 8A vs ΔUA-GlcNAc labeled by 2943



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glycosides (Figure 2); 3. when CHO-pgsA745 cells were treated with Ebselen β-D-xyloside along with phenyl-β-Dxyloside, we found that both xylosides not only primed GAGs based on monosaccharide quantification of media and cell associated GAGs (Figure 3) but also shared similar disaccharide compositions based on LC−MS analysis of the GAGs after chondroitinase ABC and heparinase I, II, III digestion (Figure 6); 4. GAGs could be detected inside of CHO-pgsA745 cells only in Ebselen β-D-xyloside treated but not phenyl-β-D-xyloside treated cells by fluorescence-facilitated confocal microscope analysis (Figures 4 and 5), indicating Ebselen contributed to the unique property of intracellular localization of the primed GAGs.

Figure 6. continued 5D-PMP from the control cells (m/z = 718.29). LOD means limitation of detection.

theoretically represented nonsulfated ΔUA-GlcNAc tagged by two molecules of 5D-PMPs (z1 718.23) from cells treated with 0.1% DMSO was undetectable. These observations indicated that both 8A and phenyl-β-D-xyloside initiated HS synthesis in CHO pgsA-745 cells.

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DISCUSSION In the current study, the xylosides were dissolved in DMSO and then diluted in cell culture medium before adding to cells. A final concentration of 0.1% DMSO was used in all cell culture experiments including the negative, positive, and 8A-treated cells.39 The reason behind this is that DMSO at high concentration can lyse cells, and therefore, the xylosides dissolved in DMSO should not be added directly to cells. DMSO is also known to cause differentiation of certain cells, which may be associated with a change in GAG biosynthesis.1 It has been reported that fully acetylated xylosides are easier to cross the plasma membrane and get into the endoplasmic reticulum/Golgi network for efficient GAG priming.40,41 Furthermore, we have synthesized over 51 different xylosides with novel aglycones structures during the past and found out that the acetylated compounds had almost identical activity as those deacetylated compounds. Combined with the published knowledge and our previous research, we intentionally left all Ebselen-β-D-glycosides fully acetylated for our current study. Xylosides are powerful tools in studying GAG biosynthesis. Historically, D-xyloside was identified as an initiator of glycosaminoglycans (GAGs) production by the discovery of its ability to restore CS biosynthesis inhibited by puromycin in embryonic chick epiphyseal cartilage.42 Subsequently, it was reported that β-D-xylosides, p-nitrophenyl-β-D-xylose, and 4methylumbelliferyl-β-D-xylose are more effective than D-xylose in enhancing the production of CS.43 By competing with proteoglycan core protein-linked xylose, β-D-xylosides induce the secretion and biosynthesis of protein-free CS chains.44 Similar to the proteoglycan core proteins, the structure of aglycones play an important role in regulating GAG biosynthesis in cells. Indeed, the aglycone moiety of xyloside is largely responsible for the size, type, and fine structure of the primed GAG chains. For initiating CS biosynthesis, phenyl O-β-Dxyloside and S-β-D-xyloside are superior compared to Nxyloside and homo-C-xyloside.45 For initiating HS biosynthesis, 3-estradiol-β-D-xyloside and a series of synthetic and natural βD-xylosides carrying two or more fused aromatic rings as aglycones are potent inducers of both HS and CS biosynthesis in Chinese hamster ovary cells.18,46,47 However, most xylosides reported so far have no cytotoxicity. The reported cytotoxic β-D-xyloside Xyl-2-Nap-6-OH19 has an IC50 value of 130 ± 20 μM vs 7.0 ± 0.37 μM for 8A (Table 1) observed in our study, which indicated 8A was a unique cytotoxic xyloside. We concluded that the aglycone Ebselen and β-D-xyloside primed GAGs co-contributed to the observed cytotoxicity based on the following facts: 1. instead of causing cell cycle arrest at S phase by Ebselen, Ebselen β-D-xyloside induced G2/M cell cycle arrest (Figure 1); 2. Ebselen β-Dxyloside significantly increased early and late phase cancer cell apoptosis compared to Ebselen and other Ebselen β-D-

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CONCLUSION In the current study, we conjugated the known cytotoxic compound Ebselen to D-xylose and three other monosaccharides to synthesize Ebselen β-D-glycosides. We found only Ebselen β-D-xyloside (8A) showed significant cytotoxicity. Most importantly, using the novel immunostaining methods, we discovered for the first time that the aglycone and β-Dxyloside primed glycosaminoglycan chains co-contributed to the Ebselen β- D-xyloside-induced cytotoxicity. We also employed the stable isotope PMP-labeling plus LC−MS analytical approach to quantitatively compare the HS and CS disaccharide structures detected in the xyloside-treated and untreated CHO pgsA-745 cells. The methods and conclusions drawn from current study should be insightful for the development of a novel antitumor strategy by targeting GAGs based on novel cytotoxic β-D-xylosides.

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EXPERIMENTAL SECTION

Chemistry. Commercial reagents and solvents were purchased from Sinopharm Chemical Reagent Company (Shanghai, PRC) and directly used without further purification. All reactions were carried out in glassware, magnetically stirred, and monitored by analytical thin-layer chromatography (TLC) using precoated silica-gel 60 F254 plates (E. Merck). All newly synthesized compounds were characterized with 1H NMR, 13C NMR, and high resolution mass spectrometry. 1H NMR and 13C NMR spectra were obtained on a Propulse 500 MHz spectrometer (Agilent) with tetramethylsilane (Me4Si) as the internal standard, and chemical shifts were recorded in δ values. Mass spectra were recorded on a Q-TOF Global mass spectrometer. Analysis of sample purity was performed on a Waters 2998 Photodiode Array Deteter HPLC system with a YMC Pack ODS-A (5 μm; 10 × 250 mm + guard column). HPLC conditions were as follows: solvent A = water, solvent B = methanol, and flow rate = 1.0 mL/min. Compounds were eluted with a gradient of A/B = 10:90 at 0 min to 100:0 at 25 min. Purity was determined via integration of UV spectra at 254 nm, and all tested compounds have a purity of >95% (Table S2). Synthesis of 2,3,4-Tri-O-acetyl-1-deoxy-β-D-xylosaminitol, 3A. The hydroxyl groups of xylopyranose were protected by acetyl, and 2A was synthesized through bromination and azidation. Then, 2A was reduced by H2 with the catalysis of Pd/C to give 3A as a white powder. The other glycosyl donors 2,3,4,6-tetra-O-acetyl-1-deoxy-β-D-glucamine (3B), 2,3,4,6-tetra-O-acetyl-1-deoxy-β-D-galactosamine (3C), and 2,3,6,2′, 3′, 4′, 6′-heptakis-O-acetyl-1-deoxy-β-D-lactosamine (3D) were also synthesized through acetylation, bromination, azidation, and reduction from starting monosaccharides. The yields of glycosyl donors from starting monosaccharides were 27.0% (3A), 28.3% (3B), 23.7% (3C), and 21.5% (3D). Synthesis of Ebselen β-D-Glycocosides (4−7): Synthesis of 2, 2′Bisselanyldibenzoic Acid (6). Anthranilic acid (4) was generated from phthalimide and 1,3-dihydro-1,3-dioxoisoindole through the reaction in sodium hydroxide and bromine with a yield of 85.5%. Selenium 2944



Article

80.60, 72.50, 71.19, 69.68, 68.00, 62.07, 21.01, 20.93, 20.86, 20.75. ESI MS m/z: 530.0551 [M + H]+, 552.0364 [M + Na]+. Compound 8D 1H NMR (CDCl3). δ 8.05−7.44 (m, 4H, Ar-H), 5.91 (d, 1H, J = 8.7 Hz, C1−H), 5.39 (m, 2H, Lac-H), 5.15 (m, 2H, LacH), 4.99 (dd, 1H, J = 10.6 Hz, Lac-H), 4.53 (d, 1H, J = 8.3 Hz, LacH), 4.51 (d, 1H, J = 12.4 Hz, Lac-H), 4.17−4,08 (m, 3H, Lac-H), 3.91−3.88 (m, 3H, Lac-H), 2.17−1.92 (s, 21H, Ac-H). Compound 8D 13C NMR (CDCl3). δ 170.4, 170.3, 170.2, 170.1, 169.7, 169.5, 169.0, 167.6, 138.6,132.9, 129.1, 126.4, 126.3, 124.2, 101.1, 80.6, 75.9, 75.4, 73.1, 71.7, 70.9, 70.8, 69.0,66.6, 61.8, 60.8, 20.9, 20.8, 20.7, 20.7, 20.6, 20.6, 20.5. ESI MS m/z: 818.1397 [M + H]+. Biology. Cell Culture. The GAG-deficient Chinese hamster ovary cells (CHO) mutants pgsA-745 (xylosyltransferase-deficient) were a gift of Professor Jeffrey D. Esko.33 CHO pgsA-745 cells were grown in F-12 medium supplemented with 5% fetal bovine serum (Hyclone, Salt Lake City, UT), 100 U/mL of penicillin, 0.1 mg/mL of streptomycin, and 100 mg/L sodium sulfate at 37 °C under an atmosphere of 5% CO2 in air and 100% relative humidity. Cells were passaged with trypsin every 3−4 days and revived periodically from frozen stocks. Human lung cancer cell lines A549 and H1299 were obtained from the Chinese Academy of Sciences (Shanghai, China). They were maintained in RPMI 1640 media supplemented with 5% FBS, 100 U/ mL penicillin, and 0.1 mg/mL streptomycin at 37 °C with 5% CO2. Human colon cancer cell lines HCT 116 and HT 29 were obtained from the Chinese Academy of Sciences (Shanghai, China). They were maintained in 5A media supplemented with 5% FBS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin at 37 °C with 5% CO2.48 Cells were passaged with trypsin every 3−4 days and revived periodically from frozen stocks. Cell Proliferation Assay. A549, H1299, HCT116, or HT29 (2000 cells/well) cells were seeded in 96-well plates. After 24 h incubation, the cells were treated with 200 μL of complete media containing serial Ebselen, 8A−D dilutions, or 0.1% DMSO as negative control. After 48 h treatments, to each well was added 20 μL of resazurin (2 mg/mL). The fluorescent signal was monitored using 544 nm excitation and 595 nm emission wavelengths by Spectramax M5 plate reader (Molecular Devices).49 The assay was performed in triplicate for each 8A treatment along with control. The same experiment was repeated twice. Cell Cycle Analysis. HCT116 cells (6 × 104 cells/well, 2 mL/well) were seeded in six-well plates. After 24 h, cells were treated with different concentrations of Ebselen, 8A−D, or 0.1% DMSO in 2 mL of complete media. After another 24 h, media were collected and combined with adherent cells that were detached by brief trypsinization. Cell pellets were resuspended in 70% ethanol at 4 °C overnight. After centrifugation, the supernatant was removed and cells were incubated with 0.5 mL of propidum iodide (PI)/RNase Staining Buffer (BD Biosciences) for 15 min at room temperature. Cell cycle was analyzed using the Beckman cell analyzer FC500-mpl.50 Detection of Apoptosis. Apoptotic cells were quantified by Annexin V/propidium iodide (PI) double staining assay kit (BD Biosciences, San Diego CA, USA). Briefly, HCT116 cells were treated with or without Ebselen and 8A−D in cell culture media containing 0.1% DMSO and harvested as described for the cell cycle analysis. After washing with binding buffer, cells were suspended in 100 μL of binding buffer containing Annexin V and PI, and incubated for 15 min at room temperature. The apoptotic cells were identified using the Beckman cell analyzer FC500-mpl. Confocal Microscopic Analysis of cellular GAGs by Immunostaining. For confocal imaging, CHO pgsA-745 cells were seeded on glass coverslips in complete cell culture media overnight before treated with phenyl-β-D-xyloside or 8A at the concentration of 10 μM with 0.1% DMSO in cell culture media for 24 h at 37 °C. After that, the media were removed, and the cells were fixed with 4% formaldehyde in complete media for 15 min. The fixed cells were washed twice and incubated in PBS (8 g/L NaCl, 0.2 g/L KCl, 1.44 g/L Na2HPO4, 0.24 g/L KH2PO4), 1 mU chondroitinase ABC (BIOKANGTAI, PRC) or 1 mU heparinase I, II, III (BIOKANGTAI, PRC), respectively, for 20 min at 37 °C. After washing with PBS, the cells were incubated in 0.5%

powder (1.6 g) was mixed with cetyltrimethylammonium bromide (CTAB) and 8% sodium hydroxide (in water, 10 mL). NaBH4 solution (in water, 4%, 3 mL) that contained NaOH (0.08 g) was added dropwise into the selenium mixture in nitrogen atmosphere in an ice bath. The reaction was stirred at room temperature for 1 h and then at 90 °C for 0.5 h in Na2Se2. Water (30 mL) and hydrochloric acid (7 mL) were added into anthranilic acid (4, 20 mmol), into which NaNO2 (1.8 g in 5 mL water) was added in an ice bath. The mixture was stirred for 1.5 h to give the desired diazonium salt. Freshly prepared Na2Se2 was mixed with NaOH (2.8 g in 3 mL water), followed by adding diazonium salt solution dropwise under ice bath, after which the reaction was stirred at 40 °C for 2 h. The mixture was then filtered and 6 was precipitated by adding excess HCl (yield 93.2%). Synthesis of 2-Chloroselenobenzoyl Chloride (7). 2,2′-Bisselanyldibenzoic acid (6) was mixed with SOCl2 (30 mL) and heated to 80 °C for 4 h. The excessive SOCl2 was removed by evaporation under vacuum, while the residue was extracted three times with anhydrous nhexane. The organic phases were combined, after which the solvent was evaporated under vacuum to give 7 as a yellow solid (10.1 mmol, yield 54.3%). Synthesis of 2-(2,3,4-Tri-O-acetyl-1-deoxy-β-D-xylopyranosyl)benzo[d][1,2]selenazol-3(2H)-one (8A). 2,3,4-Tri-O-acetyl-1-deoxyβ-D-xylosaminitol (3A, 4 mmol) was dissolved in dry THF (40 mL) by stirring. 2-Chloroselenobenzoyl chloride (7, 4 mmol) and dry Et3N (8 mmol) were dissolved in dry THF (20 mL), respectively. Et3N solutions and 7 were simultaneously added into 3A dropwise and cooled in an ice bath at the same rate, which took approximately 30 min. The reaction was warmed to room temperature followed by overnight stirring. The reaction mixture was filtered and then concentrated, followed by a silicon column to give the final product 8A, a faint yellow powder (1.5 mmol, yield 36.7%, mp: 182−183 °C). Compound 8A 1H NMR (500 MHz, DMSO-d6). δ 8.05−8.04 (d, J = 8.1 Hz, 1H, Ar-H). 7.86−7,84 (d, J = 7.7 Hz, 1H, Ar-H), 7.64−7.61 (t, J = 7.6 Hz, 1H, Ar-H), 7.43−7.40 (t, J = 7.5 Hz, 1H, Ar-H), 5.92−5.90 (d, J = 9.0 Hz, 1H, C1-H), 5.45 (t, J = 9.5 Hz, 1H, Xyl-H), 5.18 (t, J = 9.2 Hz, 1H, Xyl-H), 5.05 (td, J = 10.2, 5.6 Hz, 1H, Xyl-H), 4.02 (dd, J = 11.1, 5.6 Hz, 1H, Xyl-H), 3.76 (t, J = 10.9 Hz, 1H, Xyl-H), 2.02 (s, 3H, Ac-H), 1.98 (s, 3H, Ac-H), 1.81 (s, 3H, Ac-H). Compound 8A 13C NMR (126 MHz, DMSO-d6). δ 170.23, 170.16, 169.39, 167.69, 140.62, 132.92, 128.34, 127.92, 126.69, 126.54, 81.14, 73.07, 72.18, 68.99, 64.59, 21.15, 21.00, 20.80. ESI MS m/z: 458.0339 [M + H]+, 480.0153 [M + Na]+. Compounds 8B, 8C, and 8D were prepared according to the procedure described for the synthesis of 8A by starting from 7 (4 mmol) and Et3N (8 mmol) and added into corresponding glycosyl donors 3B, 3C, or 3D (4 mmol) in dry THF (40 mL). The yields were 42.5% (8B), 39.3% (8C), 31.8% (8D). Compound 8B 1H NMR (500 MHz, DMSO-d6). δ 8.05 (d, J = 8.1 Hz, 1H, Ar-H), 7.85 (d, J = 7.3 Hz, 1H, Ar-H), 7.63 (t, J = 7.6 Hz, 1H, Ar-H), 7.42 (t, J = 7.4 Hz, 1H, Ar-H), 6.05 (d, J = 9.0 Hz, 1H, C1-H), 5.52 (t, J = 9.5 Hz, 1H, Glc-H), 5.17 (t, J = 9.3 Hz, 1H, Glc-H), 5.10 (t, J = 9.8 Hz, 1H, Glc-H), 4.29 (m, 1H, Glc-H), 4.10 (dd, J = 6.9, 3.7 Hz, 2H, Glc-H), 2.02 (s, 6H, Ac-H), 1.95 (s, 3H, Ac-H), 1.82 (s, 3H, Ac-H). Compound 8B 13C NMR (126 MHz, DMSO-d6). δ 170.51, 169.97, 169.88, 169.28, 167.48, 140.48, 132.78, 128.17, 127.81, 126.55, 126.41, 80.12, 73.25, 73.07, 71.97, 68.33, 62.35, 21.04, 20.93, 20.78, 20.65. ESI MS m/z: 530.0548 [M + H]+, 552.0361 [M + Na]+. Compound 8C 1H NMR (500 MHz, DMSO-d6). δ 8.02 (d, J = 8.1 Hz, 1H, Ar-H), 7.85 (d, J = 7.0 Hz, 1H, Ar-H), 7.64 (t, J = 7.6 Hz, 1H, Ar-H), 7.43 (t, J = 7.8 Hz, 1H, Ar-H), 6.00 (d, J = 8.8 Hz, 1H, C1−H), 5.44 (dd, J = 10.1, 3.4 Hz, 1H, Gal-H), 5.35 (d, J = 2.9 Hz, 1H, GalH), 5.31−5.26 (m, 1H, Gal-H), 4.53 (t, J = 6.3 Hz, 1H, Gal-H), 4.11 (dd, J = 11.4, 5.6 Hz, 1H, Gal-H), 4.00 (dd, J = 11.4, 7.0 Hz, 1H, GalH), 2.18 (s, 3H, Ac-H), 1.99 (s, 3H, Ac-H), 1.93 (s, 3H, Ac-H), 1.84 (s, 3H, Ac-H). Compound 8C 13C NMR (126 MHz, DMSO-d6). δ 170.39, 170.36, 169.93, 169.38, 167.33, 140.26, 132.81, 128.19, 127.83, 126.46, 126.45, 2945



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primary monoclonal antibody 2B6 (AMSBIO, UK) for CS or monoclonal antibody 3G10 (AMSBIO, UK) for HS overnight at 4 °C followed by incubation with 0.1% Alexa Fluor 488 labeled secondary antibodies (Beyotime, PRC). The nuclei were stained with DAPI for 15 min. Double-labeled cells were preserved in PBS at room temperature and observed and photographed at 459 and 488 nm excitation and emission wavelengths by a laser scanning confocal microscope (Zeiss LSM 710, GER). Detection of GAGs with Immunostaining by Flow Cytometry. CHO pgsA-745 cells were seeded on glass coverslips in complete cell culture media overnight before treated with phenyl-β-D-xyloside or 8A at the concentration of 10 μM with 0.1% DMSO in cell culture media for 24 h at 37 °C. After that, the media were removed, and the cells were harvested by brief trypsinization and fixed with 4% formaldehyde in complete media for 15 min. The fixed cells were washed twice and incubated in PBS, 1 mU chondroitinase ABC (BIOKANGTAI, PRC), or 1 mU heparinase I, II, III (BIOKANGTAI, PRC), respectively, for 20 min at 37 °C. After washing with PBS, the cells were incubated in 0.5% primary monoclonal antibody 2B6 (AMSBIO, UK) for CS or monoclonal antibody 3G10 (AMSBIO, UK) for HS overnight at 4 °C followed by incubation with 0.1% Alexa Fluor 488 labeled secondary antibodies (Beyotime, PRC). After 15 min of incubation, the cells were washed twice with 1 mL of PBS and resuspended in 100 μL of PBS. Flow cytometry was performed with Beckman cell analyzer FC500mpl. Detection of FGF7/GAGs/FGFR2B Ternary Complex Formation by Confocal Analysis. CHO pgsA-745 cells were seeded on glass coverslips in complete cell culture media overnight before being treated with phenyl-β-D-xyloside or 8A at the concentration of 10 μM with 0.1% DMSO in cell culture media for 24 h at 37 °C. After that, the media were removed, and the cells were fixed with 4% formaldehyde in complete media for 15 min. The fixed cells were washed twice and incubated in PBS or sequentially in FGFR2β/Fc, FGF7, and Protein A-Alexa Fluor 647. FGFR2B-Fc is a soluble protein containing extracellular domain of FGFR2B and human Fc domain of IgG1 (Pro100-Lys333) that can be recognized by florescence-labeled protein A or florescence-labeled antihuman IgG antibody. The fluorescence-tagged FGFR2β/Fc was observed and photographed at 665 nm excitation wavelength bya laser scanning confocal microscope (Zeiss LSM 710, GER). Glycochemistry. Purification of GAGs. CHO pgsA-745 cells were seeded on 15 cm diameter tissue culture dishes. After 24 h, cells were treated with phenyl-β-D-xyloside or 8A at the concentration of 10 μM in cell culture media containing 0.1% DMSO for 48 h at 37 °C. Cells were lysed in 100 mM sodium hydroxide for 8 h at 4 °C. After neutralization, the amount of protein was determined by BCA Protein Assay Kit,51 and each sample was digested with 0.83 g/L engineering protease (ChongQingTianShi, PRC) in pH 6.0 for another 8 h. GAGs were isolated by anion exchange resin and precipitated in 80% alcohol. Mono- and Disaccharide Analysis. For monosaccharide analysis, each purified GAG sample was hydrolyzed in 500 μL of 6 M hydrochloric acid sealed in an ampule at 100 °C for 3 h to generate respective monosaccharides. The hydrolysates were tagged with 1phenyl-3-methyl-5-pyrazolone (PMP) (Sigma) for 1 h at 70 °C. The labeled samples are analyzed by HPLC (Agilent) with ZORBAX SBC18 column. Labeled monosaccharide samples were eluted by using 83% 0.1 M phosphate buffer (13.6 g of KH2PO4, 1.8 g of NaOH, pH 6.7) and 17% acetonitrile. The UV signal of PMP labeled monosaccharide was detected at 245 nm. For disaccharide analysis, each purified GAGs sample was digested by 1 mU chondroitinase ABC (BIOKANGTAI, PRC) or 1 mU heparinase I, II, III (BIOKANGTAI, PRC) in a final volume of 50 μL in 5 mM NH4Ac, pH 7.0, at 37 °C for 24 h. The digested GAGs were labeled by D-PMP in ammonium hydroxide, and the phenyl-β-D-xyloside or 8A treated ones were labeled by PMP. In order to make a quantitative analysis, a portion of GAGs corresponding to 1 mg of cellular total proteins was measured out from every PMP labeled xyloside-treated sample and mixed with D5-PMP labeled control samples corresponding to the same amount of cellular proteins. The mixed differentially labeled GAGs were made up to constant volume and monitored with LC−MS (Thermo)

equipped with Agilent 1200 infinity capillary liquid chromatography system and a ZORBAX Stable Bond chromatogram column (5 μm, 0.5 × 250 mm). Solvent A was 10 mM ammonium acetate (pH 5.0); solvent B was 100% acetonitrile. For HS analysis, the gradient used was 17−22% B in 60 min and 23% B for another 15 min and equilibrated with 17% B. For CS analysis, the gradient used was 13% B in 30 min and 20% B for 10 min, and equilibrated with 13% B. The flow rate was 10 μL/min. The UV absorbance of PMP labeled products was monitored at 245 nm by using an online UV detector, and the total ion chromatogram (TIC) was collected using a Thermo LTQ-XL mass spectrometer operating in negative ion mode by scanning the m/z ranging from 300−2000. The data were analyzed in Xcalibur software (Thermo).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b01835. Supporting Tables; HPLC, NMR, and HRMS spectra (PDF) SMILES (CSV)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel: +86 532 82033054 *E-mail: [email protected]. Tel: +86 532 82031615. ORCID

Yang Tang: 0000-0002-4635-1223 Tao Jiang: 0000-0002-6590-5041 Author Contributions ⊥

Y.T. and S.Z. contributed equally to this work. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Professor Jeffrey D. Esko of UCSD for providing us with phenyl-β-D-xyloside and CHO pgsA-745 cells. This work was supported by the National Natural Science Foundation of China (Grants 21171154 and 81672585), Key Technology Fund of Shandong Province (Grant 2016ZDJS07A07), the Taishan Scholar Fellowship, and the “Double First Class fund” of Shandong Province in China to L.Z.

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ABBREVIATIONS USED GAG, glycosaminoglycan; HS, heparan sulfate; CS, chondroitin sulfate; GlcN, glucosamine; GlcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine; GlcA, glucuronic acid; IdoA, iduronic acid; Gal, galactose; Xyl, xylose; DUA, 4,5-unsaturated hexuronic acid; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; PMP, 1-phenyl-3-methyl-5-pyrazolone; 5D-PMP, 1-(phenyl-2,3,4,5,6-deuterated)-3-methyl-5-pyrazolone; DMSO, dimethyl sulfoxide; DAPI, 4′,6-diamidino-2phenylindole; FITC, fluorescein isothiocyanate; PI, propidium iodide 2946



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